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Maternal-embryonic interactions during early pregnancy in cattle

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Maternal-embryonic interactions during early pregnancy in cattle
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Binelli, Mario, 1968-
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English
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xiii, 291 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Antibodies ( jstor )
Cattle ( jstor )
Embryos ( jstor )
Endometrium ( jstor )
Oviducts ( jstor )
Phosphorylation ( jstor )
Pregnancy ( jstor )
Receptors ( jstor )
Secretion ( jstor )
Uterus ( jstor )
Animal Science thesis, Ph.D ( lcsh )
Cattle -- Pregnancy ( lcsh )
Dissertations, Academic -- Animal Science -- UF ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1999.
Bibliography:
Includes bibliographical references (leaves 264-290).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Mario Binelli.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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MATERNAL-EMBRYONIC INTERACTIONS DURING
EARLY PREGNANCY IN CATTLE











By

MARIO BINELLI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
























Dedicated to Mario Rodolpho Giovanni Binelli (late) and Arnaldo Monteiro

de Oliveira (Grandfathers), Guilherme Jose Binelli (Father), Ricardo Binelli

(Brother), Luiz Alberto de Oliveira (Uncle), Cicero Spiritus, Paul Campbell, Zilmar

Ziller Marcos, H. Allen Tucker and William W. Thatcher (Mentors)

To my family and friends

and

To an enlightened humankind













ACKNOWLEDGMENTS


I would like to express my greatest appreciation to Dr. William Thatcher,

my supervisory committee chair, for giving me the honor of being his student and

friend. His dedication, generosity, enthusiasm, sincerity, intelligence and ability

to excel under a lot of pressure are remarkable and a great source of inspiration.'

I am proud of being a member of one of the most respected reproductive

physiology laboratories in the world. I also want to thank him for his patience

when I was away from the lab pursuing extracurricular activities. My gratitude is

extended to the other members of my committee: Dr. William Buhi, for changing

my abstract conception of what was a protein to something more real like a spot

in a 2-D gel; Dr. Peter Hansen, for being my "second advisor", sharing his

knowledge and laboratory; Dr. Howard Johnson, for asking me "so, what is

novel?" when I first described my dissertation project to him, and for letting me

work in his laboratory during the beginning of my program; and to Dr. Frank

Simmen, for his advice on molecular matters and for the being such an example

of a humble personality behind a powerful mind.

I am also indebted to Dr. Prem Subramaniam, Dr. Lokenga Badinga, Dr.

Rosalia Simmen, Dr. Naser Chegini, Dr. Joel Yelich, Dr. Maarten Drost, Dr.

Herbert Head, Dr. Michael Fields and Dr. Nancy Denslow for sharing their








laboratory and/or scientific expertise.

I wish to thank Dr. Michael Roberts, Dr. Thomas Hansen and Dr. Douglas

Leaman for providing important reagents used for the research described in this

dissertation.

I want to express my eternal gratitude to Dr. Thais Diaz, Dr. Eric Schmitt,

Dr. Alice de Moraes, Fabiola Paula-Lopes, Dan Arnold and Ricardo Mattos for

closely supporting me through the struggles of graduate school and for providing

their sincere friendship and scientific support.

I am grateful to fellow graduate students, post-docs, visitors and friends in

the departments of Dairy and Poultry and Animal Science, including Dr. Luzbel

de la Sota, Dr. Divakar Ambrose, Dr. Joan Burke, Dr. Maria de Fatima Pires, Dr.

Sandra Coelho, Ellen Van de Leemput, Jim Hampton, Nina Nusbaum, Monte

Meyer, Frederico Moreira, Aydin Guzeloglu, Metin Pancarci, Flavia Lopes,

Cassia Orlandi, Jan Vonk, Arthur Araujo, Dr. Carlos Arechiga, Dr. Lannett

Edwards, Morgan Peltier, Yaser AI-Katanani, Rocio Rivera, Saban Tekin,

Andrew Majewski, Inseok Kwak, Jason Blum, Max Huidsen, Tomas Belloso, Dr.

Alfredo Garcia, Dr. Rafael Roman, John Fike, Dr. Maria Cadario, Andres

Kowalski, Dr. Karen Reed, Dr. Michael Green, Michael Porter and Andy Kouba.

From helping me to solve some unearthly statistics to sharing a smile their

contributions will not be forgotten.

I want to thank, for their technical, scientific and professional expertise,

Marie-Joelle Thatcher, Idania Alvarez, Frank Michel, Jesse Johnson, Jennifer








Williams, Lauren Knickerbocker, Mary Ellen Hissen, Susan Gottshall, Larry

Eubanks, Eddie Fredriksson, Mary Russell, Dale Hissen, James Lindsey, Chris

Wilson, Joyce Hayen, Werner Collante, Kathy Austin, Debbie Akin, Stephany

Suggs, Peggy Briggs, Melissa Thomas, Patricia Hancock, the Dairy Research

Unit crew, the Large Animal Clinic crew, students and residents at the School of

Veterinary Medicine and the Meat's Laboratory crew.

As a important part of my University of Florida experience I wish to

express thanks for the generosity of Ms. Louise Curtelis, the hard work and

comradery of fellow officers and friends at the Graduate Student Council and the

Brazilian Student Association and the financial support from the Animal

Molecular and Cell Biology program during the first year of my graduate studies.

One true friendship is worth one thousands PhDs, so I want to show my

sincere gratefulness to all friends in Gainesville, the ones mentioned above and

also Mauricio, Ilka, Carlos da Costa, Patricia, Dirceu and Ana Clara, Daniella and

Fabiano, Ronaldo and Valeria, Cleisa and Cartaxo, Lawry, Claudio and

Cristiane, Tom e Jose Melvin, Ricardo Harakava, Cristina and Warley, Annie e

Wigberto, Debbie and Ken, Joe and Kirsten, Karina e Uilson, Liana and Silvano,

Deise and Alfredo, Raul and Helena, Michelle, Mary Duryea, Paul and Joan and

my friends at the School of Tai Chi Chuan, Marcus Harvey, Dorota Porazinska,

Stacey Chestain, Joe Sadek and Jackie Wilson .

Distance makes one appreciate the value of a family. I would have never

been able to complete my graduate studies without the endless support of Nice,








Arnaldo, Aurora, Lola, Dinda, Dindo, Carmen, Maga, Guilherme, Paula, Neco,

Ana, Rico, Beto, Aldo, Ines, Janete, Maria Helena, Farjala (late), Lenita, Lucia,

Elisa, Leandro, Lucio, Elisa Kampf, Lolo Kampf, Claudia, Gabi e Yara. I Thank

them for their dedication, faith, love, patience, inspiration, support and

encouragement.

Most specially of all, with my whole heart I wish to thank my wife Nana, for

helping me to externalize the best of my being and for being side-by-side with

me on this long road.














TABLE OF CONTENTS


ACKNOWLEDGMENTS ........................................... iii

ABSTRACT ............................................... xi

CHAPTERS

1 INTRODUCTION ................... ..... .. ............ 1*

2 LITERATURE REVIEW ..................... ............. 3

Maternal-embryonic Communication as a Requirement for Successful
Pregnancy ........................................... 3
Communications Between Gametes and Maternal Units ....... 4
Communications Between Conceptus and Maternal Units ...... 6
Problems Associated with Fertilization Failure and Embryonic Mortality in
Cattle .......................... ... ............. 9
Susceptible Periods During Pregnancy ..................... 9
Causes of Fertilization Failure and Embryonic Mortality ....... 10
Oviductal Function and Reproductive Failure in Cattle ............. 16
The Oviduct Environment ................ .......... 16
Steroid Regulation and Protein Synthesis .................. 20
Regulation of Reproductive Processes Occurring in the Oviduct
.............. .............................. 23
Oviductal Function and Reproductive Failure in Cattle ........ 25
Uterine Function and Reproductive Failure in Cattle ............... 27
The Uterine Environment .............................. 27
Regulation of Reproductive Processes Occurring in the Uterus
....... .................................... 31
The JAK-STAT Pathway .............................. 47
Uterine-Conceptus Interactions and Reproductive Failure in Cattle
.. ................................ 70
Manipulating Uterine Function to Minimize Embryo Mortality ... 73
Objectives of This Dissertation ............................. 75









3 PERSISTENT DOMINANT FOLLICLE ALTERS PATTERN OF
OVIDUCTAL SECRETARY PROTEINS FROM COWS AT ESTRUS .. 76

Introduction ............................ .. ............ 76
Materials and Methods ................... ........... 79
Materials .......... ........... .... ............. 79
Preparation of Medium ............................ 80
Animals and Treatments ............................. 80
Tissue Culture ................ .................. 82
Two-Dimensional Electrophoresis ..................... 83
Densitometry .................... ... ............. 83
Hormone Assays .................................. 84
Statistical Analysis ............................ 85
Results ............................................. 85
Ultrasonography and Hormone Measurements ............. 85
Incorporation Rate ................................. 88
Fluorography and Densitometry ....................... 88
Discussion .................. .. ...... ............ 95

4 EFFECTS OF BOVINE INTERFERON-TAU ON THE JAK-STAT SIGNAL
TRANSDUCTION PATHWAY AND SYNTHESIS OF PROTEINS IN
BOVINE ENDOMETRIUM AND ON THE MECHANISM OF
GENERATION OF PROSTAGLANDIN F2a IN ENDOMETRIAL
EPITHELIAL CELLS ..................... .............. 103

Introduction ..................... ................... 103'
Materials and Methods ............................ .. 105
Materials ........... ........ .. .... ............ 105
Experiment 1 .......... ............. ............ 107
Experiment 2 ........................ ............ 114
Statistical Analysis ........................... 117
Results ............ .................. ............. 117
Experiment 1 ........................... ...... 117
Experiment 2 .......... ............ ............. 121
Discussion .......... ......... .. .... .............. 123

5 BOVINE INTERFERON-TAU STIMULATES THE JAK-STAT PATHWAY
IN BOVINE ENDOMETRIAL EPITHELIAL CELLS ............... 133

Introduction ..................... ................... 133
Materials and Methods ....................... .... 135
Materials ................... ... .. ............ 135

viii








Cell Culture and Cell Extracts .......................... 136
Immunoprecipitation ............................... 142
Immunoblots .................. ..... ........... 142
Nature of BEND Cells ................................ 143
Dose Response to bIFN-r ........................... 144
Time Response to blFN-c ............................. 145
Validation of Immunoprecipitation and Immunoblots Procedures
............................................ 145
Nuclear Translocation of STATs ........................ 146
Coimmunoprecipitation of STATs ....................... 147
Densitometric Analysis .................. ......... 147
Statistical Analysis ............................... 148
Results .................................... ...........148
Nature of BEND Cells ................................ 148
Validation of Immunoprecipitation and Immunoblots ......... 151
Dose Response to blFN-r ........................... 151
Time Response to bIFN-r ............................. 156
Nuclear Translocation of STATs ........................ 156
Validation of Time Responses to bIFN-T ................. 167
Coimmunoprecipitation of STATs ....................... 167
Discussion ................ ........................... 173

6 BOVINE INTERFERON-TAU STIMULATES BINDING OF STAT
PROTEIN COMPLEXES TO DNA AND STIMULATES SYNTHESIS OF
INTERFERON RESPONSE FACTOR-1 (IRF-1) PROTEIN IN BOVINE
ENDOMETRIAL (BEND) CELLS ............................ 187

Introduction .............. ............ ...... ... .... 187
Materials and Methods .................................. 188
Materials ................ ...................... 188
Probes .......................... ................. 190
Electrophoretic Mobility Shift Assays .................... 192
Immunoblotting for IRF-1 ............................. 195
Statistical Analysis ................................ 196
Results ...................................... ........... 196
Electrophoretic Mobility Shift Assays .................... 196.
Immunoblotting for IRF-1 ............................. 201
Discussion ................... ........... ............ 201






ix








7 INTERFERON-TAU MODULATES PHORBOL ESTER-INDUCED
SECRETION OF PROSTAGLANDIN AND PROTEIN EXPRESSION OF
PHOSPHOLIPASE-A2 AND CYCLOOXYGENASE-2 FROM BOVINE
ENDOMETRIAL (BEND) CELLS ...........................208

Introduction .............................. ............ 208
Materials and Methods ................ ..................... 210
Materials ........................................ 210
Cell Culture and Sample Collection ................... 211-
Radioimmunoassay ............... ............... 212
Preparation of Extracts ............................ 214
Immunoblotting ................................. 214
Experimental Designs ............................. 215
Statistical Analysis ............................... 216
Results ............. ..................... .......... 219
Experiment 1 .................................... 219
Experiment 2 ..................................... 223
Experiment 3 ........................ .............. 227
Experiment 4 ........................ .............. 232
Discussion .............................. ..... ........ 234

8 GENERAL DISCUSSION .................................. 246

LIST OF REFERENCES ........................................ 264

BIOGRAPHICAL SKETCH ................ .................... 291













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


MATERNAL-EMBRYONIC INTERACTIONS
DURING EARLY PREGNANCY IN CATTLE

By

Mario Binelli

August 1999


Chairperson: William W. Thatcher
Major Department: Animal Science

Maternal-embryonic physiological communications are an important

feature of processes in the reproductive cycle. Communications occurring during

oviductal transit of gametes/embryos and during maternal recognition of

pregnancy for maintenance of the corpus luteum (CL) were studied in cattle.

Objectives were 1) to study the distribution pattern of oviductal secretary proteins

secreted by cows bearing persistent or fresh dominant follicles (PDF or FDF,

respectively); 2) to examine the signal transduction system stimulated by bovine

interferon-r (blFN-t) in endometrium; and 3) to characterize the effects of blFN-T

on prostaglandin F,(PGF,) production by bovine endometrial (BEND) cells.

Presence of PDF reduces fertility in cattle. Proteins synthesized from








infundibulum, ampulla and isthmus from oviducts ipsilateral and contralateral to

CL of cows bearing PDF or FDF were examined by two-dimensional

fluorography. Presence of PDF altered distribution of secretary proteins in a

side- and region-specific manner. Changes in the oviductal environment may

contribute to decreased fertility of cows bearing a PDF. Conceptus-produced

blFN-t suppresses endometrial PGF2, pulses in vivo, and is required for

maintenance of pregnancy. The hypothesis was that blFN-r stimulated

synthesis of endometrial proteins through the jak kinases (JAK)-signal

transducer and activator of transcription (STAT) pathway of signal transduction.

Presence of and bIFN-r-induced tyrosine phosphorylation of STAT proteins were

demonstrated via immunoprecipitation (IP) and immunoblotting (IB) techniques,

while blFN-r-induced secretary proteins were measured by fluorography in

endometrial explants obtained from day 15 cyclic cows. BEND cells were used

for remaining experiments. Presence of STATs, tyrosine-phosphorylation, dimer

complex formation and nuclear translocation were measured through IP, co-IP

and IB. Binding of activated STAT complexes to cis-acting elements present in

the regulatory region of interferon-inducible genes was determined using

electrophoretic mobility shift assays. Bovine IFN-T stimulated synthesis of

interferon-regulatory factor-1 (IRF-1) in BEND cells as determined by IB. The

blFN-t regulates synthesis of phorbol 12,13 dibutyrate (PDBu)-induced PGF2,,

as measured through radioimmunoassays (RIA). Regulation was associated








with blFN-t -induced suppression of phospholipase-A2 and cyclooxygenase-2

protein expression and enzymatic activity measured through IB and RIA.

Collectively, experiments elucidated mechanisms that are involved in maternal-

conceptus crosstalk required for successful reproductive outcome.













CHAPTER 1
INTRODUCTION


The world's human population is increasing at a fast rate, and

consequently the need for basic nutrients, including carbohydrates, lipids,

proteins and minerals, is also increasing. Animal agriculture historically has

been one of the most important sources of nutrients for humans. Dairy cows

efficiently metabolize feed nutrients and synthesize milk, which provides protein

and energy in a suitable form for human consumption. Lactation is a final step in

the reproductive cycle which is dependent on successful production of viable

gametes, conception, pregnancy and parturition. First insemination conception

rates for dairy cattle is 52 to 57% (Mawhinney and Roche, 1978). Improvements

on such rates will be required for dairy products to continue being a viable

source of nutrients for humans.

Classically, study of animal performance has focused on genetic and

environmental effects on a given production trait, such as meat or milk

production. Reproduction poses an interesting scenario, in which reproductive

processes are modulated by the interactions between both the maternal and

embryonic genomes. Moreover, reproductive processes are influenced by

external environment, but more importantly, by the internal environment (i.e., the










reproductive tract). Both maternal and embryonic units influence such internal

environment, and physiological crosstalk is a hallmark of the process. As a

result, a complex set of coordinated interactions takes place and it is these

interactions that will dictate a successful reproductive outcome. My thesis is that

failure in maternal-embryonic communications leads to decreased reproductive

rates.

This dissertation examines the role of maternal-embryonic

communications during two physiological windows of the reproductive cycle and

their importance on the overall reproductive process. Chapter 3 describes

steroid hormone-modulation of protein synthesis and secretion in the oviduct,

where final stages of gamete maturation, fertilization and early embryo

development occur. Chapters 4, 5, 6 and 7 characterize changes in intracellular

and secretary processes of the maternal endometrium in response to a

conceptus-secreted factor, interferon-r, that is essential for maintenance of

pregnancy.

A greater understanding of the basic mechanisms regulating reproductive

processes, specially mechanisms involving maternal-embryonic interactions, is

necessary to improve conception rates in cattle.













CHAPTER 2
LITERATURE REVIEW



Maternal-embryonic Communication as a Requirement for Successful Pregnancy



Communications of a physiological nature are very common between

embryonic (and pre-embryonic) and maternal units from the development of the

oocyte (i.e., before fertilization; Eppig et al., 1997a) to parturition (Bazer and

First, 1983; Fuchs and Fields, 1999) and lactation (Thatcher et al., 1980). The

maternal unit constitutes all tissues in the female reproductive tract that directly

or indirectly interacts with gametes or concepts conceptss = embryo and

associated embryonic membranes). Appropriate exchange of hormonal signals

between the two units is required for successful establishment and completion of

several windows of the reproductive cycle. Moreover, each window requires

unique signals that have been studied in detail and that have unique implications

on the outcome of the reproductive process (i.e., live, viable offspring). A

general review of the literature on several critical windows on the reproductive

cycle will be presented. A more detailed review will be offered on embryonic-

maternal interactions during the time of embryo transit through the oviduct and

during the window of maternal recognition of pregnancy occurring at around day

3








4

17 of pregnancy. The focus of this review will be on the bovine species, but data

from other species will be presented whenever appropriate.


Communications Between Gametes and Maternal Units


Gametes are differentiated cells with the specific function of conveying

genetic information from each paternal and maternal unit to a zygote during the

process of fertilization. To ultimately undergo fertilization, both paternal and

maternal gametes interact with somatic cells in maternal reproductive tissues.

Eqq-oocvte. During development of follicles, oocytes change the program

of granulosa cells in the follicle, so that the default program of mural granulosa

cell differentiation is suppressed. As a consequence, cells surrounding the

oocyte become more specialized in functions that favor development of the

oocyte. In mature Graafian follicles, this layer of differentiated granulosa cells

forms a small pedicle of cells, the cumulus oophurus, which contains the oocyte

and protrudes towards the interior of the antrum. Eppig and coauthors (1997a)

hypothesized that oocytes control their own microenvironment by regulating

differentiation of the supporting cells that are in direct communication with them.

For example, expression of luteinizing hormone (LH)-receptors in cumulus cells

is abrogated by presence of the oocyte (Eppig et al., 1997b).

Oocyte-oviduct. In cattle, the oviduct is divided in three functional regions,'

namely infundibulum (INF), ampulla (AMP) and isthmus (IST; Hafez, 1993a).

The INF is opened to the peritoneal cavity of the body, the IST connects the










oviduct with the uterus, and the AMP is localized between INF and IST. The

initial interaction of the oocyte with the oviduct takes place at the INF. The INF

"picks up" the ovulated oocyte and initiates its transport towards the uterus.

Overall flow of oviductal fluid is towards the body cavity (Hafez, 1993a).

Therefore, in order for the oocyte to be transported to the site of fertilization

(AMP), it must interact with ciliated epithelial cells lining the oviductal lumen.

Balance between oviductal fluid flow and ciliary beating towards the uterus yields

a net movement of the oocyte towards the AMP and ultimately, uterus. During

this trajectory towards the site of fertilization, the oocyte is under the influence of

products secreted by the oviduct which could modulate its development and the

process of fertilization (Buhi et al., 1997a). For example, Kouba and coworkers

(1999) determined that a major secretary protein of the AMP, named POSP

(porcine oviductal secretary protein), has a role in decreasing occurrence of

polyspermy during fertilization in pigs. This supports the findings of Nancarrow

and Hill (1995) that an estrus-associated glycoprotein, a protein homologous to

POSP in sheep, increased blastocyst formation after in vitro fertilization.

Furthermore, Staros and Killian (1998) identified six proteins in oviductal fluid,

including a POSP-like protein that are associated with bovine oocytes in vitro.

Sperm-oviduct. Austin (1951) and Chang (1951) independently reported

that freshly ejaculated rat and rabbit spermatozoa were incapable of penetrating

an oocyte. The ability of fertilization was only acquired after the sperm spent a

period of time in the female reproductive tract, a process called sperm










capacitation. These early findings supported the idea of the necessity of

interaction between male gametes and products of the oviduct prior to

fertilization. In fact, incubation of sperm cells with oviductal fluid capacitated and

sustained sperm mobility in vitro (Parrish et al., 1989; McNutt and Killian, 1991).

Moreover, Boatman and Magnoni (1995) identified and purified an oviductal

factor (oviductin) that acts to enhance sperm penetration in follicular oocytes.


Communications Between Conceptus and Maternal Units


Conceptus-oviduct. Following fertilization, the concepts continues to

interact with the AMP and IST before it reaches the uterus. During this period,

the concepts undergoes initial cell divisions, and there is a possibility of a

continual influence of oviductal products on the concepts. For example, Buhi

and coworkers (1993) showed that gold particles immunoreactive with porcine

oviductal secretary protein are associated with flocculent material in the

perivitelline space surrounding the blastomeres and in the zona pelucida of

embryos from the four-cell stage to blastocysts. Moreover, semi-purified

oviductal specific protein improved cleavage rates of embryos fertilized and

developed in vitro (Hill et al., 1996). In contrast, at least in pigs, presence of

gametes or embryos did not affect production of specific oviductal proteins,

suggesting that a regulation of oviductal function by gametes or embryos is

probably not important during this stage of the reproductive cycle (Buhi et al.,

1990).










A series of growth factors have been identified in the oviduct during the

estrous cycle and early pregnancy in different species (summarized in Buhi et

al., 1997a). For example, Paula-Lopes and coworkers (1999) reported synthesis

and secretion of interleukin-1 p both in oviduct and uterus of cyclic cows.

Moreover, interleukin-1 p stimulated in vitro development of embryos when added

before day 5, which suggests a oviductal effect (Paula-Lopes et al., 1998).

Although limited research has been performed to date to elucidate specific roles

of growth factors in fertilization and early embryo development, it is possible that

growth factors act in an autocrine and paracrine fashion to influence these

processes (Chegini, 1996).

Conceptus-uterus. In cattle, it takes 72 to 84 h from the time of ovulation

to the time embryos enter the uterus (Betteridge and Flechon, 1988). From entry

into the uterus to parturition there are multiple examples of interactions between

the concepts and the maternal unit, which will be discussed elsewhere in this

review. A striking example of such interactions is the process of maternal

recognition of pregnancy associated with maintenance of the CL, which will also '

be presented in detail afterwards. Briefly, maintenance of pregnancy requires a

steady supply of progesterone (P4) from the corpus luteum (CL). In contrast, in

cycling animals, it is necessary that P4 concentrations decrease so that animals

can return to estrus. This decrease is accomplished in response to pulses of

uterine prostaglandin-F2,(PGF2,,) that have lytic actions on the CL. Uterine










physiology must be changed to suppress this pulsatile release of PGF2afor the

concepts to survive. The conceptus-secreted interferon-r (IFN-t) interacts with

the uterine tissue to decrease production of PGF2,, thereby allowing for

maintenance of CL and consequent sustained elevated P4 concentrations.

The process of luteolysis includes the action of follicular estrogen on a P4-

primed uterus, which is capable of secreting PGF2,. Driancourt and coworkers

(1991) formulated the hypothesis that one possible aspect of the antiluteolytic

mechanism induced by concepts could involve attenuation in development of

follicles on the ovary adjacent to the pregnant uterine horn. They determined

that the number of follicles greater that 7 mm was reduced in the ovary

containing the CL after day 22 of pregnancy. Moreover, size of the largest

follicle was greater on the ovary contralateral to the pregnant uterine horn. An

additional study comparing follicular development in pregnant versus

hysterectomized cows indicated that products of pregnancy, either secreted

directly or induced by the concepts, decreased intraovarian follicular

development in a local manner (Thatcher et al., 1991). This could enhance

embryonic survival by attenuating luteolytic mechanisms (Thatcher et al., 1994b).

The examples above illustrate the common theme of maternal-gametic

and maternal-embryonic interactions, and their occurrences throughout the

reproductive cycle. Failure of appropriate communication between maternal and

embryonic units can lead to disruption of the reproductive cycle and termination

of pregnancy. Next I will examine the issue of embryonic mortality in cattle. In










later sections I will discuss the implications of a failure in maternal-embryonic

cross-talk on embryonic mortality.


Problems Associated with Fertilization Failure and Embryonic Mortality in Cattle



Susceptible Periods During Pregnancy


Calving rates to a single insemination are reported to be 52 to 57% for

dairy cows (Mawhinney and Roche, 1978) despite fertilization rates of about 89%

(Henricks et al., 1971). Diskin and Sreenan (1980) utilized beef heifers to

determine embryo survival during discrete periods within pregnancy. They

reported up to 93% survival rates to day 8, 66% to day 16 and 58% to day 42.

These data indicate that minor losses are due to fertilization and embryonic

death before day 8, which encompasses the period of embryo permanence in

the oviduct and development to the blastocyst stage in the uterus. However, in a

group of infertile cows, there are appreciably greater losses (-40%) due to failure

of fertilization (Tanabe and Cassida, 1949) and additional losses by day 35

(40%; Ayalon 1978). In normal cows, a large percentage of embryos is lost

between days 8 and 16 of pregnancy, which is the period of embryonic

elongation and maternal recognition of pregnancy associated with CL

maintenance. The substantial losses of pregnancies during the first 16 days of

pregnancy has obvious economic impacts in the livestock industry, and

represent an opportunity for animal scientists and reproductive physiologists to










improve calf crops. This prompted a great quantity of research to understand

mechanisms involved in the processes of fertilization failure and embryonic

mortality. Moreover, understanding the mechanisms underlying these alterations

in reproductive development will provide the basis for creation of technologies

aimed to attenuate fertility problems in the field.


Causes of Fertilization Failure and Embryonic Mortality


Inadequate embryo unit. Both genetic and environmental effects can

account for early embryonic deaths. There is an estimated 7.5% death of early

bovine embryos because of occurrence of chromosomal abnormalities (Wilmut et

al., 1986). Such abnormalities may be inherited or arise during meiosis,

fertilization or early cleavage stages (King, 1985).

Environmental effects such as heat stress also decrease the ability of

embryos to develop properly. For example, Putney and coworkers (1989)

exposed superovulated heifers to hyperthermic conditions for 10 hours after the

onset of estrus. There was no difference in the rate of fertilization between heat-

stressed and non-heat stressed heifers (control). However, only 12% of

embryos recovered from stressed heifers were normal vs. 69% of embryos

recovered from control heifers. Since the period of heat stress was administered

prior to ovulation and fertilization, it was hypothesized that the detrimental effect

of the heat stress was exerted on the oocyte within the follicular environment. In

an attempt to investigate the effects of heat stress on embryonic survival from










the time of ovulation to 7 days after estrus, Putney and coworkers (1988a) kept

heifers either in thermoneutral or hyperthermic conditions starting 30 hours after

onset of estrus. They found similar results to the ones described above, where

heat stress increased the proportion of abnormal embryos compared to controls.

To pinpoint critical stages of susceptibility of embryos to elevated maternal

temperatures, Ealy and coworkers (1993) submitted cows to heat stress on days

1, 3, 5 or 7 of pregnancy. Embryos recovered on day 8 were compared to

embryos from cows not heat stressed. Only heat stress at day 1 caused

decreased development. Collectively, data from these three heat stress

experiments suggest that environmental factors can affect embryo development

in multiple stages. Alternatively, it is possible that the toxic effects of heat stress

are exerted in the oviductal and uterine environments, which could become sub-

optimal for fertilization and embryo development, resulting in abnormal embryos.

As a final example of environmental effects on embryo development, Putney and

coworkers (1988b) incubated day 17 conceptuses at normal (39 C, 24 hours) or

high (39 C, 6 hours; 43 C, 18 hours) temperature regimens and measured de

novo protein synthesis by these conceptuses. They found that heat stress not

only decreased overall protein synthesis, but more importantly, decreased

secretion of IFN-T. This indicates that under the influence of heat stress,

embryos are less capable to sending appropriate signals to the uterus, which are

required for maintenance of an environment conducive to pregnancy.








12

Inadequate maternal unit. As mentioned above, results from the work of

Putney and coworkers (1988a; 1989) suggest that the elevated number of

abnormal embryos in heifers that underwent heat stress could be a result of the

effect of high temperatures on the maternal unit, and not a direct effect on the

embryo. In that regard, embryos produced by in vitro maturation/fertilization

techniques that are exposed to elevated temperatures (40.5 C) for 12 hours had

development comparable to that of controls (56% blastocyst formation; Rivera,

Lopes and Hansen, personal communication), supporting the concept that heat

stress effects on maternal units may create a toxic environment that is conducive

to development of abnormal embryos. Moreover, in the experiment of Putney

and others (1998b), they incubated endometrium explants removed from cows at

day 17 of the estrous cycle at 39 or 43 C as described above, and measured

secretion of PGF2 in the medium. Heat stress caused a pronounced increase in

PGF2 production over time compared to controls. This finding suggests that

heat stress favors luteolysis and consequent loss of pregnancies.

Measurements of P4 concentrations in milk following insemination of dairy

cows revealed that inseminated-pregnant cows had slightly higher P4 compared

to inseminated-non-pregnant cows (Lamming et al., 1989). This finding

prompted the hypothesis that luteal insufficiency could be a cause of increased

embryonic mortality in lactating dairy cows. Possible causes of decreased luteal

function include (1) poor development of the ovulatory follicle, resulting in a low

quality CL (i.e., low weight and consequent low P4 secretion) and (2) insufficient .








13

luteinizing hormone (LH) support of continuous luteal P4 secretion. Strategies to

provide supplemental P4 through administration of exogenous P4 (Van Cleef et

al., 1996), use of human chorionic gonadotropin (hCG; Schmitt et al., 1996a),

gonadotropin releasing hormone (GnRH) injections (Schmitt et al., 1996b), and

GnRH implants (Ambrose et al., 1998) have increased circulating concentrations

of P4 but yielded mixed results on pregnancy rates.

Asynchronv between embryonic and maternal units. Embryo survival may

be impaired because of failure in some aspects of the relationship between the

embryonic and maternal units, despite the fact that both are normal (Wilmut et

al., 1986; Thatcher et al., 1994b). An example of such failures is lack of

synchrony between uterus and embryo. During early pregnancy, embryo

development depends upon a sequence of changes in the uterine secretions,

which in turn is dependent on progressive changes in the maternal hormonal

milieu. This phenomenon became established when it was observed that

embryos transferred between animals that were not in estrus at the same time

caused abnormal development and death of the embryo (Wilmut and Sales,

1981). Moreover, when cows were treated with P4 from days 1 to 5 of the

estrous cycle and received a day-8 embryo on day 5, pregnancy was

maintained, indicating that uterine development had been advanced as a result

of the exogenous P4 (Geisert et al., 1991). A condition that can cause

asynchrony is exposure of cows to heat stress. Biggers and others (1987)

determined that high environmental temperatures between days 8 and 16 of








14

pregnancy caused a 50% reduction in weight of concepts compared to control

cows. These retarded embryos may not be able to send the appropriate

antiluteolytic signals to the maternal endometrium, thereby allowing luteolysis

and consequent loss of pregnancy to occur.

Inadequate manipulations of the system. As a means to improve

conception rates in livestock operations, researchers have developed several

management practices that often include manipulations of the endocrine system

of animals. Such practices may sometimes yield unexpected results, including

decreased fertility due to disruption of appropriate maternal-embryonic

communications. One widespread practice is synchronization of estrous cycles.

Estrus synchronization systems are used for artificial insemination, timed

insemination and embryo transfer. Most commonly, synchronization is achieved

with combinations of treatments with PGF2a, progestins and gonadotropin

releasing hormone (GnRH; Thatcher et al., 1996). Synchronization with

progestins is based on the principle that exogenous progestins, such as

progesterone delivered by a controlled internal drug release (CIDR) device, can

maintain a sub-luteal concentration of progestin in blood during a period which

permits CL regression. In the absence of a CL, removal of the progestin source

will result in a synchronized estrus (Macmillan and Peterson, 1993). However,

sub-luteal concentrations of progesterone increase LH pulse frequency, which

stimulates continuous growth of a dominant follicle (Cooperative Regional

Research Project, NE-161, 1996; Savio et al., 1993a; Savio et al., 1993b). This










"persistent" dominant follicle (PDF) is estrogenic, and subsequent fertility, as

measured by conception rate at first service (number of pregnancies / number of

animals inseminated), is lower compared to animals bearing normal DFs [37.1%

vs. 64.8% in heifers, (Savio et al., 1993b); 23.6% vs. 58.2% for cows and heifers

(Cooperative Regional Research Project, NE-161, 1996). Possible explanations

for reduced fertility include alterations in the oocyte and /or in the oviductal

environment. In a study by Ahmad et al. (Ahmad et al., 1995), embryos

recovered at Day 6 of pregnancy from cows bearing PDF were less developed

(i.e., were less able to reach the 16-cell stage) than embryos from cows

ovulating a fresh dominant follicle (FDF). In addition, Revah and Butler (1996)

showed that oocytes recovered from PDF showed expanded cumulus cells and

condensed chromatin dispersed in their ooplasm. In contrast, compact cumulus

cells and intact germinal vesicles were found in oocytes from FDF. Thus, the

PDF may induce premature oocyte maturation and/or alter oviduct function,

which could affect early embryonic development and decrease fertility.

Processes of sperm capacitation, fertilization and early embryonic development

in this altered oviductal environment can contribute to decreased fertility

experienced by cows developing persistent follicles.










Oviductal Function and Reproductive Failure in Cattle



The Oviduct Environment


The oviduct environment can be simplistically described as presenting

physical and chemical characteristics which are conducive to the reproductive

processes occurring within the oviduct. Physical and chemical characteristics

are described below in the sub-sections "Functional anatomy and morphology"

and "The oviduct fluid", respectively. To exemplify the functions of these

characteristics, Rieger and others (1995) examined development of embryos in

vitro, either in coculture with oviductal cells (both physical and chemical

influences) or in serum-free medium pre-conditioned by oviductal cells (chemical

influences, only). In both systems, embryos reached the 4-cell stage in 48

hours. However, embryos developing in the coculture system reached the

blastocyst stage 24 h before the others and also had significantly more cells.

There was no treatment where only physical factors were present, but the

conclusion from their data is that probably both physical and chemical

characteristics are necessary for best embryo development.

The ovarian cycle. There is a close association between oviduct function

and concentrations of circulating ovarian steroid hormones. Therefore, it is

appropriate to describe the changes occurring in such hormones during the








1T

estrous cycle. Other aspects of the estrous cycle will be discussed elsewhere in

this chapter.

The ovarian cycle consists of cyclic growth and demise of two ovarian

structures, the follicle and the CL. Considering one estrous cycle the period

comprised between two ovulations, two to three follicular waves of dominant

follicle growth occur (Savio et al., 1988; Sirois and Fortune, 1988). Each

follicular wave is comprised of periods of recruitment, selection, dominance and

turnover or atresia. The ovulatory follicle generated in the last wave does not

turn over, but ovulates. The main steroid secretary product from follicles are

estrogens, such as E2. There is a positive relationship between size of follicles

and E2 concentration in the circulation. Since maximum growth of follicles occurs

during the dominance phase, the peri-ovulatory period is characterized by

highest concentrations of circulating E2 during the estrous cycle. The ovulated

follicle undergoes functional and structural changes to form a CL. The CL grows

at a rapid rate to reach a maximum size in about 11 days, remains at its

maximum size until about day 16 of the estrous cycle and then regresses (the

process of luteolysis). Parallel to changes in CL size are changes in secretion of

luteal P4. Turnover of the dominant follicle is associated with high concentration

of P4, typical of mid-cycle. In contrast, final differentiation and growth of the

ovulatory follicle prior to ovulation only occurs in a low P4 environment.

Functional anatomy and morphology. The oviducts are suspended in the

mesosalpynx, a peritoneal fold of the broad ligament. As mentioned earlier, the










oviduct can be divided into three functional regions: the funnel shaped

abdominal opening near the ovary INF, which terminates in the fringe-like

fimbriae; the more distal dilated AMP and the IST, the narrow proximal portion of

the oviduct, connecting to the uterus (Hafez, 1993a). The oviduct can be simply

described as a muscular tube with a mucosal lining. There are two muscle

coats: an external longitudinal and an internal circular coat (Leese, 1988).

Thickness of the musculature increases from the ovarian to the uterine end of

the oviduct. Muscular contractions function to mix oviductal contents, aid sperm

transport, help denudate the egg, promote fertilization and regulate egg

transport. Patterns of oviduct muscular contractions vary with the stage of the

estrous cycle, indicating hormonal regulation of this process. Before ovulation,

contractions are gentle, but become more vigorous at ovulation. Muscular

contractions in the ovarian direction are more common than in the uterine

direction (Hafez, 1993a). The oviductal mucosa possesses characteristic folds,

with high, branched folds in the AMP and decreasing heights towards the IST to

become low ridges. The mucosa consists of one layer of columnar epithelial

cells, underlined by a submucosa containing smooth muscle fibers and

connective tissue. The oviductal epithelium contains both ciliated and non-

ciliated, secretary cells. Ciliated cells are most abundant in the INF and least in

the IST. Rate of cilia beating is affected by levels of ovarian hormones, with

maximal activity occurring at the periovulatory period. Cilia beating is

synchronized and toward the uterus. The opposite direction of coordinated cilia








19

beating (towards the uterus) and oviduct muscular contractions (towards ovary)

maintain eggs in constant rotation, which is essential for fertilization and to

prevent oviduct implantation (Hafez, 1993a). Non-ciliated epithelial cells have

primarily a secretary function. They contain secretary granules at their apical

aspect, and these accumulate during the follicular phase of the estrous cycle,

and are released into the lumen after ovulation (Murray, 1992). Treatment of

ovariectomized sheep with E2 stimulates hypertrophy of secretary organelles and

accumulation of granules in non-ciliated cells of the AMP (Murray, 1995).

Oviductal secretions contribute to the formation of the oviductal fluid, discussed

next.

The oviduct fluid. Reproductive processes occurring in the oviduct are

exposed and subjected to regulatory influences of ingredients in the oviductal

fluid. Chemical analyses of the oviductal fluid indicated that it is a mixture of

constituents derived from the plasma, through selective transudation, plus

specific proteins synthesized and secreted by the oviductal epithelium (Leese,

1988). The major classes of components are water, gases (02), electrolytes (Ca,

Na, K, Cl), non-electrolytes (glucose, fructose, complex carbohydrates) and

proteins. Some proteins originate from serum (albumin, immunoglobulins) while

others are synthesized de novo in the oviduct [plasminogen activator inhibitor

(Kouba et al., 1997) and bovine oviductal glycoprotein (Boyce et al., 1990)].

Functions of oviductal fluid electrolytes and non-electrolytes are reviewed in

Leese (1988). De novo synthesized oviductal proteins may affect reproductive










processes such as fertilization, and early embryonic development (Buhi et al.,

1997a). More importantly, changes in the optimal milieu of de novo synthesized,

secretary oviductal proteins may lead to sub-optimal micro-environments

conducive to reproductive failure (Binelli et al., 1999, Chapter 3).


Steroid Regulation and Protein Synthesis


Macromolecules present in oviductal fluid have been suggested to serve

important roles in sperm capacitation (Anderson and Killian, 1994), fertilization

(Boatman and Magnoni, 1995), and early embryo development (Gandolfi et al.,

1989). Therefore, alterations in oviductal biosynthetic activity and protein

synthesis and secretion may affect conception rate. Steroid modulation of

oviductal synthesis and secretion of proteins has been characterized in sheep

(Buhi et al., 1991, Murray, 1993), baboon (Verhage and Fazleabas, 1988) and

swine (Buhi et al., 1989; Buhi et al., 1990). Buhi and others (1989) measured

the biosynthetic capacity of the oviduct (i.e., rate of incorporation of radiolabeled

amino acid precursor into newly synthesized protein) of pigs during the estrous

cycle, early pregnancy and in ovariectomized animals following steroid

replacement (Buhi et al., 1992). These studies indicated that bioactivity of the

oviduct is related to the hormonal status of animals. For example, incorporation

rate of radiolabeled leucine was greater when ovariectomized animals were

treated with E2 compared to P4. These findings were consistent with what was










found with intact animals, where a greater incorporation rate was found in the

periestrus stage of the estrous cycle.

It is important to keep in mind that functional regions within the oviduct

have specific roles probably associated with particular arrays of secretary

products. Thus, it is expected that different steroid environments (e.g., estrous

cycle vs. pregnancy) have distinct effects on each oviductal region,

characterizing a biosynthetic gradient of proteins across regions. For example,

in studies with bulls (Anderson and Killian, 1994), it has been demonstrated that

culture medium conditioned by IST tissue at estrus capacitated more sperm than

did medium conditioned by AMP. This increase was abolished by heating the

conditioned medium and inactivating proteins before incubation with sperm.

Staros and Killian (1998) showed that four unidentified oviductal proteins and a

P1-like protein (Boice et al., 1990; Binelli et al., 1999; Chapter 3) from non-luteal

oviductal fluid would associate with the zona pellucida, suggesting a modulation

of sperm/egg binding or embryonic development by oviduct-derived proteins.

Biosynthetic protein gradients have been reported in the pig and sheep (Buhi et

al., 1992; Buhi et al., 1996; 28, DeSouza and Murray, 1995; Murray, 1993).

Moreover, DeSouza and Murray (1995) reported differential secretion of a

chitinase-like protein, similar to P1 in response to steroid treatments in sheep,

while Buhi et al. (1996) showed differential expression POSP mRNA among

oviductal regions in pigs.










While some evidence has accumulated for roles of oviductal secretary

proteins on reproductive products, much less is known about roles of embryonic

secretary products. To the best of my knowledge, no reports have focused on

effects of presence of embryo on the pattern of secretary proteins from the

bovine oviduct. Buhi and coworkers (1989), working with porcine oviductal

secretary proteins, failed to demonstrate differences in rate of incorporation of

non-dialyzable, 3H-leucine labeled molecules between pregnant and cyclic

oviducts. Moreover, one-dimensional SDS-PAGE analysis of secretary proteins

did not indicate changes in patterns of de novo synthesized, secretary proteins.

This indicates that presence of the embryo had little effect on modulating

secretion of macromolecules from the oviductal epithelium. In contrast, a recent

report (Wakuda et al., 1999) showed that presence of embryos in mice which

had their uterotubal junction ligated on day 1 of pregnancy, enhanced

implantation rate of embryos transferred to the uterus. This was in comparison

with pseudopregnant mice, which had uterotubal junction ligated before or after

mating with vasectomized males, and, also mated females which had uterotubal

junction ligated before mating (all mice had blastocysts transferred to uterus on

day 4). Embryo-dependent factors have not been identified, but clearly

influenced embryo development in that species.










Regulation of Reproductive Processes Occurring in the Oviduct


As summarized by Hafez (1993b) and discussed by Harper (1982) and

Anderson (1991), transport of unfertilized and fertilized eggs and sperm in the

oviduct is regulated by four primary forces: (1) frequency and force of

contractions of the oviductal musculature, influenced by endocrine and neural

mechanisms; (2) direction and intensity of beating of cilia, which conditions

movement of oviductal fluids; (3) secretary activity of non-ciliated cells, which is

dependent on the E2/P4 ratio; and (4) hydrodynamic properties of luminal fluids.

Changes in these factors are modulated by concentrations of ovarian steroids.

The outcome of these activities is efficient transport of gametes and embryos

and fertilization. Next, I will emphasize the concerted actions of these factors for

the mechanisms of egg pick up and fertilization.

Ega Dick up. At the time of ovulation, there is a noticeable increase in

frequency and amplitude of contractions in the smooth musculature supporting

the oviduct. Contractions of the mesotubarium superior and mesosalpinx draw

the oviduct in a crescent shape and slide the fimbriae over the surface of the

ovary. The fringe-like folds in the INF contract rhythmically to repeatedly touch

the ovarian surface (Hafez, 1993a). This pattern of movements constitutes an

efficient mechanism to pick up ovulated oocytes. Moreover, maximum density of

ciliated cells in the oviduct occur in the INF. During ovulation, the strokes of cilia

in the fimbriated portion of the oviduct are synchronized to propel the oocyte










towards the oviductal lumen. Furthermore, volume of oviductal fluid sharply

increases 2 days before estrus to reach maximum rate one day after estrus,

which coincides with the period of ovulation and reception of oocyte by the INF

(Perkins et al., 1965). Muscular contractions, cilia beating and fluid secretion are

controlled by ovarian steroids, being stimulated by high periovulatory E2

concentrations.

Fertilization. In the cow, oocytes are transported rapidly to the site of

fertilization, above the isthmoampullar junction, where they spend most of their

time in the oviduct (Aref and Hafez, 1973), and then are transported rapidly

through the IST into the uterus (Anderson, 1991). A balance among the effects

of cilia beating, muscular contractions modulated by catecholamines and fluid

flow rate cause this egg "lock up" at the site of fertilization. It has been proposed

that the IST of the cow is contracted throughout estrus, and that norepinephrine

release after estrus causes relaxation of the IST to allow embryo transit into the

uterus (Isla et al., 1989). This is supported by data from EI-Banna and Hafez

(1970), who showed a dramatic change in the surface area of the IST lumen

from estrus to 3 days after estrus (0.06 mm2 to 1.89 mm2). Meanwhile, if the

animal had been inseminated, sperm are migrating up the reproductive tract and

arrive at the IST portion of the oviduct, where their movement is slowed (Hunter

and Wilmut, 1982). It is hypothesized that biochemical and biophysical

properties of the IST may work to impede upward migration of spermatozoa,

including narrow isthmic lumen, viscous isthmic mucous and oviductal








25

musculature contractions (Ellington, 1991; Hafez 1993b). Within the IST, sperm

undergo hyperactivation, which is required for final sperm transport, completion -

of sperm capacitation and the acrosome reaction. Eventually, spermatozoa

become exposed to ampullary fluid, detach from the IST epithelium and continue

migration towards the site of fertilization. The control of concerted, opposite

direction-movement of sperm and eggs at similar times in the oviduct is

intriguing. Perhaps the isthmoampullary junction acts to retain oocytes in the

ampulla, while spermatozoa are allowed to enter the IST (Anderson, 1991). Low

doses of estrogen cause "tube locking", retaining ova at the isthmoampullary

junction, while larger doses promote quick movement through the isthmus and to

the uterus (Hawk, 1988).


Oviductal Function and Reproductive Failure in Cattle


The fact that oviductal function is regulated in the multi-factorial,

integrated fashion described above could lead one to hypothesize that

perturbations in the system could easily lead to reproductive failure. However, in

normal cattle, embryonic losses occurring during the time when the embryo is in

the oviduct are small, relative to other phases, as described above. This could

be interpreted at least in two ways. First, one could say that the oviduct plays

only a passive role on the processes of gamete transport, fertilization and early

embryonic development. In this view, the oviduct would keep default modes of

function (i.e., similar in presence or absence of an embryo), modulated by








26

patterns of steroids. Gametes and embryos would tolerate mild perturbations in

the system and the reproductive processes would be carried out by internal, pre-

determined programs, modestly influenced by the oviductal environment. The

oviduct would basically provide a physical substratum for events to occur. Leese

(1988) suggested that this possibility could be appropriately tested by trying to

culture embryos on an epithelium anatomically related to the oviduct, such as the

trachea (i.e., ciliated, secretary, containing active chloride ion pump). To support-

this first possibility, there is the fact that embryos can be matured and fertilized in

vitro in the absence oviductal cells, tissue or conditioned medium. Alternatively,

one could say that oviduct-gametes/embryos relationships have been optimized

in the course of evolution, to become a robust system, with little chance for

failure. Specific interactions would be required for success of reproductive

processes, including synthesis and secretion of oviductal proteins in a regional

and timely fashion. Moreover, such unique set of proteins would interact with

gametes/embryos to maximize reproductive output. To test this last possibility,

secretary proteins in the oviduct would first need to be identified. Then,

removing specific proteins from the system with use of immunoneutralization,

knockouts, transgenics and anti-sense models for example, should provide

evidence for their importance. For example, addition of specific antibodies for a

hamster E2 -dependent oviduct protein prevents in vitro fertilization (Sakai et al,

1988).










Uterine Function and Reproductive Failure in Cattle



As mentioned before, there is approximately a 30% rate of embryonic

losses occurring from days 8 to 16 of early pregnancy, which represents a period

of uterine localization of the embryo. In this section I will attempt to describe key

aspects of uterine physiology that can be considered when trying to solve the

problem of embryonic mortality during this period.


The Uterine Environment


The uterus is considered to be an extension of the oviduct (Bartol, 1999),

therefore, several of the principles regarding biophysical and biochemical

properties discussed for the oviduct also will apply here. Compared to the

oviduct, bovine embryos will spend a much longer interval of time in the uterus

(280 days vs 4 days, on average; Catchpole, 1991) which permits a much

broader set of communications between the maternal and embryonic units. To

support this notion is the fact that although there are not remarkable embryo-

induced changes between the cyclic and the pregnant oviduct, this is the

opposite for the uterus. The thesis of this section is that presence of the embryo

conditions the uterine environment, to support embryonic development.

The uterine cycle. Similar to the oviduct, the uterus also undergoes

changes dependent on the stage of the estrous cycle in response to changes in

concentrations of ovarian steroids. However, the uterus has the unique role of










controlling length of ovarian cycles (described above) and, as a consequence,

its own uterine cycle. The uterine cycle can be divided into a long progestational

phase and a short, estrogen-dominated phase (Hansel and Convey, 1983). At

the end of progestational phase, the uterus gains the ability to produce and

secrete PGF2,, which acts to cause structural and functional demise of the CL

(McCracken et al., 1971). An immediate consequence of PGF2,actions is a

decrease in circulating concentrations of P4 (Nett et al., 1976). This initiates the

estrogen dominated period that lasts until the next ovulation and formation of

new CL. Controlling CL life span, the uterus controls the ovarian cycle. During

pregnancy, presence of the concepts blocks luteolytic mechanisms so that the

CL remains functional and the uterus remains in a progestational stage until

parturition (McCracken et al., 1984). Similar to oviducts, uterine morphology and

secretary activity are modulated by ovarian steroids, as discussed below.

Functional anatomy and morphology. The uterus is suspended in the

pelvis by the mesometrium, a caudal division of the broad ligament. In cows, the

uterus can be described anatomically in two continuous portions, the gestational

part of the uterus (consisting of uterine horns and uterine body), and the cervix.

Similar to the oviduct, the uterus is a tube-shaped organ, which contains a lumen

(Bartol, 1999). Histologically, a cross section of the uterus reveals an inner

mucosal layer, the endometrium, an adluminal layer of smooth musculature, the

myometrium and an outer, serious peritoneal coat of the uterus, the perimetrium

(Bartol, 1999). For the remaining of this discussion, I will focus on characteristics










and processes occurring in the endometrium. The endometrium is lined with a

single layer of epithelial cells and contains simple, coiled, tubular glands. Glands

are relatively straight at estrus, but become more coiled and complex as

progesterone levels rise as the estrous cycle progresses (Hafez, 1993a).

Glandular secretions, the hystotroph, constitute a nutrient-rich mixture required

for development of the concepts (Bazer and First, 1983), and will be discussed

next. Underneath the luminal epithelium and around the glands is the

endometrial stroma, composed of stromal cells distributed in greater or lower

density patterns, depending on the location. In the cow, between 100 to 150

aglandular ridges are present, the caruncles (Flood, 1991). During pregnancy,

caruncles become attached to specialized areas of the allantochorion of the

concepts, the cotyledons, to form placentomes. Placentomes are units for

exchange of gas and nutrients between maternal and embryonic units. A better

description on the process of attachment and formation of placentomes is given

afterwards in this review. A highly dynamic and organized microvasculature

supplies myometrial and endometrial tissues. They originate from uterine

branches of the ovarian arteries (supply uterine body and uterine horns), uterine

arteries (supply uterine body and uterine horns) and urogenital artery (supplies

caudal uterus and cervix). In the cow, uteroovarian relationships exist in that

demise of the CL (luteolysis) is regulated by the uterine horn adjacent to the

ovary containing CL. Luteolysis is accomplished by countercurrent exchange of

the uterine produced luteolysin, PGF2,, between the uteroovarian vein and the










ovarian artery. The later is coiled about the surface of the uteroovarian vein

(Bartol, 1999). Countercurrent exchange of PGF2,was demonstrated elegantly

by Knickerbocker and coworkers (1996). The authors sampled blood originating

from uterine branch of ovarian artery(UBOA) and facial artery (FA), and

measured changes in PGF2,concentrations in response to a challenge with E2.

There was a greater concentration of PGF2a in UBOA compared to FA, indicating

existence of local countercurrent exchange between uterine venous drainage

and ovarian artery.

The hystotroph. Hystotroph is the secretions present within the uterine

lumen for nourishment of the developing concepts (Roberts and First, 1983). A

broader definition should also include functions such as paracrine regulation of

concepts physiology and development and protection of the concepts from the

maternal immune system. Solymosi and Horn (1994) measured protein content

in uterine milk (i.e., hystotroph) of cows and determined that 73% of the dry

matter content was composed proteinaceous material. Information on nature of

proteins contained in uterine milk is limited in cattle. Electrophoretic analysis

reveled at least nine proteins, seven minor and 2 major, which were identified as

lactoferrin and acid phosphatase (Bazer and First, 1983). Lactoferrin may have

a bacteriostatic function in the uterine luminal environment. The acid

phosphatase has basic PI, which is similar to uteroferrin in pigs. Uteroferrin is

involved in iron transport to the concepts (Roberts and Bazer, 1988), but

whether bovine acid phosphatase has the same role in cattle is unknown.








31

MacKenzie and coauthors (1997) reported expression of retinol-binding protein

(RBP) in bovine uterus, and monitored steroid modulation of expression of this

protein during estrous cycle and early pregnancy. The hypothesized role of RBP

is to regulate transport of vitamin A to the concepts. Finally, expression of

growth factors involved in the growth hormone (GH)-insulin-like growth factor

(IGF) axis were examined by Kirby and others (1996). They reported expression

of IGF-1, IGF-binding protein (IGFBP)-2 and 3 and GH receptor in the uterus.

Collectively, uterine milk has functions similar to those of the oviductal fluid, to

provide an adequate microenvironment for concepts development. As the

embryo develops, it starts to contribute with its own secretions to the pool of

molecules composing the uterine fluid, which becomes more complex and may

exert regulatory functions that influence both the concepts and the uterus.


Regulation of Reproductive Processes Occurring in the Uterus


As mentioned previously, the uterus plays specific roles both during the

estrous cycle and during pregnancy. During the estrous cycle, the uterus

regulates ovarian function and the ovarian cycle and the uterus in turn is

regulated by actions of ovarian steroids. During pregnancy, functions of the

uterus include transport, storage and maturation of spermatozoa, recognition

and reception of embryos, provision of an embryotrophic environment for

concepts development during gestation, and expulsion of fetus and placenta at

parturition (Bartol, 1999). At this physiological state, uterine function is regulated








32

both by ovarian steroids and embryonic bioactive molecules. During pregnancy,

misregulation of uterine function may lead to embryonic mortality. For the

remaining of this section I will focus on the mechanisms regulating PGF2,

production from the cyclic uterus, and on mechanisms of maternal recognition of

pregnancy related to CL maintenance occurring in the pregnant uterus.

Cyclic uterus. The reason for the uterine cycle is to provide repeated

opportunities for pregnancy at relatively short intervals. In practice, the turning

point in the uterine cycle is the commitment to either luteolysis or pregnancy. In

the absence of pregnancy, the progestational stage is finished by the uterus-

induced demise of the P4 source. Demise of the CL is accomplished by pulsatile

secretion of uterine PGF,(Nancarrow et al., 1973; Kindahl et al., 1976). Then,

the estrogen-dominated uterus prepares for reception of sperm, initially, and

reception of the early developing embryo. This cycle repeats until successful

establishment of pregnancy. However, early pregnancies will be terminated if

the uterine cycle is not interrupted.

Secretion of PGF,,. During the estrous cycle, the presence of two distinct

patterns of PGF, release is easily distinguishable: a basal release and a

stimulated, pulsatile release. Initial measurements of PGF2were performed in

samples collected from the venous drainage of the uterus, which required

surgical cannulation (Nancarrow et al., 1973). Measuring peripheral

concentrations of 15-keto-13, 14-dihidroprostaglandin F2,, (PGFM) the main

metabolite of PGF2,found in the circulation, facilitated study of such patterns








33

(Kindahl et al., 1976). It was determined that for most of the estrous cycle, basal

secretion of PGFM ranged from 25 to 70 pg/ml in one heifer and from 60-100

pg/ml in a second heifer. However, around the time of luteolysis, four peaks of

about 500 pg/ml and four peaks of about 250 pg/ml were observed for the first

and second heifers, respectively (Kindahl et al., 1976). Despite the clear among

animal variability in this small experiment, there was an evident decrease in P4

concentrations, from ~5 ng/ml to less than 1 ng/ml, within 24 hours after the first

PGFM peak for both heifers.

Pulsatile release of PGF,,. Generation of PGF2, pulses requires presence

of a stimulatory signal and a responsive uterus. A responsive uterus contains

receptors for the stimulatory signal, functional intracellular pathways to transduce

the stimulus into a secretary pulse and adequate amounts of substrate for PGF2,

synthesis.

In cattle, nature of the stimulatory signal for production of luteolytic pulses

of PGF2, remains unclear. It has been accepted generally that oxytocin is the

major stimulator of PGF, secretion in cattle. Armstrong and Hansel (1959)

demonstrated that exogenous oxytocin caused luteolysis in heifers. Moreover,

injections of oxytocin increased concentrations of PGF2. in the uterine vein

(Milvae and Hansel, 1980) and increased concentrations of PGFM in peripheral

circulation (Lafrance and Goff, 1985) in cows. However, while oxytocin is able to

stimulate PGF2 secretion in these experiments, it remains unclear whether

oxytocin is in fact required for the process of luteolysis. In a recent report,










Kotwica and others (1997) demonstrated that administration of an efficacious

oxytocin receptor antagonist (CAP-527) failed to block normal luteolysis in cows.

Moreover, treatment of endometrial explants from days 16-17 cyclic cows with

oxytocin failed to stimulate secretion of PGF2,, both in static (Arnold et al., 1999)

and in perifusion (Del Vecchio et al., 1990) culture systems. This raises the

possibility that perhaps ligands other than oxytocin are required for luteolysis in

cattle. Alternatively to oxytocin, possible stimulators of pulsatile PGF2.secretion

include E2 and LH.

Irradiation of ovarian follicles, which reversibly eliminates production of

follicular E2, delays luteolysis and extends the length of the estrous cycle

(Hughes et al., 1987). Thus, follicular E2 plays a major role in these events. In

heifers, Thatcher and coworkers (1986) demonstrated that injections of E2 on

day 13 of the estrous cycle stimulated release of PGFM starting 3 hours after the

injection, peaking at 6 hours and returning to basal levels by 10 hours. In

addition, heifers injected with E2 underwent luteolysis 96 hours after injections,

while it took 125 hours for control heifers. To confirm that the PGFM increases

measured in the peripheral circulation represent PGF2 of uterine origin,

Knickerbocker and others (1986) measured a sharp increase in PGF2, in the

uterine vein of cows treated with E2. Collectively, these findings indicate that

both endogenous and exogenous E2 are able to stimulate secretion of PGF2,

and to cause luteolysis.








35

A novel concept regarding control of luteolysis involves the actions of LH

in the endometrium. Friedman et al. (1995) reported the presence of LH binding

sites in bovine endometrium that were maximal in endometrium from days 15 to

17 which corresponds to the time of luteolysis. In addition, production of PGF2,,

was stimulated when endometrial cells from days 15 to17 of the estrous cycle

were treated with LH in vitro. In a series of preliminary experiments (Fields,

personal communication) ovariectomized, P4-treated cows were injected with

either saline or E2 and 4 hours later injected with either saline or human chorionic

gonadotropin (hCG, a long half life LH analog). Concentrations of PGFM in

plasma were elevated only for groups pre-treated with E2. In addition, hCG

injection elicited a pronounced release of PGFM compared to saline. It can be

concluded that exposure to E2 is required for the endometrium to secrete PGFM

in response to LH. Physiologically, it could be hypothesized that E2 acts at the

endometrium to enhance responsiveness to circulating LH, thereby evoking

PGF2, secretion during luteolysis. Indeed, a decline in P4 will elevate plasma LH

that may contribute to a continued secretion of PGF2,to re-enforce the luteolytic

process. Mechanistically, this could be accomplished by increasing

concentration of LH receptors in the endometrium or by connecting intracellular

pathways stimulated by LH with the PGF2, secretary machinery.

In summary, it is doubtful that oxytocin is the sole stimulator of pulsatile

secretion of PGF,,. It is more probable that other effectors such as E2 and LH

act in concert with oxytocin to stimulate luteolysis.










Since the dominant paradigm in the field of luteolysis has been that

oxytocin is the major stimulator of PGF2, pulsatile secretion, experiments to test

presence of a responsive uterus had as an endpoint development of oxytocin

receptors and secretion of PGFa in response to oxytocin. Based on this

paradigm, it has been well established that exposure of the uterus to

progesterone is required for the uterus to acquire responsiveness to oxytocin.

McCracken (1980) proposed that P4 has the ability to inhibit synthesis of E2

receptors, and synthesis of oxytocin receptors is an E2 -dependent process. As

long as P4 inhibits synthesis of E2 receptors, E2 is unable to stimulate synthesis

of oxytocin receptors. Moreover, McCracken and others (1984) suggested that

the uterus eventually becomes refractory to inhibitory effects of P4, allowing

oxytocin receptors to be expressed, which leads to pulses of PGF,. Lafrance

and Goff (1988) treated long-term ovariectomized heifers with P4 for 0, 7, 14 or

21 days then measured PGFM in response to an oxytocin challenge. After 7, 14

or 21 days of P4 -priming there was a significant increase in plasma PGFM after

oxytocin injection, but no increase was noticed in animals that did not receive P4.

Silvia and coauthors (1991) put forth the question of whether requirement for

long term exposure to P4 is due to stimulatory effects that take at least 10 days

to build up (7 days in Lafrance and Goff, 1988) or to slow development of the

condition whereby the uterus is desensitized to P4 inhibitory effects. Collectively,

the concept of a responsive uterus means that this organ has been primed by P4,

and as a result, the uterus becomes responsive to the luteolytic stimulus.










I will next review intracellular pathways for generation of the pulsatile

secretion of PGF2a. Little experimentation has been done to uncover potential

intracellular pathways activated by estradiol or LH, so focus is on the well studied

and established pathway of oxytocin stimulation (Flint et al., 1986; Burns et al.,

1997; Thatcher et al., 1997). Oxytocin receptors start to increase in the P4 -

primed, responsive uterus. Oxytocin originating from the neurohypophyseal lobe

of the pituitary gland binds to the seven transmembrane-domain, G protein-

coupled receptors and activates phospholipase C (PLC). The PLC cleaves

membrane phosphotydilinositol bisphosphate, yielding inositol trisphosphate (IP3)

and diacylglycerol (DAG). The IP3 binds to specific receptors in the endoplasmic

reticulum resulting in release of calcium from internal stores into the cytosolic

compartment. The DAG activates protein kinase C (PKC), leading to serine

phosphorylation of cytosolic, calcium-dependent phospholipase A2 (PLA),

probably through a MAP-kinase dependent pathway (Lin et al., 1993). The IP3-

stimulated increase in cytosolic calcium acts to further stimulate PLA, activity

(Clark et al., 1991). Stimulated PLA2 translocates to the membrane where

phospholipid substrates are located (Clark et al., 1991). Activated, membrane-

bound PLA2 cleaves arachidonic acid (AA) from phospholipids. Free AA is

converted to prostaglandin H2 (PGH2) by the enzyme cyclooxygenase-2 (COX-

2). Prostaglandin F2,synthase converts PGH2into PGF2a, which is then released

into the uterine circulation. In the endometrium, this process occurs

preferentially in epithelial cells compared to stromal cells (Danet-Desnoyers et








38

al., 1994). As discussed beforehand, PGF2,gains access to the ovary through a

counter-current mechanism. Binding of PGF2,to receptors in the CL stimulates

release of luteal oxytocin that in turn binds oxytocin receptors in the

endometrium to elicit further release of PGF,,, characterizing a positive feedback

loop.

Arnold and coauthors (1999) demonstrated that a responsive uterus is not

necessarily a oxytocin-responsive uterus. They incubated endometrial explants

obtained from day 17 cyclic cows with oxytocin or with intra-cellular stimulators of

the PGF2 -generating cascade described above. They showed that despite

oxytocin failure to stimulate PGF2, secretion, the stimulator of PKC activity,

phorbol 12, 13 dibutyrate (PDBu), and the stimulators of PLA2 activity, calcium

ionophore and melittin, were able to induce PGF2, release acutely. This supports

the notion that alternative ligands to oxytocin can play a role on pulsatile

secretion of PGF2,.

A final comment on the role of P4 -priming of the uterus for pulsatile PGF2,

secretion, relates to P4 ability to induce accumulation of lipid droplets in bovine

uterine epithelial cells (Brinsfield and Hawk, 1973). In mice, such lipid droplets

contain phospholipids (Silvia et al., 1991), which are substrate for PLA2 and

source of AA, as mentioned above. Progesterone also induces synthesis of

COX-2 (Raw et al., 1988).

Pregnant uterus. In cattle, the vast majority of embryos are found in the

uterine horn ipsilateral to the ovary where ovulation occurred, indicating a readily










"attachable" embryo, which only migrates minimally within the uterus (Flood,

1991). Embryos undergo rapid morphological changes in the first 3 weeks of

pregnancy. After a series of cellular divisions, formation and hatching of

blastocysts (day 9-10 after ovulation; Betteridge and Flechon, 1988),

conceptuses start to elongate on day 12 (Betteridge et al., 1980), to occupy the

whole length of the uterine horn ipsilateral to the CL by day 17 and to reach the

tip of the contralateral horn on day 21 (Kastelic et al., 1988; Flood, 1991). The

first intimate connection between the concepts and the uterus occurs between

days 18 and 20 of pregnancy, when numerous papillae penetrate the openings

of uterine glands (Guillomot et al., 1981). There is intimate contact of matemal

and embryonic tissues starting with apposition of apical cell membranes of

aligned epithelia from both units. Actual adhesion begins around day 22 and is

completed on day 27 after insemination. Adhesion is characterized by

interdigitation of embryonic and maternal microvilli (Flood, 1991). The next

series of events include development of placentomes and growth of placental

tissues.

Maternal recognition of pregnancy associated with CL maintenance

As mentioned previously, the turning point in the uterine cycle is the

commitment to either luteolysis or pregnancy. In cattle, commitment to

pregnancy is only accomplished if adequate signaling exists between maternal

and embryonic units. Maternal recognition of pregnancy has been defined as

the process by which the periattachment concepts signals its presence to the








40

maternal unit, as reflected by maintenance of the CL (Short, 1969; reviewed in

Hansen, 1991). More specifically, the process of maternal recognition of

pregnancy requires that embryonic molecules interact with the uterine

endometrium and change its program, so that pulsatile secretion of PGF2'is

blocked and thereby luteolysis is impeded. The net result is continuous

secretion of P4 by the CL, which is required for continuation of pregnancy. Roles

of P4 include continuous stimulation of uterine secretions and inhibition of

smooth muscle contractions (Hafez, 1993a). In cattle, the critical period for

maintenance of pregnancy is around day 17 of the estrous cycle. Betteridge and

others (1980) transferred embryos to synchronized recipients and demonstrated

that pregnancy was only maintained if embryos were transferred prior to day 17.

Moreover, inter-estrus interval increased from 20 to 25 days when conceptuses

were removed on day 17 vs. day 15 of pregnancy (Northey and French, 1980).

Based on the model proposed above, pregnancy effects on suppression

of pulsatile PGF2, could be exerted at several levels: (1) suppression of the

PGF2,-releasing stimulus (i.e., oxytocin, LH, E2), (2) alterations of the P4-primed

uterus (i.e., PGF,,-synthesizing machinery), and (3) decrease in substrate

required for PGF,2synthesis (i.e., AA). Another possibility is presence of an

conceptus-induced luteoprotective action, where CL would become less

susceptible to luteolytic effects of PGF2,,. However, since PGF2,,pulses are

effectively blocked during early pregnancy, this possibility will not be considered

in this discussion.










There is good evidence for pregnancy-induced suppression of luteolytic

stimulus in cattle relative to the attenuation of E2 effects. Pregnant cows have

reduced circulating concentrations of E2 (Pritchard et al., 1994), probably as a

result of reduced folliculogenesis (total production of follicles) and decreased

production of E2 per follicle (decreased aromatase activity; Thatcher et al., 1991).

Moreover, in day 18 pregnant cows, administration of E2 stimulates only a

modest increase in PGF2a secretion, indicating that presence of the concepts

attenuates E2 effects (Thatcher et al., 1984).

Regarding alterations on the P4 -primed uterus, Arnold and others (1999)

incubated endometrial explants obtained from day 17 cyclic or day 17 pregnant

cows with intracellular stimulators of PGF2, synthesis, and measured

concentrations of PGF2 secreted into the culture medium. Melittin, PDBu and

calcium ionophore each stimulated release of PGF,2 from explants of cyclic cows

compared to control treatment (medium alone). In contrast, all stimulators

mentioned above failed to induce release of PGF2, in explants originated from

pregnant cows. This indicated that pregnancy affected the intracellular PGF,,-

generating machinery to suppress its ability to stimulate PGF2,,. Interpretation of

these data suggests that pregnancy may have inhibitory effects at each of the

steps stimulated by treatments, which include PKC (PDBu) and PLA2 (melittin,

ionophore). Alternatively, pregnancy may affect a distal, convergence point in

the pathway, for example, at the COX-2 level. Effects on the enzymatic

machinery can be to decrease expression and/or activity of PKC, PLA2and COX-








42

2. In fact, existence of a pregnancy-induced inhibitor of COX-2 activity has been

found in the endometrium of cows (Basu and Kindahl, 1987; Gross et al, 1988).

Danet-Desnoyers and others (1993) identified linoleic acid as the active molecule

in bovine endometrium which acted to decrease COX-2 activity. Moreover,

linoleic acid acted as a competitive inhibitor of AA on a PGF2, generator assay

(Thatcher et al. 1994b). It is possible that altered lipid metabolism in the

pregnant endometrium increases availability of linoleic acid to inhibit COX-2

activity and thereby decrease PGF2, production.

Finally, pregnancy could change lipid composition and metabolism in the

endometrium to inhibit PGF2,synthesis. Thatcher and others (1995) compared

concentrations of free linoleic and free AA in endometrial microsomes from day

17 cyclic and pregnant cows. They found that pregnancy decreased

concentrations of AA and increased concentrations of linoleic acid compared to

estrous cycle, to result in a change of the ratio of linoleic to AA of 0.6 to 2.4 in

endometrium between cyclic and pregnant cows.

Effectors of maternal recognition of pregnancy: IFN-T

A considerable amount of research focused on identification and

purification of concepts products with the PGF2,-secretion inhibitory activity

required for maintenance of pregnancy. A family of molecules has been

identified as the embryonic antiluteolytic factor in ruminants, named IFN-r

(Thatcher, 1999). For a historical prospective, see Martal et al. (1979), Godkin et










al. (1982), Bartol et al. (1985), Helmer et al. (1987), Imakawa et al. (1989) and

Roberts et al. (1992). Isoforms of blFN-T are glycosylated, have molecular

weights between 22 and 24 kD and vary in isoelectric forms between PI 6.3 and

6.8 (Helmer et al., 1987; Anthony et al., 1988).

Antiluteolytic effects of bovine bIFN-t (blFN-r) have been examined both

in vivo and in vitro. Intrauterine infusions of highly enriched blFN-t complex

(Helmer et al., 1989b) and recombinant blFN-r (Meyer et al., 1995) extended

lifespan of CL in cows, compared to control infusions. Moreover, PGF,, release

in response to an oxytocin injection was suppressed in day 17 cyclic cows

infused with recombinant blFN-r compared to controls (Meyer et al., 1995).

Danet-Denoyers and others (1994) tested the ability of blFN-r to suppress

basal and oxytocin-stimulated secretion of PGF,, from primary cultures of

endometrial epithelial cells obtained from day 15 cyclic cows (Danet-Desnoyers

et al., 1994). Twenty four hour-incubation with bIFN-t reduced both basal and

oxytocin-stimulated secretion of PGF2,,. This agrees with data from Meyer and

coworkers (1996) and Xiao et al. (1999). Meyer and others (1996) reported that

endometrial epithelial cells obtained from cows which received intrauterine

infusions of blFN-T secreted less basal and oxytocin-stimulated PGF2, compared

with cows infused with a control protein. Moreover, Xiao and coworkers (1999)

cultured endometrial epithelial cells obtained from cows of days 1 to 4 of the

estrous cycle in presence of oxytocin or a combination of oxytocin and bIFN-r.










Similar to the data described above, blFN-T effectively reduced both oxytocin-

and phorbol ester-stimulated PGF2, secretion. In contrast, Asselin and others

(1998) showed that blFN-T increased secretion of PGF2,from endometrial

epithelial cells from days 1 to 5 of the estrous cycle. However this effect was

only significant when extremely high doses of blFN-r (20 ~gg/ml) were used.

Collectively, these data support the concept that blFN-T interacts with

endometrial epithelium and affects the PGF2,-generating machinery to decrease

PGF2. production. To further test this possibility, Arnold and others (1999)

infused either blFN-r or a control protein (bovine serum albumin) in the uterus of

cows from days 14 to 17 of the estrous cycle. Secretion of PGF2,was measured

in medium conditioned by endometrial explants cultured in presence of specific

intracellular stimulators of PGF2,synthesis. Incubations with calcium ionophore

and PDBu stimulated PGF2,secretion compared to medium alone in

endometrium from control cows, but not from blFN-T -treated cows. In contrast,

melittin stimulated secretion of PGF2from explants originating from blFN-r -

infused-cows. Overall, their data indicated that in vivo treatment with blFN-T

attenuated PGF2, production probably at the level of PKC, since PDBu

stimulation of PGF2,was reduced by blFN-r, whereas melittin stimulated PGF2,

secretion. This is in variance with the ubiquitous inhibitory effects of pregnancy

on stimulated PGF2 secretion (mentioned above), suggesting that other products










of pregnancy, and not blFN-t alone, probably also operate to inhibit PGF2,

production.

In an effort to pinpoint specific enzymes that blFN-r altered in the PGF2,-

generating cascade, Xiao and others (1999) measured messenger ribonucleic

acid (mRNA) and protein expression for COX-2 in endometrial epithelial cells

treated with oxytocin and with oxytocin in combination with blFN-r. Oxytocin

maximally stimulated COX-2 mRNA and protein from 3 to 24 hours compared to "

controls. Treatment with blFN-T reduced this effect of oxytocin, and this was

consistent with a reduction in PGF2,secretion in medium. In contrast, Asselin

and coworkers (Asselin et al., 1997) found that blFN-T actually stimulated

expression of COX-2, which would contradict the antiluteolytic role of blFN-r.

However, they also reported that blFN-T stimulated expression of an endometrial

prostaglandin E2-9-ketoreductase, which catalyzes the conversion of PGF, into

PGE2 (Asselin and Fortier, 1998). Since PGE2 has been shown to have luteo-

protective actions (Pratt et al., 1977), they proposed a model whereby blFN-T

actually-stimulates the PGF, -generating machinery, but a conversion of PGF2,

to PGE2 at the end of the cascade would support an antiluteolytic effect of blFN-

T.

It is expected that in order to stimulate intracellular changes resulting in

decreased PGF2,production, blFN-T needs to stimulate a receptor-mediated

mechanism of signal transduction. Such a mechanism should evoke intracellular








46

second messengers to ultimately regulate molecules involved in the generation

of PGF.,, Such regulation could involve synthesis of proteins inhibitory to the

PGF2 production cycle, or, could acutely activate molecules already present in

the cell to suppress PGF2,stimulatory actions. There is limited information on

the nature of IFN-r receptors. Knickerbocker and Niswender (1989) measured

numbers of unoccupied binding sites for IFN-r in endometrium of cyclic and

pregnant sheep. Number of unoccupied binding sites decreased for both cyclic

and pregnant ewes from day 4 to day 12. Then it increased for cyclic animals,

but was still decreased for pregnant ewes, indicating that blFN-T binding sites

were possibly being occupied by conceptus-secreted IFN-r. Interestingly, affinity

for binding sites increased after day 12 for pregnant ewes but decreased for

cyclic ewes.

Hansen and coauthors (1989) reported use of cross-linking experiments

to characterize association of iodinated ovine IFN-r to membrane peptides.

They identified binding of IFN-T to both 100 and 70 kD membrane polypeptides.

Comparison of binding kinetics of IFN-T with IFN-a in this experiment suggested

existence of different receptors for these two ligands. However, Li and Roberts

(1994) showed a reciprocal displacement of IFN-T and IFN-a from bovine

endometrial cell membranes, suggesting that binding sites for these two

molecules were the same. Recently, Han and Roberts (1998) reported cloning

and characterization of receptors for IFN-T in cattle endometrium. Sequences of








47

receptor subunits IFNaR1 and IFNaR2 are similar to ones utilized by other type I

interferons such as IFN-a. However, these receptors were not linked with

functional data, to demonstrate that such subunits are necessary and sufficient

to suppress PGF,,synthesis.


The JAK-STAT Pathway


The observations above lead to the assumption that blFN-t stimulated a

signal transduction system, the JAK-STAT pathway, similar to other type I

interferons (Schindler et al., 1992; Darnell et al., 1994; Darnell, 1997; Figure 2-

1). In this paradigm, interferon receptors do not contain intrinsic kinase activity,

but they are physically associated with protein tyrosine kinases from the Janus

family (JAK kinases). Binding of interferon to its receptor causes

phosphorylation of tyrosine residues in the JAK kinases and in the cytoplasmic

tail of the receptor. The tyrosine phosphorylated receptor attracts signal

transducer and activation of transcription, or STAT, proteins to close contact.

Members of the STAT family of proteins then become phosphorylated on

tyrosine residues and form homo- and hetero-dimers. Dimerized STATs migrate

to the nucleus where they bind to the specific regulatory elements located in the

promoter region of interferon-regulated genes. In this manner, STAT proteins
































5 6
R1 R2


Nucleus



ISGF-3 A


IISRE
IFN-lnducble gene
STAT-I STAT-2



Figure 2-1. The JAK-STAT pathway of signal transduction and gene activation.
1) Binding of type I interferon (IFN) to the interferon-a-receptor (R)-2 chain causes
recruitment of R1; 2) dimerization of IFN receptor complex causes reciprocal tyrosine
phosphorylation of associated JAK kinases (tyk-2 and jak-1); 3) JAK kinases phosphorylate
receptor subunits in tyrosine residues; 4) unphosphorylated, cytosolic STAT proteins bind
receptor complex through SH2 domains present in STAT proteins; 5) JAK kinases
phosphorylate tyrosine residues in bound STATs, STATs dissociate from receptor complex
and associate in a heterodimer (STATs 1 and 2); 6) dimerized STATs translocate to the
nucleus, bind to the DNA binding protein p48, forming the ISGF-3 transcription complex,
which stimulate synthesis of IFN-inducible genes.










stimulate transcription of genes and synthesis of interferon-specific proteins. I

will next examine characteristics of molecules involved in this pathway, and then

describe evidence for existence of this pathway in the bovine endometrium.

Type I interferon receptors

Type I interferon receptor consists of two chains, IFNaR1 and IFNaR2,

which can be presented in different forms. The IFNaR1 is present as a full chain

(IFNaRla) and as a shorter splice variant (IFNaRls). The IFNaR2 chain exists

in soluble, short and long forms, designated IFNaR2a, IFNaR2b and IFNaR2c

respectively. Probably IFNaRla and IFNaR2c are the predominant forms

(Petska, 1997). Petska (1997) reviewed a series of experiments where the

different IFNaR chains were expressed in Chinese hamster ovary cells, and

ability of different type I interferons to signal through the different chain

combinations was evaluated. There is a remarkable diversity of such

interactions, in which specific interferons can only signal through specific

combinations of chains, but not others. Petska (1997) proposes that differential

expression of individual chains and ability of individual interferons to signal

through specific chain arrangements confers tissue-specific responsiveness to

interferons. For example, Platanias and coworkers (1996a) reported that IFN-p

signaling requires association of IFNaR1 with p100, a tyrosil phosphoprotein,

which was later identified as a particular chain of the interferon receptor complex.

To the best of my knowledge, these types of experiments have not been










conducted in bovine reproductive tissue, to test signaling ability of blFN-r.

Instead of the antiviral assays used in the experiments mentioned above

(Pestka, 1997), functional assays measuring suppression in synthesis of PGF2,

from endometrial cells would be in order. Moreover, existence of a blFN-r-

specific receptor chain remains elusive.

Colamonici and coworkers (1994a; 1994b) demonstrated that the tyrosine

kinase p135t*2, or tyk-2 is associated physically with the IFNaR1 chain of the

interferon receptor. Immunoblots revealed the ability of monoclonal antibodies to

IFNaR1 and to tyk-2 to reciprocally coimmunoprecipitate both proteins.

Association of tyk-2 was mapped to a 46-amino acid juxtamembrane region of

the IFNaR1 chain. Furthermore, they demonstrated that tyk-2 could directly

phosphorylate tyrosine residues in the IFNaR1 chain after stimulation with IFN-a

(Colamonici et al., 1994b). Besides binding to extracellular interferons and

associating with JAK kinases, the interferon receptor complex also has other

functions in the JAK-STAT pathway. The unstimulated IFNaR2 chain may

contain-associated unphosphorylated STAT proteins (Li et al., 1997). Binding of

interferon brings IFNaR2 and IFNaRI, which contains tyk-2, together.

Dimerization of receptor chains elicits transfer of STATs to the IFNaR1, where

STATs become tyrosine phosphorylated. This confirms the previous finding that

tyrosine 466 in the chain of IFNaR1 acts as a docking site for association of the

SH2 domain of STAT-2, and such binding is required for tyrosine








51

phosphorylation of STAT-2 by tyk-2 (Yan et al., 1996). Similar to STAT-2, STAT-

3 activation also requires binding to IFNaR1 (Yang et al., 1996). Furthermore, it

has been demonstrated that phosphotyrosine modules (i.e., sequence of amino

acids surrounding the tyrosine residue in the receptor chain) play a major role in

selecting which STAT binds (Gerhartz et al., 1996). The authors demonstrated

that a two point mutation in the phosphotyrosine module changed the specificity

of interferon-gamma receptor from STAT-1 to STAT-3.

Chains of the interferon receptor may also play roles independent of the

JAK-STAT pathway. For example, Abramovich and others (1997) reported

binding of a protein-arginine methyltransferase to the IFNaR1 chain. This finding

suggests that methylation of proteins may be a signaling mechanism

complementary to tyrosine phosphorylation, and methylation may be required for

full stimulation by interferons. In fact, cells deficient in this methylase activity by

antisense become less sensitive to the antiproliferative effect of interferons.

Finally, Platanias and coauthors (1996b) reported that the interferon receptor

mediates tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2). The

IRS-2 molecules associate with IFNaR1 and become phosphorylated by tyk-2.

Moreover, phosphorylated IRS-2 associates with the p85 regulatory subunit of

the phosphatydilinositol 3'-kinase, suggesting that this kinase participates in the

interferon signaling cascade downstream from IRS-2. Collectively, the examples

presented above illustrate actions of the multifunctional interferon receptor. It is










tempting to speculate that some of such actions may be required for the

antiluteolytic roles of bIFN-r in the endometrium.

JAK kinases

Janus kinases or JAKs tyk-2 and jak-1 are associated respectively with

IFNaR1 and IFNaR2 and are involved in tyrosine phosphorylation of STAT

proteins. The carboxy-terminal domains of the jak kinases share considerable

sequence homology with the catalytic domains of other protein tyrosine kinases.

The amino-terminal half of the jaks contains regions of sequence homology to

other members of the jak family and the extreme amino-terminal domain

probably is involved in association with interferon receptor chains (Williams and

Haque, 1997). Ligand-mediated dimerization of interferon receptor chains is

required for interferon-stimulated signal transduction. Dimerization evokes

reciprocal tyrosine phosphorylation and consequent activation of JAKs

associated with interferon receptor chains (Ihle et al., 1995). Phosphorylation of

the kinase is the first of three tyrosine phosphorylations culminating in STAT

activation. Activated JAKs phosphorylate tyrosine residues on the interferon

receptor chains, which serve as docking sites for STATs, as mentioned above.

Lastly, STATs are phosphorylated by the JAKs (Darnell, 1997). Activated JAKs

are not specific for particular STATs. Different receptors can activate the same

STATs through different JAKs. Moreover, STAT docking sites can be

interchanged between different cytokine receptors, and the STAT specific for the










docking site present will be activated by binding of the ligand specific for the

extracellular domain of the receptor (Stahl et al., 1995). Therefore, STAT

activation is determined more by specific interactions between STATs and their

receptors than by specific JAKs associated with receptor chains.

STATs

Unlike other common intracellular second messengers, STATs not only

convey the extracellular signal to the interior of the cell, but they themselves

carry such a signal to the nucleus, acting as transcription factors to activate

transcription of genes induced by particular ligands. I will focus this discussion

on STATs 1, 2 and 3, although STATs 4 to 6 have been described (Darnell,

1997). STAT-la and 1p are encoded by alternative splicing of a single mRNA

transcript. Human STAT-la consists of 750 amino acids, while the extreme

carboxy-terminal 38 amino acids are missing for STAT-11. STAT-2 is composed

of 851 amino acids. STATs 1, 2 and 3 have significant sequence homology (Fu

et al., 1992; Zhong et al., 1994). The domain distribution in the STAT molecule

includes a centrally-located DNA-binding domain, a carboxy-terminal

transcription activation domain, and SH2 and SH3 domains located in between

them (Fu, 1992; Figure 2-2). The SH2 domain allows docking to tyrosine

phosphorylated sites in the IFN and cytokine receptors, as discussed above, and

also STAT dimerization. SH2 domain sequences are specific for each STAT, but

mutant STATs 1 and 3, in which SH2 domains were swapped, completely









400 500 600 700

DNA
binding

Y


Figure 2-2. Domain structure of the STAT-1 protein. The diagram represents the linear
structure of STAT-1 oriented in a amino- (leftmost) to carboxy- terminus sequence. DNA
binding domain, SRC homology 2 (SH2) domain, SH3 domain and transcription activation
domain (TAD) are represented in the sequence they occur in the STAT-1 molecule. The site of
tyrosine (Y) phosphorylation is also represented.


reversed their specificity for interaction with specific phosphotyrosine motifs

(Hemmann et al., 1996). This indicates that the SH2 domain is the sole

determinant of specific STAT factor recruitment to receptors. STATs contain a

unique tyrosine residue in the carboxy-terminal region (Y701, Y690 and Y705 for

STATs 1, 2 and 3 respectively). A recently developed model for STAT activation

(Li et al., 1997; Figure 2-1) proposes that unphosphorylated STATs 1 and 2 are

associated with the IFNaR2 chain. Binding of interferon causes dimerization of

this chain with IFNaR1. Tyrosine phosphorylated residue 466 of IFNaR1 binds

the SH2 domain of STAT-2, which is then phosphorylated on tyrosine 690,

providing a docking site for the SH2 domain in STAT-1. STAT-1 is

phosphorylated on tyrosine 701, and then dimerizes with STAT-2 through

reciprocal binding of tyrosine phosphorylated residues with SH2 domains.

However, an unsolved question is what drives SH2 domains of STATs to

dissociate from a higher affinity interaction with receptor phosphotyrosine to form










dimers which association is mediated by a lower-affinity phosphotyrosil

interaction (Greenlund et al., 1995). In light of this question, Gupta and

coauthors (1996) proposed an alternative model for STAT binding and dimer

formation. After binding to the receptor phosphotyrosine motif, the STAT shifts

its target to the tyrosine motif in the tyrosine kinase. Tyrosine phosphorylation of

STAT would cause a conformational change to destabilize this interaction with

the kinase, and STATs would then be driven to form more energy-stable

interactions with other STATs and form dimers. They based this model on the

finding that SH2 domains from STATs 1 and 2 bind with high affinity to

phosphotyrosine motifs on JAK kinases.

STAT dimers are competent to bind DNA. Known DNA binding

heterodimers are STAT 1:2 and STAT 1:3 (strong binding) and STAT 2:3 (weak

binding). Homodimers are STAT 1:1 and STAT 3:3 (strong) and STAT 2:2 (form

seldom in absence of STAT-1; Darnell, 1997). In variance with the notion that

tyrosine phosphorylation is required for STAT dimerization, Stancato and

coworkers (1996) demonstrated that STAT complexes exist in the cytosol of

unstimulated cells. Moreover, such association was independent of tyrosine

phosphorylation, since the Y701F STAT-1 mutant still bound to STAT-2 in

reticulocyte lysates. Such an interaction was weak, since it was not observed in *

extracts obtained with high-salt, detergent-containing buffers.

Current models for the mechanism of STAT activation of gene transcription

propose that following dimerization, STAT complexes translocate to the nucleus.










However, mechanism of transport to the nucleus remains unclear, since STAT

proteins lack the nuclear localization signal (NLS; Johnson et al., 1998b), which

are required for nuclear transport mediated through the importin mechanism

(Gorlich and Mattaj, 1996). Johnson and coauthors (1998b) proposed an

intriguing model for nuclear translocation of STATs after activation by interferon

gamma (IFNy). Since the carboxy-terminal domain of the IFNy molecule contain a

NLS, they propose that following binding to IFNy a complex containing the IFNy-

receptor, jak kinases, STATs and the bound ligand become internalized by

endocytosis. Upon cytoplasmic localization, the NLS sequence in the IFNy

molecule could associate with the importin protein complex, which would then

catalyze the transport of this complex to the nucleus, where STAT-mediated

transcription activation would ensue. They provided evidence for actual nuclear

translocation of a peptide containing the carboxy-domain of the IFNy molecule.

Although seemingly unique, they provide evidence of over 30 cytokines and/or

their receptors, which utilize STATs as signal transducers that contain NLS in

their sequence, indicating that this ligand-receptor-assisted nuclear translocation

is a viable, and intriguing mechanism. Among such cytokines and receptors are

the human IFNa and the human IFNaR1 molecules. Data in a recent paper is in

variance with this concept (Milloco et al., 1999). Those authors engineered a

STAT-1-estrogen receptor chimera, in which the estrogen receptor ligand binding

domain was fused to the carboxy-terminus of STAT-1 molecules. After










transfection to STAT-deficient U3a cells, this "conditionally active STAT"

underwent dimerization following estrogen/tamoxifen treatment. Moreover, these

chimeras were able to undergo nuclear translocation and activated transcription

of interferon-induced genes such as IRF-1. The authors concluded that tyrosine

phosphorylation of STAT is probably only a trigger for dimerization, since

dimerized, non-phosphorylated STAT chimeras also were able to stimulate

interferon-specific gene activation. Furthermore, since the estrogen receptor

domain used in the chimera did not contain any NLS, dimerization alone was

sufficient to promote nuclear translocation, sequence-specific DNA binding and

transcription activation functions of the chimeric STATs. A study conducted by

Strehlow and Schindler (1998) indicated that the amino-terminal 100 amino acids

of particular STATs mediated their nuclear translocation activity. Chimeric

constructs in which those amino acids in STAT-1 were substituted by those of

STAT-2 abolished nuclear translocation of STAT-1, while other functions were

maintained, such as activation by receptor, dimerization and DNA binding.

Collectively, it is fair to say that the mechanism of STAT nuclear translocation

remains unclear. Although the work of Johnson et al. (1998b) puts forth an

exciting proposition for such a mechanism, data from Milocco and others (1999)

argues against the requirement of a ligand-receptor-assisted transport

mechanism. However, existence of both mechanisms is feasible in vivo.










Interferon-directed gene activation

After translocation to the nuclear compartment, STAT complexes can act

as transcription factors, to direct expression of interferon-induced genes. The

best studied transcription activation complex containing STAT dimers is called

interferon-stimulated gene factor 3 (ISGF-3), which is composed of a STAT 1:2

dimer and a nuclear DNA binding protein, p48 (Darnell et al., 1994; Bluyssen et

al., 1996). ISGF-3 was first identified in electrophoretic mobility shift assays as a

complex induced by interferon treatment. It was formed independent of protein

synthesis, and was found to bind to consensus sequences on the regulatory

region of interferon-stimulated genes (Kessler et al., 1988). Consensus

sequences are known as interferon-stimulus response elements (ISREs).

Williams and Haque (1997) present a summary of sequences of ISREs of known

interferon-induced genes. A second interferon-induced transcription-activation

complex also was identified and named ISGF-2 (Kessler et al., 1988). Such a

complex is formed contingent on protein synthesis, presents different pattern of

migration in mobility shift assays and was later identified as the transcription

factor interferon regulatory factor 1 (IRF-1; Parrington et al., 1993). Interestingly,

IRF-1 and p48 are from the same family of proteins and can bind to the same

promoter elements (i.e., ISREs) in the regulatory region of interferon-stimulated

genes (Kessler et al., 1988; Parrington et al., 1993). The p48 and STAT 1:2

dimer do not associate in a stable manner to form the ISGF-3 complex in the

absence of DNA. However, contacts of amino acids 150 to 250 in the STAT-1








59

molecule with the carboxy-terminal portion of DNA-bound p48 stabilizes ISGF-3

(Horvath et al., 1996). Vickenmeier and coworkers (1996) reported direct binding

of recombinant, tyrosine phosphorylated STAT-1:1 dimers to tandem DNA

sequences. STAT-2 also forms homodimers, but requires p48 for strong

transactivation of transcription (Bluyssen and Levy, 1997). However, interactions

with DNA were not stable. Addition of STAT-1 increased the affinity and altered

sequence selectivity of p48-DNA interactions. In this scenario, ISGF-3 assembly

involves p48 functioning as an adaptor protein to recruit STAT-1 and STAT-2 to

an ISRE, STAT-2 contributes with potent transactivation but is unable to directly

contact DNA, while STAT-1 stabilizes the complex by contacting DNA directly.

Alternatively to transcription-induction through ISRE binding, interferons also

induce genes like IRF-1 which lack ISREs. Such genes are induced through

sequences named Inverted Repeats, present in their promoters (Haque and

Williams, 1994).

JAK-STAT pathway regulation

As in other tyrosine-phosphorylation-induced signaling systems, biological

responses resulting from activation of the JAK-STAT pathway are transient (Shuai

et al., 1992). Although the pathway of activation via the JAK-STAT pathway is

well established, few molecules have been identified that switch the signal off

(Starr and Hilton, 1999). Intuitively, one would predict that regulation of a tyrosine

phosphorylation pathway could occur through the actions of phosphatases, to

inactivate phosphotyrosil groups on receptors, JAKS and STATs, and proteases,










to degrade activated complexes. There is evidence for occurrence of both

mechanisms of regulation in the JAK-STAT pathway (i.e., phosphatases and

proteases), but more recent data indicate presence of novel regulatory molecules

also playing a role. Callus and Mathey-Prevot (1998) showed that treatment of

Ba/F3 cells with a specific proteasome inhibitor led to stable tyrosine

phosphorylation of the interleukin-3 (IL-3) receptor and STAT-5, after stimulation.

with IL-3. Further investigation revealed that stable phosphorylation events were

due to prolonged activation of JAKs. Moreover, Kim and Maniatis (1996)

demonstrated that after activation with interferon-y, STATs became ubiquitinated

and quickly degraded. In contrast with data from Kim and Maniatis (1996), but in

agreement with data from Callus and Mathey-Prevot (1998), Haspel and others

(1996) reported that proteasome inhibitors increased time of activation of STAT-1

by prolonging signals from the receptor (i.e., preventing degradation of receptor-

JAKs complexes), but not by blocking removal of phosphorylated STATs. This

was based on the finding that 35S-labeled STAT-1 translocated to the nucleus

upon tyrosine phosphorylation and later returned to the cytoplasm in non-

phosphorylated configuration. Data from Strehlow and Schindler (1998) agrees

and expands these findings, in that chimeric STATs with mutated amino-terminal

domains exhibited defects in nuclear translocation and deactivation, indicating

that these two events might be linked (i.e., deactivation may be dependent on

previous nuclear localization). Indeed, David and others (1993) demonstrated

that a nuclear tyrosine-phosphatase is responsible for deactivation of








61

phosphorylated STATs. To support the existence of a mechanism for regulating

activity of STATs based on phosphatases, Haque and coauthors (1995) reported

that treatment of cells with orthovanadate, molybdenate and tungstate, which are

effective inhibitors of protein-tyrosine phosphatases, resulted in accumulation of

interferon-y-induced phosphorylated STATs. Involvement of novel molecules in

the regulation of the JAK-STAT pathway was reviewed by Starr and Hilton (1999).

They propose a model in which suppressers of cytokine signaling (SOCS)

proteins such as SOCS1 bind directly to JAKs to inhibit their catalytic activities.

Another protein, CIS, binds to activated receptors to prevent docking of STATs.

SH2-domain phosphatase-1 (SHP-1) dephosphorylates JAKs or activated

receptors. Finally, a protein inhibitor of activated STAT (PIAS) inactivates STAT -

dimers. Song and Shuai (1998) demonstrated that SOCS 1 and SOCS3 inhibited

interferon-mediated antiviral and antiproliferative activities in HeLa cells. This

was linked with abolished tyrosine phosphorylation and nuclear translocation of

STAT-1 in response to interferon-a. Chung and others (1997a) reported that

PIAS3 directly interacted with STAT-3 and inhibited DNA binding of both STAT-

3:3, STAT-1:3 dimers. Binding of STAT-1 homodimers was not affected.

Moreover, cotransfections of both STAT-3 and PIAS3 showed a decrease in

luciferase activity from an IRF-1 reporter gene with increasing amounts of PIAS3.










Specificity of interferon signaling

Taken together, information presented in previous sections offers several

opportunities for occurrence of specific cellular responses to interferons. Such

opportunities include: (1) milieu of subtypes of interferons present at the receptor,

in which for example, different iso-forms of ovine IFN-T have different abilities to

extend estrous cycle length in ewes (Ealy et al., 1998); (2) composition of the

receptor complex, where recruitment of particular subunits may affect which

STATs are recruited; (3) amino acid context of the phosphotyrosine module on

the receptor chain, and amino acid context of the SH2 domain on STATs will also

determine which STAT will dock to which receptor chain; (4) which STATs are

present and which dimers will form upon ligand binding; (5) mechanism of nuclear

translocation of STATs, since whether STATs translocate as dimers alone or in

combination with ligand-receptor complexes may influence the configuration and

specificity of the transcriptional activation complex; (6) formation of single or

multiple transcription activation complexes, which will depend on nature of dimers

and interacting nuclear proteins; (7) dynamics of downregulation of JAK-STAT

pathway, in which specific branches of the pathway may be inhibited while others

may remain active to elicit specific responses; finally (8) interactions with other

cellular pathways, which will be discussed next.

Cross talk with other intracellular pathways

The best known cross-talk between JAK-STAT and other signaling

pathways is that represented by serine and threonine phosphorylation of STAT








63
residues, both constitutively and in response to ligands (see Leaman et al., 1996

for review). Such phosphorylation events are important, since treatment of cells

with kinase inhibitors disrupts STAT-3:3 DNA complexes. A mitogen-activated

protein kinase (MAPK) may be involved in phosphorylation of serine residues of

STAT-1, because the serine 727 lies in a consensus sequence for MAPK

phosphorylation. In fact, Stancato and coworkers (1997) proposed a model in

which activation of MAPK was dependent on activated JAK kinases. Binding of

interferon-a/p induced tyrosine phosphorylation of JAK-1, which stimulated

activity of membrane bound Raf-1. Activated Raf-1 phosphorylates MEK and

activates MAPK. MAPK in turn phosphorylates serine residues on STAT-1,

contributing to modulation of activity for this signal transducer. However,

modulation of STAT activities by MAPK may be stimulatory or inhibitory. For

example, Chung and others (1997b) reported serine phosphorylation of STAT-3

by growth factors, while STAT-1 was poor substrate for several MAPK tested.

Interestingly, serine phosphorylation of STAT-3 negatively modulated tyrosine

phosphorylation of this protein, and consequently inhibited dimerization, nuclear

translocation and gene activation.

Signal transducers such as IRS-1 and IRS-2 that are activated in response

to insulin, IL-2, IL-4 etc, are tyrosine phosphorylated by JAK-1. Epidermal growth

factor(EGF) is able to activate tyrosine phosphorylation of STATs 1 and 3 (David

et al., 1996). Interestingly, this does not require presence of JAKs. Moreover,

truncated receptor constructs containing the intrinsic kinase activity but lacking










the autophosphorylation domains were also effective in phosphorylating STATs.

This indicates that an alternative mechanism, where docking through SH2 domain

of STATs is not required for phosphorylation, is in place for EGF-induced STAT

phosphorylation.

The obligatory intracellular bacterium of macrophages, Ehrlichia

chaffeensis, blocked tyrosine phosphorylation of STAT-1, JAK-1 and JAK-2 in

response to IFN-y within 30 minutes of infection (Lee and Rikihisa, 1998). Also, .

PKA activity was increased 25 fold after infection. Inhibitors of PKA activity

partially abrogated the E chaffeensis-induced inhibition of STAT-1 tyrosine

phosphorylation, suggesting negative regulation of the JAK-STAT pathway by the

PKA-dependent mechanisms.

Another interesting theme is the occurrence of synergistic effects as a

result of coactivation of cellular pathways involving the JAK-STAT system. For

example, cooperation of interferon-y and tumor necrosis factor (TNF) during

inflammatory responses is a result of cooperation between STAT-1 and the

transcription factor NF-Kp. Synergistic expression of several genes involved in

the inflammatory process was contingent on presence of both transcription

factors (Ohmori et al., 1997). Stimulation by oncostatin M (OSM) induces

expression of matrix metalloproteinases (MMPs). Analysis of the regulatory

region of MMP-1 gene revealed presence of an AP-1 site as well as a STAT

binding element. Korzus and coworkers (1997) reported enhancement of MMP










expression due to synergistic actions of AP-1 and STAT-1. Such an effect was

Ras-dependent, which implies crosstalk between the MAPK and the JAK-STAT

pathways of signal transduction.

Yet another example of crosstalk is between the JAK-STAT pathway and

the PI 3' kinase, which has both lipid and serine kinase activities. Pfeffer and

others (1997) reported that PI 3' kinase is tyrosine phosphorylated through the

JAK-STAT pathway. Tyrosine phosphorylated STAT-3 proteins, bound to the

IFNaR1 chain of the interferon receptor, serve as a docking site for PI 3' kinase,

which couples its SH2 domain to tyrosine phosphorylated residues in the STAT

molecule. Upon docking, the PI 3' kinase is activated by JAKs, which then

promotes serine phosphorylation of STAT-3 to increase STAT-3 activity. In

another study (Uddin et al., 1997) interferon-a stimulated serine kinase activity of-

PI 3' kinase, which in turn activated the signal transducer IRS-1. Moreover,

stimulation with interferon p caused activation of MAPK, and such stimulation was

inhibited by Wortmannin, an inhibitor of PI 3' kinase activity. This suggests

involvement of the PI 3' kinase on MAPK activation. In contrast with data from

Pfeffer and others (1997), wortmannin failed to inhibit formation of the ISGF3

complex and interferon-mediated induction of ISG-15, indicating that the PI 3'

kinase probably is not required for interferon effects.

Finally, the work of Flati and others (1996) indicates that stimulation of

cells with interferon-a causes activation of PLA2, as measured by release of AA in








66

culture medium. PLA2 was associated with JAK-1, and inhibitors of PLA, activity

prevented formation of active ISGF3 transcription complexes. However, such

inhibition did not block binding of activated STAT-1 to inverted repeat sequences,

such as present in the regulatory region of IRF-1. Moreover, treatment of cells

with interferon-a stimulates tyrosine phosphorylation of PLA2. The authors argue

for a structural role of PLA2, which may be required for correct assembly of the

ISGF3 transcription complex.

The JAK-STAT pathway in bovine endometrium

In addition to the blFN-T receptor data mentioned above, very little has

been done on elucidation of the signal transduction system activated by blFN-T in

the endometrium. In fact, one of the main objectives of this dissertation was to

provide evidence of existence, as well as details on the blFN-r -activated,

endometrial JAK-STAT pathway.

Spencer and others (1998) conducted two studies to detect induction of

interferon-stimulated transcription factors, IRF-1 and IRF-2. Both factors were

absent in cyclic ewes and present in pregnant ewes (days 11 and 13, cyclic and

days 13, 15 and 17, pregnant). In cyclic ewes with ligated uterine homes,

unilateral infusion ovine IFN-T induced expression of IRF-1 and IRF-2 but not the

uterine horn receiving a BSA infusion. Since expression of these factors is

contingent on a functional JAK-STAT pathway, these data support existence of

such a pathway in the endometrium. Bathgate and coworkers (1998) also










reported existence of IRFs in endometrium of pregnant cows. Perry and

coauthors (1999) reported presence of STATs 1 and 2 and IRF-1 in the nucleus

of BEND cells stimulated with blFN-T. More detailed evidence for the JAK-STAT

pathway (i.e., existence, tyrosine phosphorylation, nuclear translocation, dimer

formation of and gene activation via STAT proteins) in bovine endometrium is

presented in Chapters 4, 5 and 6.

Bovine blFN-T -simulated protein synthesis in the endometrium

A main proposition of this dissertation is that proteins synthesized or

suppressed as a result of activation of the JAK-STAT pathway interact with the

PGF, synthesizing machinery to inhibit PGF2, secretion in the endometrium. In

this section, I will describe the data available on proteins synthesized in the

endometrium in response to blFN-T and their possible influences in the PGF2,

system.

Rueda and coworkers (1993) reported secretion of 12 and 28 kD proteins

both from pregnant endometrial explants and cyclic endometrial explants

stimulated with blFN-r in vitro. In a subsequent paper, Naivar and others (1995)

further characterized those proteins and discovered a novel, 16 kDa secretary

protein (P16). Endometrium explants were obtained from day 18 pregnant cows

and incubated in presence or absence of blFN-r. Both basal and stimulated

secretion of all three proteins increased in culture medium in a time-dependent

manner. More importantly, the 12 kD protein (Rueda et al., 1993), now renamed










as P8, was induced only in response to blFN-t, but not in response to IFN-a,

suggesting the possibility of blFN-r eliciting specific signal transduction and

protein synthesis. Moreover, P8 but not P16 secretion could be stimulated by

phorbol ester (Staggs et al., 1998). Amino acid analysis of the P8 revealed

identity with the alpha chemokine family: 92-100% identity with bovine bGCP-2

(Teixeira et al., 1997). Functions of bGCP-2 remain elusive, but it has been

suggested (Hansen et al., 1999) that being a chemokine, bGCP-2 may attract

concepts cells to attachment sites in the endometrium. Also, bGCP-2 may

attract cells from the immune system, to release cytokines beneficial to embryonic

development. P16 was identified as a bovine ubiquitin-cross reactive protein

(Austin et al., 1996a,b). The bUCRP mRNA (Hansen et al., 1997) and protein

(Austin et al., 1996b) are induced by blFN-r, and sequence analysis of the

bUCRP gene revealed presence of a conserved ISRE in the promoter region,

indicating putative activation by blFN-r (Perry et al., 1997). Analysis of the

primary structure of bUCRP revealed presence of critical amino acids and

domains implicated in functions of ubiquitin, such as conjugating with other

proteins. However, bUCRP lacked residues required for targeting proteins to

proteasomal degradation (Austin et al., 1996a). Therefore, it was proposed that a

possible role for bUCRP was to modify uterine proteins during early pregnancy

(Hansen et al., 1999). In fact, Johnson and others (1998a) reported that specific

conjugates of bUCRP and endometrial cytosolic proteins were formed in








69

response to treatment with blFN-r. Moreover, such complexes were distinct from

complexes containing ubiquitin, indicating a blFN-T-induced, specific action.

Although proteins present in the bUCRP conjugates have not yet been identified,

an attractive hypothesis is that blFN-, induces conjugation of bUCRP to proteins

involved in the cascade of PGF2,production in the endometrium. Such targeting

could modify function of such proteins to make them less able to stimulate PGF2,

production.

Research from Spencer and coworkers (1998) also showed that

endometrial estrogen receptors and oxytocin receptors were reduced in the

uterine horns infused with ovine IFN-r, and this was negatively correlated with

observed increase in IRF-1 and IRF-2 expression. Since IRF-2 has been

implicated as an inhibitor of gene transcription (Harada et al., 1994), the authors

hypothesized that perhaps interferon-induced IRFs were involved in inhibition of

gene transcription for estrogen and oxytocin receptors. In fact, Fleming and

coworkers (1998) cloned the ovine estrogen receptor gene and discovered IRF

response element (IRE) consensus sequences in the promoter region, further

supporting the hypothesis of interferon modulation of estrogen receptor

expression. Deletion constructs of the estrogen receptor promoter linked to

luciferase reporter gene were transfected into endometrial cells. Treatment of

these cells with ovine IFN-r caused reduction in luciferase expression only in

constructs containing the IREs. Using the same rationale, Bathgate and others








70

(1998) sequenced the bovine oxytocin receptor gene and also found IREs in the

regulatory region, and such sites bound bovine IRF-1 and -2. Again, the

suggestion is that perhaps blFN-T-induced transcription repressors may

downregulate expression of oxytocin receptors, to ultimately decrease PGF2,

secretion in the pregnant uterus.

Hypothetical model for blFN-T -mediated suppression of PGF,, secretion in the

endometrium

The hypothetical model shown in Figure 2-3 depicts some of the

possibilities discussed thus far.


Uterine-Conceptus Interactions and Reproductive Failure in Cattle


Thus far, this review has illustrated the enormous amount and intricacy of

interactions that need to occur between embryonic and maternal uterine tissues

in order for a successful pregnancy to be established. Given the high

percentage of embryonic mortality occurring during early pregnancies, it

becomes apparent that a precise program of interactions must be followed, and

that deviations from such a program may lead to pregnancy termination. Such a

program includes both embryonic and maternal components. For example, the

embryonic unit must be able to effectively interact with maternal endometrium,

undergo elongation and send antiluteolytic signals to the maternal unit in order to

survive. The maternal unit should provide a quiescent and nutritive environment,.












Uterine Lumen
INF-r


Figure 2-3. Hypothetical model of interferon-t (IFN-r)-stimulated gene activation and effects on
molecules involved in the PGF2a synthetic pathway. Estrogen receptors (ER) are up-regulated
before luteolysis and stimulate synthesis of oxytocin (OT) receptors (OTR). Oxytocin binding to
OTR stimulates phospholipase C (PLC), which cleaves phsphatydilinositol (PI) yielding inositol
trisphosphate (IP3) and diacylglycerol (DAG). The IP3 stimulates release of Ca" from
intracellular stores, and DAG activates protein kinase C (PKC). The PKC activates
phospholipase A2 (PLA,) which, in the presence of Ca+, cleaves arachidonic acid (AA) from
membrane phospholipids. Molecules of AA and linoleic acid (LA) regulate the enzyme
prostaglandin synthase (PGS) to produce PGF2a. In the pregnant cow, embryonic trophoblastic
cells secrete IFN-t into the uterine lumen. Receptors on endometrial epithelial cells bind IFN-T,
and dimerize. Dimerization of receptors promote phosphorylation (represented by a circled "p") of
associated tyrosine kinases from the JAK family, such as tyk-2 and jak-1. Phosphorylated
receptors attract signal transducer and activators of transcription (STAT) proteins. The STAT
proteins are phosphorylated in tyrosine residues by the JAK kinases and form a complex that
migrates to the nucleus. In the nucleus, the complex associates with a 48 kD DNA-binding
protein, and this new complex binds to interferon-stimulated response elements (ISRE) in the
regulatory region of interferon-induced genes, activating transcription of such genes and
synthesis of proteins. Synthesized proteins may act to specifically block one or more steps on the
PGF,2 synthetic pathway (arrows with [-] signs; see text for details and abbreviations).








72

conducive for embryonic attachment, and should bear intracellular mechanisms

to receive and transduce antiluteolytic signals from the concepts that ultimately

inhibit the default, PGF2,-secretory pathway of the uterus. Thatcher and Hansen.

(1992) reported that day 17 conceptuses varied in size from 15 to 250 mm.

Since inhibition of PGF2.is probably dependent on total amount of blFN-r

secreted and on area of endometrium occupied by the concepts, smaller

conceptuses would have already a smaller chance of survival. Environmental

effects such as heat stress (discussed above) decrease concepts development

and apparently compromises ability of the concepts to secrete blFN-r, leading

to failure in pregnancy recognition. There is also evidence for a role of the

uterus to stimulate secretion of blFN-r by concepts. Hernandez-Ledezma and

coworkers (1992) cultured IVF (in vitro fertilization)-produced embryos to

blastocyst stage and either continued in vitro culture or transferred conceptuses

to synchronized recipient cows. Embryos were recovered 4 days later, placed in

culture dishes and secretion of blFN-r was quantified. Secretion of blFN-e was

highly stimulated by exposure to the uterine environment, indicating that optimal

production of the antiluteolytic signal by the concepts is not solely determined

by the concepts. Stojkovic and coworkers (1999) reported that bovine embryos

derived by embryo flushing and in vitro production produced more blFN-r in long

term culture than embryos derived from nuclear transfer or embryo splitting Such

differences may contribute to lower pregnancy rates following embryo transfer to










recipients. Failure of cows to extend CL lifespan in response to blFN-T have

been reported (Helmer et al., 1989b; Meyer et al., 1995). This indicates failure in

the interferon receptor system, JAK-STAT-mediated signal transduction, post-

signaling mechanisms within the endometrium or a combination of these factors.

These responses have not been examined in a population of cows and warrant

further investigation.


Manipulating Uterine Function to Minimize Embryo Mortality


Bovine IFN-r administration

Based on the variation of concepts size and consequent capacity to

secrete at the critical time of maternal recognition of pregnancy for CL

maintenance, it is reasonable to propose that supplementing blFN-- at that

critical period may decrease embryonic losses. The rationale is that a slightly

underdeveloped concepts that may be unable to deliver the appropriate

antiluteolytic signal may be rescued by exogenous blFN-r administered at

around day 17 after insemination. Lack of availability of recombinant blFN-r and

structural similarity with blFN-a prompted Newton and others (1990) to test

fertility effects of blFN-a. Interferon-a extended CL lifespan but caused side

effects such as increased body temperature. Barros and coworkers (1992)

conducted a field experiment where blFN-a was administered daily from days 14

to 17 of pregnancy or as a single injection on day 13. Conception rates were










actually decreased by about 10% compared to control animals. This was

attributed to bIFN-a-induced side effects such as hyperthermia and acute drops

in P4 concentration. Alternative delivery systems and use of actual blFN-T may

still make this technology useful in the field (see Thatcher et al., 1994a for

discussion).

Fat feeding

Another possible manipulation of this system consists of attempting to

make the uterus less luteolytic, by changing the proportion of luteolytic AA to

antiluteolytic linoleic acid. For example, Thatcher and others (1997) described

an experiment in which Menhaden fish meal was fed to cows for 25 days. Fish

meal contains both eicosapentanoic and docosahexaenoic fatty acids, which had

been shown to be able to decrease PGF2 secretion. Indeed, cows fed fish oil

had a much attenuated secretion of PGFM in response to an oxytocin challenge.

Prevention of heat stress

One single environmental challenge that has negative effects both in the

embryonic and maternal units during the period of maternal recognition of

pregnancy is heat stress, as discussed previously. Therefore, strategies to

reduce effects of high environmental temperatures, such as observed in tropical

and subtropical latitudes, warrant investigation and application.








75

Objectives of This Dissertation



1) To study the distribution pattern of oviductal secretary proteins secreted

by cows bearing persistent or fresh dominant follicle;

2) To examine the signal transduction system stimulated by blFN-r in

endometrium;

3) To characterize the effects of blFN-T on PGF, production by BEND

cells.













CHAPTER 3
PERSISTENT DOMINANT FOLLICLE ALTERS PATTERN OF OVIDUCTAL
SECRETARY PROTEINS FROM COWS AT ESTRUS

Introduction



Synchronization of the estrous cycle in cattle is a very important tool for

reproductive management. For example, synchronization systems are used

widely for artificial insemination, timed insemination and embryo transfer. Most

commonly, synchronization is achieved with combinations of treatments with

prostaglandin F2a (PGF2,), progestins and GnRH (Thatcher et al., 1996).

Synchronization with progestins is based on the principle that exogenous

progestins, such as progesterone delivered by a Controlled Internal Drug

Release (CIDR) device, can maintain a sub-luteal concentration of progestin in

blood during a period which permits CL regression. In the absence of a CL,

removal of the progestin source will result in a synchronized estrus (Macmillan

and Peterson, 1993).

During the estrous cycle in cattle, two to three follicular waves of dominant

follicle development occur (Savio et al., 1988; Sirois and Fortune, 1988). Each

follicular wave is comprised of periods of recruitment, selection, dominance and

turnover or atresia. The ovulatory follicle generated in the last wave does not








77

turn over, but ovulates in a low progesterone (P4) environment. Turnover of the

dominant follicle (DF) is associated with high concentrations of P4, typical of mid-

cycle, which lowers LH pulse frequency (Kinder et al., 1996). Turnover of the

first wave DF can be blocked by exogenous progestins and injection of PGF2a

(Cooperative Regional Research Project, NE-161, 1996; Savio et al., 1993a;

Savio et al., 1993b). The resulting sub-luteal concentration of progestin in

plasma permits an increase in LH pulse frequency which sustains growth of the

DF. This "persistent" DF (PDF) is estrogenic, and subsequent fertility, as

measured by conception rate at first service (number of pregnancies / number of .

animals inseminated), is lower compared to animals bearing normal DFs [37.1%

vs. 64.8% in heifers, (Savio et al., 1993b); 23.6% vs. 58.2% for cows and heifers,

(Cooperative Regional Research Project, NE-161, 1996). Fertility after Al,

however, is restored to levels comparable to controls if the PDF is turned over

and a freshly recruited follicle is allowed to ovulate. Possible explanations for

reduced fertility include alterations in the oocyte and /or in the oviductal

environment. In a study by Ahmad et al. (1995), cows ovulating a PDF had

embryos that at day 6 of pregnancy were less developed (i.e., were less able to

reach the 16-cell stage) than embryos from cows ovulating a fresh (F) DF. In

addition, Revah and Butler (1996) showed that oocytes recovered from the PDF

showed expanded cumulus cells and condensed chromatin dispersed in their

ooplasm. In contrast, compact cumulus cells and intact germinal vesicles were

found in oocytes from FDF. Thus, the PDF may affect oocyte maturation,










oviduct and uterine function, which could affect early embryonic development

and decrease fertility.

Macromolecules present in oviductal fluid have been suggested to serve

an important role in sperm capacitation (Anderson and Killian, 1994), fertilization

(Boatman and Magnoni, 1995) and early embryo development (Gandolfi et al.,

1989). Therefore, alterations in oviductal biosynthetic activity including protein

synthesis and secretion may affect conception rate.

Steroid modulation of oviductal synthesis and secretion of proteins has

been characterized in sheep (Buhi et al., 1991, Murray, 1993), baboon (Verhage

and Fazleabas, 1988) and swine (Buhi et al., 1989; Buhi et al., 1990). An altered

steroid environment, associated with development of a PDF, may alter oviductal

protein synthesis and secretion. In turn, the altered pattern of protein synthesis

and secretion could affect optimal oviductal function, fertilization and early

embryo development that contributed to reduced embryonic survival in

synchronized cows. The present experiment tested the hypothesis that the

presence of a PDF alters protein synthesis and secretion of oviductal explants

from cows at estrus.

Specific objectives were: 1) to induce a PDF or a FDF with the strategic

use of PGF2a, progesterone-containing CIDR and GnRH; 2) to compare the

biosynthetic activity and the array of secretary proteins synthesized in the

infundibulum (INF), ampulla (AMP) and isthmus (IST) at estrus in oviducts










ipsilateral (IPSI) and contralateral (CONTRA) to the DF of cows bearing a PDF

versus a FDF.


Materials and Methods



Materials


Impervo paint was from Benjamin Moore and Co. (Jacksonville, FL) and

All-weather Paintstick was from LA-CO Industries, Inc./Markal Company

(Chicago, IL). Donations of Lutalyse were made by Pharmacia-Upjohn Co.

(Kalamazoo, MI), Buserelin from Hoescht-Roussel Agri-Vet (Somerville, NJ) and

CIDR-B devices were donated by EAZI-BREEDT, InterAg (Hamilton, New

Zealand). Eagles' minimum essential medium (MEM, catalog number 86-5007),

non-essential aminoacids (100x), anti-mycotic/antibiotic solution (100x) and

MEM vitamin solution (100x) were from Life Technologies (Gibco Laboratories,

Grand Island, NY). L-[4,5-3H] leucine (leu; 159 Ci/nmol) was from Amersham

Life Sciences, Inc. (Arlington Heights, IL) and L-leu, L-methionine, L-glutamine,

D(+) glucose, bovine pancreatic insulin, riboflavin and molecular weight

standards were purchased from Sigma Chemical Co. (St. Louis, MO).

Spectra/por 3 dialysis membrane was from Spectrum Medical Industries Inc.

(Houston, TX). Acrylamide, NN'-methylenebisacrylamide, sodium dodecyl

sulphate, Nonidet-P40, urea, agarose, diallyltartardiamide were from BDH

Laboratory Supplies (Poole, England). Ampholines were from Pharmacia










(Uppsala, Sweden), TEMED and ammonium persulphate were from Bio-Rad

(Hercules, CA). Glycine was from ICN Pharmaceuticals, Inc. (Costa Mesa, CA).

Coomassie brilliant blue, fast green, bromophenol blue, P-mercaptoethanol,

hydrochloric acid, sodium hydroxide, tris hydroxymethyll) aminomethane, sodium

salicylate, acetic acid and chromatography paper were from Fisher Scientific

(Fairlawn, NJ) and X-OMAT x-ray film was from Eastman Kodak Co. (Rochester,

NY).


Preparation of Medium


Leucine-deficient modified minimum essential medium (MEM; 10% normal

concentration of leu) was prepared as described by Buhi and coworkers (Buhi et

al., 1990). Briefly, MEM was supplemented with glucose (3g/1), methionine (1.5

mg/I), leu (5.2 mg/I), sodium bicarbonate (2.2 g/l), MEM vitamins (10 ml/l), non-

essential amino acids (10 ml/I), insulin (7.41 mg/I), sterile filtered and adjusted to

pH 7.4. Before use, medium was supplemented further with glutamine (292

mg/l), methionine (13.5 mg/I) and antimycotic-antibiotic solution (10 mill).


Animals and Treatments


During the pre-treatment period, estrous cycles of six mature non-lactating

cows were synchronized (Figure 3-1). A used CIDR device containing

approximately 1.2 g (Van Cleef et al., 1992) of P4 was placed into the vagina of









81

each cow for 7 days. One day prior to CIDR removal, cows received an injection


Pre-Treatment Period


PGF2a


CIDR


0 6 7 0
Estrus


Treatment Period

GnRH
(+1-)
PGF2a PGF2a


CIDR



7 9 16 18
Estrus
Slaughter


Ultrasonography, Blood Collection

Figure 3-1. Experimental protocol (see text).


of prostaglandin-F2o (PGF2a, Lutalyse, 25 mg) to regress the CL. To aid with

estrus detection, tail heads were painted (Impervo) and chalked (All-weather

Paintstick). Cows were observed twice daily for signs of estrus, and paint scores

were assigned (Macmillan et al., 1988). The day of standing estrus was

designated experimental day 0. During the treatment period, ovaries were

examined by transrectal ultrasonography using an Aloka echo camera model

SSD 500 linear array ultrasound scanner equipped with a 7.5MHz transducer

(Aloka Co., Japan). From Days 5 to 18, follicles and CL were measured daily

and sizes recorded. In addition, blood samples were collected in heparinized

evacuated tubes (Vacutainers, Becton Dickson Vacutainer System USA,










Rutherford, NJ) by tail venipuncture and stored in an ice bath. Plasma was

harvested by centrifugation (1800 x g for 30 minutes) and stored at -200C until

assayed for estradiol-17p (E2) and P4. On day 7, all cows were injected with

PGF2a and received one used CIDR device (Savio et al., 1993b). On day 9 cows

were assigned randomly to one of two treatment groups. Cows of the FDF group

(n=3) received an injection of GnRH agonist (Buserelin, 8 mg), to induce turnover

of any large size follicles present at that time, and allow recruitment of fresh

follicles (Schmitt et al., 1996c). Cows of the PDF group (n=3) did not receive the

GnRH agonist. On day 16 CIDR devices were removed, and cows received an

injection of PGF2, (25 mg). Cows were checked for signs of estrus twice daily

and slaughtered when observed in standing estrus (day 18 or 19). The

experimental models for persistent and fresh follicles resulted in a greater

pregnancy rate for heifers inseminated at estrus induced by FDF (Savio et al.,

1993b; Schmitt et al., 1996c).


Tissue Culture


On the day of slaughter, reproductive tracts were removed aseptically,

and oviducts were identified as IPSI or CONTRA to DF, dissected, trimmed free

of mesosalpynx and divided into INF, AMP and IST regions based on gross

anatomical characteristics. Segments of tissue between IST and AMP were

discarded. Tissue from each region was cut longitudinally to expose the lumen,

and then minced into fragments of -50mm3. Tissue fragments from each










functional region were cultured (Buhi et al., 1990) in LEU-deficient minimal

essential medium supplemented with 3H-LEU in the ratio of 100 mg tissue/3 mL

medium/20 mCi 3H-LEU for 24 hours at 370C in a controlled atmosphere of

N2:02:CO, (50%:47.5%:2.5% by volume). For AMP and INF, 500 mg of tissue

were cultured per dish, while for IST variable amounts of tissue (between 140

and 290 mg) were used.


Two-Dimensional Electrophoresis


After 24 hours incubation, conditioned media were dialyzed extensively

(MW cut-off 3500) against Tris buffered saline (10 mM Tris, 150 mM NaCI) pH

7.6 (two changes of 4 liters each/24 hours) and then dialyzed against deionized

water (two changes of four liters each/24 hours). Radioactivity in the retentate

was determined by liquid scintillation spectrometry, and incorporation rate was

defined as dpm non-dialyzable macromolecules/mg wet tissue. For each

sample, a volume of dialyzed conditioned medium containing 4 x 105 DPM was

lyophilized and submitted for two-dimensional SDS-PAGE as previously

described(Buhi et al., 1991). Gels were stained with Coomassie blue, soaked in

1 M Na salicylate solution, dried and exposed to x-ray film for 35 days at -800C.


Densitometry


Fluorographs were developed, and after qualitative analysis 20 protein

spots were selected and analyzed quantitatively by densitometry (Alphalmager








84

2000, Alpha Innotech Corporation, San Leandro, CA). Since a constant amount

of DPM was loaded for all samples, the capacity of tissues to synthesize and

secrete macromolecules (DPM/mg of tissue) was not accounted for and,

therefore, unadjusted densitometric measurements were biased. Different

secretary capacities were corrected by expressing the densitometric

measurements per unit secretary tissue. In this way, densitometric

measurements from tissues with greater secretary capacity were adjusted

upwards and vice versa for tissues with lower secretary capacities. Adjustments

were calculated by the equation: adjusted Arbitrary Density Units (ADU) =

ADU/mass of tissue equivalents, where one tissue equivalent is the mass of

tissue needed to synthesize and secrete 4 x 105 DPM of labeled

macromolecules. Mass of tissue equivalents was obtained by dividing 4 x 105

DPM by incorporation rate (DPM non-dialyzable macromolecules/mg of tissue)

for individual tissue samples.


Hormone Assays


Concentrations of E2 and P4 in plasma were measured by

radioimmunoassays previously validated in our laboratory [E2:(Badinga et al.,

1992); P4:(Knickerbocker, 1986)]. Intra- and inter-assay coefficients of variation

were 15.5 and 12.4%, respectively, for E2and, 6.8 and 8.1%, respectively, for P4










Statistical Analysis


Data were analyzed by least squares analysis of variance using the

General Linear Models of Statistical Analysis System (SAS, 1988).

Concentrations of E2 and P4 in plasma and diameter of DF were analyzed by split

plot ANOVA. The mathematical model used treatment (FDF or PDF), cow

(treatment), day, treatment by day and error. Rate of incorporation of

radioactivity into oviductal tissue and natural Log of adjusted ADU measurement

of proteins were calculated and analyzed by least squares ANOVA. The

mathematical model was: treatment (FDF or PDF), cow (treatment), side (IPSI or

CONTRA to the DF), region (INF, AMP and IST), all higher order interactions

and error. Orthogonal contrasts for treatment (PDF vs. FDF), region (INF and

AMP vs. IST and INF vs. AMP), and treatment by region interactions were used

to compare means.


Results



Ultrasonographv and Hormone Measurements


Size of DF was analyzed in two phases during the treatment period: from

day 5 to day 9 (period prior to injection of GnRH) and from day 10 until day 16

(Figure 3-2). Both FDF and PDF cows had similar sizes of DF from day 5 to day *

9. However, a significant (p<0.01) treatment by experimental day interaction was













20-






010-
U-
GnRH
5. PGF2a (+/-) PGF2a


r CIDR
5 6 7 8 9 10 11 12 13 14 15 16
Day of Treatment Period

Figure 3-2. Least squares means ( SEM) of diameter of the dominant follicle (DF) of cows
bearing a fresh DF (FDF, treated with GnRH on d 9) or persistent DF (PDF, not treated with
GnRH on d 9) during the Treatment Period. Treatments with PGF,, CIDR and GnRH are
indicated. Day 0 represents day of estrus at the beginning of Treatment Period.


detected from day 10 to day 16. All cows with FDF ovulated the first wave DF

and a newly recruited DF was detected on day 11 which reached 12 mm by day

16. In contrast, the first wave DF of PDF group was sustained and reached a

size of 22 mm by day 16.

Concentrations of E2 (Figure 3-3, panel a) and P4 (Figure 3-3, panel b)

were analyzed between experimental day 7 (day of PGF2a injections) and day 18

or 19. There was a significant (p<0.01) treatment by experimental day

interaction for both E2 and P4 concentrations in plasma. After GnRH injection on

day 9, E2 concentrations decreased in plasma of FDF cows and remained

between 5 and 10 pg/ml until day 16 and increased to 22 pg/ml at day 18











-I- FDF -- PDF


I CIDR I
5 6 7 8 9 10 11 12 13 14 15 16 17 18
Day of Treatment Period
b T


6019 CIDR Y5 I
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Day of Treatment Period
Figure 3-3. Least squares means (t SEM) of concentrations of ovarian steroids in plasma
of cows bearing a fresh dominant follicle (FDF, treated with GnRH on d 9) or persistent
dominant follicle (PDF, not treated with GnRH on d 9) during the Treatment Period.
Treatments with PGF2,, CIDR and GnRH are indicated. Day 0 represents day of estrus at the
beginning of Treatment Period.
a) estradiol-17p (E2); b) progesterone (P4).




Full Text
151
Validation of Immunoprecipitation and Immunoblots
Immunoblots of STAT proteins immunoprecipitated with anti-STAT
antibodies show a clear enrichment of the appropriate STAT, suggesting
specificity (Figure 5-5). Moreover, no bands were detected when no antibody
was used in the immunoprecipitation reaction, and only non-specific bands were
present when samples were immunoprecipitated with normal rabbit serum (i.e.,
same banding pattern noted regardless of antibody used). Similarly, STAT
bands were present in immunoblots probed with anti-phosphotyrosine and anti-
STAT antibodies, but were not present when immunoblots were probed with no
antibody or with normal rabbit serum (Figure 5-6).
Dose Response to bIFN-T
Phosphorylation of STATs 1 and 2 increased with as little as 3.125 ng/ml
of bIFN-T for STAT-2 and 6.25 ng/ml for STAT-1 (Figure 5-7). There was a
gradual Increase in phosphorylation of STAT-1 that reached a maximum at a
dose of 50 ng/ml and then declined. Phosphorylation of STAT-2 did not change
significantly with doses of bIFN-t greater than 3.125 ng/ml. Abundance of
STATs 1 and 2 was unchanged across all doses. Based on these results, a
dose of 50 ng/ml was chosen to be used in all subsequent experiments.


24
towards the oviductal lumen. Furthermore, volume of ovlductal fluid sharply
Increases 2 days before estrus to reach maximum rate one day after estrus,
which coincides with the period of ovulation and reception of oocyte by the INF
(Perkins et al., 1965). Muscular contractions, cilia beating and fluid secretion are
controlled by ovarian steroids, being stimulated by high periovulatory E2
concentrations.
Fertilization. In the cow, oocytes are transported rapidly to the site of
fertilization, above the isthmoampullar junction, where they spend most of their
time in the oviduct (Aref and Flafez, 1973), and then are transported rapidly
through the 1ST into the uterus (Anderson, 1991). A balance among the effects
of cilia beating, muscular contractions modulated by catecholamines and fluid
flow rate cause this egg lock up at the site of fertilization. It has been proposed
that the 1ST of the cow is contracted throughout estrus, and that norepinephrine
release after estrus causes relaxation of the 1ST to allow embryo transit into the
uterus (Isla et al., 1989). This is supported by data from El-Banna and Hafez
(1970), who showed a dramatic change in the surface area of the 1ST lumen
from estrus to 3 days after estrus (0.06 mm2 to 1.89 mm2). Meanwhile, if the
animal had been inseminated, sperm are migrating up the reproductive tract and
arrive at the 1ST portion of the oviduct, where their movement is slowed (Hunter
and Wilmut, 1982). It is hypothesized that biochemical and biophysical
properties of the 1ST may work to impede upward migration of spermatozoa,
including narrow isthmic lumen, viscous isthmic mucous and oviductal


106
from BDH Laboratory Supplies (Poole, England). Tissue culture-treated plastic
petri dishes (100x20) were purchased from Corning (Corning Glass Works,
Corning, NY). Tris, Tris-HCI, NaCI, EDTA, NaF, glycerol, glycine, methanol,
gelatin, Tween 20 and 15 ml polypropylene conical tubes, Tris-HCI, EDTA,
Hepes, 15 ml polypropylene conical tubes were purchased from Fisher Scientific
(Pittsburgh, PA). Microcentrifuge tubes (1.5 mL) were from USA Scientific
(Ocala, FL). Aprotlnin, leupeptln, pepstatin, Na4P207, EGTA, p-
glycerophosphate, Na3V04, benzamidine, PMSF and Proteln-A agarose beads,
bovine serum albumin, Earles balanced salts and Hank's balanced salts (HBSS)
were from Sigma Chemical Co. (St. Louis, MO). Anti-STAT-1 (E-23; catalog
number SC-346, 200pg/ml) and -STAT-2 (C-20; catalog number SC-476,
200pg/ml) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz,'
CA) and antl-phosphotyrosine (PY-20, 1 mg/ml) antibody was from Transduction
Laboratories (Lexington, KY). Nitrocellulose membranes (Hybond ECL) and
Enhanced Chemiluminescence Kit were from Amersham, Buckingamshire,
England, and X-ray films were from NEN Research Products (Reflection; Boston,
MA) and from Eastman Kodak Co. (X-Omat Blue XB-1; Rochester, NY). Non-fat
dried milk was from Mid-America Farms (Springfield, MO), and p-
mercaptoethanol was from Bio-Rad (Hercules, CA). Naxcel was from
Pharmacia & Upjohn (Kalamazoo, Ml). MEM essential and non-essential amino
acids solutions were from Gibco BRL (Life Technologies, Grand Island, NY).


282
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204
present experiment, since anti-IRF-1 caused no supershift. This probably
occurred because NE used in the present study were from cells treated with
bIFN-T for only 30 minutes, which is not enough time to allow synthesis of
sufficient amounts of IRF-1 to bind to ISRE (based on Figure 6-4, maximal
protein expression of IRF-1 was not noted until 2-hour exposure to blFN-t).
In Experiment 3, blFN-x also induced formation of two specific complexes,
s4 and s5 (SIE probe). However, nature of such complexes were probably
different than those observed in Experiment 2 (ISRE probe). The SIE sequence
is present in the c-fos gene and shown to be regulated by binding of STAT-1 (Fu
and Zhang, 1993) and STAT-3 (Rajotte et al., 1996) homo-and heterodimers.
Indeed, Yang and coauthors (1996) reported binding of STAT-1 and -3, but not
STAT-2 to SIE labeled probes after incubations with NE from interferon-treated
cells. In Experiment 3, pre-incubation with anti-STAT-1 and anti-STAT-3
antibodies yielded formation of distinct supershifted complexes, indicating that
both proteins are present and have the ability to bind specific DNA sequences.
The supershifted complexes were similar to those reported by Yang and others
(1996). Pre-Incubation with anti-STAT-1 antibody caused supershift of the faster,
migrating s4c sub-complex, but the supershifted subcomplex could not be
visualized in the gel. Examination of data in Yang and others (1996) shows that
the supershifted sub-complex elicited by pre-incubation with STAT-1 antibody is
slower migrating than the supershifted sub-complex formed by pre-incubation
with STAT-3 antibody. Since the supershifted sub-complex elicited by STAT-3


233
Figure 7-11. Experiment 4. Least squares means and SE of concentrations of PGF^ in
medium conditioned by BEND cells treated with medium alone (control), phorbol 12,13
dibutyrate (100ng/ml; PDBu),blFN-x (50 ng/ml; bIFN-tau) and PDBu and blFN-x added 3 hours
after other treatments started and PDBu (see text). Samples were removed before treatments
were added (0 hours), 1, 2, 3, 4, 5 and 6 hours after.
indicated that PGF2a secretion could be represented by third order curves (R2:
.954), which confirmed changes in secretion rates during the experiment.
Equations for each treatment were: Y=-11.96+29.28 x X-6.89 x X2+0.41 x X3
(control); Y=46.55-319.2 x X+229.1 x X2-17.64 x X3 (PDBu); Y=22.86-67.02 x
X+45.2 x XM.42 x X3 (PDBu + blFN-x); Y=58.27-287.4 x X+260.05 x X2-26.92 x
X3 (PDBu + blFN-x-3 hour); where Y represents secretion of PGF2a, and X is a
given time point in the experiment. Orthogonal comparisons of curves confirmed
that PDBu stimulated PGF2asecretion, and that blFN-x attenuated that effect


34
Kotwica and others (1997) demonstrated that administration of an efficacious
oxytocin receptor antagonist (CAP-527) failed to block normal luteolysis in cows.
Moreover, treatment of endometrial explants from days 16-17 cyclic cows with
oxytocin failed to stimulate secretion of PGF2a, both in static (Arnold et al., 1999)
and in perifusion (Del Vecchio et al., 1990) culture systems. This raises the
possibility that perhaps ligands other than oxytocin are required for luteolysis in
cattle. Alternatively to oxytocin, possible stimulators of pulsatile PGF2asecretion
include E2 and LH.
Irradiation of ovarian follicles, which reversibly eliminates production of
follicular E2, delays luteolysis and extends the length of the estrous cycle
(Hughes et al., 1987). Thus, follicular E2 plays a major role in these events. In
heifers, Thatcher and coworkers (1986) demonstrated that injections of E2 on
day 13 of the estrous cycle stimulated release of PGFM starting 3 hours after the
injection, peaking at 6 hours and returning to basal levels by 10 hours. In
addition, heifers injected with E2 underwent luteolysis 96 hours after injections,
while it took 125 hours for control heifers. To confirm that the PGFM increases
measured in the peripheral circulation represent PGF2aof uterine origin,
Knickerbocker and others (1986) measured a sharp increase in PGF2 in the
uterine vein of cows treated with E2. Collectively, these findings indicate that
both endogenous and exogenous E2 are able to stimulate secretion of PGF2
and to cause luteolysis.


162
bIFN-T, 50 ng/ml
blFN-x, 50 ng/ml
Figure 5-13. Immunoblotting analysis of STATs 1 and 2 immunoprecipitated from nuclear
extracts from BEND cells treated with blFN-r for Increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine
phosphorylation of STATs 1 and 2; b) Least squares means and SE of tyrosine phosphorylation of
STATs 1 and 2 (within each variable, bars with distinct subscripts are statistically different, p<.1).


CHAPTER 6
BOVINE INTERFERON-TAU STIMULATES BINDING OF STAT PROTEIN
COMPLEXES TO DNA AND STIMULATES SYNTHESIS OF INTERFERON
RESPONSE FACTOR-1 (IRF-1) PROTEIN IN BOVINE ENDOMETRIAL (BEND)
CELLS
Introduction
It is clear from experimental responses of Chapter 5 that bovine
interferon-tau (blFN-t) stimulates tyrosine phosphorylation, formation and
nuclear translocation of multi-STAT complexes. Type I interferons stimulate
gene transcription via a classical mechanism (i.e., JAK-STAT pathway) that
includes binding of nuclear STAT complexes to specific sequences in the
regulatory region of interferon-regulated genes (Darnell et al., 1994). Sequences
include the interferon stimulus response element (ISRE, Levy et al., 1988) and
the sis-inducible element (SIE, Sadowsky et al., 1993). Binding of STATs to
ISRE and SIE stimulates transcription of genes such as ISG54, ISG15, 6-16, 9-
27 and-e-Fos (Darnell et al., 1994).
Our working hypothesis is that blFN-n-stimulated proteins act to inhibit one'
or several steps of the cascade for synthesis of PGF2c,in the endometrium
(Figure 2-3). Type I interferons direct transcription of both IRF-1 and IRF-2
(Harada et al., 1989), and IRF-1 directly activates expression of IRF-2
187


220
Time (hours)
control
bIFN-tau
PDBu
bIFN-tau+PDBu
Figure 7-1. Experiment 1. Least squares means and SE of total PGF^ in medium conditioned
by BEND cells treated with medium alone (control), blFN-i (50 ng/ml; bIFN-tau), phorbol 12,13
dibutyrate (100ng/ml; PDBu) or blFN-i and PDBu. Samples were removed before treatments
were added (0 hours), 12 and 24 hours after.
COX-2 and PLA-, immunoblottina. Incubation of membranes with rabbit
IgG yielded a clear ECL exposure, suggesting that non-specific binding was
negligible in the immunoblotting procedures used (data not-shown). There were
low abundances of both COX-2 (Figure 7-2) and PLA2 (Figure 7-3) on cells
incubated with medium alone or with blFN-x, but treatment with PDBu stimulated
synthesis of both proteins. Incubations with blFN-x in combination with PDBu


89-
3
O)
E
i
CL

B
co
o
ro
O
Q.
30000-
25000.
20000.
15000.
10000.
5000.
0.
Figure 3-4. Least squares means ( SEM) of incorporation rates of 3H-leucine into
infundibulum (INF), ampulla (AMP) and isthmus (1ST) of cows bearing a fresh dominant follicle
(FDF, treated with GnRH on d 9) or persistent dominant follicle (PDF, not treated with GnRH
on d 9) during the Treatment Period.
top left quadrant. Results of densltometric analyses of P1 to P20 are depicted
in Tables 1 and 2.
There was a trend for effect of treatment for P2 (p<0.06) and P13
(p<0.07). Interpretation of main effects of treatments on secretion of proteins
Indicate that presence of FDF stimulated greater secretion of P2 and P13
compared to tissues from PDF cows. There was a significant (p<0.05) region
effect for P1, P4-7, P12-17 and P20, and a tendency (p<0.1) for P2, P3, P11 and
P18. Effects of side were significant for P5 (p<0.05) and P19 (p<0.05), and
approached significance for P8 (p<0.1) and P15 (p<0.06). Region by treatment


11
the time of ovulation to 7 days after estrus, Putney and coworkers (1988a) kept
heifers either in thermoneutral or hyperthermic conditions starting 30 hours after
onset of estrus. They found similar results to the ones described above, where
heat stress increased the proportion of abnormal embryos compared to controls.
To pinpoint critical stages of susceptibility of embryos to elevated maternal
temperatures, Ealy and coworkers (1993) submitted cows to heat stress on days
1, 3, 5 or 7 of pregnancy. Embryos recovered on day 8 were compared to
embryos from cows not heat stressed. Only heat stress at day 1 caused
decreased development. Collectively, data from these three heat stress
experiments suggest that environmental factors can affect embryo development
in multiple stages. Alternatively, it is possible that the toxic effects of heat stress
are exerted in the oviductal and uterine environments, which could become sub-
optimal for fertilization and embryo development, resulting in abnormal embryos.
As a final example of environmental effects on embryo development, Putney and
coworkers (1988b) incubated day 17 conceptuses at normal (39 C, 24 hours) or
high (39 C, 6 hours; 43 C, 18 hours) temperature regimens and measured de
novo protein synthesis by these conceptuses. They found that heat stress not
only decreased overall protein synthesis, but more importantly, decreased
secretion of IFN-t. This indicates that under the influence of heat stress,
embryos are less capable to sending appropriate signals to the uterus, which are
required for maintenance of an environment conducive to pregnancy.


51
phosphorylation of STAT-2 by tyk-2 (Yan et al., 1996). Similar to STAT-2, STAT-
3 activation also requires binding to IFNaRI (Yang et al., 1996). Furthermore, it
has been demonstrated that phosphotyrosine modules (i.e., sequence of amino
acids surrounding the tyrosine residue in the receptor chain) play a major role in
selecting which STAT binds (Gerhartz et al., 1996). The authors demonstrated
that a two point mutation in the phosphotyrosine module changed the specificity
of interferon-gamma receptor from STAT-1 to STAT-3.
Chains of the interferon receptor may also play roles independent of the
JAK-STAT pathway. For example, Abramovich and others (1997) reported
binding of a protein-arginine methyltransferase to the IFNaRI chain. This finding
suggests that methylation of proteins may be a signaling mechanism
complementary to tyrosine phosphorylation, and methylation may be required for
full stimulation by interferons. In fact, cells deficient in this methylase activity by
antisense become less sensitive to the antiproliferative effect of interferons.
Finally, Platanias and coauthors (1996b) reported that the interferon receptor
mediates tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2). The
IRS-2 molecules associate with IFNaRI and become phosphorylated by tyk-2.
Moreover, phosphorylated IRS-2 associates with the p85 regulatory subunit of
the phosphatydilinositol 3'-kinase, suggesting that this kinase participates in the
interferon signaling cascade downstream from IRS-2. Collectively, the examples
presented above illustrate actions of the multifunctional interferon receptor. It is


92
TABLE 1: Relative molecular weights (MW0) and least squares means (LSM)
and probability (p) values of the logarithm of densitometric units of region by
treatment interactions of selected proteins (P) on 2-D gelsb.
SPOT
MW
1NF-
FDF
INF-
PDF
AMP-
FDF
AMP-
PDF
IST-
FDF
IST-
PDF
reg.by
trt.
P1
823
9.7.3
9.3
10.4
10.1
7.4
8.7
p<0.07
P2
675
7.91
8.3
7.9
6.7
5.7
4.5
NS'
P3
582
6.41.1
7.9
6.6
6.9
8.9
9.2
NS
P4
975
6.4.2
5.8
5.9
5.9
6.8
6.6
NS
P5
764
6.2.1
6.2
5.9
6.4
6.6
6.9
NS
P6
955
6.5.2
6.4
5.8
6.0
6.5
6.8
NS
P7
975
5.3.7
5.4
1.1
5.8
6.7
6.9
p<0.02
P8
804
7.3.1
7.1
7.4
7.2
7.0
7.1
NS
P9
764
7.6.1
7.7
7.9
7.7
7.6
7.8
NS
P10
723
7.5.1
7.4
7.6
7.5
7.5
7.6
NS
P11
654
7.1 .1
7.4
7.8
7.6
7.3
7.4
NS
P12
71 3
3.51
4.9
2.1
.5
4.1
4.8
NS
P13
684
6.2 2
3.6
4.7
0
0
0
NS
P14
473
6.21.2
6.2
6.2
0
0
0
p< 0.02
P15
472
5.21
6.1
5.6
7.1
9.0
8.8
NS
P16
372
7.41
7.5
9.1
8.7
2.2
5.6
NS
P17
272
7.0.3
6.3
6.4
6.0
8.1
7.7
NS
P18
272
6.9.2
6.8
6.3
6.2
6.4
6.4
NS
P19
21 1
5.91.7
5.2
4.5
5.4
6.4
6.3
NS
P20
281
9.0.2
8.4
8.2
8.2
8.7
8.9
NS
a: Relative molecular weight (Mr x 10'3; means S.E.M.).
b: LSM pooled S.E.M. of region (reg.; INF = Infundibulum; AMP = ampulla; 1ST = isthmus) by
treatment [trt.; FDF = fresh dominant follicle (DF); PDF = persistent DF] interactions,
c: Non significant (p>0.1).


270
Del Vecchio RP, Sasser RG, Randal RD. Effect of pregnancy-specific protein B
on prostaglandin F2a and prostaglandin E2 release by day 16-perifused
bovine endometrial tissue. Prostaglandins 1990; 40:271-282.
Derecka K, Pletila EM, Rajanieml HJ, Zleclk AJ. Cycle dependent LH/hCG
receptor gene expression in porcine non-gonadal reproductive tissues. J
Physiol Pharmacol 1995; 46:77-85.
DeSouza M, Murray MK. An estrogen-dependent secretory protein, which
shares Identity with chitinases, Is expressed In a temporally and regionally'
specific manner in the sheep oviduct at the time of fertilization and embryo
development. Endocrinology 1995; 136:2485-2496.
DeWitt DL. Prostaglandin endoperoxide synthase: regulation of enzyme
expression. Blochem Blophys Acta 1991; 1083:121-134.
Diskin MG, Sreenan JM. Fertilization and embryonic mortality rates in beef
heifers after artificial Insemination. J Reprod Frtil 1980; 59:463-468.
Driancourt MA, Thatcher WW, Terqui M, Andrieu D. Dynamics of ovarian
follicular development in cattle during the estrous cycle, early pregnancy
and in response to PMSG. Domest Anlm Endocrinol 1991; 8:209-221.
Dubois DH and Bazer FW. Effect of porcine conceptus secretory proteins on in
vitro secretion of prostaglandin-F2a and -E2 from luminal and myometrial
surfaces of endometrium from cyclic and pseudopregnant gilts.
Prostaglandins 1991; 41:283-301.
Ealy AD, Alexenko AP, Kelsler DH, Roberts RM. Loss of the signature six
carboxyl amino acid tail from ovine interferon-tau does not affect biological
activity. Biol Reprod 1998; 58:1463-1468.
Ealy AD, Drost M, Hansen PJ. Developmental changes in embryonic resistance
to adverse effects of maternal heat stress in cows. J Dairy Sci 1993;
76:2899-2905.
El-Banna AA, Hafez ES. Profile analysis of the oviductal wall in rabbits and
cattle. Anat Rec 1970; 166:469-478.
Ellington JE. The bovine oviduct and its role in reproduction: a review of the
literature. Cornell Vet 1991; 81:313-328.


216
Experiment 3. Twenty seven 100 mm plates were assigned randomly to
receive medium alone (control, 9 plates), PDBu (100 ng/ml, 9 plates) or PDBu in
combination with blFN-x (50 ng/ml, 9 plates). Medium was sampled (500 pi)
after 1, 2, 3, 4, 5 and 6 hours of treatment and stored at -20 C for PGF2a analysis.
After collection of 6-hour sample, 3 plates per treatment were used for protein
extracts (WCE).
Experiment 4. Twelve wells in two 6-well plates were assigned in triplicate
to receive medium alone (control), PDBu (100 ng/ml), PDBu in combination with
blFN-x (50 ng/ml) or PDBu for 3 hours and PDBu in combination with bIFN-t for
the next 3 hours. Medium was sampled (250 pi) immediately before addition of
treatments (0-h sample), after 1, 2, 3, 4, 5 and 6 hours of treatment. Medium
was stored at -20 C for PGF2o analyses. In the last treatment (PDBu+blFN-T-3h),
150 ng of blFN-x (amount required for a 50 ng/ml final concentration in the well
that contains 3 ml of medium) was added to wells mixed in 250 pi of medium
(also containing 100 ng/ml PDBu) to replace medium removed for the 3-hour
sample^ PGF2o concentrations were not adjusted for well protein content in this
experiment.
Statistical Analysis
Data were analyzed by least squares analysis of variance, using the
procedure GLM from SAS (SAS, 1988). Each experiment was analyzed


79.
¡psilateral (IPSI) and contralateral (CONTRA) to the DF of cows bearing a PDF
versus a FDF.
Materials and Methods
Materials
Impervo paint was from Benjamin Moore and Co. (Jacksonville, FL) and
All-weather Paintstlck was from LA-CO Industries, Inc./Markal Company
(Chicago, IL). Donations of Lutalyse were made by Pharmacia-Upjohn Co.
(Kalamazoo, Ml), Buserelin from Hoescht-Roussel Agri-Vet (Somerville, NJ) and
CIDR-B devices were donated by EAZI-BREED, InterAg (Hamilton, New
Zealand). Eagles' minimum essential medium (MEM, catalog number 86-5007),
non-essential aminoacids (100x), anti-mycotic/antibiotic solution (100x) and
MEM vitamin solution (100x) were from Life Technologies (Gibco Laboratories,
Grand Island, NY). L-[4,5-3H] leucine (leu; 159 Ci/nmoi) was from Amersham
Life Sciences, Inc. (Arlington Heights, IL) and L-leu, L-methionine, L-glutamine,
D(+) glucose, bovine pancreatic insulin, riboflavin and molecular weight
standards were purchased from Sigma Chemical Co. (St. Louis, MO).
Spectra/por 3 dialysis membrane was from Spectrum Medical Industries Inc.
(Houston, TX). Acrylamide, NN'-methylenebisacrylamide, sodium dodecyl
sulphate, Nonidet-P40, urea, agarose, diallyltartardiamide were from BDH
Laboratory Supplies (Poole, England). Ampholines were from Pharmacia


273
Graf GA, Burns PD, Silvia WJ. Expression of a cytosolicphospholipase A2 by
ovine endometrium on days 11-14 of a stimulated oestrus cycle. J
Reprod Frtil 1999; 115:357-363.
Gravitt KR, Ward NE, OBrian CA. Inhibition of protein kinase C by melittin:
antagonism of binding interactions between melittin and the catalytic
domain by active-site binding of MgATP. Biochem Pharmacol 1994;
47:425-427.
Greenlund AL, Farrar MA, Viviano BL, Schreiber RD. Ligand-induced IFN
gamma receptor tyrosine phosphorylation couples the receptor to its
signal transduction system (p91). EMBO J 1994; 13:1591-1600.
Greenlund AL, Morales MO, Viviano BL, Yan H, Krolewski J, Schreiber RD.
STAT recruitment by tyrosine phosphorylated receptors: an ordered,
reversible affinity driven process. Immunity 1995; 2:677-687.
Gross TS, Thatcher WW, Hansen PJ, Helmer SD. Presence of an intracellular
endometrial inhibitor of prostaglandin synthesis during early pregnancy in
the cow. Prostaglandins 1988; 35:359-378.
Guillomot M, Flechon J, Winterberger-Torres S. Conceptus attachment in the
ewe: an ultrastructural study. Placenta 1981; 2:169-182.
Gupta S, Yan H, Wong LH, Ralph S, Krolewski J, Schindler C. The SH2
domains of STAT1 and STAT2 mediate multiple interactions in the
transduction of IFN-a signals. EMBO J 1996; 15:1075-1084.
Hafez, ESE. Anatomy of female reproduction. In: Hafez ESE (ed.),
Reproduction in farm animals, 6th ed. Philadelphia: Lea & Febiger;
1993a:20-55.
Hafez, ESE. Transport and survival of gametes. In: Hafez ESE (ed.),
Reproduction in farm animals, 6lh ed. Philadelphia: Lea & Febiger;
1993b:144-164.
Han CS, Roberts RM. Cloning and characterization of interferon-x receptors.
Biol Reprod 1998; 58 (suppl 1 ):108 (abstr 119).
Hansel W, Convey EM. Physiology of the estrous cycle. J Anim Sci 1983; 57
(suppl 2):404-424.


281
Murray MK. The biosynthesis, immunocytochemical localization of an estrogen-
dependent glycoprotein and associated morphological alterations in the
sheep AMP oviduct. Biol Reprod 1992; 47:889-902.
Murray MK. An estrogen-dependent glycoprotein is synthesized and released
from the oviduct in a temporal- and region-specific manner during early
pregnancy in the ewe. Biol Reprod 1993; 48:446-453.
Murray MK, DeSouza MM, Messinger SM. Oviduct during early pregnancy:
hormonal regulation and interactions with the fertilized ovum. Microsc
Res Tech 1995; 31:497-506.
Naivar KA, Ward SK, Austin KJ, Moore DW, Hansen TR. Secretion of bovine
uterine proteins in response to type I interferons. Biol Reprod 1995;
52:848-854.
Nam MJ, Thore C, Busija D. Effects of protein kinase C activation on
prostaglandin production and cyclooxygenase mRNA levels in ovine
astroglia. Prostaglandins 1996; 51:203-213.
Nancarrow CD, Buckmaster J, Chamley W, Cox Rl, Cumming IA, Cummins L,
Drinan JP, Findlay JK, Goding JR, Restall BJ, Schneider W, Thorburn GD.
Hormonal changes around oestrus In the cow. J Reprod Frtil 1973;
32:320-321.
Nancarrow CD, Hill JL. Oviduct proteins in fertilization and early embryo
development. J Reprod Frtil 1995; 49:3-13.
Nett TM, McClellan MC, Niswender GD. Effects of prostaglandins on the ovine
corpus luteum: blood flow, secretion of progesterone and morphology.
Biol Reprod 1976; 15:66-78.
Northey DL, French LR. Effect of embryo removal and intrauterine infusion of
embryonic homogenates on the lifespan of the bovine corpus luteum. J
Anim Sci 1980; 50:298-302.
Ohmori Y, Schreiber RD, Hamilton TA. Synergy between interferon-gamma and
tumor necrosis factor-alpha In transcriptional activation is mediated by
cooperation between signal transducer and activator of transcrription 1
and nuclear factor kappa p. J Biol Chem 1997; 272:148899-14907.


198
** Wtt
(1!
NEa
-
0
30
30
30
30
30
30
30
10 x coldb
-
-
-
+
-
-
-
-
-
anti-STAT-1
-
-
-
-
+
-
-
-
-
anti-STAT-2
-
-
-
-
-
+
-
-
-
anti-STAT-3
-
-
-
-
-
-
+
-
-
rabbit IgG
-
-
-
-
-
-
-
+
-
anti-IRF-1
-
-
-
-
-
-
-
-
+
Figure 6-2. Experiment 2. Electrophoretic mobility shift assay of nuclear extracts of BEND cells
incubated with radiolabeled ISRE Ingredients listed in the first column were present (+) or absent
(-) in pre-incubation reaction (see text). Arrows indicate shifted complexes (s1, s2, S3).
Nuclear extract used: (-) none, (0) from BEND cells incubated in medium alone for 30 minutes, (30)
from BEND cells incubated with blFN-r for 30 minutes
b Presence (+) or absence (-) of 10 fold excess ISRE unlabeld probe.


CHAPTER 2
LITERATURE REVIEW
Maternal-embryonic Communication as a Requirement for Successful Pregnancy
Communications of a physiological nature are very common between
embryonic (and pre-embryonic) and maternal units from the development of the
oocyte (i.e., before fertilization; Eppig et al., 1997a) to parturition (Bazer and
First, 1983; Fuchs and Fields, 1999) and lactation (Thatcher et al., 1980). The
maternal unit constitutes all tissues in the female reproductive tract that directly
or indirectly interacts with gametes or conceptus (conceptus = embryo and
associated embryonic membranes). Appropriate exchange of hormonal signals
between the two units is required for successful establishment and completion of
several windows of the reproductive cycle. Moreover, each window requires
unique signals that have been studied in detail and that have unique implications
on the outcome of the reproductive process (i.e., live, viable offspring). A
general review of the literature on several critical windows on the reproductive
cycle will be presented. A more detailed review will be offered on embryonic-
maternal interactions during the time of embryo transit through the oviduct and
during the window of maternal recognition of pregnancy occurring at around day
3


85
Statistical Analysis
Data were analyzed by least squares analysis of variance using the
General Linear Models of Statistical Analysis System (SAS, 1988).
Concentrations of E2 and P4 in plasma and diameter of DF were analyzed by split
plot ANOVA. The mathematical model used treatment (FDF or PDF), cow
(treatment), day, treatment by day and error. Rate of incorporation of
radioactivity into oviductal tissue and natural Log of adjusted ADU measurement
of proteins were calculated and analyzed by least squares ANOVA. The
mathematical model was: treatment (FDF or PDF), cow (treatment), side (IPSI or
CONTRA to the DF), region (INF, AMP and 1ST), all higher order interactions
and error. Orthogonal contrasts for treatment (PDF vs. FDF), region (INF and
AMP vs. 1ST and INF vs. AMP), and treatment by region interactions were used
to compare means.
Results
Ultrasonography and Hormone Measurements
Size of DF was analyzed in two phases during the treatment period: from
day 5 to day 9 (period prior to injection of GnRH) and from day 10 until day 16
(Figure 3-2). Both FDF and PDF cows had similar sizes of DF from day 5 to day
9. However, a significant (p<0.01) treatment by experimental day interaction was


148
delected with an antibody specific for phosphotyrosil groups of any protein) of
STATs in the dose response, nuclear translocation and coimmunoprecipitation
experiments were analyzed by densitometry as described for Chapter 3. Density,
values for bands were adjusted for the background of each Individual lane.
Statistical Analysis
Variables analyzed by densitometry were also analyzed by least squares
analysis of variance, using the GLM procedure of SAS (SAS, 1988).
Independent variables were dose and replicate for dose response experiments
and time and replicate for time response experiments. For time response
experiments, means were compared using a series of pre-planned orthogonal
contrasts (0 vs. the average of 3, 8, 15, 30, 60 and 120 minutes; 3 vs. the
average of 8, 15, 30, 60 and 120 minutes; 8 vs. the average of 15, 30, 60 and
120 minutes; 15 vs. the average of 30, 60 and 120 minutes; 30 vs. the average
of 60 and 120 minutes; 60 vs. 120 minutes).
Results
Nature of BEND Cells
Visual evaluation of BEND cells at passage 5 suggests that cells are
largely of epithelial origin, since they are mostly round in shape (Figure 5-3,
panel a). A few elongated (perhaps of stromal origin) are also noted, and larger,'


155
121
X
*" 78-
p-STAT-2
p-STAT-1
121
78
-STAT-2
STAT-1
3.1 6.2 12.5 25 50 100 ng/ml
bIFN-x, 15 min.
0 3.1 6.2 12.5 25 50 100 ng/ml
blFN-r, 15 min.
Figure 5-7 Immunoblotting analysis of STATs 1 and 2 immunoprecipitated from whole cell
extracts from BEND cells treated with increasing doses of blFN-t for 15 minutes.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine
phosphorylation of STATs 1 and 2; b) Representative ECL exposure of abundance of STATs 1 and
2; c) Least squares means and SE of tyrosine phosphorylation of STATs 1 and 2 (within each
variable, bars with distinct subscripts are statistically different, p<0.1).


188
(Henderson et al., 1997). Conversely, IRF-2 negatively regulates expression of
IRF-1, characterizing a yin-yang type of regulatory paradigm (Harada et al.,
1989, 1994).
IRF-2 is a protein induced by ovine IFN-t in the endometrium of sheep
(Spencer et al., 1998). IRF-2 is a transcription repressor that inhibits
transcription of genes such as EBNA-1 (Zhang and Pagano, 1999) and NOS-2
(Faure et al., 1997). It is possible that, in the endometrium, IRF-2 suppresses
synthesis of proteins critical to the generation of PGF2ol. In fact, sequences of
regulatory regions of the bovine oxytocin receptor (Bathgate et al., 1998) and the
ovine estrogen receptor (Fleming et al., 1998) genes have putative sequences
for binding to members of the IRF family (IRF response elements, or IREs).
Our objectives were (1) to characterize binding of nuclear proteins from b-
IFN-t stimulated BEND cells to ISRE and SIE elements, (2) identify nature of
nuclear proteins binding ISRE and SIE elements and (3) to examine ability of
bIFN-T to induce synthesis of IRF-1 in BEND cells.
Materials and Methods
Materials
Bio-Rad protein assay, TEMED and ammonium persulphate were from
Bio-Rad Laboratories (Hercules, CA). Bovine IFN-t (dissolved in 20 mM Tris-
HCI, 1 mM EDTA, pH 8 to 200 pg/ml; 1.08 x 107 units of antiviral activity) was a


250
and reports from other laboratories. For example, putative cis-activating
elements for IRFs have been found in the regulatory region of the ovine oxytocin
receptor gene (Fleming et al., 1998) and bovine oxytocin receptor gene
(Bathgate et al., 1998). Since both receptors have presumed roles in the
process of luteolysis, a suppressive effect of bIFN-x in their expression mediated
through IRFs, would be in order with the hypothesis of antiluteolytic effects of
bIFN-x. Test of the hypothesis for a requirement of the JAK-STAT pathway for
bIFN-x -induced suppression of PGF2awill require inactivation of specific
components of the JAK-STAT pathway, such as STATs, through use of
dominant negative molecules, specific inhibitors, anti-sense technology etc.
Data from Chapters 5 and 6 provided general information on
characteristics of the JAK-STAT pathway in BEND cells, but a number of
questions remain unanswered and warrant further investigation. For example,
presence of a specific bIFN-x receptor/signal transduction pathway to decrease
PGF2a secretion remains a provoking, largely unanswered question. Data from
Staggs and co workers (1998) provide indirect evidence for existence of a bIFN-x
specific receptor/signaling system. They investigated the regulation of
endometrial bovine GCP-2 protein secretion and found that although bIFN-x was
able to induce its expression, the closely related bovine interferon-a failed to do
so. In contrast, both interferons induced secretion of another endometrial
protein, bovine UCRP. Moreover, expression of bovine GCP-2, but not bovine


141
Cells were scraped from plates in the presence of 1 ml of appropriate extraction
buffer, transferred to a microcentrifuge tube and incubated on Ice for at least 10
minutes. Subsequently, cells were aspirated back and forth 5 times through a 25
gauge needle attached to a tuberculin syringe to complete lysis. To obtain WCE,
lysates were spun in a microcentrifuge (model 235b, Fisher Scientific, Pittsburgh,
PA; 13000 x g) for 2 minutes and supernatants were used for
immunoprecipitation. To obtain CE and NE (Ghislain and Fish, 1996), lysates
were spun in a microcentrifuge (13000 x g) for 20 seconds. Appropriate volume
of 1M KCI was added to supernatants (cytosolic fractions) to adjust final
concentration to 60 mM KCI. Then supernatants were spun for 30 minutes in a
microcentrifuge (13000 x g) and used for immunoprecipitation. The pellet (from
initial 20 seconds spin; crude nuclei) was resuspended in 1 ml hypotonic buffer,
layered over 9 ml of a 30% sucrose solution of hypotonic buffer in a 100 x 16 mm
polypropylene centrifuge tube and centrifuged at 7000 x g for 8 minutes at 4 C in
a refrigerated Sorvall centrifuge (model RC-3B, equipped with a H-6000A rotor,
Du Pont Co., Wilmington, DE). The pellet (nuclear fraction) was resuspended
and incubated in a rotating device (Roto-torque", heavy duty rotator, Cole
Parmer, Chicago, IL; lever setting at low", speed setting at 7) with 150 pi high
salt buffer (hypotonic buffer containing 300mM KCI and 20% glycerol) for 30
minutes and clarified by centrifugation (20 minutes, 13000 x g). Resulting
supernatant (NE) was diluted to1 ml with hypotonic buffer, adjusted to 60 mM
KCI with appropriate volume of 1 M KCI, and used for immunoprecipitation.


50
conducted in bovine reproductive tissue, to test signaling ability of blFN-t.
Instead of the antiviral assays used in the experiments mentioned above
(Pestka, 1997), functional assays measuring suppression in synthesis of PGF2o
from endometrial cells would be in order. Moreover, existence of a blFN-t-
specific receptor chain remains elusive.
Colamonici and coworkers (1994a; 1994b) demonstrated that the tyrosine
kinase p135tyk2, or tyk-2 is associated physically with the IFNaRI chain of the
interferon receptor. Immunoblots revealed the ability of monoclonal antibodies to
IFNaRI and to tyk-2 to reciprocally coimmunoprecipitate both proteins.
Association of tyk-2 was mapped to a 46-amlno acid juxtamembrane region of
the IFNaRI chain. Furthermore, they demonstrated that tyk-2 could directly
phosphorylate tyrosine residues in the IFNaRI chain after stimulation with IFN-a
(Colamonici et al., 1994b). Besides binding to extracellular interferons and
associating with JAK kinases, the interferon receptor complex also has other
functions in the JAK-STAT pathway. The unstimulated IFNaR2 chain may
contain-associated unphosphorylated STAT proteins (Li et al., 1997). Binding of
interferon brings IFNaR2 and IFNaRI, which contains tyk-2, together.
Dimerization of receptor chains elicits transfer of STATs to the IFNaRI, where
STATs become tyrosine phosphorylated. This confirms the previous finding that
tyrosine 466 in the chain of IFNaRI acts as a docking site for association of the
SH2 domain of STAT-2, and such binding is required for tyrosine


234
(p<0.01). Moreover, curves representing secretion of PGF2ciinduced by PDBu
and PDBu + bIFN-x were different (p<0.01).
Discussion
The present chapter describes a cell culture-based model that is adequate
for studying effects of blFN-x on PGF2c, secretion in the endometrium. This
system has the basic components for such studies, which are (1)
responsiveness to a stimulator of PGF2a synthesis (i.e., PDBu) and (2),
responsiveness to blFN-x, which blocks a stimulation in PGF2cl. Using this
system, it was determined that a bIFN-x-induced suppression of PGF2a is
probably mediated through a decrease in protein expression and or activity of
two critical enzymes for synthesis of PGF2cl: COX-2 and PLA2. Moreover, blFN-x
suppression of PDBu-stimulated PGF2cl secretion can be achieved quickly (less
than 2 hours) and after PGF2osynthesis has been stimulated with PDBu.
BEND cells provide an adequate model for studying regulation of PGF2in
the endometrium. Previous studies utilized endometrial epithelial cells in primary
culture harvested from uteri collected from day 15 cyclic cows (Danet-Desnoyers
et al., 1994) and day 1 to 4 cyclic cows (Asselin et al., 1997; Asselin et al., 1998;
Xiao et al., 1999). Although day 15 cells are preferable in comparison to days 1
to 4 (i.e., cells are from a pre-luteolysls stage of the estrous cycle in which uteri
have been primed adequately by progesterone), collection procedures are time


This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
August, 1999
Dean, College of Agriculture
Dean, Graduate School


Williams, Lauren Knickerbocker, Mary Ellen Hissen, Susan Gottshall, Larry
Eubanks, Eddie Fredriksson, Mary Russell, Dale Hissen, James Lindsey, Chris
Wilson, Joyce Hayen, Werner Collante, Kathy Austin, Debbie Akin, Stephany
Suggs, Peggy Briggs, Melissa Thomas, Patricia Hancock, the Dairy Research
Unit crew, the Large Animal Clinic crew, students and residents at the School of
Veterinary Medicine and the Meats Laboratory crew.
As a important part of my University of Florida experience I wish to
express thanks for the generosity of Ms. Louise Curtelis, the hard work and
comradery of fellow officers and friends at the Graduate Student Council and the
Brazilian Student Association and the financial support from the Animal
Molecular and Cell Biology program during the first year of my graduate studies.
One true friendship is worth one thousands PhDs, so I want to show my
sincere gratefulness to all friends in Gainesville, the ones mentioned above and
also Mauricio, Ilka, Carlos da Costa, Patricia, Dirceu and Ana Clara, Daniella and
Fabiano, Ronaldo and Valeria, Cleisa and Cartaxo, Lawry, Claudio and
Cristiane, Tom e Jose Melvin, Ricardo Harakava, Cristina and Warley, Annie e
Wigberto, Debbie and Ken, Joe and Kirsten, Karina e Uilson, Liana and Silvano,
Deise and Alfredo, Raul and Helena, Michelle, Mary Duryea, Paul and Joan and
my friends at the School of Tai Chi Chuan, Marcus Harvey, Dorota Porazinska,
Stacey Chestain, Joe Sadek and Jackie Wilson .
Distance makes one appreciate the value of a family. I would have never
been able to complete my graduate studies without the endless support of Nice,
v


203
for STAT-1 elicited formation of the s2 complex, but band was faint and its
significance must be further confirmed. Experiments with alternative conditions
during the pre-incubation period may yield more definitive results (e.g., different
temperatures). In contrast, antibody for STAT-2 caused complete displacement
of s1. This could be due to two reasons. First, binding of STAT-2 antibody to
STAT-2 on the ISGF-3 complex may cause conformation changes that prevent
appropriate binding of ISGF-3 to the ISRE probe. A second possibility is that the
a supershifted complex was formed, but became too bulky to be resolved in a
6% acrylamide gel. In fact, other reports demonstrating supershifted ISGF-3
complexes used lower percentage acrylamide gels (4.5%; Ghislain and Fish,
1996; 5%; Yang et al., 1996). This preferred alternative is substantiated by the
formation of s3 complex (lane 6, Figure 6-2). Since a s3-like complex also was
formed in the absence of NE (lane 1, Figure 6-2), one could argue that s3 is non
specific. A follow up experiment demonstrated that formation of s3-like complex
in the absence of NE in Experiment 2 was probably an artifact, because no such
complex was present in the absence of NE, but was present in NE pre-incubated
with anti-STAT-2 (Figure 6-3). Lack of alteration on s1 by anti-STAT-3 antibody
or rabbit IgG further confirmed specificity of the ISRE probe to the ISGF-3
complex that does not contain STAT-3. Absence of supershift in response to
anti-STAT-3 also was noted by Yang and coauthors in Daudi cells (1996). IRF-1
can stimulate transcription of the 9-27 interferon-inducible gene through binding
at the ISRE of that gene (Henderson et al., 1997). This was not noted in the


274
Hansen PJ. Rescue of the corpus luteum from luteolysis by bovine trophoblast
protein-1: an example of maternal recognition of pregnancy. Revista
Brasileira de Reproducao Animal (1991); 3(suppl 1):42-65.
Hansen PJ. Interactions between the immnune system and the bovine
conceptus. Theriogenology 1997; 42:121-130.
Hansen TR, Austin KJ, Johnson GA. Transient ubiquitin cross-reactive protein
gene expression in the bovine endometrium. Endocrinology 1997;
138:5079-5082.
Hansen TR, Austin KJ, Perry DJ, Pru JK, Teixeira MG, Johnson GA. Mechanism'
of interferon-tau action in the uterus during early pregnancy. J Reprod
Frtil 1999; (suppl).
Hansen TR, Kazemi M, Keisler DH, Malathy P-V, Imakawa K, Roberts RM.
Complex binding of the embryonic interferon, ovine trophoblast protein-1,
to endometrial receptors. J Interferon Res 1989; 9:215-225.
Haque SJ, Flati V, Deb A, Williams BRG. Roles of protein-tyrosine
phosphatases in STATIa -mediated cell signalling. J Biol Chem 1995;
270:25709-25714.
Haque SJ, Williams BRG. Identification and characterization of an interferon
(IFN)-stimulated response element-IFN-stimulated gene factor 3-
independent signaling pathway. J Biol Chem 1994; 269:19523-19529.
Harada H, Fujita T, Miyamoto M, Kimura Y, Maruyama M, Furia A, Miyata T,
TaniguchiT. Structurally similar but functionally distinct factors, IRF-1 and
IRF-2, bind to the same regulatory elements of IFN and IFN-inducible
genes. Cell 1989; 58:729-739.
Harada H, Takahashi E, Itoh S, Harada K, Hori TA, Taniguchi T. Structure and
regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2
genes: implications for a gene network in the interferon system. Mol Cell
Biol 1994; 14:1500-1509.
Harper MJK. Sperm and egg transport. In: Austin CR, Short RV (eds.),
Reproduction in mammals, Book 1: Germ cells and fertilization, 2nd ed.
Cambridge: Cambridge University Press; 1982:102-127.
Hartley HO. The maximum F-ratio as a short-cut test for heterogeneity of
variance. Biometrika 1950; 37:308-312.


137
centrifuged in a Dynac non-refrigerated bench centrifuge (Parsippany, NJ) with
the dial speed set to 70 (approximately 700xg) for 5 minutes. Supernatant was
discarded and cells were resuspended in 5 to 10 ml of medium. About one fifth
of this suspension (~1 x 106 cells; determined by hematocytometer count) was
transferred to a fresh culture flask containing 50 ml of complete culture medium .
to maintain a stock of growing cells for subsequent experiments. Remaining cell
suspension was divided according to the number of plates needed in a particular
experiment. For freezing, cells were diluted to 4 x 10s cell /ml of complete
culture medium. Then to 500 pi of that suspension, 500 pi of freezing medium
(20% DMSO in complete culture medium) were added dropwise and transferred
to a cryovial. Cryovials were stored at -20 C for 2 hours, transferred to -80 C
overnight and finally stored in a liquid nitrogen tank. Cell morphology was
observed in each passage using a Nikon (model TMS) inverted microscope, and
photographed using an Olympus inverted microscope (model CK2, Japan)
equipped with a closed circuit TV camera (model DCE-107; Sony Corp., Japan)
and connected to a color video copy processor (model CP110U; Mitsubishi
Corp., Japan). Cells were obtained from repeated passages until cells started to
change morphology (see Results) and growth rate decreased. When decreased
growth rate was noticed, cells from a frozen stock were thawed (quickly,
submerging cryovial in a 37C water bath), plated (50 ml complete culture
medium) and upon reaching confluence, were used as a stock.


96'
biosynthetic activity and protein synthesis and secretion from different functional
regions of the oviduct. This altered pattern of protein synthesis and secretion in
cows bearing PDFs may contribute to the lower fertility of this group of animals
compared to cows ovulating FDFs (Savio et al., 1993b). Normal embryos
transferred 7 days after estrus to uteri of cows that ovulated a PDF had no
difference in pregnancy rates compared to controls (Wehrman et al. 1997). This
supports the concept that low fertility associated with PDF may be due to an
inappropriate oviductal, not uterine, environment before day 7, or an abnormal
embryo (Ahmad et al., 1995; Mihm et al., 1994; Revah and Butler, 1996).
Both ultrasonography and hormonal data indicated that the first wave DF of the
experimental period was ovulated after injection of GnRFI agonist at day 9 (FDF
cows) while no injection of GnRFI agonist (PDF cows) permitted sustained
development of the first wave DF. Continued growth of the first wave DF
occurred in the low P4 environment supported by the Cl DR in the PDF group.
The PDF maintained high concentrations of estradiol in plasma. Cows of the
FDF group ovulated the first wave DF in response to GnRFI, and the resulting CL
secreted increasing amounts of P4 after day 13. A newly selected FDF was
detected by day 11. Changes in E2 and P4 between PDF and FDF groups
reflected the differences in CL and follicle dynamics have been reported
previously (Schmitt et al., 1996c). The acute increase in E2 concentration in the
FDF group was associated with development of a DF during the pro-estrous
period following injection of PGF2a and withdrawal of the CIDR.


25-i
86
FDF PDF
Day of Treatment Period
Figure 3-2. Least squares means ( SEM) of diameter of the dominant follicle (DF) of cows
bearing a fresh DF (FDF, treated with GnRH on d 9) or persistent DF (PDF, not treated with
GnRH on d 9) during the Treatment Period. Treatments with PGF2a, CIDR and GnRH are
indicated. Day 0 represents day of estrus at the beginning of Treatment Period.
detected from day 10 to day 16. All cows with FDF ovulated the first wave DF
and a newly recruited DF was detected on day 11 which reached 12 mm by day
16. In contrast, the first wave DF of PDF group was sustained and reached a
size of 22 mm by day 16.
Concentrations of E2 (Figure 3-3, panel a) and P4 (Figure 3-3, panel b)
were analyzed between experimental day 7 (day of PGF2a injections) and day 18
or 19. There was a significant (p<0.01) treatment by experimental day
interaction for both E2 and P concentrations in plasma. After GnRFI injection on
day 9, E2 concentrations decreased in plasma of FDF cows and remained
between 5 and 10 pg/ml until day 16 and increased to 22 pg/ml at day 18


a
230
| 78-
s 39.5-
- COX-2
1 2 3 4 5 6 7 8
b
Figure 7-9. Experiment 3. Immunoblotting analysis of COX-2 in whole cell extracts from BEND
cells treated with medium alone (control), phorbol 12,13 dibutyrate (100ng/ml; PDBu) or bIFN-t
(50 ng/ml; bIFN-tau) and PDBu for 6 hours.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of
COX-2 (control: lanes 1 and 2; PDBu: lanes 3, 4 and 5; blFN-i and PDBu: lanes 6, 7 and 8); b)
Least squares means and SE of abundance of COX-2 arbitrary densitometric units (ADU).


14
pregnancy caused a 50% reduction in weight of conceptus compared to control
cows. These retarded embryos may not be able to send the appropriate
antiluteolytic signals to the maternal endometrium, thereby allowing luteolysis
and consequent loss of pregnancy to occur.
Inadequate manipulations of the system. As a means to improve
conception rates in livestock operations, researchers have developed several
management practices that often include manipulations of the endocrine system
of animals. Such practices may sometimes yield unexpected results, including
decreased fertility due to disruption of appropriate maternal-embryonic
communications. One widespread practice is synchronization of estrous cycles.
Estrus synchronization systems are used for artificial insemination, timed
insemination and embryo transfer. Most commonly, synchronization is achieved
with combinations of treatments with PGF2o, progestins and gonadotropin
releasing hormone (GnRH; Thatcher et al 1996). Synchronization with
progestins is based on the principle that exogenous progestins, such as
progesterone delivered by a controlled internal drug release (CIDR) device, can
maintain a sub-luteal concentration of progestin in blood during a period which
permits CL regression. In the absence of a CL, removal of the progestin source
will result in a synchronized estrus (Macmillan and Peterson, 1993). However,
sub-luteal concentrations of progesterone increase LH pulse frequency, which
stimulates continuous growth of a dominant follicle (Cooperative Regional
Research Project, NE-161, 1996; Savio et al., 1993a; Savio et al., 1993b). This


120
a
cow
2215
5497
blFN-x, 50 ng/ml
b
control interferon
T 71
2215 5354 5497
Figure 4-2. Fluorographic analysis of newly synthesized intracellular
proteins from endometrial explants of cows 2215, 5354 and 5497, incubated
for 24 h in presence (+) or absence (-) of blFN-r.
a) Representative fluorograph of one dimensional SDS-PAGE
analysis of intracellular proteins (arrow indicates differentially expressed 75
kD protein (P75); b) Densitometric analysis of treatment by cow interaction of
abundance of P75 (least squares meansSEM).


128
proteins are functional GTPases that are strongly induced by type I interferons in
response to viral infections (Pavlovic et al., 1993). Ott and coworkers (1998)
showed a presence of Mx mRNA in endometrium of day 13 to day 19 pregnant
sheep, which coincide with maximum expression of IFN-x by ovine conceptuses
(Roberts et al., 1992). However, function of Mx protein in the endometrium
remains elusive.
Visual evaluation of fluorographs of both secretory and intracellular
proteins indicate that there is a differential responsiveness for different cows, in
regard to proteins induced by blFN-x. Cow 2215 appears to be the most, while
cow 5497 the least responsive to blFN-x. This phenomenon of non-uniform
responsiveness to blFN-x was also reported by Helmer and others (1989b).
They measured concentrations of plasma progesterone from cows that received
intra-uterine infusions of either bovine serum albumin or blFN-x, as a measure of
CL lifespan. Although the overall lifespan of CLs was greater for blFN-x-treated
compared to BSA-treated-cows (26 vs. 19.5 days, respectively), CL lifespan for
individual cows receiving blFN-x varied from 21 to 28 days. Thatcher and
coauthors (1997) estimated that around 20% of cows did not respond to blFN-x
in different experiments. This variability may be associated with embryo losses
observed during the period of maternal recognition of pregnancy in cattle.
Possible causes are lack of functional blFN-x receptors or failure of blFN-x to


10
improve calf crops. This prompted a great quantity of research to understand
mechanisms involved in the processes of fertilization failure and embryonic
mortality. Moreover, understanding the mechanisms underlying these alterations'
in reproductive development will provide the basis for creation of technologies
aimed to attenuate fertility problems in the field.
Causes of Fertilization Failure and Embryonic Mortality
Inadequate embrvo unit. Both genetic and environmental effects can
account for early embryonic deaths. There is an estimated 7.5% death of early
bovine embryos because of occurrence of chromosomal abnormalities (Wilmut et
al., 1986). Such abnormalities may be inherited or arise during meiosis,
fertilization or early cleavage stages (King, 1985).
Environmental effects such as heat stress also decrease the ability of
embryos to develop properly. For example, Putney and coworkers (1989)
exposed superovulated heifers to hyperthermic conditions for 10 hours after the
onset of estrus. There was no difference in the rate of fertilization between heat-
stressed and non-heat stressed heifers (control). However, only 12% of
embryos recovered from stressed heifers were normal vs. 69% of embryos
recovered from control heifers. Since the period of heat stress was administered
prior to ovulation and fertilization, it was hypothesized that the detrimental effect
of the heat stress was exerted on the oocyte within the follicular environment. In
an attempt to investigate the effects of heat stress on embryonic survival from


192
placing the tube in a speed vacuum apparatus for 10 minutes at room
temperature. Dried pellet was resuspended in 300 pi TE (hereafter referred to as
"labeled probe). Two pi of labeled probe were measured in duplicate to
determine specific activity. Specific activity of labeled probe was 140000 cpm/pl.
Radiolabeled probe was stored at -20 C until use in assay for electrophoretic
mobility shift assays (EMSA).
Probes for SIE were prepared by fill-in. A dNTP mix was prepared mixing
10 pi each of 10 mM dATP, dTTP and dGTP, resulting in a mix containing 3.33
mM of each deoxyribonucleotide. Two hundred ng of double stranded (i.e.,
annealed) SIE DNA (2 pi of stock solution) were mixed with 4pl of 10X Klenow
polymerase buffer, 16pl of water, 6 pi of dNTP mix, 10 pi of alpha 32P-dCTP (100
pCi) and 2 pi of Klenow in a 500 pi microfuge tube and incubated for 30 minutes
at 37 C. The remainder of the procedure was as described for labeling of the
ISRE probe. Specific activity of labeled probe was 42000 cpm/pl. Labeled
probe was stored at -20 C until used for EMSA.
Electrophoretic Mobility Shift Assays
General. A 5X EMSA buffer (10 mM Tris -HCI pH 7.4, 60 mM KCI, 1 mM
MgCI2, 0.5 mM EDTA, 0.5 DTT, 10% glycerol, 4 pg herring sperm DNA) was
prepared and stored at -20 C. Ten fold excess cold probes were prepared as
follows: based on volume of labeled probe added per tube, mass of DNA present


214
Preparation of Extracts
At the end of cultures, plates were transported to a cold room (4 C) and
cells were washed twice in ice cold PBS. Whole cell extracts (WCE) were
obtained as described in Chapter 5. Protein concentrations were determined by
the Bradford method, as described in Chapter 4, In WCE diluted 1:50 (20 pi
sample in 980 pi PBS).
Immunoblottina
Volumes of WCE from each plate corresponding to 20 pg of protein were
loaded onto duplicate 7.5% acrylamide gels, submitted to SDS-PAGE, and
electrophoretic transfer of proteins to nitrocellulose membranes as described In
Chapter 5. Membranes were separately probed with antibodies against either
COX-2 [1:500 dilution in Tris-buffered saline (TBS)] and PLA2 (1:250 dilution in
TBS). Secondary antibody was anti-rabbit IgG (1:5000 dilution in TBS containing
.1% tween 20). Proteins were detected by ECL and analyzed by densitometry.
To validate specificity of antibodies for PLA2 and COX-2, samples used in
Experiment 3 (positive for PLA2 and COX-2; see below) were submitted to SDS-
PAGE and transferred to a nitrocellulose membrane. Membrane was blocked
with 5% non-fat dried milk In TBS and incubated with 8 pg of -rabbit IgG
(equivalent amount of IgG present in 1:250 dilution of anti-PLA2 antibody; diluted


101
preferentially alter synthesis of selected proteins. In the present study, P5, P6,
P8, P9 and P11 were reduced In the IPSI side compared to the CONTRA side In
FDF cows, whereas for PDF cows, abundance was similar regardless of the
side. This indicates that PDF overrode the side-dependent regulation of
secretion of P5, P6, P8, P9 and P11 that occurs normally in FDF cows. Perhaps
sustained exposure to E2 downregulates E2 receptors that abolished the
decrease in protein secretion associated with E2.
In summary, FDF and PDF regulation of protein synthesis and secretion in
the oviduct is protein, region and side specific. This suggests that several
mechanisms are involved in the complex regulation of oviduct function.
Possible mechanisms include: E2 and P4 receptor regulation; differential action of
E2 and P4 (i.e., stimulatory vs. inhibitory) depending on protein, side and region;
effects of autocrine and paracrine factors; cross-talk between E2- and P4-induced
signal transduction and other intracellular effector systems.
In addition to steroids, it is possible that other effectors may control
oviductal protein synthesis and secretion. A low P4 environment elicited by a
progestin-containing device in PDF cows is associated with higher LH pulse
frequency compared to FDF cows (5, 6). Derecka et al. (1995) reported the
presence of LH receptor mRNA in porcine oviduct tissue. Recently, LH receptor
protein and mRNA transcripts were described in bovine oviductal epithelial cells
(1997). Moreover, these authors reported that hCG treatment of bovine
oviductal epithelial cells in vitro induced time- and dose-dependent secretion of a


Since the dominant paradigm in the field of luteolysis has been that
oxytocin is the major stimulator of PGF2o pulsatile secretion, experiments to test
presence of a responsive uterus had as an endpoint development of oxytocin
receptors and secretion of PGF2otin response to oxytocin. Based on this
paradigm, it has been well established that exposure of the uterus to
progesterone is required for the uterus to acquire responsiveness to oxytocin.
McCracken (1980) proposed that P4 has the ability to inhibit synthesis of E2
receptors, and synthesis of oxytocin receptors is an E2 -dependent process. As
long as P4 inhibits synthesis of E2 receptors, E2 is unable to stimulate synthesis
of oxytocin receptors. Moreover, McCracken and others (1984) suggested that
the uterus eventually becomes refractory to inhibitory effects of P4, allowing
oxytocin receptors to be expressed, which leads to pulses of PGF2a. Lafrance
and Goff (1988) treated long-term ovariectomized heifers with P4 for 0, 7, 14 or
21 days then measured PGFM in response to an oxytocin challenge. After 7,14
or 21 days of P4 -priming there was a significant increase in plasma PGFM after
oxytocin injection, but no increase was noticed in animals that did not receive P4.
Silvia and coauthors (1991) put forth the question of whether requirement for
long term exposure to P4 is due to stimulatory effects that take at least 10 days
to build up (7 days in Lafrance and Goff, 1988) or to slow development of the
condition whereby the uterus is desensitized to P4 inhibitory effects. Collectively,
the concept of a responsive uterus means that this organ has been primed by P4,
and as a result, the uterus becomes responsive to the luteolytic stimulus.


279
Leese, HJ. The formation and function of oviduct fluid. J Reprod Frtil 1988;
82:843-856.
Li J and Roberts RM. Interferon-t and interferon-a interact with the same
receptors in the bovine enndometrium. J Biol Chem 1994; 269:13544-
13550.
Li X, Leung S, Kerr IM, Stark GR. Functional subdomains of STAT2 required for
preassociation with the alpha interferon receptor and for signaling. Mol
Cell Biol 1997; 17:2048-2056.
Lin L-L, Lin AY, Knopf JL. Cytosolic phospholipase A2 is coupled to hormonally
regulated release of arachidonic acid. Proc Natl Acad Sci USA 1992;
89:6147-6151.
Lin L-L, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA2 is
phosphorylated and activated by MAP kinase. Cell 1993; 72:269-278.
MacKenzie SH, Roberts MP, Liu KH, Dore JJ, Godkin JD. Bovine endometrial
retinol-binding protein secretion, messenger ribonucleic acid expression,
and cellular localization during the estrous cycle and early pregnancy.
Biol Reprod 1997; 57:1445-1450.
Macmillan KL, Peterson AJ. A new intravaginal progesterone releasing device
for cattle (CIDR-B) for oestrus synchronization, increasing pregnancy
rates, and the treatment of post-partum anestrus. Anim Reprod Sci 1993;
33:1-9.
Macmillan KL, Taufa VK, Barnes DR, Day AM, Flenry R. Detecting estrus in
synchronized heifers using tailpaint and an aerosol raddle.
Theriogenology 1988; 30:1099-1114.
Martal J, Lacroix M-C, Lourdes C, Saunier M, Wintenberger-Torres M.
Trophoblastin, an antiluteolytic protein present in early pregnancy in
sheep. J Reprod Frtil 1979; 56:63-73.
Mawhinney S, Roche JM. Factors involved in oestrous cycle control in the
bovine. In: Sreenan, JM (ed.), Control of reproduction in the cow.
Luxembourg: EEC: 1978; 511-530.
Mayer RJ, Marshall LA. New insights on mammalian phospholipase A2(s);
comparison of arachidonyl-selective and -nonselective enzymes. FASEB .
J 1993; 7:339-348.


132
to be an adequate system for such studies. However, they could be used to
verify steady state levels of endometrial epithelial proteins. The newly developed
BEND cell line (Austin et al., 1996b) may prove to be a useful model to conduct
studies in the signal transduction system employed by bIFN-x in the
endometrium.


229
c
Q>
*-*
O
L_
CL
Ol
m
CL
a
CM
LL
O
CL
TO
o
Figure 7-8. Experiment 3. Least squares means and SE of concentrations of PGF!a in medium
conditioned by BEND cells treated with medium alone (control), phorbol 12,13 dibutyrate
(100ng/ml; PDBu) or blFN-x (50 ng/ml; bIFN-tau) and PDBu. Samples were removed before
treatments were added (0 hours), 1, 2, 3, 4, 5 and 6 hours after.
X2-5.4 x X3+0.35 x X4 (PDBu); Y=6.88-10.46 x X+6.34 x XM.21 x X3+0.08 x X4
(PDBu + blFN-x); where Y represents secretion of PGF2a, and X is a given time
point in the experiment. Orthogonal comparisons of curves confirmed that PDBu
stimulated PGF2asecretion, and that bIFN-t attenuated that effect (p<0.01).
COX-2 and PLA, immunoblottina. In agreement with previous
experiments, there was only a low, basal level of COX-2 (Figure 7-9) and PLA;


222
a
ro

X
s"
1 2 3 4 5 6 7 8
b
Figure 7-3. Experiment 1. Immunoblotting analysis of PLA-2 in whole cell extracts from
BEND cells treated with medium alone (control), blFN-t (50 ng/ml; bIFN-tau), phorbol 12,13
dibutyrate (100ng/ml; PDBu) or blFN-t and PDBu for 24 hours.
a) Enhanced chemiluminescence (ECL) exposure of abundance of PLA-2 (control:
lanes 1 and 2; PDBu: lanes 3 and 4; bIFN-T lanes 5 and 6; blFN-i and PDBu: lanes 7 and 8);
b) Least squares means and SE of abundance of PLA-2 arbitrary densltometric units (ADU).


67
reported existence of IRFs in endometrium of pregnant cows. Perry and
coauthors (1999) reported presence of STATs 1 and 2 and IRF-1 in the nucleus
of BEND cells stimulated with blFN-x. More detailed evidence for the JAK-STAT
pathway (i.e., existence, tyrosine phosphorylation, nuclear translocation, dimer
formation of and gene activation via STAT proteins) in bovine endometrium is
presented in Chapters 4, 5 and 6.
Bovine blFN-x -simulated protein synthesis in the endometrium
A main proposition of this dissertation is that proteins synthesized or
suppressed as a result of activation of the JAK-STAT pathway interact with the
PGF2a synthesizing machinery to inhibit PGF2cl secretion in the endometrium. In
this section, I will describe the data available on proteins synthesized in the
endometrium in response to blFN-x and their possible influences in the PGF2
system.
Rueda and coworkers (1993) reported secretion of 12 and 28 kD proteins
both from pregnant endometrial explants and cyclic endometrial explants
stimulated with blFN-x in vitro. In a subsequent paper, Naivar and others (1995)
further characterized those proteins and discovered a novel, 16 kDa secretory
protein (P16). Endometrium explants were obtained from day 18 pregnant cows
and Incubated in presence or absence of blFN-x. Both basal and stimulated
secretion of all three proteins increased in culture medium in a time-dependent
manner. More importantly, the 12 kD protein (Rueda et a!., 1993), now renamed


99
(Slayden and Brenner, 1994) also change in response to manipulations of the
steroid environment and steroid binding to their receptors. Therefore,
concentrations of circulating E2 and P4 for FDF and PDF cows may have
differentially regulated numbers of E2 and P4 receptors In the oviduct, and
consequently, expression of steroid-responsive proteins (as illustrated by
changes In oviductal function). Alternative explanations for the discrepancy
between incorporation rate and abundance of specific protein spots are: (1) in
FDF cows, incorporation may have been greater in proteins of higher (>97kD) or
lower (<20 kD) Mr which were not resolved and would be undetectable in gel
analyzes; or, (2) FDF may have induced a higher turnover of proteins and the
resulting partially degraded proteins were not resolved by electrophoresis (i.e.,
MW between 3.5 and 20 kD).
Several studies in cattle suggest the importance of the oviductal region
and protein milieu in reproductive processes. In studies with bulls (Anderson
and Killian, 1994), it has been demonstrated that culture medium conditioned by '
1ST tissue at estrus capacitated more sperm than did medium conditioned by
AMP. This increase was abolished by heating the conditioned medium and
inactivating proteins before incubation with sperm. Staros and Killian (1998)
showed that four unidentified oviductal proteins and a P1-llke protein from non-
luteal oviductal fluid would associate with the zona pellucida, suggesting a
modulation of sperm/egg binding or embryonic development by oviduct-derived
proteins. In the present experiment, of 20 proteins analyzed by densitometry, P2


Arbitrary Densitometric Units x 10
172
121-
* 78- t¡¡ It 11
-STAT-2
STAT-1
5121-
S 78
: -?m
p-STAT-3
5121-
s 78-
-STAT-3
0 1 3 8 15 30 60 120 min.
bIFN-T, 50 ng/ml
coip-STAT-1 coip-STAT-2
min.
blFN-x, 50 ng/ml


143
immunoblots). Membranes were washed for 15 minutes in TBST and incubated
with the appropriate primary antibody. For phosphotyrosine immunoblots,
membranes were incubated for 1 hour in a 1:1000 dilution of anti-
phosphotyrosine monoclonal antibody (PY-20) in 2% gelatin in TBS, washed 4
times for 10 minutes in TBST, incubated for 1h in a 1:8000 dilution of horse
radish peroxidase (HRP)-linked anti-mouse antibody diluted in TBST containing
2% gelatin, washed once for 15 minutes and four times for 5 minutes with TBST.
Tyrosine-phosphorylated proteins on membranes were detected with an
enhanced chemiluminescence (ECL) kit with exposures varying from 10
seconds to 10 minutes. For STATs 1, 2 and 3, membranes were incubated for 2
hours in a 1:1000 dilution each of either anti-STAT-1 and anti-STAT-2, or, anti-
STAT-3 polyclonal antibodies in 5% non-fat dried milk diluted in TBS, washed
once for 15 minutes and 2 times for 5 minutes in TBST, incubated for 1 hour in a
1:8000 dilution of horse radish peroxidase (FIRP)-linked anti-rabbit antibody in
TBST containing 5% non-fat dried milk, washed once for 15 minutes and four
times for 5 minutes with TBST. STAT proteins on membranes were detected
with an ECL kit, with exposures varying from 5 seconds to 5 minutes.
Nature of BEND Cells
To confirm that the BEND cells were primarily from epithelial origin, WCEs
were obtained from BEND cells at the second (P2), fourth (P4), ninth (P9) and
thirteenth(P13) passages. Increasing amounts of protein from extracts of each


212
free medium was mixed with appropriate treatments and added to cells in the
same volumes listed above. Samples of medium (500 pi in Experiment s 1, 2,
and 3; 250 pi in Experiment 4) were collected at specified times (see
Experimental designs) and stored at -20 C. After sampling, the same volume of
medium removed was replaced with medium containing the appropriate
treatment, so that a constant volume was maintained throughout each
experiment. Concentrations of PGF2awere measured in medium as described
below.
Radioimmunoassay
Concentrations of PGF2awere measured in medium undiluted (control-,
blFN-t- and PDBu + blFN-x-treated cells) or diluted 1:2 in medium (PDBu-treated
cells). Twenty five pi of each sample were further diluted in 75 pi of 50 mM Tris-
HCI, pH 7.5 (Tris buffer). Therefore, total volume of diluted sample for assay
was 100 pi. Radioimmunoassay procedure was described by Danet-Desnoyers
and coworkers (1994). Assay was validated for serum-free medium adding 25
pg/.1 ml PGF2oto medium. Average recovered PGF2awas 25.11.42 pg/.1 ml,
which yielded a calculated recovery of 100.25%. To prepare standard curves,
known amounts of non-radioactive PGF2 (1.25 to 1000 pg/tube) were diluted in
Tris buffer (100 pi). Anti-PGF2o antiserum (characterized by Dubois and Bazer,
1991) was diluted 1:5000 (Tris buffer; 100 pl/tube). Final volume of sample was


73
recipients. Failure of cows to extend CL lifespan in response to blFN-x have
been reported (Helmer et al., 1989b; Meyer et al., 1995). This indicates failure in
the interferon receptor system, JAK-STAT-mediated signal transduction, post
signaling mechanisms within the endometrium or a combination of these factors.
These responses have not been examined in a population of cows and warrant
further investigation.
Manipulating Uterine Function to Minimize Embryo Mortality
Bovine IFN-t administration
Based on the variation of conceptus size and consequent capacity to
secrete at the critical time of maternal recognition of pregnancy for CL
maintenance, it is reasonable to propose that supplementing blFN-x at that
critical period may decrease embryonic losses. The rationale is that a slightly
underdeveloped conceptus that may be unable to deliver the appropriate
antiluteolytic signal may be rescued by exogenous blFN-x administered at
aroundjday 17 after insemination. Lack of availability of recombinant blFN-x and
structural similarity with bIFN-a prompted Newton and others (1990) to test
fertility effects of bIFN-a. Interferon-a extended CL lifespan but caused side
effects such as increased body temperature. Barros and coworkers (1992)
conducted a field experiment where bIFN-a was administered daily from days 14
to 17 of pregnancy or as a single injection on day 13. Conception rates were


184
NE is inconclusive (Figure 5-13 represents the only experiment where
phosphorylation of nuclear STAT-2 was detected). However, there is an
indication of increased phosphorylation after 15 minutes. Moreover, there is
clear increase in abundance of STAT-2 in the nucleus, which occurs earlier than
maximum appearance of STAT-1, indicating earlier nuclear translocation (Figure
5-11). Similar to STAT-1 and -3, abundance of STAT-2 in NE was still higher at
120 minutes compared to 0 minutes. This suggests that STATs could be
continuously transported to the nucleus, even after 120 minutes. This is
consistent with data from Haspel and others (1996) which showed a continuous
influx of STAT-1 to the nucleus up to 2.5 hours after interferon treatment.
In contrast to STATs 1 and 2, changes in phosphorylation of STAT-3 in
the CE (Figure 5-16) and NE (Figure 5-17) were almost parallel, indicating a
ready translocation of phosphorylated STAT-3 to the nuclear compartment.
Similar to STAT-1, there is an abrupt decrease in phosphorylation between 30
and 60 minutes in both the CE and NE, but such a decrease is more
accentuated in the CE. Since this decrease is not followed by a concomitant
change in abundance of STAT-3 (Figure 5-14), it suggests a faster removal of
phosphorylated STAT-3 in the CE compared to the NE.
In the coimmunoprecipitation experiments, there was an overall trend of
reciprocal changes in abundance of STATs in NE compared to CE (Figures 5-19
to 5-22). Therefore, an increase in abundance of STATs in NE was usually
paralleled by a decrease in the CE, and abundance in the CE returned to control


211
mM EDTA, pH 8 to 200 pg/ml; 1.08 x 107 units of antiviral activity) was a
generous gift from Dr. Michael Roberts, University of Missouri. Isotopically
labeled [5, 6, 8, 11, 12, 14, 15-3H]-PGF2a (specific activity=212 Ci/mole) was from
Amersham Corp. (Arlington Heights, IL). Tris-HCI, Tween 20, isopropyl alcohol
and chloroform were from Fisher Scientific (Pittsburgh, PA). Anti-COX-2
polyclonal antibody was from Cayman Chemical (Ann Arbor, Ml; lyophilized anti-
COX-2 antibody was reconstituted in 200 pi sterile water, aliquoted in 20 pi
portions and stored at -20 C). Cytosolic PLA2 polyclonal antibody (N-216;
catalog number SC-438, 200pg/ml) was purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Material used for cell culture, whole cell
extracts (WCE) and for immunoblotting procedures were as described for
Chapter 5.
Cell Culture and Sample Collection
BEND cells were plated on 100 mm tissue culture-treated petri dishes
(8.5 x 105 cells/plate; 20 ml of medium, Experiment 1; 10 ml of medium,
Experiments 2, and 3) or on 35 mm wells on 6-well plates (2 x 105 cells /well; 3
ml of medium, Experiment 4). Cells were grown to confluency, washed in serum-
free medium (20 ml, Experiment 1; 10 ml, Experiments 2 and 3; 3 ml Experiment
4) and cultured for 24 hours (same volumes used for washes) in serum-free
medium. After this time, a sample of medium was collected and stored at -20 C.
This was designated 0" hour sample. Then cells were washed again and serum



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150
Figure 5-4. Immunoblotting analysis of cytokeratin and vimentin in whole cell
extracts from BEND cells of different passages (P). Increasing concentrations
of proteins from extracts (2.5, 5, 10 pg, lanes 1 and 4, 2 and 5, 3 and 6
respectively) were loaded.
a) Cytokeratin, P9 (lanes 1-3), P2 (lanes 4-6); b) cytokeratin, P4 (lanes
1-3), P13 (lanes 4-6); c) Vimentin, P9 (lanes 1-3), P2 (lanes 4-6); d) vimentin,
P4 (lanes 1-3), P13 (lanes 4-6).


287
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226
5121
= 78
cPLA-2
*# i'*
123 456 789
Figure 7-6. Experiment 2. Immunoblotting analysis of PLA-2 in whole cell extracts from
BEND cells treated with medium alone (control), phorbol 12,13 dlbutyrate (100ng/ml; PDBu)
or blFN-t (50 ng/ml; bIFN-tau) and PDBu for 12 hours.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of
PLA-2 (control: lanes 1 and 2; PDBu: lanes 3, 4 and 5; bIFN-T and PDBu: lanes 6, 7, 8 and
9); b) Least squares means and SE of abundance of PLA-2 arbitrary densltometrlc units
(ADU).


167
Validation of Time Responses to blFN-x
In the absence of blFN-x, there was no change in phosphorylation of
STATs 1, 2 or 3 overtime (Figure 5-18). Basal levels of phosphorylation
associated with STAT proteins were noted, but there was no change In
phosphorylation associated with time of exposure to medium alone.
Coimmunoprecipitation of STATs
Coimmunoprecipitation of STAT-1 with STAT-3 (immunoblot analysis of
STAT-1 after immunoprecipitation of extracts with STAT-3 antibody). In CE,
abundance of STAT-1 complexed with STAT-3 decreased during the 30 and 60
minutes samples, but Increased again by 120 minutes (Figure 5-19). There was
an Increase in abundance of STAT-1 associated with STAT-3 In the nucleus after
8 minutes of exposure to blFN-x (Figure 5-20). STAT-1 remained elevated up to
30 minutes and then declined after that.
Coimmunoprecipitation of STAT-2 with STAT-3. In the cytosol,
abundance of STAT-2 gradually decreased to reach Its minimum level at 30
minutes, but Increased to control levels at 120 minutes (Figure 5-19). In NE,
bIFN-T stimulated a time-dependent association of STAT-2 with STAT-3 that
increased after 8 minutes to reach a maximum at 15 minutes and decline
thereafter (Figure 5-20).


29
and processes occurring in the endometrium. The endometrium is lined with a
single layer of epithelial cells and contains simple, colled, tubular glands. Glands
are relatively straight at estrus, but become more coiled and complex as
progesterone levels rise as the estrous cycle progresses (Hafez, 1993a).
Glandular secretions, the hystotroph, constitute a nutrient-rich mixture required
for development of the conceptus (Bazer and First, 1983), and will be discussed
next. Underneath the luminal epithelium and around the glands is the
endometrial stroma, composed of stromal cells distributed in greater or lower
density patterns, depending on the location. In the cow, between 100 to 150
aglandular ridges are present, the caruncles (Flood, 1991). During pregnancy,
caruncles become attached to specialized areas of the allantochorion of the
conceptus, the cotyledons, to form placentomes. Placentomes are units for
exchange of gas and nutrients between maternal and embryonic units. A better
description on the process of attachment and formation of placentomes is given
afterwards in this review. A highly dynamic and organized microvasculature
supplies myometrlal and endometrial tissues. They originate from uterine
branches of the ovarian arteries (supply uterine body and uterine horns), uterine
arteries (supply uterine body and uterine horns) and urogenital artery (supplies
caudal uterus and cervix). In the cow, uteroovarlan relationships exist in that
demise of the CL (luteolysis) is regulated by the uterine horn adjacent to the
ovary containing CL. Luteolysis is accomplished by countercurrent exchange of
the uterine produced luteolysin, PGF2o, between the uteroovarian vein and the


286
Shuai K, Schindler C, PreziosoVR, Darnnell jr JE. Activation of transcription by
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74
actually decreased by about 10% compared to control animals. This was
attributed to blFN-a-lnduced side effects such as hyperthermia and acute drops
in P4 concentration. Alternative delivery systems and use of actual blFN-x may
still make this technology useful in the field (see Thatcher et al., 1994a for
discussion).
Fat feeding
Another possible manipulation of this system consists of attempting to
make the uterus less luteolytic, by changing the proportion of luteolytic AA to
antiluteolytic linoleic acid. For example, Thatcher and others (1997) described
an experiment in which Menhaden fish meal was fed to cows for 25 days. Fish
meal contains both elcosapentanoic and docosahexaenoic fatty acids, which had
been shown to be able to decrease PGF2asecretion. Indeed, cows fed fish oil
had a much attenuated secretion of PGFM in response to an oxytocin challenge.
Prevention of heat stress
One single environmental challenge that has negative effects both in the
embryonic and maternal units during the period of maternal recognition of
pregnancy is heat stress, as discussed previously. Therefore, strategies to
reduce effects of high environmental temperatures, such as observed in tropical
and subtropical latitudes, warrant investigation and application.


144
passage were loaded in duplicate 7.5% acrylamide mini-gels. Proteins were
transferred to nitrocellulose membranes, which were separately immunoblotted .
with monoclonal antibodies against vimentin (a marker for stromal cells; 1:2000
final dilution) and cytokeratin (a marker for epithelial cells; 1:2000 final dilution).
Similar immunoblotting procedures as described above were used.
Dose Response to blFN-x
To test for specificity of blFN-x on STAT phosphorylation and in order to
choose a dose to use in subsequent experiments, seven plates of BEND cells
were assigned randomly to receive 0, 3.125, 6.25, 12.5, 25, 50 or 100 ng/ml b-
IFN-x for 15 minutes. In the present and subsequent experiments, blFN-x was
diluted from a stock containing 200 pg/ml blFN-x. Total mass of blFN-x required
for a given plate was calculated (e.g., 50 ng/ml x 20 ml of medium in a well =
1000 ng blFN-x), and the volume of stock containing the mass of blFN-x required
was calculated [e.g., 1000 ng blFN-x needed/(200ng/pl) stock = 5pl stock] and
mixed with medium to a final volume of 1 ml, which was added to the culture
dish. Whole cell extracts were obtained and immunoprecipitated with anti-STAT-
1 and -2 antibodies and analyzed by immunoblotting for anti-phosphotyrosine
and for anti-STAT-1 and -2.


1
2
48
C Kk-O
R1
w
\J
w
1
STAT-2
6
Figure 2-1. The JAK-STAT pathway of signal transduction and gene activation.
1) Binding of type I interferon (IFN) to the interferon-a-receptor (R)-2 chain causes
recruitment of R1; 2) dimerization of IFN receptor complex causes reciprocal tyrosine
phosphorylation of associated JAK kinases (tyk-2 and jak-1); 3) JAK kinases phosphorylate
receptor subunits in tyrosine residues; 4) unphosphorylated, cytosolic STAT proteins bind
receptor complex through SH2 domains present in STAT proteins; 5) JAK kinases
phosphorylate tyrosine residues in bound STATs, STATs dissociate from receptor complex
and associate in a heterodimer (STATs 1 and 2); 6) dimerized STATs translocate to the
nucleus, bind to the DNA binding protein p48, forming the ISGF-3 transcription complex,
which stimulate synthesis of IFN-inducible genes.


42
2. In fact, existence of a pregnancy-induced inhibitor of COX-2 activity has been
found in the endometrium of cows (Basu and Kindahl, 1987; Gross et al, 1988).
Danet-Desnoyers and others (1993) identified linoleic acid as the active molecule
in bovine endometrium which acted to decrease COX-2 activity. Moreover,
linoleic acid acted as a competitive inhibitor of AA on a PGF2o[ generator assay
(Thatcher et al. 1994b). It is possible that altered lipid metabolism in the
pregnant endometrium increases availability of linoleic acid to inhibit COX-2
activity and thereby decrease PGF2ci production.
Finally, pregnancy could change lipid composition and metabolism in the
endometrium to inhibit PGF2(J synthesis. Thatcher and others (1995) compared
concentrations of free linoleic and free AA in endometrial microsomes from day
17 cyclic and pregnant cows. They found that pregnancy decreased
concentrations of AA and increased concentrations of linoleic acid compared to
estrous cycle, to result in a change of the ratio of linoleic to AA of 0.6 to 2.4 in
endometrium between cyclic and pregnant cows.
Effectors of maternal recognition of pregnancy: IFN-t
A considerable amount of research focused on identification and
purification of conceptus products with the PGF2a-secretion inhibitory activity
required for maintenance of pregnancy. A family of molecules has been
identified as the embryonic antiluteolytic factor in ruminants, named IFN-t
(Thatcher, 1999). For a historical prospective, see Martal et al. (1979), Godkin et


284
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golden hamster. J Reprod Immunol 14:177-189.


107
Scalpel blades (number 20) were from Feather Safety Razor Co., Ltd, (Japan).
Donations of Lutalyse were made by Pharmacla-Upjohn Co. (Kalamazoo, Ml).
Experiment 1
Animals. Multiparous non-lactating dairy cows (n=3, 24 hour incubation
experiment; n=3, 2 hour incubation experiment) were synchronized with one
injection of PGF2c, and observed for estrus, using chalk-assisted estrus detection
as described in Chapter 3 (Macmillan et al., 1988). Transrectal ultrasonography
was used to confirm ovulations (48 hours after standing heat) and presence of a
healthy CL prior to slaughter (day 16 of the synchronized estrous cycle) using an
Aloka echo camera model SSD 500 linear array ultrasound scanner equipped
with a 7.5MHz transducer (Aloka Co., Japan). At day 16 (2 hour incubations) or
day 17 (24 hour incubations) of the estrous cycle, a blood sample was collected
from each cow by tail venipuncture and cows were slaughtered at the Meat
Laboratory of the Department of Animal Science, University of Florida.
Reproductive tracts were removed Immediately and transported to the laboratory
on ice. Ovaries were examined for presence of CL and follicles. Endometrium
was dissected from myometrium using scissors, and strips of tissue were placed
in a 100x20 plastic tissue culture-treated petri dish containing modified minimum
essential medium [(MEM, prepared as described in Chapter 3, but supplemented
with 46.8 mg/I leucine (leu)] to prevent dehydration. Explants of endometrium


161
a
S 121
X
= 78
M
"4
M MM* -
8 15 30 60 min.
'-P-STAT-2
STAT-1
bIFN-T, 50 ng/ml
8 15 30 60 120
min.
blFN-x, 50 ng/ml
Figure 5-12. Immunoblotting analysis of STATs 1 and 2 immunoprecipitated from cytosolic
extracts from BEND cells treated with blFN-t for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine
phosphorylation of STATs 1 and 2; b) Least squares means and SE of tyrosine phosphorylation of
STATs 1 and 2 (within each variable, bars with distinct subscripts are statistically different, p<. 1).


31
MacKenzie and coauthors (1997) reported expression of retinol-binding protein
(RBP) in bovine uterus, and monitored steroid modulation of expression of this
protein during estrous cycle and early pregnancy. The hypothesized role of RBP
is to regulate transport of vitamin A to the conceptus. Finally, expression of
growth factors involved in the growth hormone (GH)-insulin-like growth factor
(IGF) axis were examined by Kirby and others (1996). They reported expression
of IGF-1, IGF-binding protein (IGFBPJ-2 and 3 and GH receptor in the uterus.
Collectively, uterine milk has functions similar to those of the oviductal fluid, to
provide an adequate microenvironment for conceptus development. As the
embryo develops, it starts to contribute with its own secretions to the pool of
molecules composing the uterine fluid, which becomes more complex and may
exert regulatory functions that influence both the conceptus and the uterus.
Regulation of Reproductive Processes Occurring in the Uterus
As mentioned previously, the uterus plays specific roles both during the
estrous cycle and during pregnancy. During the estrous cycle, the uterus
regulates ovarian function and the ovarian cycle and the uterus in turn is
regulated by actions of ovarian steroids. During pregnancy, functions of the
uterus include transport, storage and maturation of spermatozoa, recognition
and reception of embryos, provision of an embryotrophic environment for
conceptus development during gestation, and expulsion of fetus and placenta at
parturition (Bartol, 1999). At this physiological state, uterine function is regulated


205
was located at the very top of the EMSA gel, it is presumed that the STAT-1
antibody supershlfted sub-complex was of higher molecular weight and therefore
did not enter the gel. Alternatively, STAT-1 antibody may have Interfered with
binding of STAT-1-containing complexes (i.e., STAT 1:1 and STAT 1:3) to the
SIE probe. This would explain why s4a was more intense when anti-STAT-1
was added to the pre-incubation mixture (lane 8) compared to lane 6. Since
binding of STAT complexes to the labeled SIE is probably competitive, the s4a
complex, probably formed by STAT 3:3 homodimer, had a greater chance to
bind to the SIE, causing increase in intensity of that sub-complex.
Collectively, EMSA experiments confirmed and expanded findings of
Chapters 4 and 5. STAT proteins indeed migrated to the nucleus upon
activation with blFN-x. More Importantly, these activated STATs formed
complexes with the ability to bind sequences of DNA contained in interferon-
regulated genes. This supports the concept that blFN-x stimulates synthesis of
proteins through the JAK-STAT pathway. Our working hypothesis is that blFN-x-
induced proteins act to inhibit the pathway of PGF2a production in the
endometrium. In fact, one such protein is the bovine ubiquitin cross-reactive
protein (bUCRP; Perry et al., 1997), which promoter region contains five putative
ISRE elements. For example, UCRP forms conjugates with cytosolic proteins,
possibly altering their cellular activity (Johnson et al., 1998a). Conjugated
proteins may include proteins involved in the synthesis of PGF2a, such as the


80
(Uppsala, Sweden), TEMED and ammonium persulphate were from Bio-Rad
(Hercules, CA). Glycine was from ICN Pharmaceuticals, Inc. (Costa Mesa, CA).
Coomassie brilliant blue, fast green, bromophenol blue, p-mercaptoethanol,
hydrochloric acid, sodium hydroxide, tris (hydroxymethyl) aminomethane, sodium
salicylate, acetic acid and chromatography paper were from Fisher Scientific
(Fairlawn, NJ) and X-OMAT x-ray film was from Eastman Kodak Co. (Rochester,
NY).
Preparation of Medium
Leucine-deficient modified minimum essential medium (MEM; 10% normal
concentration of leu) was prepared as described by Buhi and coworkers (Buhi et
al., 1990). Briefly, MEM was supplemented with glucose (3g/l), methionine (1.5
mg/I), leu (5.2 mg/I), sodium bicarbonate (2.2 g/l), MEM vitamins (10 ml/I), non-
essential amino acids (10 ml/l), insulin (7.41 mg/I), sterile filtered and adjusted to
pH 7.4. Before use, medium was supplemented further with glutamine (292
mg/I), methionine (13.5 mg/I) and antimycotic-antibiotic solution (10 ml/I).
Animals and Treatments
During the pre-treatment period, estrous cycles of six mature non-lactating
cows were synchronized (Figure 3-1). A used CIDR device containing
approximately 1.2 g (Van Cleef et al., 1992) of P4 was placed into the vagina of


84
2000, Alpha Innotech Corporation, San Leandro, CA). Since a constant amount
of DPM was loaded for all samples, the capacity of tissues to synthesize and
secrete macromolecules (DPM/mg of tissue) was not accounted for and,
therefore, unadjusted densitometrlc measurements were biased. Different
secretory capacities were corrected by expressing the densitometric
measurements per unit secretory tissue. In this way, densitometric
measurements from tissues with greater secretory capacity were adjusted
upwards and vice versa for tissues with lower secretory capacities. Adjustments
were calculated by the equation: adjusted Arbitrary Density Units (ADU) =
ADU/mass of tissue equivalents, where one tissue equivalent is the mass of
tissue needed to synthesize and secrete 4 x 105 DPM of labeled
macromolecules. Mass of tissue equivalents was obtained by dividing 4 x 105
DPM by incorporation rate (DPM non-dialyzable macromolecules/mg of tissue)
for individual tissue samples.
Hormone Assays
Concentrations of E2 and P4 in plasma were measured by
radioimmunoassays previously validated in our laboratory [E2:(Badlnga et al.,
1992); P4:(Knickerbocker, 1986)]. Intra- and inter-assay coefficients of variation
were 15.5 and 12.4%, respectively, for E2and, 6.8 and 8.1%, respectively, for P4


159
121
78
9MUM
-STAT-2
-STAT-1
i 8 15 30 60 min.
bIFN-T, 50 ng/ml
blFN-x, 50 ng/ml
Figure 5-10. Immunoblotting analysis of STATs 1 and 2 immunoprecipitated from
cytosolic extracts from BEND cells treated with blFN-x for increasing Intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of STATs
tip
1 and 2; represents positive control for STATs 1 and 2; b) Least squares means and SE of
abundance of STATs 1 and 2.


100
and P13 showed differences in synthesis between treatments. Both proteins
were inhibited by PDF, suggesting an overall down-regulation. The strong effect
of region for 16 of 20 spots measured suggested a biosynthetic gradient in which
the secretion was greater or less for the 1ST depending upon the protein. Such a
gradient has been reported in the pig and sheep (Buhi et al., 1992; Buhi et al.,
1996; DeSouza and Murray, 1995; Murray, 1993). Moreover, DeSouza and
Murray (1995) reported differential secretion of a chitinase-like protein, similar to
P1 in response to steroid treatments in sheep, while Buhi et al. (1996) showed
differential expression POSP mRNA among oviductal regions in pigs.
Treatment by region interactions indicate that the steroid milieu generated
by PDF vs. FDF modulated synthesis and secretion of particular proteins
differently, depending on the oviductal region. As an example, PDF abolished
synthesis of P14 in the AMP, while it was absent in the 1ST and present in the
INF regardless of the treatment. In contrast, P7 synthesis was induced by PDF
in the AMP, although P7 was present in similar amounts for both treatments in
the INF and 1ST. It is likely that optimal function of each region is achieved when
the oviductal micro-environment includes the appropriate secretory proteins.
Treatment by side interactions indicated that the oviduct adjacent to the
ovary bearing the DF responded differently depending upon follicular status (FDF
vs. PDF). Ireland et al. (1984) demonstrated that blood drainage from the ovary '
containing the DF contained higher concentrations of E2 compared to the
CONTRA ovary. Exposure to higher concentrations of E2 may therefore


207
a response takes place in blFN-i -stimulated BEND cells. Alternatively, IRF-1
could regulate transcription of unrecognized genes with potential effects on the
PGF2(1generation cascade.
The working hypothesis of this dissertation is that bIFN-T acts through the '
JAK-STAT pathway to reduce production of PGF2a in the endometrium.
Chapters 4, 5 and 6 demonstrated the presence and functionality of this pathway
In BEND cells. Next chapter will examine whether blFN-x has the ability to
reduce PGF.
In BEND cells.


217
individually. For PGF2a, data were analyzed as a split-plot design. Independent
variables in the mathematical models were: treatment, dish (or well) within
treatment, time, treatment by time and residual error. Effects of treatment were
tested using dish within treatment as the error term. Heterogeneity of variance
among treatments was tested by the maximum F-ratio test (f max; Hartley, 1950)
and found significant in all experiments. Since this invalidates assumptions for
analysis of variance, data were transformed to log10 and re-tested for f max.
Transformations effectively eliminated heterogeneity of variance for Experiments
2, 3 and 4, but not for Experiment 1. On that experiment, the Box-Cox
procedure for data transformation was applied (Peltier et al., 1998) and a A, value
of 0.4 was found to eliminate heterogeneity of variance. Data were analyzed
after appropriate transformations. However, for the sake of clarity, data are
presented as untransformed values. Means were compared as a series of pre
planned orthogonal contrasts.
In Experiment 1, only 12 and 24 hour data were used for analysis. Due its
factorial nature, contrasts were blFN-x and blFN-x+PDBu vs. control and PDBu '
(tests the effects of blFN-x), PDBu and blFN-x+PDBu vs. control and blFN-x
(tests the effects of PDBu) and, control and blFN-x+PDBu vs. blFN-x and PDBu
(tests the interaction of PDBu and blFN-x effects). Contrasts were calculated
using dish within treatment as the error term. Treatment by time contrasts were
also calculated.


134
conducted to study the signal transduction system activated by blFN-x. Initial
work of our laboratory (Binelli et al., 1996) suggests that blFN-x activates the
JAK-STAT signal transduction pathway (Darnell et al., 1994; Darnell, 1997) in
bovine endometrial cells. Our hypothetical model (Figures 2-1 and 2-3) is that
binding of blFN-x to its receptor elicits tyrosine-phosphorylation of STAT proteins
in the cytosol of endometrial epithelial cells (Levy et al., 1989). Phosphorylated
STATs (STAT-1, -2 and -3) form homo- and heterodimers (Schindler and Darnell,
1995; Darnell, 1997) that translocate to the nucleus where they activate
transcription of blFN-x induced genes and protein synthesis (Schindler et al.,
1992). Synthesized proteins could act to decrease synthesis of PGF2o. Binelli
and coworkers (1996; Chapter 4) showed that STAT-1 is present in bovine
endometrial explants and that blFN-x is able to induce tyrosine-phosphorylation
of STAT-1. The present Chapter contains results from a series of studies
designed to gain further knowledge on the signal transduction pathway
stimulated by blFN-x in bovine endometrium. Our model of study was a recently
developed line of bovine endometrial epithelial cells (BEND cells), obtained from
day 14 cyclic cows (Austin et al., 1996b). Specific objectives were: (1) to test the
dose responsiveness of bIFN-x-induced phosphorylation of STAT-1, and -2, (2)
to study the time responsiveness of bIFN-x-induced phosphorylation of STAT-1,
-2, and -3; (3) to verify whether blFN-x induces nuclear translocation of STAT
proteins; and, (4) to examine the formation of STAT-STAT complexes.


Figure 5-21. Immunoblotting analysis of STAT-3 co-immunoprecipitated with STATs 1 and 2
from cytosolic extracts from BEND cells treated with blFN-t for Increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of STAT-3;
b) Representative ECL exposure of tyrosine phosphorylation of STATs 1 and 2; c) Representative
ECL exposure of abundance of STATs 1 and 2; d) Least squares means and SE of abundance of
STAT-3 (bars with distinct subscripts are statistically different, p<.1).


20
processes such as fertilization, and early embryonic development (Buhi et al.,
1997a). More importantly, changes in the optimal milieu of de novo synthesized,
secretory oviductal proteins may lead to sub-optimal micro-environments
conducive to reproductive failure (Binelli et al., 1999, Chapter 3).
Steroid Regulation and Protein Synthesis
Macromolecules present in oviductal fluid have been suggested to serve
important roles in sperm capacitaron (Anderson and Killian, 1994), fertilization
(Boatman and Magnoni, 1995), and early embryo development (Gandolfi et al.,
1989). Therefore, alterations in oviductal biosynthetic activity and protein
synthesis and secretion may affect conception rate. Steroid modulation of
oviductal synthesis and secretion of proteins has been characterized in sheep
(Buhi et al., 1991, Murray, 1993), baboon (Verhage and Fazleabas, 1988) and
swine (Buhi et al., 1989; Buhietal., 1990). Buhi and others (1989) measured
the biosynthetic capacity of the oviduct (i.e., rate of incorporation of radiolabeled
amino acid precursor into newly synthesized protein) of pigs during the estrous
cycle, early pregnancy and in ovariectomized animals following steroid
replacement (Buhi et al., 1992). These studies indicated that bioactivity of the
oviduct is related to the hormonal status of animals. For example, incorporation
rate of radiolabeled leucine was greater when ovariectomized animals were
treated with E2 compared to P4. These findings were consistent with what was


35
A novel concept regarding control of luteolysls involves the actions of LH
in the endometrium. Friedman et al. (1995) reported the presence of LH binding
sites in bovine endometrium that were maximal in endometrium from days 15 to
17 which corresponds to the time of luteolysis. In addition, production of PGF2a
was stimulated when endometrial cells from days 15 to17 of the estrous cycle
were treated with LH in vitro. In a series of preliminary experiments (Fields,
personal communication) ovariectomized, P4-treated cows were injected with
either saline or E2 and 4 hours later injected with either saline or human chorionic
gonadotropin (hCG, a long half life LH analog). Concentrations of PGFM in
plasma were elevated only for groups pre-treated with E2. In addition, hCG
injection elicited a pronounced release of PGFM compared to saline. It can be
concluded that exposure to E2 is required for the endometrium to secrete PGFM
in response to LH. Physiologically, it could be hypothesized that E2 acts at the
endometrium to enhance responsiveness to circulating LH, thereby evoking
PGF2o secretion during luteolysis. Indeed, a decline in P4 will elevate plasma LH
that may contribute to a continued secretion of PGF2oto re-enforce the luteolytic
process. Mechanistically, this could be accomplished by increasing
concentration of LH receptors in the endometrium or by connecting intracellular
pathways stimulated by LH with the PGF2ot secretory machinery.
In summary, it is doubtful that oxytocin is the sole stimulator of pulsatile
secretion of PGF2a. It is more probable that other effectors such as E2 and LH
act in concert with oxytocin to stimulate luteolysis.


with blFN-t -induced suppression of phospholipase-A2 and cyclooxygenase-2
protein expression and enzymatic activity measured through IB and RIA.
Collectively, experiments elucidated mechanisms that are involved in maternal-
conceptus crosstalk required for successful reproductive outcome.


177
a *
1121 ra
= 78-¡ !, II idI-3
b
| 121 ^
s- 7g ^ -p-STAT-1
c
-121- i
*
. -STAT-2
= STAT-1
0 1 3 8 15
30 60 120 min.
blFN-x, 50 ng/ml
d
blFN-r, 50 ng/ml


116
medium was discarded and cells were resuspended in 500 pi of whole cell
extract (WCE) buffer, and WCE were obtained as described in Chapter 5.
Immunoblottina. To evaluate the nature of cells scraped from
endometrium (described above), 20 pg of WCE from each cow were loaded onto
duplicate 7.5% acrylamide gels, submitted to SDS-PAGE and electrophoretic
transfer of proteins to nitrocellulose membranes as described for Experiment 1.
Membranes were probed separately for cytokeratin (1:2000 dilution) and
vimentin (1:2000 dilution) as described in Chapter 5. Proteins were detected
using ECL.
Volumes of WCE from each cow corresponding to 25 pg of protein were
loaded onto 7.5% acrylamide gels, submitted to SDS-PAGE and electrophoretic
transfer of proteins to nitrocellulose membranes as described for Experiment 1.
Membranes were separately probed with antibodies against COX-2 and PLA2 as
described in Chapter 7. Proteins were detected by ECL and analyzed by
densitometry.
Use of densitometry to evaluate abundance of protein signals obtained
after chemiluminescence were validated. Serial dilutions of WCE positive for
COX-2 were loaded onto a 7.5% acrylamide gel and submitted to electrophoresis
and immunoblotting for COX-2. The ECL exposure was analyzed by
densitometry.


112
bottom, removal and discard of supernatant, addition of 1 ml ice-cold WCE
buffer, and resuspension of beads in this buffer by gently hand mixing. Fifty pi of
slurry were added to 4000 gg of protein per sample. WCE buffer was added up
to a final reaction volume of 1 ml. Samples were immunoprecipitated overnight
at 4 C in a rotating device, washed thrice in WCE buffer and once with 62.5 mM
Tris-HCI pH 6.8. After this last buffer was removed, tubes were pulse-spun once
more and residual liquid was gently aspirated with a 25 gauge needle
(PrecisionGlide, Becton Dickinson and Co., Franklin Lakes, NJ) attached to a
tuberculin syringe (Becton Dickinson and Co., Franklin Lakes, NJ). Sixty gl of 1x
Laemmli buffer was added in each tube, gently mixed with the tip of a pipette
and proteins were solubilized by incubating beads for 5 minutes in a heating
block (Dry bath incubator, Fisher Scientific, Pittsburgh, PA) at 100 C. Samples
were pulse-spun in a microcentrifuge and supernantants were used for
immunoblots.
Immunoblots. Immonoprecipitated proteins were separated in 7.5%
acrylamide mini-gels by 1-dimensional SDS-PAGE. Mini-gels were incubated in
Towbin transfer buffer (25 mM Tris, 192 mM Glycine, 20% methanol) and
proteins transferred to nitrocellulose membranes using a mini-gel transfer tank
apparatus (Hoeffer Scientific Instruments, TE series transphor electrophoresis
unit, San Francisco, CA) for 4 hours at 1000V and 200mA. After transfer,
membranes were blocked for 2 hours in 200 ml of 2% (w/v) gelatin in Tris-
buffered saline (TBS, 10 mM Tris, 150 mM NaCI, pH 7.6) supplemented with


CHAPTER 3
PERSISTENT DOMINANT FOLLICLE ALTERS PATTERN OF OVIDUCTAL
SECRETORY PROTEINS FROM COWS AT ESTRUS
Introduction
Synchronization of the estrous cycle in cattle is a very important tool for
reproductive management. For example, synchronization systems are used
widely for artificial insemination, timed insemination and embryo transfer. Most
commonly, synchronization is achieved with combinations of treatments with
prostaglandin F2o (PGF2a), progestins and GnRH (Thatcher et al., 1996).
Synchronization with progestins is based on the principle that exogenous
progestins, such as progesterone delivered by a Controlled Internal Drug
Release (CIDR) device, can maintain a sub-luteal concentration of progestin in
blood during a period which permits CL regression. In the absence of a CL,
removal of the progestin source will result in a synchronized estrus (Macmillan
and Peterson, 1993).
During the estrous cycle in cattle, two to three follicular waves of dominant
follicle development occur (Savio et al., 1988; Sirois and Fortune, 1988). Each
follicular wave is comprised of periods of recruitment, selection, dominance and
turnover or atresia. The ovulatory follicle generated in the last wave does not
76


232
(Figure 7-10) in cells incubated with medium alone. Presence of PDBu
treatment stimulated synthesis of both proteins (p<.01 and p<.08; contrast c1 for
COX-2 and PLA2 respectively at 6 hours). However, adding blFN-t markedly
reduced PDBu-induced expression of COX-2 (p<.01; contrast c2) and a
numerical but non-significant decrease in PLA2 (p>.2; contrast c2).
Experiment 4
Secretion of PGF-,~ Patterns of PGF2o, secretion into culture medium over
time were similar to ones described in Experiment 3 for controls, PDBu and
PDBu + blFN-x treatments (Figure 7-11). Treatment with PDBu stimulated
PGF2 secretion through 6 hours and this response was attenuated markedly
with the concurrent addition of blFN-x. Addition of blFN-x after a 3-hour
exposure to PDBu caused a decrease in PDBu-stimulated PGF2ctsecretion,
which is noticeable at the 5-hour sample and becomes even more pronounced at-
the 6-hour sample. Moreover, rate of accumulation of PGF2ain medium between
4 and 6 hour is greater in absence of blFN-x is (1201 pg/ml/2 hours for PDBu vs.
442 pg/ml/2 hours for PDBu + blFN-x-3h). Furthermore, when PDBu and PDBu
+ blFN-x-3h treatments were analyzed without other treatments, there was a
significant time by treatment interaction (p<0.01), indicating that blFN-x
effectively suppressed PDBu-stimulated PGF2asecretion, even when added after
PDBu treatment had been initiated. Analysis of homogeneity of regression


127
presence of a region of pronounced growth of capillaries in the endometrium of
cows which received intrauterine infusions of blFN-x. Chemokines may recruit
specific populations of lymphocytes to the endometrium, which could act to
secrete cytokines beneficial to development of the conceptus (for reviews see
Hansen, 1997; Robertson et al., 1994 ). Protein P16 was identified as bovine
ubiquitin-cross-reactive protein (bUCRP; Austin et al., 1996b). Ubiquitin
conjugates with proteins to modulate their cellular activities and also to target
them to proteasomal degradation (Finley and Chau, 1991). The bUCRP cross-
reacts with antibodies against ubiquitin in immunoblots (Austin et al., 1996b) and
sequence analysis revealed that bUCRP maintains critical domains and residues
involved in conjugation with other proteins (Hansen et al., 1999). Moreover,
Johnson and coworkers (1998a) demonstrated the ability of bUCRP to form
conjugates with an array of cytosolic endometrial proteins following treatment
with blFN-x. It is possible that bUCRP plays a role on the antiluteolytic actions of
blFN-x in the endometrium, perhaps targeting proteins involved in the pathway of
PGF2a production to alter their cellular activity and ultimately decrease secretion
of PGF2a.
Examination of proteins in whole tissue extracts indicated presence of a
75 kD protein (P75) induced by blFN-x in endometrium. Review of the literature
on interferon-induced proteins revealed presence of a ovine IFN-x-induced
protein of 70 to 80 kD (Pavlovic et al., 1993) in the endometrium of sheep,
designated Mx protein (Charleston and Stewart, 1993; Ott et al., 1998). Mx


BIOGRAPHICAL SKETCH
Mario Binelli was born March 4, 1968, to Carmem Lucia Rezende de
Oliveira and Guilherme Jose Binelli in Rio de Janeiro, Brazil. He is a stepchild to
Maria Estela Machado Binelli. He is the oldest of three children. He is married
to Eliana Kampf Binelli. In 1990 he received his Bachelor of Science in
agronomy from the "Escola Superior de Agricultura Luiz de Queiroz, which is
the College of Agriculture in the University of Sao Paulo in Piracicaba, Brazil.
From 1990 to 1991 he worked as manager in a dairy and beef farm in Bofete,
Brazil. In January 1992 the author enrolled in a masters degree program at the
Department of Animal Science at Michigan State University in East Lansing, Ml
under the supervision of Dr. H. Allen Tucker, and received his title in 1993. He
started his PhD program in the Department of Dairy and Poultry Sciences at the
University of Florida in August 1994. His career goal is to become a faculty
member of a federal or state university in Brazil and to conduct research in the
area of animal physiology.
291


238
compared to controls, and this was noticed as early as 6 hours (Experiment 3)
and lasted for at least 24 hours (Experiment 1). Increased abundance of these
enzymes was probably related to their increased cellular activity [abundance (pg
of protein) x specific enzymatic activity (activity/pg of protein)], as evaluated
through increased PGF2asecretion in medium. Presence of bIFN-x attenuated
PDBu-stimulated PLA2 and COX-2 protein expression, but such effects were
time dependent. Responses of PLA2 were somewhat variable, but consistently
lower than PDBu treatment alone. Although treatment by experiment
interactions were not examined (experiments were analyzed independently),
visual inspection of data suggests an increase in the ratio of PDBu- to PDBu +
blFN-x-induced PLA2 from 6 (Experiment 3) to 24 hours (Experiment 1).
Interestingly, the opposite happened for COX-2 abundance. Apparently, bIFN-x
gradually lost its ability inhibit COX-2 expression overtime, and no difference was
detected between PDBu and PDBu + bIFN-x after 24-hour treatments. When
related to time trends of PGF2asecretion, these results suggest that differential
increases in COX-2 protein expression are associated with increasing PGF2a
accumulation overtime as observed for cells treated with the combination of
PDBu and bIFN-x. However, COX-2 levels alone do not explain differences in
PGF2c,secretion between PDBU and PDBU + bIFN-x treatments. The
proportional decrease in PGF2Js much greater than the proportional decrease in
COX-2 abundance for cells treated with PDBu in presence vs. absence of bIFN-


Figure 5-22. Immunoblotting analysis of STAT-3 co-immunoprecipitated with STATs 1 and 2
from nuclear extracts from BEND cells treated with blFN-r for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of STAT-3;.
b) Representative ECL exposure of tyrosine phosphorylation of STATs 1 and 2; c) Representative
ECL exposure of abundance of STATs 1 and 2; d) Least squares means and SE of abundance of
STAT-3 (bars with distinct subscripts are statistically different, p<0.1).


244
UCRP an unlikely mediator of early bIFN-x suppression of PGF2a. Other possible
mediators of bIFN-x actions include the interferon-induced transcription factors
IRF-1 and IRF-2 (Harada et al., 1989; Spencer et al., 1998; Chapter 6). IRFs are
rapidly synthesized in response to bIFN-x (detected after 1 hour exposure to
bIFN-x, Chapter 6) and could induce synthesis of specific proteins that could act
to suppress PGF2o,synthesis. Again, the short time frame of bIFN-x-mediated
suppression of PGF2a makes this possibility questionable. Therefore, I would like
to postulate a protein synthesis-independent mode of action for bIFN-x in the
endometrium. Bovine IFN-x could activate intracellular second messengers
other than STAT proteins, which may have modulatory effect on the PGF2ct
synthesizing machinery. For example, Stancato and others (1997) proposed that
binding of interferon a to its receptor can activate the ras-raf pathway, resulting
in activation of MAP kinases. Moreover, Pfeffer and coauthors reported a
pathway involving PI-3' kinase activation of MAP kinase, stimulated by interferon
p. The MAP kinases can modulate a series of Intracellular responses and
regulation of PGF2a synthesis is a plausible possibility and testable hypothesis.
Finally, PLA2 becomes associated with JAK-1 after stimulation of cells with
Interferon-cc (Flatl et al., 1996). It is possible that such an association either
sequesters this enzyme or modifies its activity to attenuate its normal function.
Collectively, these and other mechanisms may explain protein synthesis-


189
generous gift from Dr. Michael Roberts (University of Missouri). Complementary
oligodeoxyribonucleotides corresponding to the interferon stimulus response
element (ISRE; double stranded, 100 ng/pl solution) and to the sis-inducible
element (SIE; sense and anti-sense strands, 100 ng/pl solutions) were obtained
as a gift from Dr. Douglas Leaman (The Cleveland Clinic Foundation, OH).
Sequences of the sense (5' to 3') strands were as follows: ISRE, 5-
TTTACAAACAGCAGGAAATAGAAACTTAAGAGAAATACA-3', from the 9-27
gene; SIE, 5'-AGCTTCATTTCCCGTAAATCCCTA-3', from the c-Fos gene. T4
Polynucleotide kinase (100 units in a 10 units/gl solution; supplied with 10 X
kinase reaction buffer), Klenow kit (contains 150 units Klenow in a 5 units/pl
solution, 10 X polymerase reaction buffer and 500 pi each 10 mM dATP, dCTP,
d GTP, dTTP) herring sperm DNA were from Promega Corp (Madison, Wl).
Gamma 32P-ATP (5 mCi in 30 pi; specific activity: 7000 Ci/mMol) and alpha 32P-
dCTP (1 mCi in 100 pi; specific activity: 3000 Ci/mMol) were from ICN (Costa
Mesa, CA). Tris, EDTA, phenol, borosilicate Pasteur pipettes (5.75"), ammonium
acetate^ tris-HCI, KCI, MgCI2, DTT, glycerol, xylene cyanol and Whatman filter
paper were purchased from Fisher Scientific (Pittsbugh, PA). Sephadex G-10
beads, rabbit IgG and tRNA were from Sigma Chemical Co. (St. Louis, MO).
Acrylamide and NN'-methylenebisacrylamide were from BDH Laboratory
Supplies (Poole, England). Poly dldC (10 A260 units diluted in 400 pi of sterile
water) and ATP were from Boehringer Mannheim Co. (Indianapolis, IN). X-ray


80
224
Figure 7-4. Experiment 2. Least squares means and SE of concentrations of PGF^in medium
conditioned by BEND cells treated with medium alone (control), phorbol 12,13 dibutyrate
(100ng/ml; PDBu) or blFN-t (50 ng/ml; bIFN-tau) and PDBu. Samples were removed before
treatments were added (0 hours), 3, 6, 9 and12 hours after.
COX-2 and PLA, immunoblotting. Similar to Experiment 1, there were
very low abundances of both COX-2 (p<0.01; Figure 7-5) and PLA2 (p<0.04;
Figure 7-6) in cells incubated with medium alone. However, treatment with
PDBu stimulated synthesis of both proteins. In comparison with Experiment 1,
treatment with blFN-x in combination with PDBu decreased abundance of both
COX-2 (p<0.01) and PLA2 (p<0.05) compared to PDBu alone at 12 hours.


243
induced PKC effects were suppressed (i.e., there was still PGF2a production,
even in presence of bIFN-x) it is unlikely that blFN-x effects were exerted
upstream from PKC (e.g., a block on PDBu ability to bind PKC). It is likely that
blFN-x blocked some, but not all PDBu effects downstream from PKC.
Regarding the modes of action of blFN-x, the fact that attenuation of
PGF2asynthesis occurred so quickly suggests that blFN-x may act through a
pathway alternative to the classical JAK-STAT pathway. The JAK-STAT
pathway involves phosphorylation and nuclear translocation of cytoplasmic STAT
proteins, as a result of blFN-x binding to its receptor (Darnell et al., 1994;
Chapter 5). In the nucleus, STAT proteins act as transcription factors to
stimulate expression of interferon-stimulated genes. Interferon-induced proteins
may act in a variety of ways to produce the interferon-induced phenotype.
Presence and function of this classical mode of action in the endometrium has
been the prevalent dogma of laboratories studying effects of blFN-x on
production of PGF2aduring maternal recognition of pregnancy in cattle (Austin et
al., 1996b; Binelli et al., 1996; Hansen etal., 1997; Perry etal., 1998; Chapter 5
and 6) and sheep (Spencer et al., 1998). For example, ubiquitin cross-reactive
protein Is induced by blFN-x in endometrial explant culture (Austin et al., 1996b;
Staggs et al., 1998). This protein forms complexes with other cytosolic proteins,
and could modulate their activity and turnover rate (Johnson et al., 1998a).
However, complex formation requires 12-hour exposure to blFN-x, which makes


245
independent actions of bIFN-t observed in the present study. Definitive studies
with the critical use of protein synthesis inhibitors warrant investigation.


78
oviduct and uterine function, which could affect early embryonic development
and decrease fertility.
Macromolecules present in oviductal fluid have been suggested to serve
an important role in sperm capacitation (Anderson and Killian, 1994), fertilization
(Boatman and Magnoni, 1995) and early embryo development (Gandolfi et al.,
1989). Therefore, alterations in oviductal biosynthetic activity including protein
synthesis and secretion may affect conception rate.
Steroid modulation of oviductal synthesis and secretion of proteins has
been characterized in sheep (Buhi et al., 1991, Murray, 1993), baboon (Verhage
and Fazleabas, 1988) and swine (Buhi et al., 1989; Buhi et al., 1990). An altered
steroid environment, associated with development of a PDF, may alter oviductal
protein synthesis and secretion. In turn, the altered pattern of protein synthesis
and secretion could affect optimal oviductal function, fertilization and early
embryo development that contributed to reduced embryonic survival in
synchronized cows. The present experiment tested the hypothesis that the
presence of a PDF alters protein synthesis and secretion of oviductal explants
from cows at estrus.
Specific objectives were: 1) to induce a PDF or a FDF with the strategic
use of PGF2a, progesterone-containing CIDR and GnRFI; 2) to compare the
biosynthetic activity and the array of secretory proteins synthesized in the
infundibulum (INF), ampulla (AMP) and isthmus (1ST) at estrus in oviducts


47
receptor subunits IFNctRI and IFNaR2 are similar to ones utilized by other type I
interferons such as IFN-a. However, these receptors were not linked with
functional data, to demonstrate that such subunits are necessary and sufficient
to suppress PGF2asynthesis.
The JAK-STAT Pathway
The observations above lead to the assumption that blFN-t stimulated a
signal transduction system, the JAK-STAT pathway, similar to other type I
interferons (Schindler et al., 1992; Darnell et al., 1994; Darnell, 1997; Figure 2-
1). In this paradigm, interferon receptors do not contain intrinsic kinase activity,
but they are physically associated with protein tyrosine kinases from the Janus
family (JAK kinases). Binding of interferon to its receptor causes
phosphorylation of tyrosine residues in the JAK kinases and in the cytoplasmic
tail of the receptor. The tyrosine phosphorylated receptor attracts signal
transducer and activation of transcription, or STAT, proteins to close contact.
Members of the STAT family of proteins then become phosphorylated on
tyrosine residues and form homo- and hetero-dimers. Dimerized STATs migrate
to the nucleus where they bind to the specific regulatory elements located in the
promoter region of interferon-regulated genes. In this manner, STAT proteins


140
Grow cells to 90%confluency
I
Starve cells for 45 min.
1
Add 50 ng/ml blFN-x for 0,1,3, 8,15, 30 or 60 min.
Wash cells in ice-cold PBS
I
Lyse cells (hypotonic buffer)
1
Spin lysate, collect supernatant and save pellet
i I
Supernatant:
Adjust to 60 mM KCL
Spin 30 min.
Supernatant is cytosolic extract (CE)
Pellet:
Resuspend in hypotonic buffer
Spin over sucrose cushion
Incubate pellet in high salt buffer
Spin 30 min.
Supernatant is nuclear extract (NE)
1 1
Cytosolic extracts Nuclear extracts
Immunoprecipitate overnight with o -STAT-1 and a -STAT-2 or a -STAT-3 abs
1
SDS-PAGE and transfer to nitrocellulose membrane
4
Western blot 1: a -phospho-tyrosine
Strip and neutralize membrane
1
Western blot 2: a -STAT-1 and a -STAT-2 or a-STAT-3
Figure 5-2. Outline of experimental sequence involving immunoprecipitation of cytosolic and
nuclear extracts of BEND cells treated with blFN-t for increasing intervals of time. See text for
details.


193
was estimated, and a 10 fold excess of unlabeled probe solution was prepared
by diluting appropriate oligodeoxyribonucleotides (i.e., ISRE or SIE) in water. Six
% acrylamide, large format gels were set up and allowed to polymerize
overnight. Samples were prepared in 500 pi microfuge tubes, and included
appropriate nuclear extracts (NE), 8pl 5X EMSA buffer, 2pl poly dldC and sterile
water brought up to a final volume of 40pl. When required, a 10 fold excess of
unlabeled probe, specific antibodies (1 pi each of anti STAT-1, anti-STAT-2 or
anti-IRF-1 or 10 pi of anti-STAT-3) or rabbit IgG (1 pi of a 200 pg/ml solution)
were added. Volume of water was calculated so that final volume of the reaction
mix was 40 pi. This mix was incubated at room temperature (Experiment 1) or at
37 C (Experiments 1, 2 and 3) for 30 minutes (pre-incubation period).
Subsequently, specific labeled probe was added to the reaction mix (100000
cpm) and incubated at 37 C for a 30 minute binding period (ISRE, Experiments 1
and 2; SIE, Experiment 3). During pre-incubation and binding periods, gels were
pre-electrophoresed, at maximum voltage and constant 30 mA current per gel.
Five pi of gel dye (40% glycerol, 60% water, 0.01% xylene cyanol) were added in
each tube, mixed and loaded onto gel. Gels were run for 5 to 6 hours at
constant 30 mA. After electrophoresis, a sheet of Whatman filter paper was
placed in contact with surface of gel, and gel was relocated from glass plate to
adhere to filter paper. Gel was covered with plastic wrap and dried on a on a
slab gel drier (model SE 1150, Floeffer Scientific Instruments, San Francisco,


37
I will next review intracellular pathways for generation of the pulsatile
secretion of PGF2a. Little experimentation has been done to uncover potential
intracellular pathways activated by estradiol or LH, so focus is on the well studied
and established pathway of oxytocin stimulation (Flint et al., 1986; Burns et al.,
1997; Thatcher et al., 1997). Oxytocin receptors start to increase in the P4 -
primed, responsive uterus. Oxytocin originating from the neurohypophyseal lobe
of the pituitary gland binds to the seven transmembrane-domain, G protein-
coupled receptors and activates phospholipase C (PLC). The PLC cleaves
membrane phosphotydilinositol bisphosphate, yielding inositol trisphosphate (IP3)
and diacylglycerol (DAG). The IP3 binds to specific receptors in the endoplasmic
reticulum resulting in release of calcium from internal stores into the cytosolic
compartment. The DAG activates protein kinase C (PKC), leading to serine
phosphorylation of cytosolic, calcium-dependent phospholipase A2 (PLA2),
probably through a MAP-kinase dependent pathway (Lin et al., 1993). The IP3-
stimulated increase in cytosolic calcium acts to further stimulate PLA2 activity
(Clark et al., 1991). Stimulated PLA2 translocates to the membrane where
phospholipid substrates are located (Clark et al., 1991). Activated, membrane-
bound PLA2 cleaves arachidonic acid (AA) from phospholipids. Free AA is
converted to prostaglandin H2 (PGH2) by the enzyme cyclooxygenase-2 (COX-
2). Prostaglandin F2a synthase converts PGH2into PGF2( which is then released
into the uterine circulation. In the endometrium, this process occurs
preferentially in epithelial cells compared to stromal cells (Danet-Desnoyers et


52
tempting to speculate that some of such actions may be required for the
antiluteolytic roles of blFN-x in the endometrium.
JAK kinases
Janus kinases or JAKs tyk-2 and jak-1 are associated respectively with
IFNaRI and IFNaR2 and are involved in tyrosine phosphorylation of STAT
proteins. The carboxy-terminal domains of the jak kinases share considerable
sequence homology with the catalytic domains of other protein tyrosine kinases.
The amino-terminal half of the jaks contains regions of sequence homology to
other members of the jak family and the extreme amino-terminal domain
probably is involved in association with interferon receptor chains (Williams and
Flaque, 1997). Ligand-mediated dimerization of interferon receptor chains is
required for interferon-stimulated signal transduction. Dimerization evokes
reciprocal tyrosine phosphorylation and consequent activation of JAKs
associated with interferon receptor chains (Ihle et al., 1995). Phosphorylation of
the kinase is the first of three tyrosine phosphorylations culminating in STAT
activation. Activated JAKs phosphorylate tyrosine residues on the interferon
receptor chains, which serve as docking sites for STATs, as mentioned above.
Lastly, STATs are phosphorylated by the JAKs (Darnell, 1997). Activated JAKs
are not specific for particular STATs. Different receptors can activate the same
STATs through different JAKs. Moreover, STAT docking sites can be
interchanged between different cytokine receptors, and the STAT specific for the


122
0.5 min 1 min A 3 min
Figure 4-4. Validation of densitometry. Densitometrio values of COX-2 abundance in
serially diluted samples. Values were obtained after enhanced chemiluminescence
exposures of 0.5, 1 and 3 minutes.
WCE from scraped cells from all cows showed a strong staining for cytokeratin
(Figure 4-5). In contrast, staining for vimentin was absent for all BSA-treated
cows while two blFN-i-treated cow showed staining for vimentin (Figure 4-6).
tfnmunoblotting for COX-2 and PLA,. Abundances of COX-2 (Figure 4-7)
and PLA2 (Figure 4-8) in WCE from scraped endometrial cells were similar for
cows treated with either BSA or blFN-x in vivo.


CHAPTER 8
GENERAL DISCUSSION
Distinct from other areas within the greater field of physiology, the study of
physiology of reproduction Is complicated by the fact that a large number of
processes In the reproductive cycle are affected by two beings, the developing
conceptus and the conceptus-bearing maternal unit. The Immediate
consequence of this scenario is that studies in this area must take into
consideration not only the Individual units Involved, but much more Importantly,
the interactions taking place between the two units. As exemplified in the review
of literature (Chapter 2) and further stressed by the specific examples studied in
all chapters of this dissertation, successful outcome of the reproductive process
is dependent on successful exchange of concerted physiological signals
between maternal and embryonic units. This dissertation described examples of
communications in the physiological windows of the periestrual period and
maternal recognition of pregnancy. To each window there is an associated
percentage of embryonic losses in cattle, which calls for further research, if ones
goal is to understand causes of such mortality to be able to propose solutions
and improve overall pregnancy rates.
246


4
17 of pregnancy. The focus of this review will be on the bovine species, but data
from other species will be presented whenever appropriate.
Communications Between Gametes and Maternal Units
Gametes are differentiated cells with the specific function of conveying
genetic information from each paternal and maternal unit to a zygote during the
process of fertilization. To ultimately undergo fertilization, both paternal and
maternal gametes interact with somatic cells in maternal reproductive tissues.
Eaa-oocvte. During development of follicles, oocytes change the program
of granulosa cells in the follicle, so that the default program of mural granulosa
cell differentiation is suppressed. As a consequence, cells surrounding the
oocyte become more specialized in functions that favor development of the
oocyte. In mature Graafian follicles, this layer of differentiated granulosa cells
forms a small pedicle of cells, the cumulus oophurus, which contains the oocyte
and protrudes towards the interior of the antrum. Eppig and coauthors (1997a)
hypothesized that oocytes control their own microenvironment by regulating
differentiation of the supporting cells that are in direct communication with them.
For example, expression of luteinizing hormone (LH)-receptors in cumulus cells
is abrogated by presence of the oocyte (Eppig et al., 1997b).
Oocyte-oviduct. In cattle, the oviduct is divided in three functional regions;
namely infundibulum (INF), ampulla (AMP) and isthmus (1ST; Hafez, 1993a).
The INF is opened to the peritoneal cavity of the body, the 1ST connects the


124
o
X
204-
121
78-
39.5-
30.7
Figure 4-6. Immunoblotting analysis of vimentin in whole cell extracts from endometrial
epithelial cells from cows treated in vivo with placebo or blFN-x. Representative enhanced
chemiluminescence (ECL) exposure of abundance of vimentin for control cows (lines 1, 3, 5, 6
and 9) and for blFN-x-treated cows (lines 2, 4, 7, 8, and 10).
by treatment of endometrial explants in vitro with bIFN-t, of molecular weights 8
(P8), 16 (P16) and 28 kD (P28). Although the calculated molecular weight of the
proteins found In our study were 12 and 19 kD, It Is likely that they correspond to
P8 and P16 In their study. The lowest (I.e., fastest migrating) molecular weight
standard marker used in the electrophoresis procedure of the present
experiment Is 19 kD. Sizes of proteins with lower molecular weights were
calculated based on a regression equation that might have slightly overestimated
the calculated molecular weights of P19 and P12. Protein P8 was identified as
bovine granulocyte chemotactlc proteln-2, a member of the alpha chemokine
family (Teixelra et al., 1997). Chemoklnes are potent chemo-attractants for cells
of the immune system and have been implicated In cell adhesion, Inflammatory
and angiogenic processes (Oppenhelm et al., 1991). In support of a possible
angiogenic role of the chemokine, Arnold and coworkers (1999) observed


71
Figure 2-3. Hypothetical model of interferon-T (IFN-t)-stimulated gene activation and effects on
molecules involved in the PGF2a synthetic pathway. Estrogen receptors (ER) are up-regulated
before luteolysis and stimulate synthesis of oxytocin (OT) receptors (OTR). Oxytocin binding to
OTR stimulates phospholipase C (PLC), which cleaves phsphatydilinositol (PI) yielding inositol
trisphosphate (IP3) and diacylglycerol (DAG). The IP3 stimulates release of Ca*'1' from
intracellular stores, and DAG activates protein kinase C (PKC). The PKC activates
phospholipase A2 (PLAJ which, in the presence of Ca++, cleaves arachidonic acid (AA) from
membrane phospholipids. Molecules of AA and linoleic acid (LA) regulate the enzyme
prostaglandin synthase (PGS) to produce PGF2n. In the pregnant cow, embryonic trophoblastic
cells secrete IFN-t into the uterine lumen. Receptors on endometrial epithelial cells bind IFN-t,
and dimerize. Dimerization of receptors promote phosphorylation (represented by a circled "p") of"
associated tyrosine kinases from the JAK family, such as tyk-2 and jak-1. Phosphorylated
receptors attract signal transducer and activators of transcription (STAT) proteins. The STAT
proteins are phosphorylated in tyrosine residues by the JAK kinases and form a complex that
migrates to the nucleus. In the nucleus, the complex associates with a 48 kD DNA-binding
protein, and this new complex binds to interferon-stimulated response elements (ISRE) in the
regulatory region of interferon-induced genes, activating transcription of such genes and
synthesis of proteins. Synthesized proteins may act to specifically block one or more steps on the
PGF2o synthetic pathway (arrows with [-] signs; see text for details and abbreviations).


283
Perry DJ, Austin KJ, Hansen TR. Cloning of interferon stimulated gene 17: the
promoter and nuclear proteins that regulate transcription. Mol Endocrinol
1999; 13:1197-1206.
Pestka S. The Interferon receptors. Semin Oncol 1997; 24 (suppl 9):s918-s940.
Pfeffer LM, Mullersman JE, Pfeffer SR, Murti A, Shi W, Yang CH. STAT3 as an
adapter to couple phosphatydilinoositol 3-kinase to the IFNAR1 chain of
the type I interferon receptor. Science 1997; 276:1418-1420.
Plante C, Hansen PJ, Martinod S, Siegenthaler B, Thatcher VWV. Effect of
intrauterine and intramuscular administration of recombinant bovine
interferon ct1 on luteal lifespan in cattle. J Dairy Sci 1989; 72:1859-1865.
Plante C, Thatcher WW, Hansen PJ. Alteration of estrous cycle length, ovarian
function and oxytocin-induced release of prostaglandin F2a by intrauterine
and intramuscular administration of recombinant bovine interferon-a to
cows. J Reprod Frtil 1991; 93:375-384.
Platanias LC, Uddin S, Domanski P, Colaminici OR. Differences in interferon
alpha and beta signaling. Interferon beta selectively induces the
interaction of the alpha and betaL subunits of the type I interferon
receptor. J Biol Chem 1996a; 271:23630-23633.
Platanias LC, Uddin S, Yetter A, Sun XJ, White WF. The type I interferon
receptor mediates tyrosine phosphorylation of insulin receptor substrate 2.
J Biol Chem 1996b; 271:278-282.
Pratt BR, Butcher RL, Inskeep EK. Antiluteolytic effect of the conceptus and of
PGE2in ewes. J Anim Sci 1977; 45:784-791.
Pritchard JY, Schrick FN, Inskeep EK. Relationship of pregnancy rate to
peripheral concentrations of progesterone and estradiol in beef cows.
Therlogenology 1994; 42: 247-259.
Putney DJ, Drost M, Thatcher WW. Embryonic development in superovulated
dairy cattle exposed to elevated ambient temperatures between days 1 to
7 post insemination. Therigenology 1988a; 30:195-209.
Putney DJ, Malayer JR, Gross TS, Thatcher WW, Hansen PJ, Drost M. Heat
streess-induced alterations in the synthesis and secretion of proteins and
prostaglandins by cultured bovine conceptuses and uterine endometrium.
Biol Reprod 1988b; 39:717-728.


194
CA; 50 minutes at 80 C). Dried gels were exposed to X-ray films overnight (all
experiments) and for 7 hours (Experiment 3) at -80 C and developed in a Konica
-X-ray film processor model QX-70 (Konica Corp., Japan).
Experimental designs. Experiment 1 was designed to define ideal
experimental conditions to be used in subsequent experiments. Two pre
incubation/binding temperatures (room temperature vs. 37 C) and two amounts
of nuclear protein (10 vs. 20 pg) were tested'for nuclear extracts obtained from
BEND cells treated in presence or absence of blFN--t. Experiment 2 was
designed to identify nature of proteins contained in complexes induced by bIFN-T
and bound to an ISRE labeled probe, through use of specific antibodies against
STATs 1, 2 and 3 and against IRF-1. Specificity of antibody action was verified
incubating a sample with rabbit IgG. Protein from nuclear extracts (10 pg) was
used in each pre-incubation reaction. Pre-incubation and binding reactions were
carried out at 37 C. Experiment 3 was designed to identify nature of proteins
contained in complexes induced by blFN-x, and bound to an SIE labeled probe,
through use of specific antibodies against STATs 1 and 3. Specificity of antibody
action was verified incubating a sample with rabbit IgG. Protein from nuclear
extracts (7 pg) was used in each pre-incubation reaction. Pre-incubation and
binding reactions were carried out at 37 C.


70
(1998) sequenced the bovine oxytocin receptor gene and also found IREs in the
regulatory region, and such sites bound bovine IRF-1 and -2. Again, the
suggestion is that perhaps blFN-x-induced transcription repressors may
downregulate expression of oxytocin receptors, to ultimately decrease PGF2a
secretion in the pregnant uterus.
Hypothetical model for bIFN-x -mediated suppression of PGF,. secretion in the
endometrium
The hypothetical model shown in Figure 2-3 depicts some of the
possibilities discussed thus far.
Uterine-Conceotus Interactions and Reproductive Failure in Cattle
Thus far, this review has illustrated the enormous amount and intricacy of
interactions that need to occur between embryonic and maternal uterine tissues
in order for a successful pregnancy to be established. Given the high
percentage of embryonic mortality occurring during early pregnancies, it
becomes apparent that a precise program of interactions must be followed, and
that deviations from such a program may lead to pregnancy termination. Such a
program includes both embryonic and maternal components. For example, the
embryonic unit must be able to effectively interact with maternal endometrium,
undergo elongation and send antiluteolytic signals to the maternal unit in order to
survive. The maternal unit should provide a quiescent and nutritive environment,.


251
UCRP could be stimulated by phorbol ester, indicating complex regulation of
these proteins. The bovine UCRP gene has five ISREs in its regulatory region,
which explains its regulation through the classical JAK-STAT-ISGF-3 pathway
(Darnell et al., 1994). However, promoter sequence of bovine GCP-2 has not
been published. Collectively, these data indicate that in addition to regulation
through the classical JAK-STAT pathway, blFN-x may be able to use alternative,
specific signaling pathways. For example, binding of blFN-x could cause
recruitment of a specific STAT protein or another transcription factor, recruitment
of a specific polypeptide chain to the receptor complex or of another kinase.
Han and Roberts (1998) cloned various forms of type I interferon receptor from
ovine and bovine endometrium cDNA libraries and failed to identify novel
receptor chains that could be specific for blFN-x. They concluded that blFN-x
utilizes standard IFNaRI and IFNaR2 for signaling. They used a homology
screening method which would not be appropriate to detect a peptide with a
unknown sequence (I.e., a novel receptor chain with a unknown sequence). It is
my contention that the hypothesis of a unique receptor for blFN-x is still open for
testing. Additionally, as mentioned above, removal of critical components of the
JAK-STAT pathway Is an appropriate way to test their requirement for blFN-x
functional regulation. The bIFN-x-regulated suppression in PDBu-induced PGF2a
secretion model, as described in Chapter 7, would be appropriate to conduct
such experiments.


104
(Danet-Desnoyers et al., 1994). However, the nature of such intracellular events
within the endometrium is unknown. Interferon-alpha (IFN-a) shares sequence .
homology with bIFN-x (Roberts et al., 1992), efficiently competes with bIFN-x for
binding to endometrial epithelium (Li and Roberts 1994), and also has
antiluteolytic effects in cattle (Plante et al., 1989; Plante et al., 1991). Studies
with human cells showed that IFN-a stimulates transcription of IFN-responsive
genes through phosphorylation of specific transcription factors identified as
"Signal Transducers and Activators of Transcription" (STAT) proteins (STAT-1,
84/91 kD; STAT-2, 113 kD; Darnell et al., 1994; Fu etal., 1992; Schindler et al.,
1992). Moreover, ovine IFN-x also stimulates transcription in Daudi cells
through phosphorylation of STAT proteins (Subramaniam et al., 1995). It is
hypothesized that bIFN-x stimulates bovine endometrial cells through a similar
signal transduction mechanism. An initial series of experiments was designed
(1) to verify that bIFN-x stimulated synthesis of specific intracellular and
secretory endometrial proteins; (2) to examine whether STAT proteins are
present in bovine endometrium; (3) to test whether bIFN-x causes
phosphorylation of STAT proteins in endometrial explants.
Arnold and coworkers (1999) incubated endometrial explants from cows
infused in vivo with bIFN-x or a control protein (bovine serum albumin, BSA) with
intracellular stimulators of PGF2o synthesis. They found that in vivo treatment
with bIFN-x inhibited the ability of phorbol 12, 13 dibutyrate (PDBu) and a


271
Eppig JJ, Chesnel F, Hirao Y, OBrien MJ, Pndola FL, Watanabe S,
Wigglesworth K. Oocyte control of granulosa cell development: how and
why. Hum Reprod 1997a; 12:127-132.
Eppig JJ, Wigglesworth K, Pndola F, Hirao Y. Murine oocytes suppress
expression of luteinizing hormone receptor messenger ribonucleic acid by
granulosa cells. Biol Reprod 1997b; 56:976-984.
Farin CE, Imakawa K, Hansen TR, McDonnell, Murphy CN, Farin PW, Roberts
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Biol Reprod 1990; 43:210-218.
Faure V, Courtols Y, Goureau O. Inhibition of inducible nitric oxide synthase
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epithelial cells. J Biol Chem 1997; 19:32169-32175.
Finley D, Chau V. Ubiquitination. Annu Rev Cell Biol 1991; 7:25-69.
Flati V, Haque SJ, Williams BRG. Interferon-cx-induced phosphorylation and
activation of cytosolic phospholipase A2 is required for the formation of
interferon-stimulated gene factor 3. EMBO J 1996; 15:1566-1571.
Fleming JGW, Spencer TE, Bazer FW. Cloning and analysis of the 5' flanking
region of the ovine estrogen receptor alpha gene. Biol Reprod 1998; 58
(Suppl. 1):402 (Abstr.).
Flint APF, Leat WMF, Sheldrlck EL, Stewart HJ. Stimulation of phosphoinisitide
hydrolysis by oxytocin and the mechanism by which oxytocin controls
prostaglandin synthesis in the ovine endometrium. J Biochem 1986;
237:797-805.
Flood PF, The development of the conceptus and its relationship to the uterus.
In: Cupps PT (ed), Reproduction in domestic animals, fourth ed. San
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Friedman S, Gurevich M, Shemesh M. Bovine cyclic endometrium containshigh-
affinity luteinizing hormone/human chorionic gonadotropin binding sites.
Biol Reprod 1995; 52:1020-1026.
Fu X-Y. A transcription factor with SH2 and SH3 domains is directly activated by
an interferon alpha-induced cytoplasmic protein tyrosine kinase(s). Cell
1992; 70:323-335.


Figure 5-6. Validation of immunoblotting procedure on whole cell extracts of BEND cells
incubated for 15 minutes in presence (+) or absence (-) of blFN-t.
a) Enhanced chemiluminescence (ECL) exposure of abundance of STATs 1,2 and 3; b) ECL
exposure of tyrosine phosphorylation of STATs 1, 2 and 3.
a Antibody used for immunoprecipitation (1=anti-STAT-1; 2=anti-STAT-2; 3=anti-STAT-3).
b First antibody used for immunoblotting (1=anti-STAT-1; 2=anti-STAT-2; 3=anti-STAT-3; p-Y=anti-
phosphotyrosine; NRS=normal rabbit serum; n=nothing).
c Second antibody used for immunoblotting (r=antl-rabbit IgG; m=anti-mouse IgG; n=nothing).


163
s 121
x
s 78
a
^STAT-3
0 1 3 8 15 30 60 120 min.
bIFN-x, 50 ng/ml
b
STAT-3
bIFN-x, 50 ng/ml
Figure 5-14. Immunoblotting analysis of STATs 3 immunoprecipitated from cytosolic
extracts from BEND cells treated with blFN-r for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of STAT-3;
b) Least squares means and SE of abundance of STAT-3 (bars with distinct subscripts are
statistically different, p<0.1).


13
luteinizing hormone (LH) support of continuous luteal P4 secretion. Strategies to
provide supplemental P4 through administration of exogenous P4 (Van Cleef et
al., 1996), use of human chorionic gonadotropin (hCG; Schmitt et ai., 1996a),
gonadotropin releasing hormone (GnRH) injections (Schmitt et al., 1996b), and
GnRH implants (Ambrose et al., 1998) have increased circulating concentrations
of P4 but yielded mixed results on pregnancy rates.
Asvnchronv between embryonic and maternal units. Embryo survival may
be impaired because of failure in some aspects of the relationship between the
embryonic and maternal units, despite the fact that both are normal (Wilmut et
al., 1986; Thatcher et al., 1994b). An example of such failures is lack of
synchrony between uterus and embryo. During early pregnancy, embryo
development depends upon a sequence of changes in the uterine secretions,
which in turn is dependent on progressive changes in the maternal hormonal
milieu. This phenomenon became established when it was observed that
embryos transferred between animals that were not in estrus at the same time
caused abnormal development and death of the embryo (Wilmut and Sales,
1981). Moreover, when cows were treated with P4 from days 1 to 5 of the
estrous cycle and received a day-8 embryo on day 5, pregnancy was
maintained, indicating that uterine development had been advanced as a result
of the exogenous P4(Geisert et al., 1991). A condition that can cause
asynchrony is exposure of cows to heat stress. Biggers and others (1987)
determined that high environmental temperatures between days 8 and 16 of


210
coauthors (1999) demonstrated that treatment of day 15 endometrial explants
with phorbol 12,13 dibutyrate (PDBu; an stimulator of PKC activity), but not with
oxytocin, stimulated production of PGF2o. Working with endometrial cells
collected on days 1 to 4 of the estrous cycle, Xiao and coworkers (1999)
determined that phorbol 12-myristate 13-acetate (PMA) stimulated both secretion
of PGF2oand expression of COX-2 protein. In addition, these authors reported
that bIFN-x reduced both of these PMA-induced effects. Objectives of the
present study were (1) to test whether PDBu could stimulate PGF2a secretion
from BEND cells and whether bIFN-x could block such an effect; (2) to study the
time-response dynamics of PDBu-induced PGF2asecretion in presence and
absence of bIFN-x; (3) to examine the effects of PDBu on PLA2 and COX-2
protein expression in presence and absence of bIFN-x, and (4) to investigate
whether bIFN-x could inhibit PGF2osecretion in BEND cells previously stimulated
with PDBu.
Materials and Methods
Materials
Tissue culture treated, polystyrene Costar 6 well plates were from
Corning Inc. (Corning, NY). The PDBu (diluted to 1 mg /ml in 100% ethanol) and
rabbit IgG (diluted to 1 mg/ml in TBS; stored at -20 C) were from Sigma
Chemical, St. Louis, MO. Recombinant bIFN-x (dissolved in 20 mM Tris-HCI, 1


199
a
s1-
H M
ISRE
+
+
+
-
-
-
-
-
-
-
SIE
-
-
-
+
+
+
+
+
+
+
NE
-
30
30
-
0
30
30
30
30
30
10 x cold SIE
-
-
-
-
-
-
+
-
-
-
anti-STAT-1
-
-
-
-
-
-
-
+
-
-
anti-STAT-2
-
-
+
-
-
-
-
-
-
-
anti-STAT-3
-
-
-
-
-
-
-
-
+
-
rabbit IgG
-
-
-
-
-
-
-
-
-
+
b
s4a -
s4b-^
s4c-*-
Figure 6-3. Experiment 3. Electrophoretic mobility shift assay of nuclear extracts of BEND cells
incubated with radiolabeled ISRE or SIE.
a) Ingredients listed in the first column were present (+) or absent (-; see text). Arrows
indicate shifted complexes (s1, s4, s5, s6); b) Detail of panel a obtained at a lower exposure time.
Partition of complex s4 into 3 sub-complexes, s4a, s4b, s4c.
1 Nuclear extract used: (-) none, (0) from BEND cells incubated in medium alone for 30 minutes, (30)
from BEND cells incubated with blFN-t for 30 minutes
2 Presence (+) or absence (-) of 10 fold excess SIE unlabeld probe.


258
picture of in vivo events. Also, endometrial explants proved to be a useful
system to study secretory and intracellular proteins synthesized de novo in
response to blFN-x. However, explants proved to be a wrong choice for the
study of the JAK-STAT pathway. It was very challenging to detect tyrosine
phosphorylation of proteins in explants. This is probably because during the
process of homogenization, phosphatases, previously compartmentalized in
intact cells, were mixed with phosphorylated STATs. This may have caused
removal of phosphate groups, even in the presence of phosphatase inhibitors in
the homogenization buffer. Moreover, immunoprecipitation of STATs was only
possible after endogenous IgGs were removed by pre-incubation of tissue
extracts with protein A-sepharose beads. In the whole tissue extracts, presence
of stromal cell proteins diluted epithelial cell proteins, which decreased the ability
to detect epithelial cell-specific effects of blFN-i. Second in the rank of a
physiologically significant model, the use of scraped cells from the endometrial
lumen was attempted as described in Chapter 4. This procedure was described
by Charpigny and others (personal communication) for sheep endometrium, and
presented an attractive alternative to explants, since epithelial cells could be
easily isolated (strong cytokeratin staining; Chapter 4), and used immediately,
which would minimize loss of in vivo phenotype. This approach would avoid
protease digestion, as normally used for preparation of cells for primary culture,
minimize loss of membrane receptors and allow immediate use of cells in
experiments. Charpigny and co-workers were able to plate cells scraped from


183
STATs in the CE. Other reports support the concept that STAT molecules cycle
to the nucleus as tyrosine phosphorylated molecules, reaching a maximum at 20
to 30 minutes, and later return quantitatively to the cytoplasm as non-
phosphorylated molecules (Haspel et al., 1996)
Phosphorylation of STAT-1 increased and peaked sooner in CE than in
NE, suggesting a delayed translocation of STAT-1, initially phosphorylated in the
cytoplasm, to the nuclear compartment (Figures 5-12 and 5-13). The elevated .
phosphorylation in control extracts (0 minutes blFN-x) is due to a high
background that was consistently found in those samples. Although there was
no distinguishable band at the region corresponding to the MW of STAT-1, high
pixel reading accounted for the elevated densitometric units. These findings are
also true for other STATs. After maximum phosphorylation of STAT-1 at 30
minutes in the nucleus there was a steep decrease in phosphorylation observed
at 60 minutes (Figure 5-13). During the same time frame, abundance of STAT-1
in the nucleus decreased proportionally less (Figure 5-11). This indicates that
dephosphorylation of STAT-1 occurs while the protein is still in the nucleus.
This concept is supported by the work of David and others (1993). These
authors identified a nuclear tyrosine phosphatase, which deactivated interferon-
regulated STATs and down-regulated transcription of interferon-activated genes.
The faster increase and decrease in phosphorylation of STAT-2 in CE
compared to STAT-1 suggests a more acute regulation (i.e., phosphorylation and
dephosphorylation; Figure 5-12) of this protein. Phosphorylation of STAT-2 in


237
In summary, the experiments presented provide a useful model for
studying blFN-x regulation of PGF2asynthesis in the endometrium of cattle.
Experiments should be limited to 12-hour duration or less, since no further
accumulation of PGF2owas noted for longer time frames. Treatment-induced
differences in secretion of PGF2awere only noticed starting 3 hours after
administration of PDBu. Therefore, measurements taken before this period may
not provide useful information. Although there was no difference in protein
content among treatments, it is useful to express PGF2odata relative to some
response representative of number of cells present per plate. This cell culture
system can be used as a model to answer a number of scientific questions
regarding blFN-x effects in the endometrium. Such questions include the role of .
lipid metabolism on blFN-x antiluteolytic actions, role of the JAK-STAT pathway
and other intracellular signal transduction pathways on blFN-x -mediated PGF2cl
inhibition, blFN-x regulation of expression and activity of enzymes involved in
the synthesis of PGF2a, role of blFN-x -induced proteins on inhibition of PGF2a
synthesis, potential antiluteolytic effects of conceptus-secreted products other
than blFN-x etc.
In the present Chapter, cellular mechanisms mediating PDBu-induced
and bIFN-x-suppressed secretion of PGF2c,were studied. Specifically, protein
expression of PI_A2 and COX-2 were examined over different time frames.
Treatment with PDBu strongly stimulated synthesis of both COX-2 and PLA2


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175
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61
phosphorylated STATs. To support the existence of a mechanism for regulating
activity of STATs based on phosphatases, Haque and coauthors (1995) reported
that treatment of cells with orthovanadate, molybdenate and tungstate, which are
effective inhibitors of protein-tyrosine phosphatases, resulted in accumulation of
interferon-y-induced phosphorylated STATs. Involvement of novel molecules in
the regulation of the JAK-STAT pathway was reviewed by Starr and Hilton (1999).
They propose a model in which suppressers of cytokine signaling (SOCS)
proteins such as SOCS1 bind directly to JAKs to inhibit their catalytic activities.
Another protein, CIS, binds to activated receptors to prevent docking of STATs.
SH2-domain phosphatase-1 (SHP-1) dephosphorylates JAKs or activated
receptors. Finally, a protein inhibitor of activated STAT (PIAS) inactivates STAT
dimers. Song and Shuai (1998) demonstrated that SOCS 1 and SOCS3 inhibited
interferon-mediated antiviral and antiproliferative activities in HeLa cells. This
was linked with abolished tyrosine phosphorylation and nuclear translocation of
STAT-1 in response to interferon-a. Chung and others (1997a) reported that
PIAS3 directly interacted with STAT-3 and inhibited DNA binding of both STAT-
3:3, STAT-1:3 dimers. Binding of STAT-1 homodimers was not affected.
Moreover, cotransfections of both STAT-3 and PIAS3 showed a decrease in
luciferase activity from an IRF-1 reporter gene with increasing amounts of PIAS3.


267
Boice ML, Geisert RD, Blair RM.Verhage HG. Identification and characterization
of bovine oviductal glycoproteins synthesized at estrus. Biol Reprod
1990; 43:457-465.
Boos A. Immunohistochemical assessment of prostaglandin H-synthase in
bovine endometrial biopsy samples collected throughout the estrous
cycle. Anim Reprod Sci 1998; 51:261-273.
Brinsfield TH, Hawk HW. Control by progesterone of the concentration of lipid
droplets In epithelial cells of the sheep endometrium. J Anim Sci 1973;
36:919-922.
Buhi WC, Alvarez IM, Choi I, Cleaver BD, Simmen FA. Molecular cloning and
characterization of an estrogen-dependent porcine oviductal secretory
glycoprotein (POSP). Biol Reprod 1996; 55:1305-1314.
Buhi WC, Alvarez IM, Kouba AJ. Oviductal regulation of fertilization and early
embryonic development. J Reprod Frtil 1997a; 52 (suppl 1):285-300.
Buhi WC, Alvarez IM, Pickard AR, Mclntush EW, Kouba AJ, Ashworth CJ, and
Smith MF. Expression of tissue inhibitor of metalloproteinase-1 protein
and messenger ribonucleic acid by the oviduct of cycling, early-pregnant,
and OVX steroid-treated gilts. Biol Reprod 1997b; 57:7-15.
Buhl WC, Alvarez IM, Sudhipong V, Dones-Smith MM. Identification and
characterization of the de novo-syntheslzed porcine oviductal secretory
proteins. Biol Reprod 1990; 43:929-938.
Buhi WC, Ashworth CJ, Bazer FW, Alvarez IM. In vitro synthesis of oviductal
secretory proteins by estrogen-treated ovariectomized gilts. J Exp Zool
1992; 262:426-435.
Buhi WC, Bazer FW, Alvarez IM, Mirando MA. In vitro synthesis of oviductal
proteins associated with estrus and 17p-estradiol-treated OVX ewes.
Endocrinology 1991; 128:3086-3095.
Buhi WC, OBrien B, Alvarez IM, Erdos G, Dubois D. Immunogold localization of
porcine oviductal secretory proteins within the zona pellucida, perivitelline
space, and plasma membrane of oviductal and uterine oocytes and early
embryos. Biol Reprod 1993;48:1274-1283.


165
'P-STAT-3
3 8 15 30 60 120 min.
blFN-x, 50 ng/ml
bIFN-x, 50 ng/ml
Figure 5-16. Immunoblotting analysis of STATs 3 immunoprecipitated from cytosolic extracts
from BEND cells treated with blFN-r for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine phosphorylation
of STAX-3 ; b) Least squares means and SE of tyrosine phosphorylation of STAT-3 ( bars with
distinct subscripts are statistically different, p<.1).


6
capacitation. These early findings supported the idea of the necessity of
interaction between male gametes and products of the oviduct prior to
fertilization. In fact, incubation of sperm cells with oviductal fluid capacitated and
sustained sperm mobility in vitro (Parrish et al., 1989; McNutt and Killian, 1991).
Moreover, Boatman and Magnoni (1995) identified and purified an oviductal
factor (oviductin) that acts to enhance sperm penetration in follicular oocytes.
Communications Between Conceptus and Maternal Units
Conceptus-oviduct. Following fertilization, the conceptus continues to
interact with the AMP and 1ST before it reaches the uterus. During this period,
the conceptus undergoes initial cell divisions, and there is a possibility of a
continual influence of oviductal products on the conceptus. For example, Buhi
and coworkers (1993) showed that gold particles immunoreactive with porcine
oviductal secretory protein are associated with flocculent material in the
perivitelline space surrounding the blastomeres and in the zona pelcida of
embryos from the four-cell stage to blastocysts. Moreover, semi-purified
oviductal specific protein improved cleavage rates of embryos fertilized and
developed in vitro (Hill et al., 1996). In contrast, at least in pigs, presence of
gametes or embryos did not affect production of specific oviductal proteins,
suggesting that a regulation of oviductal function by gametes or embryos is
probably not important during this stage of the reproductive cycle (Buhi et al.,
1990).


16
Oviductal Function and Reproductive Failure in Cattle
The Oviduct Environment
The oviduct environment can be simplistically described as presenting
physical and chemical characteristics which are conducive to the reproductive
processes occurring within the oviduct. Physical and chemical characteristics
are described below in the sub-sections Functional anatomy and morphology"
and The oviduct fluid, respectively. To exemplify the functions of these
characteristics, Rieger and others (1995) examined development of embryos in
vitro, either in coculture with oviductal cells (both physical and chemical
influences) or in serum-free medium pre-conditioned by oviductal cells (chemical
influences, only). In both systems, embryos reached the 4-cell stage in 48
hours. However, embryos developing in the coculture system reached the
blastocyst stage 24 h before the others and also had significantly more cells.
There was no treatment where only physical factors were present, but the
conclusion from their data is that probably both physical and chemical
characteristics are necessary for best embryo development.
The ovarian cycle. There is a close association between oviduct function
and concentrations of circulating ovarian steroid hormones. Therefore, it is
appropriate to describe the changes occurring in such hormones during the


225
a
| 78 .. cox-2
* 39.5
1 23456789
b
20 n
Figure 7-5. Experiment 2. Immunoblotting analysis of COX-2 in whole cell extracts from BEND
cells treated with medium alone (control), phorbol 12,13 dibutyrate (100ng/ml; PDBu) or blFN-t
(50 ng/ml; bIFN-tau) and PDBu for 12 hours.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of
COX-2 (control: lanes 1 and 2; PDBu: lanes 3, 4 and 5; blFN-r and PDBu: lanes 6, 7, 8 and 9);
b) Least squares means and SE of abundance of COX-2 arbitrary densitometrlc units (ADU).


62
Specificity of interferon signaling
Taken together, information presented in previous sections offers several
opportunities for occurrence of specific cellular responses to interferons. Such
opportunities include: (1) milieu of subtypes of interferons present at the receptor,
in which for example, different iso-forms of ovine IFN-x have different abilities to
extend estrous cycle length in ewes (Ealy et al., 1998); (2) composition of the
receptor complex, where recruitment of particular subunits may affect which
STATs are recruited; (3) amino acid context of the phosphotyrosine module on
the receptor chain, and amino acid context of the SH2 domain on STATs will also
determine which STAT will dock to which receptor chain; (4) which STATs are
present and which dimers will form upon ligand binding; (5) mechanism of nuclear
translocation of STATs, since whether STATs translocate as dimers alone or in
combination with ligand-receptor complexes may influence the configuration and
specificity of the transcriptional activation complex; (6) formation of single or
multiple transcription activation complexes, which will depend on nature of dimers
and interacting nuclear proteins; (7) dynamics of down regulation of JAK-STAT
pathway, in which specific branches of the pathway may be inhibited while others
may remain active to elicit specific responses; finally (8) interactions with other
cellular pathways, which will be discussed next.
Cross talk with other intracellular pathways
The best known cross-talk between JAK-STAT and other signaling
pathways is that represented by serine and threonine phosphorylation of STAT


CHAPTER 5
BOVINE INTERFERON-TAU STIMULATES THE JAK-STAT PATHWAY IN
BOVINE ENDOMETRIAL EPITHELIAL CELLS
Introduction
In cattle, maintenance of pregnancy is accomplished by suppression of
luteolytic pulses of PGF2o from the pregnant uterus. Presence of a conceptus
maintains the corpus luteum (CL) at around day 17 of pregnancy (Thatcher et al.,
1997). Several studies indicate that bovine (b) interferon (IFN-t) is secreted by
the elongating embryo during the time of CL maintenance (Bartol et al., 1985;
Helmer et al., 1987; Farin et al., 1990). Moreover, bIFN-x is able to suppress
the oxytocin-induced release of PGF2a in vivo (Meyer et al., 1995) and in vitro
with cultured endometrial epithelial cells (Danet-Desnoyers et al., 1994; Xiao et
al., 1999) and increases CL lifespan in cows that received infusions of bIFN-x in
tero (Meyer et al., 1995). In order to exert its anti-luteolytic functions, bIFN-x
must: (1) interact with endometrial epithelial cells, since they are the major
source of uterine PGF2a (Danet-Desnoyers et al., 1994) and (2) stimulate signal
transduction mechanisms to change the function of endometrial epithelial cells to
suppress PGF2a pulses. Although binding of bIFN-x has been demonstrated in
bovine endometrial cells (Li and Roberts, 1994), very little work has been
133


laboratory and/or scientific expertise.
I wish to thank Dr. Michael Roberts, Dr. Thomas Hansen and Dr. Douglas
Leaman for providing important reagents used for the research described in this
dissertation.
I want to express my eternal gratitude to Dr. Thais Diaz, Dr. Eric Schmitt,
Dr. Alice de Moraes, Fabiola Paula-Lopes, Dan Arnold and Ricardo Mattos for
closely supporting me through the struggles of graduate school and for providing
their sincere friendship and scientific support.
I am grateful to fellow graduate students, post-docs, visitors and friends in
the departments of Dairy and Poultry and Animal Science, including Dr. Luzbel
de la Sota, Dr. Divakar Ambrose, Dr. Joan Burke, Dr. Maria de Fatima Pires, Dr.
Sandra Coelho, Ellen Van de Leemput, Jim Hampton, Nina Nusbaum, Monte
Meyer, Frederico Moreira, Aydin Guzeloglu, Metin Pancarci, Flavia Lopes,
Cassia Orlandi, Jan Vonk, Arthur Araujo, Dr. Carlos Arechiga, Dr. Lannett
Edwards, Morgan Peltier, Yaser Al-Katanani, Rocio Rivera, Saban Tekin,
Andrew Majewski, Inseok Kwak, Jason Blum, Max Huidsen, Tomas Belioso, Dr.
Alfredo Garcia, Dr. Rafael Roman, John Fike, Dr. Maria Cadario, Andres
Kowalski, Dr. Karen Reed, Dr. Michael Green, Michael Porter and Andy Kouba.
From helping me to solve some unearthly statistics to sharing a smile their
contributions will not be forgotten.
I want to thank, for their technical, scientific and professional expertise,
Marie-Joelle Thatcher, Idania Alvarez, Frank Michel, Jesse Johnson, Jennifer
IV


125
a
S 78 ->cox-2
T- ~ e *~*^~ ~111 -i 1
s 39.5-
1 23456789
b
control H interferon
Figure 4-7. Immunoblotting analysis of COX-2 in whole cell extracts from endometrial epithelial
cells from cows treated in vivo with placebo or blFN-r.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of COX-2
for control cows (lines 1 to 4) and for blFN-x-treated cows (lines 5 to 8); line 9 is a positive control
for COX-2; b) Densitometric analysis of abundance of COX-2.


110
(diluted in medium to a volume of 100 pi which was added to wells), in duplicate,
for 3,10, 30, 60 or 120 minutes. Amounts and proportions of medium and tissue
were the same as described for 24-hour cultures. Treatments were stopped by
quickly rinsing explants in fresh medium and immediately immersing in a 8 oz.
Styrofoam cup containing liquid nitrogen. Frozen tissues were stored at -70 C
until homogenization. Frozen tissues were homogenized as described above,
but a different buffer was used. Whole cell extract (WCE) buffer was comprised
of 50 mM Tris (pH 8.0), 300 mM NaCI, 20 mM NaF, 1mM Na3 V04, 1mM
Na4P207, 1mM EDTA, 1mM EGTA, 1mM DTT, 0.5 mM PMSF, 10% v/v Glycerol,
0.5% v/v NP-40 and 10pg/ml each aprotinin, leupeptin and pepstatin. Tissues
were homogenized in 3 ml of ice-cold WCE buffer. Homogenates were
incubated in ice for 15 minutes and clarified by centrifugation as described above
to obtain WCE. Extracts were pre-cleared to reduce competition for protein A in
Sepharose beads between endogenous immunoglobulins in extracts and
antibodies used for immunoprecipitation (see below). Pre-clearing was
performed by incubating 1 mL WCE with 40 pi protein A slurry (see below) in a
microcentrifuge tube at 4 C, in a rotating device (Roto-torque, heavy duty
rotator, Cole Parmer, Chicago, IL; lever setting at low, speed setting at 7) for 2
hours. The extract was spun in the microcentrifuge for 10 seconds and pre
cleared supernatant transferred to a clean tube. Removal of endogenous
immunoglobulins was verified by incubating pellet (protein A beads) with 1 X
Laemmli buffer for 5 minutes at 95 C and submitting supernatant to SDS-PAGE .


CHAPTER 1
INTRODUCTION
The worlds human population Is Increasing at a fast rate, and
consequently the need for basic nutrients, Including carbohydrates, lipids,
proteins and minerals, is also Increasing. Animal agriculture historically has
been one of the most important sources of nutrients for humans. Dairy cows
efficiently metabolize feed nutrients and synthesize milk, which provides protein
and energy in a suitable form for human consumption. Lactation is a final step in
the reproductive cycle which is dependent on successful production of viable
gametes, conception, pregnancy and parturition. First insemination conception
rates for dairy cattle Is 52 to 57% (Mawhlnney and Roche, 1978). Improvements
on such rates will be required for dairy products to continue being a viable
source of nutrients for humans.
Classically, study of animal performance has focused on genetic and
environmental effects on a given production trait, such as meat or milk
production. Reproduction poses an interesting scenario, in which reproductive
processes are modulated by the Interactions between both the maternal and
embryonic genomes. Moreover, reproductive processes are influenced by
external environment, but more importantly, by the Internal environment (i.e., the
1


252
Data from Chapter 7 challenges the concept that protein synthesis is
required for bIFN-x-mediated suppression of PGF2. The quick reduction in the
rate of PGF2ll accumulation suggests that either blFN-x stimulates very quickly
synthesis and action of proteins that are inhibitory to the PGF2c, cascade, or that
blFN-t acts in the cytosol to modulate activity of enzymes involved in PGF2a
production. This agrees with data from Chapter 4, in which abundance of COX-2
and PLA2 in endometrial epithelial cells recovered fresh were similar between
cows that received uterine infusions of control protein or blFN-x. Perhaps
modulation of activity of such enzymes, rather than their expression, was critical
for bIFN-x-induced suppression of PGF2a. Indeed, intracellular stimulator-
induced PGF2o secretion from explants recovered from these same cows was
attenuated in cows that received blFN-x in vivo. (Arnold et al., 1999). However,
critical experiments have not been performed to determine if protein synthesis-
independent mechanisms are in place for blFN-x suppression of PGF2cl. Perhaps
blFN-x stimulates both protein synthesis-dependent and -independent
mechanisms to suppress PGF2o, synthesis in the endometrium (see Figure 8-1).
Chapter 7 described experiments using a novel model system for study of
PGF2o,secretion and blFN-x effects on PGF2asecretion in the endometrium.
Such system features PDBu-induced PGF2e,accumulation in culture medium,
measurable by radioimmunoassay, and also blFN-x inhibition of PGF2a


223
decreased abundance of PLA2 compared to PDBu alone. In contrast, there was
no effect of bIFN-x on the PDBu-induced COX-2 protein expression.
Experiment 2
Secretion of PGF-. Similar to Experiment 1, treatment with PDBu
stimulated an acute release of PGF2o, compared to controls (Figure 7-4). The
PDBu-induced PGF2asecretion was noticed as early as 3 hours and increased at
a fast rate between 0 and 6 hours (47.4 pg/ pg protein/6 hours). Further
increase in PGF2o release was noticed, but occurred at a lower rate (14.7 pg/ pg
of protein/6 hours). Bovine IFN-x attenuated the PDBu-induced release of PGF2a
at 3, 6, 9 and 12 hours of treatment. Significant time by treatment contrasts [c2 x
c3, (p<0.01); c2 x c5 (p<0.04); c2 x c6 (p<0.01)] indicate that bIFN-x decreased
the rate of increase on PDBu-stimulated PGF2osecretion. Furthermore, analysis
of homogeneity of regression indicated that PGF2o secretion could be
represented by third order curves (R2: .968), which confirmed changes in
secretion rates during the experiment. Equations for each treatment were: Y
0.5808+7.3175 x X+0.21227 x X2-0.0325 x X3 (PDBu); Y=-0.1259 x X-0.1961 x .
X2+0.011 x X3 (PDBu + bIFN-x); where Y represents secretion of PGF2a, and X is
a given time point in the experiment. Orthogonal comparisons of curves
confirmed that PDBu stimulated PGF2osecretion, and that bIFN-x attenuated that
effect (p<0.01).


215
in 5% non-fat dried milk in TBS) for 2 hours. Remaining immunoblotting and
ECL procedures were conducted as described above.
Experimental Designs
Experiment 1. Sixteen 100 mm plates were assigned randomly to receive
blFN-i (0 or 50 ng/ml) or PDBu (0 or 100 ng/ml) in quadruplicate, in a two-by-two.
factorial design, for 24 hours. Medium was sampled (500 pi) immediately before
addition of treatments (0-h sample), after 12 and 24 hours of treatment.
Samples were stored at -20 C for PGF2a analysis. After the 24 h-sample,
remaining medium was discarded and cells were harvested for extraction of
proteins for immunoblots (WCE; two plates per treatment).
Experiment 2. Ten 100 mm plates were assigned randomly to receive
medium alone (control, 3 plates), PDBu (100 ng/ml, 4 plates) or PDBu in
combination with blFN-x (50 ng/ml, 4 plates) for 12 hours. Medium was sampled
(500 pi) immediately before addition of treatments (0-h sample), after 3, 6, 9 and
12 hours of treatment and stored at-20 C for PGF2a analysis. Following each
sample collection time, cells from each treatment were photographed digitally
using the imaging apparatus described on Chapter 4. After collection of 12 h-
samples, remaining medium was discarded and cells were harvested for
extraction of proteins for immunoblots with antiserum to PLA2 and COX-2 for all
plates.


Figure 5-20. Immunoblotting analysis of STATs 1 and 2 co-immunoprecipitated with STAT-3
from nuclear extracts from BEND cells treated with blFN-r for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of STATs
1 and 2; b) Representative ECL exposure of tyrosine phosphorylation of STAT-3; c) Representative-
ECL exposure of abundance of STAT-3; d) Least squares means and SE of abundance of STATs
1 and 2 (within each variable, bars with distinct subscripts are statistically different, p<.1).


.
197
s1
lr-t-
blFN-x
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
temp3
rt
rt
rt
rt
37
37
37
rt
rt
rt
rt
37
37
37
37
pg protb
10
20
10
20
10
20
20
10
20
10
20
20
10
10
20
10x cold3
-
-
+
+
-
-
+
-
-
+
+
-
-
+
+
Figure 6-1. Experiment 1. Electrophoretic mobility shift assay of nuclear extracts of BEND cells
incubated with radiolabeled ISRE in presence (+) or absence (-) of blFN-i for 30 min. Arrow
indicates a shifted complex (s1).
a Temperature of reaction (rt=room temperature; 37=37 C).
b Amount of protein loaded in pg.
0 Presence (+) or absence (-) of 10 fold excess ISRE unlabeld probe.
untreated cells were used (lane 2). The apparent s3 complex present in lane 1 is
non-specific, and was not observed in a subsequent experiment (Figure 6-3).
Formation of s1 complex was noticed when nuclear extracts from cells treated
with blFN-x were used (lane 3), and intensity of such complex was reduced when
a 10 fold excess cold ISRE probe was added to the reaction mixture (lane 4).
Addition of an anti-STAT-1 antibody caused a reduction in intensity of s1, which
may be associated with the formation of the low-intensity s2 complex. Anti-
STAT-2 antibody abolished formation of s1 complex, but stimulated formation of
a s3 complex, which was not resolved in the present gel. Formation of an anti-
STAT-2-induced s3 complex was confirmed in a subsequent experiment (Figure


Dedicated to Mario Rodolpho Giovanni Binelli (late) and Arnaldo Monteiro
de Oliveira (Grandfathers), Guilherme Jose Binelli (Father), Ricardo Binelli
(Brother), Luiz Alberto de Oliveira (Uncle), Cicero Spiritus, Paul Campbell, Zilmar
Zlller Marcos, H. Allen Tucker and William W. Thatcher (Mentors)
To my family and friends
and
To an enlightened humankind


69
response to treatment with blFN-t. Moreover, such complexes were distinct from
complexes containing ubiquitin, indicating a blFN-x-Induced, specific action.
Although proteins present in the bUCRP conjugates have not yet been identified,
an attractive hypothesis is that blFN-x induces conjugation of bUCRP to proteins
involved in the cascade of PGF2c,production in the endometrium. Such targeting
could modify function of such proteins to make them less able to stimulate PGF2o
production.
Research from Spencer and coworkers (1998) also showed that
endometrial estrogen receptors and oxytocin receptors were reduced in the
uterine horns infused with ovine IFN-t, and this was negatively correlated with
observed increase in IRF-1 and IRF-2 expression. Since IRF-2 has been
implicated as an inhibitor of gene transcription (Harada et al., 1994), the authors
hypothesized that perhaps interferon-induced IRFs were involved in inhibition of
gene transcription for estrogen and oxytocin receptors. In fact, Fleming and
coworkers (1998) cloned the ovine estrogen receptor gene and discovered IRF
response element (IRE) consensus sequences in the promoter region, further
supporting the hypothesis of interferon modulation of estrogen receptor
expression. Deletion constructs of the estrogen receptor promoter linked to
luciferase reporter gene were transfected into endometrial cells. Treatment of
these cells with ovine IFN-t caused reduction in luciferase expression only in
constructs containing the IREs. Using the same rationale, Bathgate and others


53
docking site present will be activated by binding of the ligand specific for the
extracellular domain of the receptor (Stahl et al., 1995). Therefore, STAT
activation is determined more by specific interactions between STATs and their
receptors than by specific JAKs associated with receptor chains.
STATs
Unlike other common intracellular second messengers, STATs not only
convey the extracellular signal to the interior of the cell, but they themselves
carry such a signal to the nucleus, acting as transcription factors to activate
transcription of genes induced by particular ligands. I will focus this discussion
on STATs 1, 2 and 3, although STATs 4 to 6 have been described (Darnell,
1997). STAT-1a and ip are encoded by alternative splicing of a single mRNA
transcript. Human STAT-1a consists of 750 amino acids, while the extreme
carboxy-terminal 38 amino acids are missing for STAT-ip. STAT-2 is composed
of 851 amino acids. STATs 1, 2 and 3 have significant sequence homology (Fu
et al., 1992; Zhong et al., 1994). The domain distribution in the STAT molecule
includes a centrally-located DNA-binding domain, a carboxy-terminal
transcription activation domain, and SH2 and SH3 domains located in between
them (Fu, 1992; Figure 2-2). The SH2 domain allows docking to tyrosine
phosphorylated sites in the IFN and cytokine receptors, as discussed above, and
also STAT dimerization. SH2 domain sequences are specific for each STAT, but
mutant STATs 1 and 3, in which SH2 domains were swapped, completely


173
Coimmunoprecipitation of STAT-3 with STATs 1 and 2. There was a
numerical decrease in the abundance of STAT-3 in the cytosol at 15 minutes,
but such decrease may have been due to lower densitometric reading because
of a bubble (see Figure 5-21). Abundance remained lower up to 120 minutes
(Figure 5-21). In the nucleus, STAT-3 association with STATs 1 and / or 2
increased overtime to reach maximum levels at 15 minutes and then gradually
decreased (Figure 5-22).
Discussion
Presence and activation of tyrosine phosphorylation of STAT proteins by
blFN-t has been shown previously in non-reproductive tissues (MDBK cells;
Subramaniam et al., 1995) as well as bovine uterus (Binelli et al., 1996; Perry et
al., 1999; Chapter 4). The present series of experiments demonstrate that blFN-
t can stimulate the JAK-STAT pathway of signal transduction in a line of bovine
endometrial epithelial cells (BEND cells; Austin et al., 1996b). The STAT
proteins^ 1, 2 and 3 are present, and after exposure to blFN-t become tyrosine-
phosphorylated in a dose- and time-dependent manner and associate to form
complexes. Such complexes migrate to the nucleus of cells, where they can act
as transcription factors and induce or repress expression of IFN-responsive
genes (Figure 5-23).


105
calcium ionophore to stimulate secretion of PGF2cl, compared to in vivo infusion
with BSA. Intracellularly, PDBu and the calcium ionophore stimulate activity of
protein kinase C (PKC, Silvia and Homanics, 1988; Silvia et al., 1994). The PKC
promotes phosphorylation and activation of phospholipase A2 (PLA2, Parker et
al., 1987; Lin et al., 1993), which cleaves arachidonic acid (AA) from
phospholipids in the cellular membrane (Lin et al., 1992). Arachidonic acid is a
substrate for cyclooxygenase-2 (COX-2), which directs AA to prostaglandin
synthesis (Smith et al., 1992). In order to suppress secretion of PGF2oin
response to those treatments, blFN-i must interact with the PGF2asynthesizing
machinery in the endometrium. In a second experiment, objective was to test
the hypothesis that uterine infusions of blFN-x decrease abundance of PLA2 and
COX-2 in endometrial epithelial cells.
Materials and Methods
Materials
Materials, equipment and reagents utilized for estrous cycle
synchronization, tissue culture, electrophoresis, densitometry and fluorography
were as described for Chapter 3, except when specified. Bovine IFN-t (200
pg/ml in 20 mM Tris-HCI, pH 8; 1.08 x 107 units of antiviral activity)was a gift from
Dr. Michael Roberts from the University of Missouri. Urea and Nonidet P40 were


5
oviduct with the uterus, and the AMP is localized between INF and 1ST. The
initial interaction of the oocyte with the oviduct takes place at the INF. The INF
picks up the ovulated oocyte and initiates its transport towards the uterus.
Overall flow of oviductal fluid is towards the body cavity (Hafez, 1993a).
Therefore, in order for the oocyte to be transported to the site of fertilization
(AMP), it must interact with ciliated epithelial cells lining the oviductal lumen.
Balance between oviductal fluid flow and ciliary beating towards the uterus yields
a net movement of the oocyte towards the AMP and ultimately, uterus. During
this trajectory towards the site of fertilization, the oocyte is under the influence of
products secreted by the oviduct which could modulate its development and the
process of fertilization (Buhi et al., 1997a). For example, Kouba and coworkers
(1999) determined that a major secretory protein of the AMP, named POSP
(porcine oviductal secretory protein), has a role in decreasing occurrence of
polyspermy during fertilization in pigs. This supports the findings of Nancarrow
and Hill (1995) that an estrus-associated glycoprotein, a protein homologous to
POSP in sheep, increased blastocyst formation after in vitro fertilization.
Furthermore, Staros and Killian (1998) identified six proteins in oviductal fluid,
including a POSP-like protein that are associated with bovine oocytes in vitro.
Sperm-oviduct. Austin (1951) and Chang (1951) independently reported
that freshly ejaculated rat and rabbit spermatozoa were incapable of penetrating
an oocyte. The ability of fertilization was only acquired after the sperm spent a
period of time in the female reproductive tract, a process called sperm


LD
1780
1993
,641
UNIVERSITY OF FLORIDA
3 1262 08554 4384


123
Figure 4-5. Immunoblotting analysis of cytokeratin in whole cell extracts from endometrial
epithelial cells from cows treated in vivo with placebo or blFN-t. Representative enhanced
chemiluminescence (ECL) exposure of abundance of cytokeratin for control cows (lines 1, 3, 5, 6
and 9) and for blFN-t-treated cows (lines 2, 4, 7, 8, and 10).
Discussion
Results from Experiment 1 indicated that treatment with blFN-t stimulated
synthesis of both secretory and intracellular proteins from bovine endometrium.
Moreover, the JAK-STAT pathway of signal transduction is present and
operational in bovine uterine tissue, although use of explants does not provide
an ideal model to study blFN-x-mediated signal-transduction in the endometrium.
Differential expression of at least two secretory proteins (P19 and P12)
suggests that the recombinant blFN-x used in the present experiments is
biologically active and stimulates synthesis and secretion of unique endometrial
proteins. These findings agree with those reported by Naivar and coworkers
(1995). Those authors identified three secretory proteins which were stimulated


138
Freshly harvested BEND cells (~1x106 cells)were platted In 150x25 mm
sterile, polystyrene, tissue culture dishes in 40 ml of complete culture medium.
Plates were incubated at 37 C and experiments were conducted when cells
reached 90% confluency as determined by microscopic evaluation of plates. For
a given experiment, cells were washed (20 ml) and incubated (19 ml) in serum
free medium for 45 minutes. Recombinant blFN-x treatments were diluted in 1
ml of serum-free medium and were added as described below for specific
experiments. To end bIFN-x incubations, plates were quickly transported to a
walk in cooler (4 C), where all subsequent steps of extract preparation were
conducted. Culture medium was discarded and cells were rinsed twice in ice-
cold PBS containing 1 mM Na3V04 and 5 mM NaF. As indicated below, different
experiments utilized either whole cell extracts (WCE; Figure 5-1) or cytosolic
(CE) and nuclear extracts (NE; Figure 5-2). Therefore, slight differences in
procedures were necessary, as indicated below. Cells were washed briefly in 1
ml of appropriate ice-cold extraction buffer [WCE buffer: 50 mM Tris, pH 8.0, 300
mM NaCI, 20 mM NaF, 1 mM Na3 VO, 1 mM Na4P207, 1 mM EDTA, 1 mM
EGTA, 1 mM DTT, 0.5 mM PMSF, 10% v/v Glycerol, 0.5% v/v NP-40 and 10
pg/ml each aprotinin, leupeptin and pepstatin (Dr. Douglas Leaman, personal
communication) or hypotonic buffer: 12 mM Hepes pH 7.9, 4 mM Tris pH 7.9,
,6mM EDTA, 10 mM KCI, 5 mM MgCI2, 1 mM Na3V04, 1 mM Na4P207, 1 mM
NaF, 0.5 mM PMSF, 0.6 mM DTT and 10 gg/ml each aprotinin, leupeptin,
pepstatin (Ghislain and Fish, 1996) for CE and NE] and allowed to drip dry.


146
Specificities of anti-STAT antibodies used for immunoblots were tested in
WCE of untreated cells. Whole cell extracts were immunoprecipitated with anti-
STAT-1 and -2 antibodies or anti-STAT-3 antibody (1pg each) and analyzed by
immunoblotting with no antibody, normal rabbit serum or either a combination of
anti-STAT-1 and -2 or anti-STAT-3 antibodies.
Nuclear Translocation of STATs
In order to stimulate gene transcription, STATs must first be
phosphorylated in the cytoplasm and then translocated to the nucleus. The aim
of this experiment was to study the time responsiveness of phosphorylation of
STATs to blFN-x in CE and NE obtained from the same cells. Plates of BEND
cells were assigned randomly to receive 50 ng/ml blFN-x for 1,3, 8, 15, 30, 60 or'
120 minutes, or to receive nothing (0 minutesVcontrol treatment). Cytosolic
extracts and NE from each plate were immunoprecipitated with anti-STAT-1 and
-2 antibodies and analyzed by immunoblotting for anti-phosphotyrosine and anti-
STAT-1 and -2. The same experiment was repeated but CEs and NEs were
immunoprecipitated with anti-STAT-3 antibody and analyzed by immunoblotting
for anti-phosphotyrosine and anti-STAT-3. To confirm that the time-
responsiveness to bIFN-t was specific, this experiment was repeated, except
that blFN-x was not added at any time point, and CE and NE were


268
Buhi WC, Vallet JL, Bazer FW. De novo synthesis of polypeptides from cyclic
and early pregnant porcine ovlductal tissue In explant culture. J Exp Zool
1989; 252:79-88.
Burns PD, Graf GA, Hayes SH, Silvia WJ. Cellular mechanisms by which
oxytocin stimulates uterine PGF2o, synthesis In bovine endometrium: roles
of phospholipases C and A2. DomestAnim Endocrinol 1997; 14:181-191.
Callus BA, Mathey-Prevot B. lnterleukln-3-induced activation of the JAK/STAT
pathway is prolonged by proteasome inhibitors. Blood 1998; 91:3182-
3192 .
Catchpole HR. Hormonal mechanisms in pregnancy and parturition. In: Cupps
PT (ed.), Reproduction in domestic animals, 4th ed. San Diego: Academic
Press; 1991:361-383.
Chang MC. Fertilizing capacity of spermatozoa deposited into the Fallopian
tubes. Nature 1951; 168:697-698.
Charleston B, Stewart HJ. An interferon-induced Mx protein: cDNA sequence
and high-level expression in the endometrium of pregnant sheep. Gene
1993; 137:327-331.
Charpigny G, Reinaud P, Creminon C, Tamby J-P. Increased levels of ovine
endometrial cyclooxygenase-2 correlates the increase in PGE2 and PGD2
observed in the late luteal phase. Biol Reprod 1999; (in press).
Chegini N. Oviductal-derived growth factors and cytokines: implications in
preimplantation. Semin Reprod Endocrinol 1996; 14:219-229.
Chung CD, Liao J, Liu B, Rao X, Jay P, Berta P, Shuai K. Specific inhibition of
Stat3 signal transduction by PIAS3. Science 1997a; 278:1803-1805.
Chung J, Uchida E, Grammar TC, Blenis J. STAT-3 serine phosphorylation by
ERK-dependent and -independent pathways negativeely modulates its
tyrosine phosphorylation. Mol Cell Biol 1997b; 17:6508-6516.
Clark JD, Lin L-L, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, Knopf
JL. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2t-
dependent translocation domain with homology to PKC and GAP. Cell
1991; 65:1043-1051.


95
TABLE 3: Summary of means comparison of effects of treatment3, treatment by
region6 side0 and treatment by side interactions on the logarithm of densitometric
units of selected proteins (P) in two-dimensional gelsa.
Effect
Comparison
Proteins
Treatment
FDF>PDF
P2, P13
Treatment by region
AMP-FDF>AMP-PDF
P14
AMP-PDF>AMP-FDF
P7
IST-PDFHST-FDF
P1
Side
CONTRAHPSI
P7, P8
IPSI>CONTRA
P15, P19
Treatment by side
CONTRA-FDF>CONTRA-PDF
P6, P8, P9, P11
CONTRA-PDF>CONTRA-FDF
P19
IPSI-PDF>!PSI-FDF
P5, P6, P9, P11
a: FDF=fresh dominant follicle (DF); PDF=persistent DF.
b: INF=lnfundlbulum; AMP=ampulla; IST=isthmus.
c: IPSMpsilateral to CL; CONTRA=contralateral to CL.
d: Proteins are listed for effects which were different with p<0.1.
Discussion
In the present study, we have shown that hormonal manipulations of
animals altered both their follicular and luteal functions, which provided a model
to study oviductal protein synthesis and secretion at estrus from distinctly
different periestrus hormonal environments (Figs. 2 and 3). Distinctly different
steroidal environments for cows bearing PDFs or FDFs specifically modulated


27
Uterine Function and Reproductive Failure in Cattle
As mentioned before, there is approximately a 30% rate of embryonic
losses occurring from days 8 to 16 of early pregnancy, which represents a period
of uterine localization of the embryo. In this section I will attempt to describe key
aspects of uterine physiology that can be considered when trying to solve the
problem of embryonic mortality during this period.
The Uterine Environment
The uterus is considered to be an extension of the oviduct (Bartol, 1999),
therefore, several of the principles regarding biophysical and biochemical
properties discussed for the oviduct also will apply here. Compared to the
oviduct, bovine embryos will spend a much longer interval of time in the uterus
(280 days vs 4 days, on average; Catchpole, 1991) which permits a much
broader set of communications between the maternal and embryonic units. To
support this notion is the fact that although there are not remarkable embryo-
induced changes between the cyclic and the pregnant oviduct, this is the
opposite for the uterus. The thesis of this section is that presence of the embryo
conditions the uterine environment, to support embryonic development.
The uterine cycle. Similar to the oviduct, the uterus also undergoes
changes dependent on the stage of the estrous cycle in response to changes in
concentrations of ovarian steroids. However, the uterus has the unique role of


277
Johnson HM, Torres BA, Green MM, Szente BE, Siler Kl, Larkin 3rd J,
Subramaniam PS, Hypothesis: ligand/receptor-assisted nuclear
translocation of STATs. Proc Soc Exp Biol Med 1998b; 218:149-155.
Karimi K, Lennartz MR. Protein kinase C activation precedes arachidonic acid
release during IgG-medlated phagocytosis. J Immunol 1995; 15:5786-
5794.
Kastellc JP, Curran S, Pierson RA, Ginther OJ. Ultrasonic evaluation of the
bovine conceptus. Theriogenology 1988; 29:39-54.
Kawashima M, Takahashi T, Kamiyoshi M, Tanaka K. Effects of progesterone,
estrogen, and androgen on progesterone receptor binding in hen oviduct
uterus (shell gland). Poultry Sci 1996; 75:257-260.
Kessler DS, Levy DE, Darnell jr JE. Two interferon-induced nuclear factors bind
a single promoter element In interferon-stimulated genes. Proc Natl Acad
Sci USA 1988; 85:8521-8525.
Kim TK, Manlatis T. Regulation of interferon-y-activated STAT1 by the ubiquitin-
proteasome pathway. Science 1996; 273:1717-1719.
Kinder JE, Kojima FN, Bergfeld EGM, Wehrman ME, Fike KE. Progestin and
estrogen regulation of pulsatile LH release and development of persistent
ovarian follicles in cattle. J Anlm Sci 1996; 74:1424-1440.
Kindahl H, Edqvist L-E, Granstrom E, Bane A. The release of prostaglandin F2a
as reflected by 15-keto-13,14-dlhidroprostaglandin F2Jn the peripheral
cieculation during normal luteolysis in heifers. Prostaglandins 11:871-
878.
King WA. Intrinsic embryonic factors that may affect survival after transfer.
Theriogenology 1985; 23:161-174.
Kirby CJ, Thatcher WW, Collier RJ, Simmen FA, Lucy MC. Effects of growth
hormone and pregnancy on expression of growth hormone receptor,
insulin-like growth factor-l, and insulin-like growth factor binding protein-2
and -3 genes in bovine uterus, ovary, and oviduct. Biol Reprod 1996;
55:996-1002.
Knickerbocker JJ and Niswender GD. Characterization of endometrial receptors .
for ovine trophoblast protein-1 during the estrous cycle and early
pregnancy In sheep. Biol Reprod 1989; 40:361-369.


260
treatment. However, while explants responded immediately to PDBu (within 20
minutes; Arnold et al., 1999), BEND cells need at least two hours of exposure to
PDBu to start secreting PGF2a. This is probably due to the fact that while
enzymes needed for PGF2asynthesis are already present in explants, they need
to be synthesized and activated in BEND cells. In other words, effects in
explants are probably mediated through stimulation of PLA2 and COX-2
enzymatic activity, whereas in BEND cells, stimulation of both enzymatic
synthesis and activity are required. The idea of pre-existing enzymes in explants
is corroborated by findings in Chapter 4, showing presence of PLA2 and COX-2
in freshly scraped endometrial epithelial cells. The bIFN-x effects in blocking
PGF2a secretion in BEND cells should be tested in vitro with the explant system,
to further verify the validity of this convenient and physiological cell culture
system.
Collectively, antiluteolytic mechanisms exerted by bIFN-x and pregnancy
are not understood completely, but appear to be complex. A fine, ordered and
concerted series of events involving multiple signal transduction systems,
second-messengers, and mediators, which act to modulate synthesis and activity
of enzymes involved in the production of PGF2a, probably take place in the
endometrium during maternal recognition of pregnancy associated with CL
maintenance. Such a series of events are probably stimulated not only by blFN-
x, but also by other conceptus-derived factors. Breakdown on the conceptus-
maternal signaling system during maternal recognition of pregnancy associated


MATERNAL-EMBRYONIC INTERACTIONS DURING
EARLY PREGNANCY IN CATTLE
By
MARIO BINELLI
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1999


113
0.1% v/v Tween-20 (TBST). Membranes were quickly rinsed twice, then washed
for 15 minutes in TBST and incubated for 1 hour in 10 ml of a 1:1000 dilution of
antl-phosphotyrosine monoclonal antibody (PY-20) in 2% (w/v) gelatin in TBS,
quickly rinsed twice, washed 4 times for 10 minutes in TBST, incubated for 1 h in
10 ml of a 1:8000 dilution of horse radish peroxidase (HRP)-linked anti-mouse
IgG antibody [part of the Enhanced Chemiluminescence(ECL) kit] In TBST
containing 2% (w/v) gelatin. The membrane was quickly rinsed twice, washed
once for 15 minutes and four times for 5 minutes with TBST. Membranes were
allowed to drip-dry and then were exposed to ECL kit reagents (8 ml total
volume), following the manufacturers instructions. Membranes were drip-dried
again and placed face down on a piece of plastic wrap, which was wrapped
around them. Wrapped membranes were placed in a X-ray film cassette (Kodak
X-Omatlc cassette, Eastman Kodak Co., Rochester, NY) and X-ray films were
exposed for 10 minutes to detect tyrosine-phosphorylated proteins. Exposed X-
ray films were developed in a Konica -X-ray film processor model QX-70 (Konica
Corp., Japan). Since both tyrosine phosphorylation and abundance of STATs
needed to be measured in the same sample (i.e., same membrane), membranes
were stripped of antibodies used to detect phosphorylated proteins and re-used
for a second immunoblot, to determine abundance of STATs. Membranes were
stripped in 200 ml solution of 0.1 M Glycine (pH 2.5) for 45 minutes, neutralized in
400 ml 0.1 M Tris-HCI (pH 8) for 45 minutes, and blocked for 2 hours in 200 ml of
5% (w/v) non-fat dried milk in TBST. For STATs 1 and 2 immunoblots,


164
I 121-
x
s 78-
o
a
i ¡rthMr¡.rSTAT-3
1 3 8 15 30 60 120 min.
bIFN-x, 50 ng/ml
b
STAT-3
Figure 5-15. Immunoblotting analysis of STATs 3 immunoprecipitated from nuclear extracts
from BEND cells treated with blFN-i for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of STAT-3;
b) Least squares means and SE of abundance of STAT-3 (bars with distinct subscripts are
statistically different, p<0.1).


22
While some evidence has accumulated for roles of oviductal secretory
proteins on reproductive products, much less Is known about roles of embryonic
secretory products. To the best of my knowledge, no reports have focused on
effects of presence of embryo on the pattern of secretory proteins from the
bovine oviduct. Buhi and coworkers (1989), working with porcine oviductal
secretory proteins, failed to demonstrate differences in rate of incorporation of
non-dialyzable, 3H-leucine labeled molecules between pregnant and cyclic
oviducts. Moreover, one-dimensional SDS-PAGE analysis of secretory proteins
did not indicate changes in patterns of de novo synthesized, secretory proteins.
This indicates that presence of the embryo had little effect on modulating
secretion of macromolecules from the oviductal epithelium. In contrast, a recent
report (Wakuda et al., 1999) showed that presence of embryos In mice which
had their uterotubal junction ligated on day 1 of pregnancy, enhanced
implantation rate of embryos transferred to the uterus. This was in comparison
with pseudopregnant mice, which had uterotubal junction ligated before or after
mating with vasectomized males, and, also mated females which had uterotubal
junction ligated before mating (all mice had blastocysts transferred to uterus on
day 4). Embryo-dependent factors have not been identified, but clearly
influenced embryo development in that species.


Figure 5-19. Immunoblotting analysis of STATs 1 and 2 co-immunoprecipitated with STAT-3
from cytosolic extracts from BEND cells treated with blFN-r for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of STATs
1 and 2; b) Representative ECL exposure of tyrosine phosphorylation of STAT-3; c) Representative
ECL exposure of abundance of STAT-3; d) Least squares means and SE of abundance of STATs
1 and 2 (within each variable, bars with distinct subscripts are statistically different, p<.1).


17
estrous cycle. Other aspects of the estrous cycle will be discussed elsewhere in
this chapter.
The ovarian cycle consists of cyclic growth and demise of two ovarian
structures, the follicle and the CL. Considering one estrous cycle the period
comprised between two ovulations, two to three follicular waves of dominant
follicle growth occur (Savlo et al., 1988; Slrois and Fortune, 1988). Each
follicular wave is comprised of periods of recruitment, selection, dominance and
turnover or atresia. The ovulatory follicle generated in the last wave does not
turn over, but ovulates. The main steroid secretory product from follicles are
estrogens, such as E2. There is a positive relationship between size of follicles
and E2 concentration in the circulation. Since maximum growth of follicles occurs
during the dominance phase, the perl-ovulatory period Is characterized by
highest concentrations of circulating E2 during the estrous cycle. The ovulated
follicle undergoes functional and structural changes to form a CL. The CL grows
at a rapid rate to reach a maximum size in about 11 days, remains at its
maximum size until about day 16 of the estrous cycle and then regresses (the
process of luteolysis). Parallel to changes in CL size are changes in secretion of
luteal P4. Turnover of the dominant follicle is associated with high concentration
of P, typical of mid-cycle. In contrast, final differentiation and growth of the
ovulatory follicle prior to ovulation only occurs in a low P4 environment.
Functional anatomy and morphology. The oviducts are suspended in the
mesosalpynx, a peritoneal fold of the broad ligament. As mentioned earlier, the


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
id
William W. Thatcher, Chair
Graduate Research Professor
of Animal Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is Mhradeqttate
and quality, as a dissertation for the degree of Doctor oTRfeilosophy.
William C. Buhi
Professor of Biochemistry and
Molecular Biology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Peter J.tHarisen
Professor of Dairy and Poultry
Sciences
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
-toward M. Johnson
Graduate ResearCn Professor of
Microbiology ana Cell Science
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.
Frank A. Slmmen
Professor of Dairy and Poultry
Sciences


A series of growth factors have been identified in the oviduct during the
estrous cycle and early pregnancy in different species (summarized in Buhi et
al., 1997a). For example, Paula-Lopes and coworkers (1999) reported synthesis
and secretion of interleukin-1 p both in oviduct and uterus of cyclic cows.
Moreover, interleukin-1 p stimulated in vitro development of embryos when added
before day 5, which suggests a oviductal effect (Paula-Lopes et al., 1998).
Although limited research has been performed to date to elucidate specific roles
of growth factors in fertilization and early embryo development, it is possible that
growth factors act in an autocrine and paracrine fashion to influence these
processes (Chegini, 1996).
Conceptus-uterus. In cattle, it takes 72 to 84 h from the time of ovulation
to the time embryos enter the uterus (Betteridge and Flechon, 1988). From entry
into the uterus to parturition there are multiple examples of interactions between
the conceptus and the maternal unit, which will be discussed elsewhere in this
review. A striking example of such interactions is the process of maternal
recognition of pregnancy associated with maintenance of the CL, which will also '
be presented in detail afterwards. Briefly, maintenance of pregnancy requires a
steady supply of progesterone (P4) from the corpus luteum (CL). In contrast, in
cycling animals, it is necessary that P4 concentrations decrease so that animals
can return to estrus. This decrease is accomplished in response to pulses of
uterine prostaglandin-F2a(PGF2o) that have lytic actions on the CL. Uterine


54
400
500
600
700
r
V_
Y
I

Figure 2-2. Domain structure of the STAT-1 protein. The diagram represents the linear
structure of STAT-1 oriented in a amino- (leftmost) to carboxy- terminus sequence. DNA
binding domain, SRC homology 2 (SH2) domain, SH3 domain and transcription activation
domain (TAD) are represented in the sequence they occur in the STAT-1 molecule. The site of
tyrosine (Y) phosphorylation is also represented.
reversed their specificity for interaction with specific phosphotyrosine motifs
(Hemmann et al., 1996). This indicates that the SH2 domain is the sole
determinant of specific STAT factor recruitment to receptors. STATs contain a
unique tyrosine residue in the carboxy-terminal region (Y701, Y690 and Y705 for
STATs 1, 2 and 3 respectively). A recently developed model for STAT activation
(Li et al., 1997; Figure 2-1) proposes that unphosphorylated STATs 1 and 2 are
associated with the IFNaR2 chain. Binding of interferon causes dimerization of
this chain with IFNaRI. Tyrosine phosphorylated residue 466 of IFNaRI binds
the SH2 domain of STAT-2, which is then phosphorylated on tyrosine 690,
providing a docking site for the SH2 domain in STAT-1. STAT-1 is
phosphorylated on tyrosine 701, and then dimerizes with STAT-2 through
reciprocal binding of tyrosine phosphorylated residues with SH2 domains.
However, an unsolved question is what drives SH2 domains of STATs to
dissociate from a higher affinity interaction with receptor phosphotyrosine to form


235
and resource consuming. BEND cells are spontaneously immortalized, and can
be used repeatedly for at least 15 passages (see Chapter 5; unpublished
authors observations). Moreover, BEND cells originated from day 14 cyclic
cows, which provide an adequate model to study mechanisms associated with
luteolysls and maternal recognition of pregnancy associated with CL
maintenance. Secretion of PGF2aand suppression of such secretion by
conceptus-secreted blFN-x are hallmark processes during luteolysis and
maternal recognition of pregnancy events, respectively. Therefore, an adequate
in vitro system to study such events should mimic such processes.
In all experiments reported in the present study, PDBu stimulated PGF2o
from BEND cells. Phorbol esters such as PMA have been used previously to
study regulation of PGF2asecretion in vitro, and have shown a similar stimulatory
effect (Xiao et al., 1999). In the present study, PDBu continuously stimulated
PGF2a secretion for up to 12 hours, but further increase between 12 and 24 hours
of exposure to PDBu was negligible. Further increases in PDBu-stimulated
PGF2a may have occurred due to depletion of arachidonic acid (precursor for
PGF2o synthesis), or due to down-regulation of the PDBu stimulatory system.
Experiment 2 indicated that maximum PGF2a secretion rate occurs during the
first 6 hours. From 6 to 12 hours secretion rate is less. Experiments 3 and 4
suggest that PGF2ais secreted in a constant rate between 3 and 6 hours, and
that it takes at least 2 hours for PDBu effects to be detected. This suggests that
PDBu activity is, at least initially dependent on de novo protein synthesis, and


a
202
O control | interferon
b
30
time (h)
Figure 6-4. Immunoblotting analysis of IRF-1 in nuclear extracts from BEND cells
incubated In presence (+) or absence (-) of blFN-i (50 ng/ml) for Increasing intervals of
time.
a) Representative enhanced chemiluminescence exposure of abundance of
IRF-1; b) Densltometric analysis of abundance of IRF-1 (least squares meanstSEM).


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MATERNAL-EMBRYONIC INTERACTIONS
DURING EARLY PREGNANCY IN CATTLE
By
Mario Binelli
August 1999
Chairperson: William W. Thatcher
Major Department: Animal Science
Maternal-embryonic physiological communications are an important
feature of processes in the reproductive cycle. Communications occurring during
oviductal transit of gametes/embryos and during maternal recognition of
pregnancy for maintenance of the corpus luteum (CL) were studied in cattle.
Objectives were 1) to study the distribution pattern of oviductal secretory proteins
secreted by cows bearing persistent or fresh dominant follicles (PDF or FDF,
respectively); 2) to examine the signal transduction system stimulated by bovine
interferon-T (bIFN-x) in endometrium; and 3) to characterize the effects of bIFN-x
on prostaglandin F2a(PGF2J production by bovine endometrial (BEND) cells.
Presence of PDF reduces fertility in cattle. Proteins synthesized from
XI


190
films were from NEN Research Products (Reflection; Boston, MA) and from
Eastman Kodak Co. (X-Omat Blue XB-1; Rochester, NY). Konlca -X-ray film
processor model QX-70 was from Konlca Corp.(Japan). Centricons (10.000
MWCO) were from Amicon Inc. (Beverly, MA). Anti-IRF-1 polyclonal antibody
(C-20, catalog number SC-497X, 200pg/100pl), anti-STAT-1 (E-23; catalog
number SC-346X, 200pg/100pl) and anti-STAT-2 (C-20; catalog number SC-
476X, 200pg/100pl), anti-STAT-3 (C-20; catalog number SC-482, 200pg/1ml)
were from Santa Cruz Biotechnology (Santa Cruz, CA).
Probes
For annealing, sense and anti-sense strands (25 pi each) of SIE were
mixed in a 500 pi microfuge tube and placed in a beaker containing water at 100
C. The beaker was removed from heat, and water allowed to cool to room
temperature. Annealed probes were stored at -20 C. Annealed, complementary
ollgodeoxyribonucleotides corresponding to ISRE and to SIE were labeled
radioactively for experiments. Probes for ISRE were prepared by end labeling.
Two hundred ng of double stranded DNA (2 pi of stock solution) were mixed with
5 pi of 10X polynucleotide kinase buffer, 40 pi of water, 2 pi of gamma 32P-ATP
(333 pCi) and 1 pi T4 polynucleotide kinase in a microfuge tube (500pl) and
incubated for 30 minutes at 37 C. Then, 5pl of 250 mM ATP were added to the
reaction mixture, and incubated for 10 minutes at 37 C. Finally, 10 pi of tRNA (1


56
However, mechanism of transport to the nucleus remains unclear, since STAT
proteins lack the nuclear localization signal (NLS; Johnson et al., 1998b), which
are required for nuclear transport mediated through the importin mechanism
(Gorlich and Mattaj, 1996). Johnson and coauthors (1998b) proposed an
intriguing model for nuclear translocation of STATs after activation by interferon .
gamma (IFNy). Since the carboxy-terminal domain of the IFNy molecule contain a
NLS, they propose that following binding to IFNy a complex containing the IFNy-
receptor, jak kinases, STATs and the bound ligand become internalized by
endocytosis. Upon cytoplasmic localization, the NLS sequence in the IFNy
molecule could associate with the importin protein complex, which would then
catalyze the transport of this complex to the nucleus, where STAT-mediated
transcription activation would ensue. They provided evidence for actual nuclear
translocation of a peptide containing the carboxy-domain of the IFNy molecule.
Although seemingly unique, they provide evidence of over 30 cytokines and/or
their receptors, which utilize STATs as signal transducers that contain NLS in
their sequence, indicating that this ligand-receptor-assisted nuclear translocation
is a viable, and intriguing mechanism. Among such cytokines and receptors are
the human IFNa and the human IFNaRI molecules. Data in a recent paper is in
variance with this concept (Milloco et al., 1999). Those authors engineered a
STAT-1-estrogen receptor chimera, in which the estrogen receptor ligand binding
domain was fused to the carboxy-terminus of STAT-1 molecules. After


168
E178-BBBBPWSS
-STAT-2
STAT-1
STAT-3
? 121
2 78-
feBoJ
5121-
2 78-
TP
n
0 1 3 8 15 30 60 120 min.
Figure 5-18. Verification of effects of time on phosphorylation of STATs 1 and 2 in cytosolic
(CE) and nuclear extracts (NE) from BEND cells incubated with medium alone for increasing
intervals of time.
a) Enhanced chemiluminescence (ECL) exposure of tyrosine phosphorylation of STATs 1, 2 and
3 in CE; b) ECL exposure of abundance of STATs 1, 2 and 3 in CE; c) ECL exposure of tyrosine
phosphorylation of STATs 1, 2 and 3 in NE; d) ECL exposure of abundance of STATs 1, 2 and 3
in CE.


139
Grow cells to 90%confluency
i
Starve cells for 45 min.
1
Add 50 ng/ml blFN-x for 0, 3,8,15, 30, 60 or 120 min.
1
Wash cells in ice-cold PBS
i
Lyse cells (WCE buffer)
i
Spin lysate, collect supernatant
1
Immunoprecipitate overnight with a-STAT-1 and a-STAT-2 or a-STAT-3 abs.
1
SDS-PAGE and transfer to nitrocellulose membrane
1
Western blot 1: a -phospho-tyrosine
1
Strip and neutralize membrane
i
Western blot 2: a -STAT-1 and a -STAT-2 or a-STAT-3
Figure 5-1. Outline of experimental sequence involving Immunoprecipitation of whole cell
extracts of BEND cells treated with blFN-r for increasing Intervals of time. See text for details.


a
154
CO

X
121
78-*
bIFN-x
-
IP aba
1/2
1/2
1/2
1/2
1/2
1/2
1/2
3
3
1st abb
1
1
2
2
1/2
1/2
NRS
3
3
2nd abc
r
n
r
n
r
n
r
r
n
b
bIFN-x
+
+
+
+
+
+
IP aba
1/2
1/2
1/2
3
3
3
1st abb
n
NRS
p-Y
n
NRS
p-Y
2nd abc
m
m
m
m
m
m


206
oxytocin receptor, estrogen-receptor, protein kinase C, phospholipase A2 and/or
cyclooxygenase 2.
Another way to influence negatively the PGF2o generation pathway is to
stimulate synthesis of transcription repressors. In the ewe, IFN-x induces
synthesis of IRF-2, a transcription repressor (Spencer et al., 1998). Moreover,
the promoter region of estrogen receptor gene, which is involved in luteolysis,
contains binding sites for IRFs (IRE, Fleming et al., 1998). Furthermore,
promoter deletion experiments indicated that treatment with blFN-x decreased
expression of an estrogen-receptor-luciferase reporter construct, but such a
decrease was noted only if the region containing the IRE was present (Fleming
et al., 1998). In cattle, the oxytocin receptor also is probably involved in the
luteolytic cascade. The oxytocin receptor gene has been sequenced recently
and also contains an IRE element, which binds both IRF-1 and IRF-2 (Bathgate
et al., 1998). Nuclear extracts from endometrium of pregnant cows can bind to
the IRE in mobility shift assays. However, binding can not be supershifted with
anti-IRF-1 or-2 antibodies, indicating that other members of the IRF family may
be involved (Bathgate et al., 1998).
In the present study, blFN-x induced IRF-1 protein expression in BEND
cells. Attempts were made to measure induction of IRF-2 after exposures to
blFN-x ranging from 1 to 48 hours, but failed probably due to use of heterologous
reagents (anti-human IRF-2 antibody; data not shown). However, since IRF-2
expression can be induced by IRF-1 (Henderson et al., 1997), it is possible such


195
Immunoblottina for IRF-1
To verify whether blFN-x was able to induce synthesis of the early gene
IRF-1, BEND cells were grown to 90% confluency as described in Chapter 5.
Then, cells were washed (20 ml) and incubated (19 ml) in serum-free medium for
1 hour. Nuclear extracts were obtained as described for Chapter 5 from one
plate Immediately after this 1 hour starvation, and from the remaining eight plates
after they received 0 or 50 ng/ml blFN-t for 1, 2, 4 or 6 hours. Treatments were
diluted in 1 ml of medium before adding to plates (final volume = 20 ml/per
plate). Hypotonic buffer (see Chapter 5) was added to NE to a final volume of 2
ml, and adjusted to 60 mM KCI adding 1 M KCI as needed. Diluted NEs were
then concentrated using 10000 molecular weight cutoff Centricons overnight, in
a Son/all RC-5B refrigerated superspeed centrifuge, equipped with a SM24 (Du
Pont Co., Wilmington, DE) at 5000 RPM. Retentates (approximately 50 pi) were
recovered and analyzed for protein content using the Bradford method (Bio-Rad
protein assay). Volumes corresponding to 40 pg of total protein were solubilized
in 2X Laemmli buffer (25 pi) and water (up to 50 pi final volume) and loaded onto
7.5% acrylamide mini-gels. Transfer to nitrocellulose membrane and
immunoblotting were performed as described in Chapter 5. Briefly, membrane
was blocked in 5% non-fat dried milk in TBST for 2 hours, washed, incubated
with anti-IRF-1 polyclonal antibody (2 mg/ml; 1:10000) diluted in 5% non-fat dried
milk in TBS for 2 hours, washed, incubated with anti-rabbit-IgG secondary


152
a
CO
191

^ IZ I
X
s 78 i
M
1
*
8
IP aba
1
2
1/2
3
n
n
n
n
1st abb
1
2
1/2
3
1
2
1/2
3
2nd abc
r
r
r
r
r
r
r
r
Figure 5-5. Validation of immunoprecipitation procedure on whole cell extracts (WCE) of BEND
cells.
a) Enhanced chemiluminescence (ECL) exposure of WCE Immunopreclpitated with anti-
STAT-1, anti-STAT-2 antl-STAT-3 or nothing; b) ECL exposure of WCE immunopreclpitated with
normal rabbit serum,
* Antibody used for Immunoprecipitation (1=anti-STAT-1; 2=anti-STAT-2; 3=anti-STAT-3;
NRS=normal rabbit serum; n=nothing).
b First antibody used for immunoblotting (1=anti-STAT-1; 2=anti-STAT-2; 3=anti-STAT-3.
c Second antibody used for immunoblotting (r=anti-rabbit IgG).


200
6-3, lanes 1 to 3). Anti-STAT-3, anti-IRF-1 and rabbit IgG had no effect on
migration or intensity of s1 complex.
In Experiment 3, no specific complexes were formed in the absence of
nuclear extracts (lane 4; Figure 6-3) nor when nuclear extract from untreated
cells was used (lane 5). Treatment with blFN-x induced formation of a major and
a minor complex (s4 and s5 respectively; lane 6). Formations of both s4 and s5
complexes were abolished completely when a 10-fold excess of unlabeled SIE
probe was added (lane 7). Addition of antibodies against STATs 1 and 3
revealed that s4 was composed of three distinct sub-complexes (Figure 6-3,
panel b), a faster (s4c), an intermediate (s4b) and a slower (s4a) migrating
complexes. Presence of anti-STAT-1 antibody displaced both s4b and s4c, but
intensity of s4a was actually enhanced. Pre-incubation with anti-STAT-3 caused
displacement of s4a and s4b to form the supershifted complex s6. In contrast,
s4c migration remained unaltered. The fact that s4b was displaced both by anti-
STAT-1 and -3 antibodies, suggests that complex contains both STATs, and is
probably a STAT 1:3 heterodimer. Since s4c was displaced when anti-STAT-1
antibody was used, but not anti-STAT-3 suggests it represents a STAT-1:1
homodimer. Similarly, s4a displacement with anti-STAT-3 exclusively suggests
that it is composed of STAT 3:3 homodimers. The s5 was formed of two sub
complexes that had similar alterations on migrating pattern to s4 sub-complexes
in response to STAT-1 and STAT-3 antibodies. Addition of rabbit IgG did not
alter intensity or mobility of s4, but may have affected migration properties of s5.


126-
a
1 2 3 4 5 6 7 8
b
l~l control | interferon
Figure 4-8. Immunoblotting analysis of PLA2 in whole cell extracts from endometrial epithelial
cells from cows treated in vivo with placebo or blFN-i.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of PLAj
for control cows (lines 1 to 4) and for blFN-t-treated cows (lines 5 to 8); b) Densitometric analysis
of abundance of PLAj.


32
both by ovarian steroids and embryonic bioactive molecules. During pregnancy,
misregulation of uterine function may lead to embryonic mortality. For the
remaining of this section I will focus on the mechanisms regulating PGF2a
production from the cyclic uterus, and on mechanisms of maternal recognition of
pregnancy related to CL maintenance occurring in the pregnant uterus.
Cyclic uterus. The reason for the uterine cycle is to provide repeated
opportunities for pregnancy at relatively short intervals. In practice, the turning
point in the uterine cycle is the commitment to either luteolysis or pregnancy. In
the absence of pregnancy, the progestational stage is finished by the uterus-
induced demise of the P4 source. Demise of the CL is accomplished by pulsatile
secretion of uterine PGF2a(Nancarrow et al., 1973; Kindahl et al., 1976). Then,
the estrogen-dominated uterus prepares for reception of sperm, initially, and
reception of the early developing embryo. This cycle repeats until successful
establishment of pregnancy. However, early pregnancies will be terminated if
the uterine cycle is not interrupted.
Secretion of PGF-.. During the estrous cycle, the presence of two distinct
patterns of PGF2a release is easily distinguishable : a basal release and a
stimulated, pulsatile release. Initial measurements of PGF2ctwere performed In
samples collected from the venous drainage of the uterus, which required
surgical cannulation (Nancarrow et al., 1973). Measuring peripheral
concentrations of 15-keto-13, 14-dihidroprostaglandin F2(J (PGFM) the main
metabolite of PGF2afound in the circulation, facilitated study of such patterns


129
induce appropriate intracellular signals within the endometrium, which could
result in a breakdown on the antiluteolytic mechanism.
Preliminary experiments reported by Binelli and others (1996) indicated
that STAT-1 protein is present and can be stimulated in endometrial explants
treated with bIFN-x in vitro. Data in the present report confirm and expand those
early observations. Tyrosine phosphorylation of STATs 1 and 2 occurred in a
time-dependent fashion, which was only noted in explants treated with bIFN-x.
This is in agreement with data from Silvennoinen and coworkers (1993), which
also showed induction of STAT phosphorylation to be time-responsive. Signal
transduction systems working through tyrosine phosphorylation are regulated
acutely, to provide specific responses to specific stimuli. Therefore it is not
surprising that a similar dynamic is also valid for bIFN-x-stimulated signal
transduction in the endometrium. There was no clear pattern of rise and fall of
phosphorylation of STATs over time. There was an increase to a maximum level
at 30 minutes, followed by a decrease by 60 and another increase at 120
minutes. This might reflect cycling dynamics of tyrosine phosphorylation of
STATs.
In general, it was extremely challenging to obtain consistent results of
tyrosine phosphorylation using this model of in vitro incubations of explants.
Explants were chosen initially because it was considered desirable to maintain
structural integrity of the endometrium and stroma-epithelium interactions to best
mimic the in vivo system. Most production of PGF2a in the endometrium comes


213
400 ni (100 ni Tris buffer, 100 ni diluted sample, 100 pi ab solution, 100 pi
radiolabeled PGF2ct solution). Minimum detectable concentration of PGF2[, was
3.32 pg/tube. Inter- and intra-assay coefficients of variation were 13.99 and
12.61% respectively. Since multiple samples were removed, and medium was
added back to plates, concentrations of PGF2 were adjusted to account for
PGF2aremoved from previous samples. Adjustment consisted on adding the
amount of PGF2o removed in previous samples to the amount measured In the
current sample. For example, assume samples 1, 2 and 3 (500 pi each) were
removed sequentially from the same plate (10 ml total volume), and had assay
values 10, 20 and 30 pg/.1 ml, respectively. Adjusted value for sample 2 would
be 20 pg + .5 pg (500 pi removed from 10 ml corresponds to 5% of the
concentration on sample 1), which equals 20.5 pg. Similarly, adjusted value for
sample 3 should be 30 + .5 + 1 pg (5% concentration sample 1 + 5%
concentration unadjusted sample 2) totaling 31.5 pg. Further adjustment on
concentrations of PGF2t> was performed to account for potential differences in
cell number due to treatment effects. Total cell protein was determined as
described below, and adjusted total PGF2o values per well were expressed on a
per pg of protein basis. Final values of PGF2a production per well were adjusted
for previous removal and expressed on a protein basis (total PGF2a, pg/pg
protein), were used for statistical analyses (see below).


201
Immunoblotting for IRF-1
There was a significant time by treatment interaction on abundance of
IRF-1 (Figure 6-4; p<0.03). Treatment with blFN-x, but not with medium alone,
stimulated synthesis of IRF-1 in a time dependent manner (p<0.04). IRF-1
increased after 1 hour of treatment to reach a maximum at 2 hours and gradually
decreased after 4 and 6 hours of exposure to blFN-x.
Discussion
When BEND cells were stimulated with blFN-x, nuclear proteins bound to
specific response elements (i.e., ISRE and SIE) present in the regulatory region
of genes stimulated by interferons. Moreover, synthesis of the transcription
factor IRF-1 was documented. These findings support the concept that the JAK-
STAT signal transduction system described in Chapters 4 and 5 is indeed
functional in BEND cells. Data from Experiment 2 indicated clearly the formation
of a specific complex (s1) when nuclear extracts from bIFN-x-treated cells were
used. The radiolabeled ISRE probe is from the 9-27 interferon-induced gene,
and contains the classical ISRE sequence of nucleotides (Parrington etal., 1993;
Darnell et al., 1994). Therefore, complex s1 probably is comprised of the ISGF-3
proteins: STAT-1, STAT-2 and p48 (Shindleret al., 1992; Darnell et al., 1994),
which is known to bind to such a sequence. Our attempt to elicit a supershift of
this complex with antibodies for STATs 1 and 2 yielded mixed results. Antibody


87
Day of Treatment Period
Figure 3-3. Least squares means ( SEM) of concentrations of ovarian steroids in plasma
of cows bearing a fresh dominant follicle (FDF, treated with GnRH on d 9) or persistent
dominant follicle (PDF, not treated with GnRH on d 9) during the Treatment Period.
Treatments with PGF2a, CiDR and GnRH are indicated. Day 0 represents day of estrus at the
beginning of Treatment Period.
a) estradiol-17p (E2); b) progesterone (P4).


12
Inadequate maternal unit. As mentioned above, results from the work of
Putney and coworkers (1988a; 1989) suggest that the elevated number of
abnormal embryos in heifers that underwent heat stress could be a result of the
effect of high temperatures on the maternal unit, and not a direct effect on the
embryo. In that regard, embryos produced by in vitro maturation/fertilization
techniques that are exposed to elevated temperatures (40.5 C) for 12 hours had
development comparable to that of controls (56% blastocyst formation; Rivera,
Lopes and Hansen, personal communication), supporting the concept that heat
stress effects on maternal units may create a toxic environment that is conducive
to development of abnormal embryos. Moreover, in the experiment of Putney
and others (1998b), they incubated endometrium explants removed from cows at
day 17 of the estrous cycle at 39 or 43 C as described above, and measured
secretion of PGF2Jn the medium. Heat stress caused a pronounced increase in
PGF2a production over time compared to controls. This finding suggests that
heat stress favors luteolysis and consequent loss of pregnancies.
Measurements of P4 concentrations in milk following insemination of dairy
cows revealed that inseminated-pregnant cows had slightly higher P4 compared
to inseminated-non-pregnant cows (Lamming et al., 1989). This finding
prompted the hypothesis that luteal insufficiency could be a cause of increased
embryonic mortality in lactating dairy cows. Possible causes of decreased luteal
function include (1) poor development of the ovulatory follicle, resulting in a low
quality CL (i.e., low weight and consequent low P4 secretion) and (2) insufficient


240
initiated previously by PDBu. We chose to add blFN-x after 3 hour-exposure to
PDBu because (1) PDBu-induced PGF2o secretion was increasing at a fast rate
by this time (Figure 7-8), which indicates that the PGF2a synthesizing machinery
was present and functional [also suggested by data from Xiao and coworkers
(1999), showing maximum gene expression of COX-2 after 3-hour stimulation
with oxytocin) and (2) maximum secretion rate was noticed in the first 6 hours of
PDBu stimulation (Experiment 2; Figure 7-4). Decreased rate of secretion of
PGF2a1 hour after addition of blFN-x (PDBu + blFN-x-3 h treatment, Experiment
4) indicated that blFN-x was able to quickly suppress PDBu-induced PGF2a. The
exact mechanism whereby blFN-i exerts its effects is unknown, and are under
active investigation at the moment. However it is tempting to speculate that such
a quick action is independent of protein synthesis and can occur through novel,
previously undescribed cytosolic (i.e., nuclear independent) actions of blFN-x.
Next, I will propose a mechanism of blFN-x actions in the PDBu-
stimulated PGF2a synthesis model. The following discussion makes a distinction
between blFN-x effects and blFN-x modes of action. Effects include the
changes in protein expression of PLA2 and COX-2 examined in the present
study. Modes of action include bIFN-x-stimulated signal transduction, generation
of blFN-x effector molecules and modification (i.e., activation, deactivation) of
pre-existing bIFN-x-mediator molecules.


15
persistent dominant follicle (PDF) is estrogenic, and subsequent fertility, as
measured by conception rate at first service (number of pregnancies / number of
animals inseminated), is lower compared to animals bearing normal DFs [37.1%
vs. 64.8% in heifers, (Savio et al., 1993b); 23.6% vs. 58.2% for cows and heifers
(Cooperative Regional Research Project, NE-161,1996). Possible explanations
for reduced fertility include alterations in the oocyte and /or in the oviductal
environment. In a study by Ahmad et al. (Ahmad et al., 1995), embryos
recovered at Day 6 of pregnancy from cows bearing PDF were less developed
(i.e were less able to reach the 16-cell stage) than embryos from cows
ovulating a fresh dominant follicle (FDF). In addition, Revah and Butler (1996)
showed that oocytes recovered from PDF showed expanded cumulus cells and
condensed chromatin dispersed in their ooplasm. In contrast, compact cumulus
cells and intact germinal vesicles were found in oocytes from FDF. Thus, the
PDF may induce premature oocyte maturation and/or alter oviduct function,
which could affect early embryonic development and decrease fertility.
Processes of sperm capacitation, fertilization and early embryonic development
in this altered oviductal environment can contribute to decreased fertility
experienced by cows developing persistent follicles.


ACKNOWLEDGMENTS
I would like to express my greatest appreciation to Dr. William Thatcher,
my supervisory committee chair, for giving me the honor of being his student and
friend. His dedication, generosity, enthusiasm, sincerity, intelligence and ability
to excel under a lot of pressure are remarkable and a great source of inspiration.'
I am proud of being a member of one of the most respected reproductive
physiology laboratories in the world. I also want to thank him for his patience
when I was away from the lab pursuing extracurricular activities. My gratitude is
extended to the other members of my committee: Dr. William Buhi, for changing
my abstract conception of what was a protein to something more real like a spot
in a 2-D gel; Dr. Peter Hansen, for being my "second advisor", sharing his
knowledge and laboratory; Dr. Howard Johnson, for asking me "so, what is
novel?" when I first described my dissertation project to him, and for letting me
work in his laboratory during the beginning of my program; and to Dr. Frank
Simmen, for his advice on molecular matters and for the being such an example
of a humble personality behind a powerful mind.
I am also indebted to Dr. Prem Subramaniam, Dr. Lokenga Badinga, Dr.
Rosalia Simmen, Dr. Naser Chegini, Dr. Joel Yelich, Dr. Maarten Drost, Dr.
Herbert Head, Dr. Michael Fields and Dr. Nancy Denslow for sharing their
iii


239
x. These observations are consistent with those of Xiao and others (1999).
They reported that PMA stimulated COX-2 protein expression (about 5 fold
higher than control) and that bIFN-x attenuated that response (to about 2.5 fold
higher than control). Moreover, those authors treated cells with oxytocin in
presence or absence of bIFN-x for increasing intervals of time (3 to 24 hours)
and measured secretion of PGF2o, gene and protein expression of COX-2.
Oxytocin stimulated PGF2a accumulation in culture medium throughout the
experiment. Message for COX-2 was maximum at 3 hours and gradually
decreased overtime, while protein was maximum at 12 hours. Presence of blFN-
x attenuated all responses measured at all time points. In contrast, Asselin and
others (1997) reported no changes in PLA2 gene expression after treatment of
endometrial and stromal cells with bIFN-x. Moreover, COX-2 expression was
upregulated by bIFN-x. Large doses of bIFN-x (1 to 20 pg/ml vs 50 ng/ml in the
current study) could explain this discrepancy of results. Collectively, it appears
that bIFN-x exerts a complex regulation of both PLA2 and COX-2 gene
expression and activity to modulate PGF2asecretion in BEND cells (see
Hypothetical Model below).
The fact that inhibitory effects of bIFN-x on PDBu-induced PGF2usecretion
were noted as early as 2 hours (time when first PDBu-induced rise in PGF2ais
noted; Experiment 3; Figure 7-8) prompted us to examine how quickly bIFN-x
would be able to suppress synthesis of PGF2a after PGF2ol secretion had been


65
expression due to synergistic actions of AP-1 and STAT-1. Such an effect was
Ras-dependent, which implies crosstalk between the MAPK and the JAK-STAT
pathways of signal transduction.
Yet another example of crosstalk is between the JAK-STAT pathway and
the PI 3' kinase, which has both lipid and serine kinase activities. Pfeffer and
others (1997) reported that PI 3' kinase is tyrosine phosphorylated through the
JAK-STAT pathway. Tyrosine phosphorylated STAT-3 proteins, bound to the
IFNaRI chain of the interferon receptor, serve as a docking site for PI 3' kinase,
which couples its SH2 domain to tyrosine phosphorylated residues in the STAT
molecule. Upon docking, the PI 3' kinase is activated by JAKs, which then
promotes serine phosphorylation of STAT-3 to increase STAT-3 activity. In
another study (Uddin et al., 1997) interferon-a stimulated serine kinase activity of-
PI 3' kinase, which in turn activated the signal transducer IRS-1. Moreover,
stimulation with interferon p caused activation of MAPK, and such stimulation was
inhibited by Wortmannin, an inhibitor of PI 3' kinase activity. This suggests
involvement of the PI 3' kinase on MAPK activation. In contrast with data from
Pfeffer and others (1997), wortmannin failed to inhibit formation of the ISGF3
complex and interferon-mediated induction of ISG-15, indicating that the PI 3'
kinase probably is not required for interferon effects.
Finally, the work of Flati and others (1996) indicates that stimulation of
cells with interferon-a causes activation of PLA2, as measured by release of AA in


93
TABLE 2: Relative molecular weights (MW3) and least square mean (LSM) and
probability (p) values of the logarithm of densitometrlc units side by treatment
Interactions of selected proteins (P) on 2-D gelsb.
SPOT
MW
IPSI-
FDF
CONTRA-
FDF
IPSI-
PDF
CONTRA-
PDF
side by
trt.
P1
823
9.2,2
9.4
9.4
9.3
NSC
P2
675
7.2.6
7.5
7.0
5.9
NS
P3
582
5.4.9
7.4
8.2
7.7
NS
P4
975
6.1.17
6.5
6.2
6.0
NS
P5
764
5.9.2
6.5
6.5
6.5
p<0.06
P6
955
5.8.1
6.5
6.5
6.3
p<0.05
P7
975
3.1.5
4.5
6.3
5.8
NS
P8
804
7.0.1
7.5
7.2
7.2
p<0.07
P9
764
7.6.1
7.9
7.8
7.7
p<0.05
P10
723
7.4.11
7.7
7.5
7.5
NS
P11
654
7.2.1
7.6
7.5
7.4
p<0.03
P12
713
3.0.7
2.9
4.8
2.0
NS
P13
684
3.7.8
2.7
.3
0
NS
P14
473
2.81.2
3.7
1.4
0.7
NS
P15
472
7.8.9
4.0
8.4
6.4
NS
P16
372
6.91
5.8
6.2
8.3
NS
P17
272
6.9.2
7.3
6.7
6.6
NS
P18
272
6.4.2
6.6
6.4
6.5
NS
P19
21 1
5.9.9
2.9
5.9
5.4
p<0.09
P20
281
8.4.2
8.7
8.5
8.5
NS
a: Relative molecular weight (Mrx 10'3; means S.E.M.).
b: LSM+pooled S.E.M. of side (CONTRA = contralateral to DF; IPSI = Ipsllateral to DF) by
treatment Interactions,
c: Non significant (p>0.1).


121
a
blFN-x, 50 ng/ml
Figure 4-3. Immunoblotting analysis of STATs 1 and 2 immunoprecipitated from whole tissue
extracts from endometrial explants treated with blFN-r for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine
phosphorylation of STATs 1 and 2; b) Representative ECL exposure of abundance of STATs
1 and 2
Experiment 2
Validation of densitometric technique. There was a linear decrease in
arbitrary densitometric units associated with increased dilution of WCE for all
exposure times tested (Figure 4-4).
Immunoblotting for cvtokeratin and vimentin. Although we originally
intended to use scraped endometrial luminal epithelial cells for culture, analysis
of scraped cells using trypan exclusion revealed that 99% of cells were dead at
the end of the scraping procedure. Therefore, these cells were used only for
preparation of extracts. Regardless of in vivo treatment (i.e., BSA or blFN-x),


9
later sections I will discuss the implications of a failure in maternal-embryonic
cross-talk on embryonic mortality.
Problems Associated with Fertilization Failure and Embryonic Mortality in Cattle
Susceptible Periods Purina Pregnancy
Calving rates to a single insemination are reported to be 52 to 57% for
dairy cows (Mawhinney and Roche, 1978) despite fertilization rates of about 89%
(Henricks et al., 1971). Diskin and Sreenan (1980) utilized beef heifers to
determine embryo survival during discrete periods within pregnancy. They
reported up to 93% survival rates to day 8, 66% to day 16 and 58% to day 42.
These data indicate that minor losses are due to fertilization and embryonic
death before day 8, which encompasses the period of embryo permanence in
the oviduct and development to the blastocyst stage in the uterus. However, in a
group of infertile cows, there are appreciably greater losses (~40%) due to failure
of fertilization (Tanabe and Cassida, 1949) and additional losses by day 35
(40%; Ayalon 1978). In normal cows, a large percentage of embryos is lost
between days 8 and 16 of pregnancy, which is the period of embryonic
elongation and maternal recognition of pregnancy associated with CL
maintenance. The substantial losses of pregnancies during the first 16 days of
pregnancy has obvious economic impacts in the livestock industry, and
represent an opportunity for animal scientists and reproductive physiologists to


219
approach allowed us to (1) estimate order of regression of curves for each
treatment within experiments, so we could identify changes in secretion rate
overtime, (2) determine regression equations for each treatment within
experiments, and (3) perform orthogonal comparison of PGF2cl secretion among
treatments within experiments.
For abundance of COX-2 and PLA2, mathematical model included only
the effect of treatment. Orthogonal contrasts used for mean comparisons were .
the same used for PGF2ain Experiment 1, and c1 and c2 for Experiments 2 and
3, respectively.
Results
Experiment 1
Secretion of PGF- Secretion of PGF2aat time 0 was negligible (<0.3
pg/pg protein) for all treatments (Figure 7-1). Long term treatment of BEND
cells for 12 or 24 hours with PDBu stimulated a pronounced release of PGF2a
(effect of PDBu; p<0.01). In contrast, blFN-x reduced secretion of PGF2o (effect
of blFN-x; p<0.01). Furthermore, blFN-x attenuated the PDBu-induced release
of PGF2a both at 12 and 24 hours of treatment (interaction between PDBu and
blFN-x, p<0.01). Moreover, this attenuation was less at 24 hours compared to
12 hours (treatment by time Interaction; p<0.01).


262
apt to produce offspring. During in vitro production of embryos, oocytes are
recovered from a large number of follicles in the ovary, since the ovary usually is
sliced with a scalpel blade. In theory, after fertilization of recovered oocytes, this
procedure will generate embryos from oocytes that would have never naturally
ovulated, perhaps due to incompetence of the oocyte inside. As a result,
embryos are being produced and individuals generated that would have never
been created if nature was to take its course. Holm and Callensen (1998)
summarized data comparing in vivo and in vitro produced embryos. Results are
summarized in Table 8-1.
Table 8-1. Differences in pregnancy rates, incidence of congenital defects and
birth weight of calves following transfer of in vivo and in vitro produced bovine
embryos (Holm and Caliesen, 1998)
Parameter
Vitro
Vivo
Pregnancy rates
Fresh (not frozen)
40-50%
50-75%
Abortion rates
>60 days
7-24%
3-5%
Congenial defects
Overall
3-5%
0.5-2%
Birth weights
>50 kg
>30%
<10%
Overall less favorable results characterize in vitro embryo production.
This reflects consequences of in vitro production techniques, but may also reflect
the lower adequacy of embryos artificially induced to undergo development to
term. For example, in vivo derived embryos secreted more blFN-x than embryos
produced in vitro via a variety of techniques, which may explain partially the
lower pregnancy rates presented in Table 8-1 (Stojkovic et al., 1999). Data
regarding lifelong productive and reproductive evaluation of animals derived from'


97
These endocrine environments induced distinctly different patterns of
protein synthesis and secretion by all ovlductal regions, between cows with PDF
vs. FDF. Studies in sheep (Buhi et al., 1991), baboon (Verhage and Fazleabas,
1988) and pigs (Buhi et al., 1989; Buhi et al, 1990; Buhi et al., 1997b) indicate
that specific oviductal proteins and specific mRNAs are regulated by
endogenous steroids during the estrous cycle or early pregnancy and by
exogenous steroids in ovariectomized animals. In the pig, a family of related
glycoproteins (POSP 1-3), a basic and an acidic 100,000 Mr protein and a very
acidic protein (75,000-85,000 Mr), are synthesized primarily by the AMP during
proestrus, estrus and metestrus (high E2, low P4) but not diestrus (low E2, high
P4). Consistent with POSP protein synthesis and secretion, POSP mRNA
expression is also estrogen-dependent and significantly greater in the AMP on
day 0 and day 1 of estrous cycle or pregnancy (Buhl et al., 1996). Similar to
POSP, the cow oviduct produces a basic 97 kD protein (Boyce et al., 1990)
identified as P1 in this study. Protein 1 secretion reaches a maximum at estrus
and decreases during the luteal phase of the estrous cycle. In agreement with
Boice et al. (1990), P1 was secreted by all oviductal regions in the present study.
Other proteins, such as tissue inhibitor of metalloproteinase-1, found to be
produced by the pig and cow oviduct (P20 in this study) was shown to be
expressed optimally on day 2 in the pig, when E2 and P4 were both low (Buhi et
al., 1992). Further, E2 appeared to suppress synthesis and/or secretion of
several protein complexes which were not identified (Buhi et al., 1992).


102
95 kD oviductal glycoprotein. Therefore, it Is possible that different patterns of
LH release may directly affect the oviduct and modulate differential protein
synthesis in FDF compared to PDF cows.
The present study identified a series of proteins in which synthesis and
secretion are modulated differentially according to exposure of the oviduct to the
in vivo steroid milieu. This indicates that the oviductal micro-environment is
altered. Collectively, our findings add support to the concept that a less than
optimal oviductal micro-environment may contribute to the low fertility of cows
bearing a PDF. We propose that a combination of the effects of premature
maturation of oocytes and inappropriate oviductal micro-environment is
responsible for the decreased fertility observed in cows bearing PDF.
This chapter provides clear evidence for alterations on maternal-
embryonic communications occurring in the oviduct that could lead to disruption
of the reproductive cycle. Remaining chapters will examine maternal-conceptus
communications during maternal recognition of pregnancy for maintenance of
the CL.


Cell Culture and Cell Extracts 136
Immunoprecipltatlon 142
Immunoblots 142
Nature of BEND Cells 143
Dose Response to bIFN-T 144
Time Response to bIFN-T 145
Validation of Immunoprecipltatlon and Immunoblots Procedures
145
Nuclear Translocation of STATs 146
Coimmunopreclpltation of STATs 147
Densitometrlc Analysis 147
Statistical Analysis 148
Results 148
Nature of BEND Cells 148
Validation of Immunoprecipltatlon and Immunoblots 151
Dose Response to blFN-x 151
Time Response to bIFN-T 156
Nuclear Translocation of STATs 156
Validation of Time Responses to bIFN-T 167
Colmmunoprecipitatlon of STATs 167
Discussion 173
6 BOVINE INTERFERON-TAU STIMULATES BINDING OF STAT
PROTEIN COMPLEXES TO DNA AND STIMULATES SYNTHESIS OF
INTERFERON RESPONSE FACTOR-1 (IRF-1) PROTEIN IN BOVINE
ENDOMETRIAL (BEND) CELLS 187
Introduction 187
Materials and Methods 188
Materials 188
Probes 190
Electrophoretic Mobility Shift Assays 192
Immunoblotting for IRF-1 195
Statistical Analysis 196
Results 196
Electrophoretic Mobility Shift Assays 196.
Immunoblotting for IRF-1 201
Discussion 201
IX


Figure 3-5. Representative fluorographs of two-dimensional SDS-PAGE analysis of culture
medium conditioned by oviductal explants. Proteins analyzed by densitometry (1 through 20) are
indicated. Molecular weight standards are indicated (X1 O'3), and pH gradient runs from left (pH 8)
to right (pH 4).
a) Infundibulum; b) Ampulla; c) Isthmus.


82
Rutherford, NJ) by tail venipuncture and stored in an ice bath. Plasma was
harvested by centrifugation (1800 x g for 30 minutes) and stored at -20C until
assayed for estradiol-17p (E2) and P4. On day 7, all cows were injected with
PGF2a and received one used CIDR device (Savio et al., 1993b). On day 9 cows
were assigned randomly to one of two treatment groups. Cows of the FDF group
(n=3) received an injection of GnRH agonist (Buserelin, 8 mg), to induce turnover
of any large size follicles present at that time, and allow recruitment of fresh
follicles (Schmitt et al., 1996c). Cows of the PDF group (n=3) did not receive the
GnRH agonist. On day 16 CIDR devices were removed, and cows received an
injection of PGF2a (25 mg). Cows were checked for signs of estrus twice daily
and slaughtered when observed in standing estrus (day 18 or 19). The
experimental models for persistent and fresh follicles resulted in a greater
pregnancy rate for heifers inseminated at estrus induced by FDF (Savio et al.,
1993b; Schmitt et al., 1996c).
Tissue Culture
On the day of slaughter, reproductive tracts were removed aseptically,
and oviducts were identified as IPSI or CONTRA to DF, dissected, trimmed free
of mesosalpynx and divided into INF, AMP and 1ST regions based on gross
anatomical characteristics. Segments of tissue between 1ST and AMP were
discarded. Tissue from each region was cut longitudinally to expose the lumen,
and then minced into fragments of 50mm3. Tissue fragments from each


160
blFN-x, 50 ng/ml
b
STAT-1 STAT-2
0 1 3 8 15 30 60 120 min.
blFN-x, 50 ng/ml
Figure 5-11. Immunoblotting analysis of STAXs 1 and 2 immunoprecipitated from nuclear
extracts from BEND cells treated with blFN-x for Increasing Intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposureof abundance of STATs
1 and 2 (*, positive control); b) Least squares means and SE of abundance of STATs 1 and 2
(within each variable, bars with distinct subscripts are statistically different, p<0.1).


117
Statistical Analysis
Variables analyzed by densitometry also were analyzed by least squares
analysis of variance, using the GLM procedure of SAS (SAS, 1988). In
Experiment 1, independent variables were cow, gel, treatment, cow by gel, cow
by treatment and gel by treatment. In Experiment 2, independent variables were
gel, treatment and gel by treatment.
Results
Experiment 1
Synthesis of secretory proteins from endometrial explants. Visual
inspection of fluorographs suggested presence of two proteins of 19 (P19) and
12 kD (P12) that were regulated by blFN-x (Figure 4-1, panel a). Treatment with
blFN-x enhanced synthesis and secretion of both proteins qualitatively, but
densitometric analysis indicated only a numeric difference in the intensity of
bands between treatments, both for P19 (p<0.26) and for P12 (p<0.16; Figure 4-
1, panTb). Although the effect of cow was not significant for either P19 or P12
(p>0.1), cow 5497 had a numerically reduced response to treatment with blFN-x
for both proteins. Data were re-analyzed without cow 5497 and statistical
significance for treatment changed slightly (p<0.24) for P19 but significantly
(p<0.05) for P12.


108
(500 mg) were further cut into five or six pieces and cultured for either 2 or 24
hours.
24 hour cultures. To study both intracellular and secretory proteins
stimulated by bIFN-x, endometrial explants were assigned randomly to two
treatments (0 or 50 ng/mL bIFN-x, diluted in medium to a volume of 100 pi which
was added to wells) in duplicate and cultured for 24 hours with leu-deficient MEM
supplemented with 3H-leu (500 mg tissue/5 ml medium/100 mCi 3H-leu) at 37C
in a controlled atmosphere of N2:02:C02 (50%:47.5%:2.5% by volume). Dose of
bIFN-x used was slightly higher than estimates of the Kd of the ovine IFN-x
receptor for binding of ovine IFN-x (0.2 -11.6 ng/ml; see Danet-Desnoyers et al.,
1994). After 24 hours, explants were separated from medium. Explants were
homogenized to provide samples of endometrial intracellular proteins; culture
medium was dialyzed to provide samples of secreted proteins.
Endometrial explants were washed in culture medium, blotted dry and
stored at -70 C until homogenization. Frozen explants were placed in a 10 ml
glass beaker, which was inserted halfway into a plastic 100 mL beaker
containing ground ice. Explants were homogenized in 2 ml of ice-cold
homogenization buffer (9.3M urea, 50 mM Tris-HCI (pH 7.4), 150 mM NaCI, 2
mM EGTA, 2 mM EDTA, 50 mM NaF, 20 mM p-glyceryl phosphate, 2 mM
Na3V04, benzamidine [5 mg/mL], 1 mM PMSF, 10% v/v glycerol and 1% v/v NP-
40) using a Polytron tissue homogenizer (Brinkmann, Luzern, Switzerland),


115.
were exteriorized through flank incisions and catheterized. Catheters were
exteriorized through an orifice in the left flank, filled with 20 mM Tris-HCI, pH 8.0
containing 1 mM EDTA (750 pi) mixed with antibiotic (Nacxel, 450 pi of a 50
mg/ml solution) and, placed into bags with iodine soaked gauze. After surgery,
animals were returned to their pen.
Infusions. Either BSA (1.9 mg in 1.2 ml 20 mM Tris-HCI, 1 mM EDTA, pH
8.0) or IFN-t (0.2 mg +1.7 mg BSA in 1.2 ml 20 mM Tris-HCI, 1 mM EDTA, pH
8.0) were infused every 12 hours from experimental days 14 to 17.
Slaughter and Tissue Collection. On the morning of experimental day 17,
after the blFN-x infusion, animals were slaughtered by jugular exanguination and
reproductive tracts were immediately removed and transported to the laboratory.
Uterine lumen from the horn contralateral to CL was exposed and endometrial
tissue was dissected as described for Experiment 1.
Cell extract. A technique described for ovine endometrium (Charpigny et
al., 1998) was used to obtain endometrial epithelial cells for whole cell extracts.
Briefly, endometrial lumen was irrigated with Hanks balanced salt solution and
gently scraped with a scalpel blade. Cells were placed in a 15 ml conical tube
containing Earles medium supplemented as described above. After whole
lumen had been scraped, cell suspension was centrifuged for 5 minutes (in a
Dynac non-refrigerated bench centrifuge, Parsippany, NJ) at 500 x g,
resuspended in 5 ml of Earles medium and examined for viability using the
trypan blue exclusion method. The cell suspension was centrifuged again,


CHAPTER 7
INTERFERON-TAU MODULATES PHORBOL ESTER-INDUCED SECRETION
OF PROSTAGLANDIN AND PROTEIN EXPRESSION OF PHOSPHOLIPASE-
A2 AND CYCLOOXYGENASE-2 FROM BOVINE ENDOMETRIAL (BEND)
CELLS
Introduction
Both In vivo (Meyer et al., 1995) and in vitro (Helmer et al., 1989; Danet-
Desnoyers, 1994; Xiao et al., 1999) experiments demonstrated that bIFN-x is
able to attenuate endometrial secretion of PGF2(t. A variety of experimental
models have been used to demonstrate this effect of bIFN-x, including explants
(Helmer et al., 1989), primary cultures of endometrial epithelial cells collected on
days 1 to 4 (Xiao et al., 1999) or 15 of the estrous cycle (Danet-Desnoyers,
1994). The aforementioned approaches involve synchronization and slaughter
of animals at specific stages of the estrous cycle or obtaining tissue from the
slaughterhouse, in order to collect cells to conduct experiments. These
approaches are both time and resource consuming. Moreover, secretion of
PGF2a Is highly variable among animals, which make interpretation of results
challenging. An alternative experimental model for studying effects of bIFN-x in
the endometrium is use of BEND cells. BEND cells are a line of spontaneous
replicating endometrial epithelial cells originating from day 14 cyclic cows (Austin
208


180
(Meyer et al., 1995) and others have used doses of 100 ng/ml (Xiao et al., 1999)
and up to 20 pg/ml (Asselin et al., 1998) of blFN-t for in vitro studies utilizing
endometrial epithelial cells. Our data show that bIFN-t is able to stimulate
tyrosine phosphorylation of STAT-1, but not STAT-2 in a dose dependent
manner (Figure 5-7). Perhaps if doses between 0 and 3.125 ng/ml of bIFN-t
had been used, a more gradual, dose dependent tyrosine phosphorylation of
STAT-2 also would have been observed. Therefore we concluded that 50 ng/ml
was adequate for the subsequent studies conducted. This dose effectively
suppressed basal and oxytocin-induced PGF2a secretion in primary culture of
endometrial epithelial cells (Danet-Desnoyers et al., 1994).
As in typical signal transduction paradigms mediated through tyrosine
phosphorylation, blFN-x stimulates transient phosphorylation of STAT proteins in
WCE (Sillvennoinen et al., 1993; Figures 5-8 and 5-9). The dynamics of
phosphorylation indicate the presence of a pool of STAT proteins readily
available for phosphorylation. However, after a period of maximum
phosphorylation (between 8 and 15 minutes) STATs become refractory to further
blFN-x-induced tyrosine phosphorylation, which declines. Decreased
phosphorylation of STATs after exposure to blFN-x longer that 15 minutes may
reflect degradation of STATs (Kim and Maniatis, 1996) or dephosphorylation of
STATs through the actions of phosphatases (David et al., 1993; Haque et al.,
1995). Since abundance of STATs is unchanged over time, the later hypothesis


149
a
b
Figure 5-3. Morphology of BEND cells at different stages of growth.
a) Passage 5; b) Passage 27. Note majority of cells in a" have round, compact
morphology, typical of epithelial cells, but elongated cells with stromal morphology (top center-
right) is also present in "a". Note much lower cell density and the spread-out" phenotype of
most cells in b". Note at least one cell with same morphology in a", on the center-right.
Magnification: 100X.
round and flattened cells are also present in less number. There Is a noticeable
change in cell morphology when cells of a more advanced passage are
examined (passage 27; Figure 5-3 panel b). Cells on P27 stopped growing and
were not used further for experiments. Strong immunoreactivity for cytokeratin
was noted regardless of cell passage number (Figure 5-4 panels a and b).
Staining for vimentin was always less intense than for cytokeratin (Figure 5-4
panels c and d). Staining was not observed in extracts from cells of P2, but all
other passages revealed presence of vimentin. No increase in vimentin intensity
was observed in later passages compared to earlier passages.


60
to degrade activated complexes. There is evidence for occurrence of both
mechanisms of regulation in the JAK-STAT pathway (i.e., phosphatases and
proteases), but more recent data indicate presence of novel regulatory molecules
also playing a role. Callus and Mathey-Prevot (1998) showed that treatment of
Ba/F3 cells with a specific proteasome inhibitor led to stable tyrosine
phosphorylation of the interleukin-3 (IL-3) receptor and STAT-5, after stimulation .
with IL-3. Further investigation revealed that stable phosphorylation events were
due to prolonged activation of JAKs. Moreover, Kim and Maniatis (1996)
demonstrated that after activation with interferon-y, STATs became ubiquitinated
and quickly degraded. In contrast with data from Kim and Maniatis (1996), but in
agreement with data from Callus and Mathey-Prevot (1998), Haspel and others
(1996) reported that proteasome inhibitors increased time of activation of STAT-1
by prolonging signals from the receptor (i.e., preventing degradation of receptor-
JAKs complexes), but not by blocking removal of phosphorylated STATs. This
was based on the finding that 35S-labeled STAT-1 translocated to the nucleus
upon tyrosine phosphorylation and later returned to the cytoplasm in non-
phosphorylated configuration. Data from Strehlow and Schindler (1998) agrees
and expands these findings, in that chimeric STATs with mutated amino-terminal
domains exhibited defects in nuclear translocation and deactivation, indicating
that these two events might be linked (i.e., deactivation may be dependent on
previous nuclear localization). Indeed, David and others (1993) demonstrated
that a nuclear tyrosine-phosphatase is responsible for deactivation of


a
170
O
T
X
121
78
r:rnrnn¡:r
-STAT-2
-STAT-1
b
*>
121
* 7g__> -p-STAT-3
C
121
78-
-STAT-3
0 1 3 8 15 30 60 120 min.
blFN-x, 50 ng/ml
d
coip-STAT-1 coip-STAT-2
bIFN-r, 50 ng/ml


55.
dimers which association is mediated by a lower-affinity phosphotyrosil
interaction (Greenlund et al., 1995). In light of this question, Gupta and
coauthors (1996) proposed an alternative model for STAT binding and dimer
formation. After binding to the receptor phosphotyrosine motif, the STAT shifts
its target to the tyrosine motif in the tyrosine kinase. Tyrosine phosphorylation of
STAT would cause a conformational change to destabilize this interaction with
the kinase, and STATs would then be driven to form more energy-stable
interactions with other STATs and form dimers. They based this model on the
finding that SH2 domains from STATs 1 and 2 bind with high affinity to
phosphotyrosine motifs on JAK kinases.
STAT dimers are competent to bind DNA. Known DNA binding
heterodimers are STAT 1:2 and STAT 1:3 (strong binding) and STAT 2:3 (weak
binding). Homodimers are STAT 1:1 and STAT 3:3 (strong) and STAT 2:2 (form
seldom in absence of STAT-1; Darnell, 1997). In variance with the notion that
tyrosine phosphorylation is required for STAT dimerization, Stancato and
coworkers (1996) demonstrated that STAT complexes exist in the cytosol of
unstimulated cells. Moreover, such association was independent of tyrosine
phosphorylation, since the Y701F STAT-1 mutant still bound to STAT-2 In
reticulocyte lysates. Such an interaction was weak, since it was not observed in
extracts obtained with high-salt, detergent-containing buffers.
Current models for the mechanism of STAT activation of gene transcription
propose that following dimerization, STAT complexes translocate to the nucleus.


196
antibody (1:8000) for 1 hour, washed and proteins detected using an enhanced
chemiluminescence kit, with exposures of 3 minutes. Immunoblots for IRF-1
were analyzed by densitometry as described in Chapter 5.
Statistical Analysis
Abundance of IRF-1 also was analyzed by least squares analysis of
variance using the GLM procedure of SAS (SAS, 1988). Independent variables
were gel, treatment, time, gel by treatment, gel by time and treatment by time.
Since there was only a negligible signal for IRF-1 in the absence of blFN-x, data
were re-analyzed for the effect of time on samples treated with blFN-x only.
Results
Electrophoretic Mobility Shift Assays (EMSA1
Experiment 1 indicated that incubation with NE from cells treated with
blFN-x produced slower migrating bands (shifted complexes;s1) compared to
incubation with NE from untreated cells (Figure 6-1). Reactions performed at 37
C produced stronger bands than room temperature regardless of amount of NE
protein used. Intensity of shifted complexes was reduced greatly when a 10-fold
excess of non-radioactively labeled probe was added to the incubation reactions.
Experiment 2 revealed that no specific complexes were formed in absence of
nuclear extracts (lane 1; Figure 6-2) or when nuclear extracts obtained from


231
121
78
- -cPLA-2
1'S-ra- L.
1 2 3 4 5 6 7 8
Figure 7-10. Immunoblotting analysis of PLA-2 in whole cell extracts from BEND cells
treated with medium : one (control), phorbol 12,13 dibutyrate (100ng/ml; PDBu) or blFN-t
(50 ng/ml; bIFN-tau nd PDBu for 6 hours.
a) Representative enhanced chemiluminescence (ECL) exposure of abundance of
PLA-2 (control: lanes 1 and 2; PDBu: lanes 3, 4 and 5; bIFN-T and PDBu: lanes 6, 7 and 8);
b) Least squares me-ns and SE of abundance of PLA-2 arbitrary densitometric units
(ADU).


57
transfection to STAT-deficient U3a cells, this "conditionally active STAT"
underwent dimerization following estrogen/tamoxifen treatment. Moreover, these
chimeras were able to undergo nuclear translocation and activated transcription
of interferon-induced genes such as IRF-1. The authors concluded that tyrosine
phosphorylation of STAT is probably only a trigger for dimerization, since
dimerized, non-phosphorylated STAT chimeras also were able to stimulate
interferon-specific gene activation. Furthermore, since the estrogen receptor
domain used in the chimera did not contain any NLS, dimerization alone was
sufficient to promote nuclear translocation, sequence-specific DNA binding and
transcription activation functions of the chimeric STATs. A study conducted by
Strehlow and Schindler (1998) indicated that the amino-terminal 100 amino acids
of particular STATs mediated their nuclear translocation activity. Chimeric
constructs in which those amino acids in STAT-1 were substituted by those of
STAT-2 abolished nuclear translocation of STAT-1, while other functions were
maintained, such as activation by receptor, dimerization and DNA binding.
Collectively, it is fair to say that the mechanism of STAT nuclear translocation
remains unclear. Although the work of Johnson et al. (1998b) puts forth an
exciting proposition for such a mechanism, data from Milocco and others (1999)
argues against the requirement of a ligand-receptor-assisted transport
mechanism. However, existence of both mechanisms is feasible in vivo.


227
Cell morphology. The PDBu both alone and in combination with blFN-x
Induced clear changes In cell morphology overtime (Figure 7-7). Cells became
clustered and were connected by arm-llke projections that were evident by 3
hours. In contrast, cell morphology did not change In controls. Despite such
changes, total protein content was similar among treatments (835.4, 936.2 and
787.652.3 for control, PDBu and PDBu + blFN-x respectively).
Experiment 3
Secretion of PGF-, This experiment characterized the short term rise in .
PGF2(i secretion in response to PDBu stimulation, in presence and absence of
blFN-x (Figure 7-8). Noticeable stimulation by PDBu was detected after 2 hours
of exposure. There was a steady and faster rate increase in PDBu-induced
secretion of PGF2(1 throughout the experiment compared to PDBu + blFN-x.
Significant treatment by time contrasts (d x c7, c1 vs c7, c1 x c8, d x c9 and d
x c10; p<.01) indicates that basal accumulation of PGF2awas negligible
throughout the experiment. In contrast, other treatment by time contrasts (c2 x
c9, c2 x c10, c2 x c11) were not significant (p>.1), indicating that trends in
increase of PDBu-stimulated PGF2o, were similar in presence or absence of blFN-
x. Analysis of homogeneity of regression indicated that PGF2a secretion could be
represented by fourth order curves (R2: .982), which confirmed changes in
secretion rates during the experiment. Equations for each treatment were: Y
2.51+7.66 x X-4.2 x X2+0.91 x X3-0.07 x X" (control); Y=23.59-43.3 x X+27.63 x


181
better explains the data. This agrees with data from Haspel and coworkers
(1996), where they followed 35S-labeled STAT-1 throughout an IFN-y treatment
cycle of 4 hours. Only about 10% of STATs were degraded, although 20 to 30%
of STATs were In the nucleus by 20 minutes of treatment. Recent reports have
identified tyrosine phosphatases from the families of SOCS and PIAS molecules
as negative regulators of the JAK-STAT pathway (Chung et al 1997; Sung and
Shuai, 1998; Starr and Hilton, 1999).
Our preliminary experiments with endometrial explants indicate that
STATs 1 and 2 (Chapter 4) are present and become phosphorylated In response
to blFN-x treatment. This indicates that STAT phosphorylation Is not an artifact
from BEND cells, but a real mechanism occurring In the endometrium.
Among the STATs analyzed, there seemed to be a lower abundance of
STAT-2 compared to STAT-1 and -3. This could indicate that STAT-2 is present
in lower amounts In cells, or, could be due to an artifact in the methodology used
for detection. For example, there could be a lesser affinity of antl-STAT-2
antibody for STAT-2 than the affinity of anti-STATs-1 and -3 antibodies for their
respective antigens. There is little evidence for less affinity on immunoblots,
since phosphorylation of tyrosine is also less for STAT-2 compared to other
STATs. There could be, however, a reduced affinity during Immunoprecipitatlon,
which would in turn result in less detectable STAT-2 in immunoblots.
Binding of type-l IFNs to a membrane receptor causes sequential tyrosine
phosphorylation of associated JAK kinases and the cytoplasmic tail of the


241
Effects of bIFN-T to inhibit synthesis of PGF2Jnvolving PLA2 and COX-2
can be explained in terms of attenuation in both gene expression and enzymatic
activity (Figure 7-12). Bovine IFN-x initially inhibited PDBu-induced COX-2
synthesis (Experiment 3) but lost this ability after 24 hours (Experiment 1; Figure
7-2). However, the continued suppression of PGF2asecretion (Figure 7-1)
suggests that blFN-x must have altered COX-2 enzymatic activity. Two possible
mechanisms leading to that effect are activation of blFN-t-induced COX-2
intracellular inhibitors and a decrease in the availability of the PGF2 synthesis
substrate, arachidonic acid. Regarding the first alternative, Thatcher and
coauthors (1994) reported the identification of an endometrial prostaglandin
synthesis inhibitor, which acts as a competitive inhibitor of COX-2 activity, as
measured by conversion of radiolabeled arachidonic acid into PGF2ain a
microsome assay system. This inhibitor has been identified as linoleic acid.
Therefore, one could speculate that blFN-x increases availability of linoleic acid
in BEND cells to inhibit COX-2 activity. Availability of arachidonic acid for PGF2ol
synthesis is dependent on PLA2 activity. PLA2 specifically cleaves arachidonic
acid from second position of membrane phospholipids. Bovine IFN-t
continuously suppressed PDBu-stimulated synthesis of PLA2 in the present
study. Although we have no direct measure of PLA2 enzymatic activity, it is
possible that blFN-x also suppressed activity of this enzyme as a means to
inhibit PGF2asecretion. The common pathway precursor for regulation of both


265
Asselin E, Drolet P, Fortier MA. In vitro response to oxytocin and interferon-tau
in bovine endometrial cells from caruncular and inter-caruncular areas.
Biol Reprod 1998; 59:241-247.
Asselin E, Fortier MA. Molecular cloning of a bovine endometrial 20a-
hydroxysteroid dehydrogenase (20a-FISD): regulation by blFN-x. Biol
Reprod 1998; 58 (suppl 1):86 (abstr47).
Asselin E, Lacroix D, Fortier MA. IFN-x increases PGE2 production and COX-2
gene expression in the bovine endometrium in vitro. Mol Cell Endocrinol
1997; 56:402-408.
Austin CR. Observations on the penetration of the sperm into the mammalian
egg. Aust J Sci Res B 1951; 4:581-596.
Austin KA, Pru JK, Hansen TR. Complementary deoxyribonucleic acid
sequence encoding bovine ubiquitin cross-reactive protein. Endocrine
1996a; 5:191-197.
Austin KA, Ward SK, Teixeira MG, Dean VC, Moore DW, Hansen TR. Ubiquitin
cross-reactive protein is released by the bovine uterus in response to
interferon during early pregnancy. Biol Reprod 1996b; 54:600-606.
Ayalon N. A review of embryonic mortality in cattle. J Reprod Frtil 1978;
54:483-493.
Badinga L, Driancourt MA, Savio JD, Wolfenson D, Drost M, de la Sota RL,
Thatcher WW. Endocrine and ovarian responses associated with the first-
wave dominant follicle in cattle. Biol Reprod 1992; 47:871-883.
Barros CM, Newton GR, Thatcher WW, Drost M, Plante C, Hansen PJ. The
effect of bovine interferon-a,1 on pregnancy rate in heifers. J Anim Sci
1992; 70:1471-1477.
Bartol FF. Uterus, non-human. In: Knobil E, Neill JD (eds.), Encyclopedia of
Reproduction, vol. 4. San Diego: Academic Press; 1999:950-960.
Bartol FF, Roberts RM, Bazer FW, Lewis GS, Godkin JD, Thatcher WW.
Characterization of proteins produced in-vitro by peri-attachment bovine
conceptuses. Biol Reprod 1985; 32:681-694.


114
membranes were incubated in a 5% (w/v) solution of non-fat dried milk in TBS
for2 hours in 10 ml of a 1:1000 dilution each of anti-STAT-1 and anti-STAT-2
polyclonal antibodies, quickly rinsed twice, washed once for 15 minutes and 2
times for 5 minutes in TBST, incubated for 1h in a 1:8000 dilution (10ml total) of
horse radish peroxidase (HRP)-linked anti-rabbit IgG antibody (part of the ECL
kit) in TBST containing 5% (w/v) non-fat dried milk, quickly rinsed twice, washed
once for 15 minutes and four times for 5 minutes with TBST. STAT proteins on
membranes were detected with an ECL kit with exposures of 30 seconds.
Densitometry, Enhanced chemiluminescence exposures were analyzed
by densitometry as described in Chapter 3.
Experiment 2
Details of the methodology used for estrous cycles synchronization,
surgeries and infusions are described in Arnold et al. (1999).
Estrous cycle synchronization. Non-lactating dairy cows housed at the
Dairy Research Unit (Hague, FL) received an injection of 25 mg of PGF2a
(Lutalyse). Estrus were detected as described for experiment 1, and was
designated experimental day 0.
Surgery. On experimental day 10, cows were transported to a grassy pen
at the College of Veterinary Medicine (University of Florida, Gainesville). On day
11, cows were examined by rectal palpation to confirm presence of healthy
corpora ltea (CL). Briefly, after epidural and local anesthesia, uterine horns


269
Colamonici OR, Uyttendaele H, Domanski P, Yan H, Krolewski JJ. PISS'*112, an
interferon-a-activated tyrosine kinase, is physically associated with an
interferon-a receptor. J Biol Chem 1994a; 269:3518-3522.
Colamonici OR, Yan H, Domanski P, Handa R, Smalley D, Mullersman J, Witte
M, Krishnan K, Krolewski J. Direct binding to and tyrosine
phosphorylation of the a subunit of the type I receptor by p135lyk2 tyrosine
kinase. Mol Cell Biol 1994b; 14:8133-8142.
Cooperative Regional Research Project, NE-161. Relationship of fertility to
patterns of ovarian follicular development and associated hormonal
profiles in dairy cows and heifers. J Anim Sci 1996; 74:1943-1952.
Danet-Desnoyers G, Johnson JW, OKeefe SF, Thatcher WW. Characterization
of a bovine endometrial prostaglandin synthesis inhibitor (EPSl). Biol
Reprod 1993; 48 (suppl 1):115 (abstr).
Danet-Desnoyers G, Wetzels C., Thatcher WW. Natural and recombinant
bovine interferon x regulate basal and oxytocin-induced secretion of
prostaglandins F2o and E2 by epithelial cells and stromal cells in the
endometrium. Reprod Frtil Dev 1994; 6:193-202.
Darnell jrJE. STATs and gene regulation. Science 1997; 277:1630-1635.
Darnell jr JE, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional
activation in response to IFNs and other extracellular signaling proteins.
Science 1994; 264:1415-1421.
David M, Grimley PM, Finbloom DS and Larner AC. A nuclear tyrosine
phosphatase downregulates interferon-induced gene expression. Mol
Cell Biol 1993; 13:7515-7521.
David M, Wong L, Flavell R, Thompson SA, Wells A, Larner AC, Johnson GR.
STAT activation by epidermal growth factor (EGF) and amphiregulin.
Requirement for the EGF receptor kinase but not phosphorylation sites or
JAK1. J Biol Chem 1996; 271:9185-9188.
de Moraes AS, Hansen PJ. Granulocyte-macrophage colony-stimulating factor
promotes development of in vitro produced bovine embryos. Biol Reprod
1997; 57:1060-1065.


77
turn over, but ovulates in a low progesterone (P4) environment. Turnover of the
dominant follicle (DF) Is associated with high concentrations of P4, typical of mid
cycle, which lowers LH pulse frequency (Kinder et al., 1996). Turnover of the
first wave DF can be blocked by exogenous progestins and Injection of PGF2a
(Cooperative Regional Research Project, NE-161, 1996; Savioetal., 1993a;
Savlo et al., 1993b). The resulting sub-luteal concentration of progestin in
plasma permits an Increase In LH pulse frequency which sustains growth of the
DF. This persistent DF (PDF) Is estrogenic, and subsequent fertility, as
measured by conception rate at first service (number of pregnancies / number of
animals inseminated), is lower compared to animals bearing normal DFs [37.1%
vs. 64.8% In heifers, (Savio et al., 1993b); 23.6% vs. 58.2% for cows and heifers,
(Cooperative Regional Research Project, NE-161,1996). Fertility after Al,
however, Is restored to levels comparable to controls If the PDF is turned over
and a freshly recruited follicle Is allowed to ovulate. Possible explanations for
reduced fertility Include alterations in the oocyte and /or in the ovlductal
environment. In a study by Ahmad et al. (1995), cows ovulating a PDF had
embryos that at day 6 of pregnancy were less developed (I.e., were less able to
reach the 16-cell stage) than embryos from cows ovulating a fresh (F) DF. In
addition, Revah and Butler (1996) showed that oocytes recovered from the PDF
showed expanded cumulus cells and condensed chromatin dispersed In their
ooplasm. In contrast, compact cumulus cells and intact germinal vesicles were
found in oocytes from FDF. Thus, the PDF may affect oocyte maturation,


145
Time Response to bIFN-T
Since tyrosine phosphorylation of proteins is an acutely regulated event,
this experiment was designed to study the phosphorylation response of STATs
to blFN-x overtime. Seven plates of BEND cells were assigned randomly to
receive 50 ng/ml bIFN-T for 3, 8, 15, 30, 60 or 120 minutes, or to receive nothing
("0 minutesVcontrol treatment). Whole cell extracts were immunoprecipitated
with anti-STAT-1 and -2 antibodies and analyzed by immunoblotting for anti-
phosphotyrosine and anti-STAT-1 and -2. The same experiment was repeated
but WCE were immunoprecipitated with anti-STAT-3 antibody and analyzed by
immunoblotting for anti-phosphotyrosine and anti-STAT-3.
Validation of Immunoprecipitation and Immunoblots Procedures
To verify the specificity of antibodies used for immunoprecipitation, WCE
from untreated cells were immunoprecipitated with no antibody, normal rabbit
serum, anti-STAT-1, -2 or -3 antibodies or a combination of anti-STAT-1 and -2
(1 pg each) and analyzed by immunoblotting for STATs 1, 2 or 3 as appropriate.
Specificity of anti-phosphotyrosine antibody used for immunoblots was
tested in WCE of cells that had been treated with blFN-x (50 ng/ml for 8
minutes). Whole cell extracts were immunoprecipitated with anti-STAT-1 and -2
antibodies or anti-STAT-3 antibody (1 pig each) and analyzed by immunoblotting
with no antibody, normal rabbit serum or anti-phosphotyrosine antibody.


91
20


63
residues, both constitutively and in response to ligands (see Leaman et al., 1996
for review). Such phosphorylation events are important, since treatment of cells
with kinase inhibitors disrupts STAT-3:3 DNA complexes. A mitogen-activated
protein kinase (MAPK) may be involved in phosphorylation of serine residues of
STAT-1, because the serine 727 lies in a consensus sequence for MAPK
phosphorylation. In fact, Stancato and coworkers (1997) proposed a model in
which activation of MAPK was dependent on activated JAK kinases. Binding of
interferon-a/p induced tyrosine phosphorylation of JAK-1, which stimulated
activity of membrane bound Raf-1. Activated Raf-1 phosphorylates MEK and
activates MAPK. MAPK in turn phosphorylates serine residues on STAT-1,
contributing to modulation of activity for this signal transducer. However,
modulation of STAT activities by MAPK may be stimulatory or inhibitory. For
example, Chung and others (1997b) reported serine phosphorylation of STAT-3
by growth factors, while STAT-1 was poor substrate for several MAPK tested.
Interestingly, serine phosphorylation of STAT-3 negatively modulated tyrosine
phosphorylation of this protein, and consequently inhibited dimerization, nuclear
translocation and gene activation.
Signal transducers such as IRS-1 and IRS-2 that are activated in response
to insulin, IL-2, IL-4 etc, are tyrosine phosphorylated by JAK-1. Epidermal growth
factor(EGF) is able to activate tyrosine phosphorylation of STATs 1 and 3 (David
etal., 1996). Interestingly, this does not require presence of JAKs. Moreover, '
truncated receptor constructs containing the intrinsic kinase activity but lacking


-P-STAT-2
-P-STAT-1
X
78
b
i I f T STAT-2
0 3 8 15 30 60 120 min.
bIFN-x, 50 ng/ml
Figure 5-8. Immunoblotting analysis of STATs 1 and 2 immunoprecipitated from whole cell
extracts from BEND cells treated with bIFN-x for Increasing Intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine
phosphorylation of STATs 1 and 2; b) Representative ECL exposure of abundance of STATs 1 and
2.
minute exposure to bIFN-x (Figure 5-13). Phosphorylation decreased by 60
minutes.
STAT-2. In the cytosol there was a numerical (statistically non-significant)
decrease in the abundance of STAT-2 that reached a minimum level at 30
minutes but returned to control levels by 120 minutes (Figure 5-10). Abundance
of STAT-2 in NE started to increase after only 1 minute exposure to bIFN-x and
reached a maximum at 15 minutes, remaining elevated up to 30 minutes and
then slowly decreased (Figure 5-11). Phosphorylation of STAT-2 in the cytosol
increased after 3 minutes, remained elevated between 3 and 15 minutes and
gradually decreased to levels lower than control levels in all other time points
(Figure 5-12). Due to lack of sensitivity, phosphorylation of STAT-2 in the
121-
s 78-


236
not on stimulation of activity of pre-existing enzymes involved in PGF2c,
production. This agrees with data from Nam and others (1996), which reported
that a protein synthesis inhibitor, cyclohexamide, blocked the ability of PDBu to
stimulate PGF2ri production in astroglial cells. The PDBu had a acute effect on
morphology of BEND cells. Flowever, this did not seem to be associated with
increased cell mortality, at least based on total protein measurements, which
were similar among treatments at the end of cultures.
When added alone to BEND cells, bIFN-x had negligible effect on PGF2o,
secretion (Experiment 1). Therefore, this treatment was excluded from
subsequent experiments. In contrast, blFN-x effectively suppressed the PDBu-
induced release of PGF2ain all experiments. However, blFN-t was never able to
completely suppress PDBu-stimulated PGF2cl. This could indicate that other
conceptus molecules are required to abolish PGF2cl completely in vivo. These
findings confirm and expand those of Xiao and coworkers (1999). Those authors
reported a 4-fold, PMA-induced (2.9 ng/ gg DNA) stimulation of PGF2asecretion
compared to controls (0.75 ng/ gg DNA) in a 12-hour experiment. In combination
with PMA, blFN-x attenuated the PGF2aincrease (2 ng/ gg DNA), but only to
about 2.7 fold of control values. PGF2 attenuation in the present study was
much more dramatic (presence of blFN-x reduced PDBu-stimulated PGF2ato one
third; Experiment 2, 12 hours).


94
interactions were significant for P7 and P14 (p<0.02), and tended to be
significant for P1 (p<0.07). As suggested by significant treatment by region
interactions, protein synthesis and secretion in response to treatments varied
according to region. In the AMP, P7 was stimulated by PDF whereas P14 was
stimulated in FDF cows. Protein 14 was present in the INF and absent in the 1ST
regardless of treatment, but ampullary P14 was abolished by PDF. In the 1ST,
synthesis of P1 was stimulated by PDF. Proteins with significant side by
treatment interactions were P6 (p<0.05), P9 (p<0.05), P11 (p<0.03), while P5
(p<0.06), P8 (p<0.07), and P19 (p<0.09) only approached significance.
Treatment by side interactions indicate a differential response of IPSI and
CONTRA sides to FDF compared to PDF. For FDF cows, abundance of P5,
P6,P8, P9 and P11 was reduced in the IPSI compared with the CONTRA side.
In contrast, abundance of these same proteins was similar across sides for PDF
cows. Protein 19 was secreted in similar amounts at the IPSI side for both
treatments. In the CONTRA side however, PDF maintained, while FDF reduced
abundance of P19 compared to the IPSI side. Treatment by side by region
interaction was significant for P19 (p<0.01) and tended to be significant for P2
(p<0.1) and P13 (p<0.06).
A summary of mean comparisons of effects of treatment, side, treatment
by region and treatment by side for individual spots is presented in Table 3.


242
Endometrial Epithelial Cell
Figure 7-12. Hypothetical model for actions of phorbol 12,13 dibutyrate (PDBu) and bovine
Interferon-T (blFN-t) In BEND cells. Phospholipase K¡ (PLA2) cleaves arachldonic add (AA)
from phospholipids In the cell membrane. Cyclooxygenase-2 (COX-2) converts AA Into PGF2a.
The PDBu stimulates protein kinase C (PKC) enzymatic activity, which induced synthesis of
both PLA2 and COX-2 proteins and synthesis of PGF^was stimulated as a result. Presence of
blFN-t inhibited protein expression of PLA2 and COX-2, and COX-2 enzymatic activity to
suppress synthesis of PGF2i in BEND cells.
PLA2 and COX-2 is PKC. Phorbol esters mimic the action of diacylglycerol, a
product of phospholipase-C activity, which has the effect of activating PKC
activity. The PKC may stimulate PGF2ol secretion via stimulation synthesis and/or
activity of both PLA2 (Mayer and Marshall, 1993; Karimi and Lennartz, 1995) and
COX-2 (DeWitt, 1991; Vezza et al., 1996). Therefore, effects of blFN-t can be at
the level of PKC, to inhibit the ability of this enzyme to stimulate PGF2a synthesis
through modulation of its mediators, PLA2 and COX-2. Since not all PDBu-


Figure 8-1. Hypothetical model of actions of bovine interferon-tau (blFN-x) to prevent protein
kinase C (PKC)-induced production of PGF2ain endometrial epithelial cells. Activation of PKC
stimulates synthesis (green squares) and enzymatic activity (yellow diamonds) of specific
endometrial proteins, such as phospholipase A2 (PLA2)and cyclooxygenase-2 (COX-2) to
ultimately stimulate synthesis of PGF2a. PKC may stimulate synthesis or activity of other cellular
mediators, such as kinases, lipases and transcription factors to further enhance synthesis and
activity of enzymes directly involved in the PGF2a production. Binding of blFN-x to the type I
receptor, here represented with a hypothetical, blFN-t-specific chain (Rt), may have protein
synthesis-dependent (through activation of the JAK-STAT pathway; right side of diagram) or -
independent (left side of diagram) actions to decrease production of PGF2a. STAT dimers may
translocate to the nucleus to stimulate transcription of interferon-inducible genes and to repress
transcription of genes related to synthesis of PGF2 The blFN-t-induced proteins (red circles)
may act as transcription factors to regulate transcription of genes, or may have cytosolic actions
to regulate activity of enzymes involved in production of PGF2. Alternatively, blFN-x may
regulate activity of pre-existing cellular mediators (blue circles) to negatively affect the PGF2a
synthesizing machinery.


7 INTERFERON-TAU MODULATES PHORBOL ESTER-INDUCED
SECRETION OF PROSTAGLANDIN AND PROTEIN EXPRESSION OF
PHOSPHOLIPASE-A2 AND CYCLOOXYGENASE-2 FROM BOVINE
ENDOMETRIAL (BEND) CELLS 208
Introduction 208
Materials and Methods 210
Materials 210
Cell Culture and Sample Collection 211-
Radioimmunoassay 212
Preparation of Extracts 214
Immunoblotting 214
Experimental Designs 215
Statistical Analysis 216
Results 219
Experiment 1 219
Experiment 2 223
Experiment 3 227
Experiment 4 232
Discussion 234
8 GENERAL DISCUSSION 246
LIST OF REFERENCES 264
BIOGRAPHICAL SKETCH 291
x


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43
al. (1982), Bartol et al. (1985), Helmer et al. (1987), Imakawa et al. (1989) and
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2
reproductive tract). Both maternal and embryonic units influence such internal
environment, and physiological crosstalk is a hallmark of the process. As a
result, a complex set of coordinated interactions takes place and it is these
interactions that will dictate a successful reproductive outcome. My thesis is that
failure in maternal-embryonic communications leads to decreased reproductive
rates.
This dissertation examines the role of maternal-embryonic
communications during two physiological windows of the reproductive cycle and
their importance on the overall reproductive process. Chapter 3 describes
steroid hormone-modulation of protein synthesis and secretion in the oviduct,
where final stages of gamete maturation, fertilization and early embryo
development occur. Chapters 4, 5, 6 and 7 characterize changes in intracellular
and secretory processes of the maternal endometrium in response to a
conceptus-secreted factor, interferon-x, that is essential for maintenance of
pregnancy.
A greater understanding of the basic mechanisms regulating reproductive
processes, specially mechanisms involving maternal-embryonic interactions, is
necessary to improve conception rates in cattle.


142
Immunoorecipitation
Immunoprecipitations were conducted as described in Chapter 4, except
when noted. Briefly, protein-A agarose beads were prepared as described in
Chapter 4 and incubated with anti- STAT-1, -STAT-2 and / or -STAT-3 antibodies
(1 pg each/sample) overnight. Beads were then washed thrice in WCE or
hypotonic buffer, as appropriate, resuspended in the same buffer and added (50
pi of slurry/sample) to WCE, CE or NE as appropriate. Samples were
immunoprecipitated overnight at 4 C, washed thrice in 1 ml of the appropriate
buffer and once with 62.5 mM Tris-HCI pH 6.8. Proteins were solubilized by
Incubating beads for 5 minutes at 100 C with reducing Laemmli buffer. Samples
were pulse-spun in microcentrifuge and supernatants used for immunoblots.
Immunoblots
Immunoblotting was conducted as described in Chapter 4. Briefly,
immunoprecipitated proteins were separated in 7.5% acrylamide gels by 1-
dimensional SDS-PAGE (150 V, 100 mA for 50 minutes). Gels were incubated
in transfer buffer and proteins were transferred to nitrocellulose membranes
using a mini-gel transfer tank apparatus (350 V, 100 mA for 60 minutes, Bio-Rad
apparatus or maximum voltage, 400 mA for 4 hours, Hoffer apparatus). After
transfer, membranes were blocked for 2 hours in 2% (w/v) gelatin in TBST
(phosphotyrosine immunoblots) or 5% (w/v) non-fat dried milk (STAT


49
stimulate transcription of genes and synthesis of interferon-specific proteins. I
will next examine characteristics of molecules involved in this pathway, and then
describe evidence for existence of this pathway in the bovine endometrium.
Type I interferon receptors
Type I interferon receptor consists of two chains, IFNaRI and !FNaR2,
which can be presented in different forms. The IFNaRI is present as a full chain
(IFNaRI a) and as a shorter splice variant (IFNaRI s). The IFNaR2 chain exists
in soluble, short and long forms, designated IFNaR2a, IFNaR2b and IFNaR2c
respectively. Probably IFNaRla and IFNaR2c are the predominant forms
(Petska, 1997). Petska (1997) reviewed a series of experiments where the
different IFNaR chains were expressed in Chinese hamster ovary cells, and
ability of different type I interferons to signal through the different chain
combinations was evaluated. There is a remarkable diversity of such
interactions, in which specific interferons can only signal through specific
combinations of chains, but not others. Petska (1997) proposes that differential
expres.sion of individual chains and ability of individual interferons to signal
through specific chain arrangements confers tissue-specific responsiveness to
interferons. For example, Platanias and coworkers (1996a) reported that IFN-p
signaling requires association of IFNaRI with p100, a tyrosil phosphoprotein,
which was later identified as a particular chain of the interferon receptor complex.
To the best of my knowledge, these types of experiments have not been


209
et al., 1996b). Our goal was to characterize the effect of blFN-x on secretion of
PGF2ofrom BEND cells, and our working hypothesis was that blFN-x should have
an inhibitory effect on stimulated PGF2a synthesis.
Oxytocin stimulates secretion of PGF2<1 from primary endometrial epithelial
cells in culture (Danet-Desnoyers et al., 1994). Oxytocin binds to a seven
transmembrane-domain, G protein-coupled receptors and activates
phospholipase C (PLC). The PLC cleaves membrane phosphotydilinositol
bisphosphate, yielding inositol trisphosphate (IP3) and diacylglycerol (DAG). The
IP3 binds to specific receptors in the endoplasmic reticulum resulting in release of
calcium from internal stores into the cytosolic compartment. The DAG activates
protein kinase C (PKC), leading to serine phosphorylation of cytosolic, calcium-
dependent phospholipase A2 (PLA2), probably through a MAP-kinase dependent
pathway (Lin et al., 1993). The IP3-stimulated increase in cytosolic calcium acts
to further stimulate PLA2 activity (Clark et al., 1991). Stimulated PLA,
translocates to the membrane where phospholipid substrates are located (Clark
et al., 1991). Activated, membrane-bound PLA2 cleaves arachidonic acid (AA)
from phospholipids. Free AA is converted to prostaglandin H2 (PGH2) by the
enzyme cyclooxygenase-2 (COX-2). Prostaglandin F2a synthase converts PGH2
into PGF2o, which is then released into the uterine circulation (Smith et al., 1991).
However, in a preliminary experiment (Binelli, Arnold and Thatcher, unpublished
results) it was determined that one hour treatment with oxytocin failed to
stimulate secretion of PGF2

119
Synthesis of intracellular proteins from endometrial explants. Visual
inspection of fluorographs suggested presence of a 75 kD protein (P75)
regulated by bIFN-x (Figure 4-2, panel a). Treatment with bIFN-x enhanced
synthesis and secretion of P75 qualitatively, but densitometric analysis indicated
only a numeric difference in the intensity of bands between treatments (p<0.25;
Figure 4-2, panel b). There was a significant effect of cow (p<0.05). Cow 2215
had higher synthesis of P75 compared to the other cows.
2 hour cultures. Treatment of endometrial explants with bIFN-x stimulated
tyrosine phosphorylation of both STAT-1 and STAT-2 (Figure 4-3, panel a),
despite a similar abundance of both proteins over time (Figure 4-3, panel b).
Phosphorylation increased after 10 minutes of exposure to bIFN-x and reached a
maximum after 30 minutes. Then, phosphorylation apparently decreased by 60
minutes to increase again after 120 minutes. Changes in phosphorylation
occurred despite a similar abundance of STATs 1 and 2 for all treatmentintervals
(except for a reduced abundance of STAT-2 at the 0 minute sample; Figure 4-3,
panel b) Overall, it was difficult to detect tyrosine phosphorylation in extracts
from explants. There was consistently a high background on ECL exposures
and intensity of bands was always low. Data from only one cow is presented
because it was not possible to detect phosphorylation in response to bIFN-x for
the other cows.


TABLE OF CONTENTS
ACKNOWLEDGMENTS iii
ABSTRACT xi
CHAPTERS
1 INTRODUCTION T
2 LITERATURE REVIEW 3
Maternal-embryonic Communication as a Requirement for Successful
Pregnancy 3
Communications Between Gametes and Maternal Units 4
Communications Between Conceptus and Maternal Units 6
Problems Associated with Fertilization Failure and Embryonic Mortality in
Cattle 9
Susceptible Periods During Pregnancy 9
Causes of Fertilization Failure and Embryonic Mortality 10
Ovlductal Fnction and Reproductive Failure in Cattle 16
The Oviduct Environment 16
Steroid Regulation and Protein Synthesis 20
Regulation of Reproductive Processes Occurring In the Oviduct
23
Oviductal Function and Reproductive Failure in Cattle 25
Uterine Function and Reproductive Failure in Cattle 27
The Uterine Environment 27
Regulation of Reproductive Processes Occurring in the Uterus
31
The JAK-STAT Pathway 47
Uterine-Conceptus Interactions and Reproductive Failure in Cattle
70
Manipulating Uterine Function to Minimize Embryo Mortality ... 73
Objectives of This Dissertation 75
vii


289
Thatcher WW, Staples CR, Danet-Desnoyers G, Oldick B, Schmitt E J-P.
Embryo health and mortality in sheep and cattle. J Anim Sci 1994b; 72
(suppl 3):16-30.
Thatcher WW, Terqui M, Thimonier J, Mauleon P. Effects of estradiol-17p on
peripheral plasma concentration of 15-keto-13,14-dihydro PGF2aand
luteolysis in cyclic cattle. Prostaglandins 1986; 31:745-756.
Thatcher WW, Wilcox CJ, Collier RJ, Eley DS, Head HH. Bovine conceptus-
maternal interactions during pre- and postpartum periods. J Dairy Sci
1980; 63:1530-1540.
Thatcher WW, Wolfeson D, Curl JS, Rico LE, Knickerbocker JJ, Bazer FW, Drost
M. Prostaglandin dynamics associated with development of the bovine
conceptus. Anim Reprod Sci 1984; 7:149-176.
Uddin S, Fish EN, Sher DA, Gardziola C, White MF, Platanias LS. Activation of
the phosphtydilinositol 3-kinase serine kinase by IFN-a. J Immunol 1997;
158:2390-2397.
Van Cleef JK, Lucy MC, Wilcox CJ, Thatcher WW. Plasma and milk
progesterone and plasma LH in ovariectomized lactating cows treated
with new or used controlled internal drug release devices. Anim Reprod
Sci 1992; 27:91-106.
Van Cleef JK, Macmillan KL, Drost M, Lucy MC, Thatcher WW. Effects of
administering progesterone at selected intervals after insemination of
synchronized heifers on pregnancy rates and resynchronization of returns,
to service. Theriogenology 1996; 46:1117-1130.
Verhage HG, Fazleabas AT. In vitro synthesis of estrogen-dependent proteins
by baboon (Pap/'o anubis) oviduct. Endocrinology 1988; 123:552-558.
Vezza R, Habib A, Li H, Lawson JA, FitzGerald GA. Regulation of
cyclooxygenase by protein kinase C. J Biol Chem 1996; 271:30028-
30033.
Vickenmeier U, Cohen SL, Moareffi I, Chait BT, Kuriyan J, Darnell jr JE. DNA
binding of in vitro activated STAT1 alpha, STAT 1 beta and truncated
STAT 1: interaction betweenNH2-terminal domains stabilizes binding of
two dimers to tandem sites. EMBO J 1996; 15:5616-5626.


261
with CL maintenance may explain the large percentage of embryonic loss during
this physiological window. Losses can be attributed to a failure of conceptus to
send signals, and to failure of the maternal unit to transduce signals that
ultimately suppresses synthesis of PGF2o. Differences in developmental stages
of the conceptus collected at day 17 of pregnancy (critical day for release of
antiluteolytic signals; Thatcher and Hansen, 1992) can explain conceptus
failures. Conversely, cow to cow variation in ability to extend estrous cycle in
response to blFN-x treatment (Helmer et al., 1989b; Meyer et al., 1995) explains
maternal unit failure. However, whether poor responsiveness to bIFN-x is
consistent for particular cows remains to be determined.
This leads to my final point of discussion. The main justification for the
kind of research presented throughout this dissertation is that a better
understanding of mechanisms controlling reproduction should lead to better
ways to manipulate cow/conceptus physiology to ultimately increase pregnancy
rates (i.e., decrease embryonic mortality). An important philosophical question
is: should animal scientists be trying to rescue embryos fated to death, due to
their own survival incompetence or inadequate maternal environment? Will
rescued embryos produce healthy offspring and healthy adult animals? A good
assessment of these questions was discussed in Holm and Caliesen (1998),
where they compared embryos produced in vivo with embryos produced in vitro.
During a normal estrous cycle several follicles grow and undergo atresia, but
only one ovulates. This dominant-ovulatory follicle is considered to be the most


40
maternal unit, as reflected by maintenance of the CL (Short, 1969; reviewed In
Hansen, 1991). More specifically, the process of maternal recognition of
pregnancy requires that embryonic molecules interact with the uterine
endometrium and change its program, so that pulsatile secretion of PGF2ois
blocked and thereby luteolysis is Impeded. The net result is continuous
secretion of P by the CL, which is required for continuation of pregnancy. Roles
of P include continuous stimulation of uterine secretions and inhibition of
smooth muscle contractions (Hafez, 1993a). In cattle, the critical period for
maintenance of pregnancy is around day 17 of the estrous cycle. Betterldge and
others (1980) transferred embryos to synchronized recipients and demonstrated
that pregnancy was only maintained if embryos were transferred prior to day 17.
Moreover, inter-estrus interval increased from 20 to 25 days when conceptuses
were removed on day 17 vs. day 15 of pregnancy (Northey and French, 1980).
Based on the model proposed above, pregnancy effects on suppression
of pulsatile PGF2a could be exerted at several levels: (1) suppression of the
PGF2a-releasing stimulus (i.e., oxytocin, LH, E2), (2) alterations of the P4-primed
uterus (i.e., PGF2a-synthesizing machinery), and (3) decrease in substrate
required for PGF2asynthesis (i.e., AA). Another possibility is presence of an
conceptus-induced luteoprotective action, where CL would become less
susceptible to luteolytlc effects of PGF2a. However, since PGF2a pulses are
effectively blocked during early pregnancy, this possibility will not be considered
in this discussion.


44
Similar to the data described above, bIFN-x effectively reduced both oxytocin-
and phorbol ester-stimulated PGF2 secretion. In contrast, Asselin and others
(1998) showed that bIFN-x increased secretion of PGF2afrom endometrial
epithelial cells from days 1 to 5 of the estrous cycle. However this effect was
only significant when extremely high doses of bIFN-x (20 pg/ml) were used.
Collectively, these data support the concept that bIFN-x interacts with
endometrial epithelium and affects the PGF2a-generating machinery to decrease
PGF2aproduction. To further test this possibility, Arnold and others (1999)
infused either bIFN-x or a control protein (bovine serum albumin) in the uterus of
cows from days 14 to 17 of the estrous cycle. Secretion of PGF2awas measured
in medium conditioned by endometrial explants cultured in presence of specific .
intracellular stimulators of PGF2ctsynthesis. Incubations with calcium ionophore
and PDBu stimulated PGF2asecretion compared to medium alone in
endometrium from control cows, but not from bIFN-x -treated cows. In contrast,
melittin stimulated secretion of PGF2afrom explants originating from bIFN-x -
infused-cows. Overall, their data indicated that in vivo treatment with bIFN-x
attenuated PGF2a production probably at the level of PKC, since PDBu
stimulation of PGF2awas reduced by bIFN-x, whereas melittin stimulated PGF2a
secretion. This is in variance with the ubiquitous inhibitory effects of pregnancy
on stimulated PGF2osecretion (mentioned above), suggesting that other products


166
a
I 121
X
s 78
P-STAT-3
0 1 3 8 15 30 60 120 min.
bIFN-x, 50 ng/ml
b
p-STAT-3
ab
Figure 5-17. Immunoblotting analysis of STATs 3 immunoprecipitated from nuclear extracts
from BEND cells treated with blFN-t for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine
phosphorylation of STAT-3; b) Least squares means and SE of tyrosine phosphorylation of STAT-3
(bars with distinct subscripts are statistically different, p<0.1).


109
intermittently pulsed for 5 seconds every 10 seconds, for a total of 1 minute, or
until most tissue was homogenized and no large tissue fragments were visible.
Homogenates were transferred to a 15 ml polypropylene conical tube and kept
on ice. Homogenates were centrifuged in a refrigerated Sorvall centrifuge
(model RC-3B, equipped with a H-6000A rotor, Du Pont Co., Wilmington, DE) at
5000 RPM for 10 minutes. Supernatants were transferred to microcentrifuge
tubes and spun for 15 minutes at 14000 RPM in a microcentrifuge (model 235b,
Fisher, Pittsburgh, PA) at 4C. Supernatants were dialyzed as described in
Chapter 3 and concentration of radioactivity measured in aliquots from dialyzed
extracts and volumes corresponding to 500000 DPM were lyophilized (Labconco
Corp., Kansas City, MO, connected to a vacuum pump, Precision, model D150,
Chicago, IL), reconstituted with 1X Laemmli buffer (Laemmli, 1970; 62.5 mM
Tris-HCI pH 6.8, 2% SDS, 10% v/v glycerol, 5% v/v p-mercaptoethanol, 0.001%
v/v bromophenol blue) and stored at -20 C until SDS-PAGE.
Culture medium was dialyzed, as described in Chapter 3, to remove
unincorporated 3H-leu and degraded peptides. Concentration of radioactivity in
the dialyzed samples was determined. Volumes of each dialyzed sample
containing 500000 DPM were lyophilized, reconstituted in 1X Laemmli buffer and
stored at -20C.
2 hour cultures. To examine presence and phosphorylation of STAT
proteins 1 and 2 in the endometrium, explants were pre-incubated in medium
alone for 2 hours and then randomly assigned to receive 0 or 625 ng/mL blFN-x


228
12h
Figure 7-7. Experiment 2. Changes in morphology over time (vertical axis, hours) of BEND cells
treated with medium alone (control), phorbol 12,13 dibutyrate (PDBu; 100ng/ml) or blFN-r (50
ng/ml) and PDBu (horizontal axis). Magnification: 100x.
Control
PDBu
PDBu+blFN-x


285
SAS. SAS/STAT users guide (Release 6.03). Cary, NC: SAS institute Inc.;
1988.
Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant
follicles during the oestrous cycle in heifers. J Reprod Frtil 1988; 83:663-
671.
Savio JD, Thatcher WW, Badinga L, de la Sota RL Wolfeson D. Regulation of '
dominant follicle turnover during the oestrous cycle in cows. J Reprod
Frtil 1993a; 97:197-203.
Savio JD, Thatcher WW, Morris GR, Entwistle K, Drost M, Mattiacci MR. Effects
of induction of low progesterone concentrations with a progesterone
releasing intravaginal device on follicular turnover and fertility in cattle. J
Reprod Frtil 1993b; 98:77-84.
Schindler C, Darnell jr JE. Transcriptional responses to polypeptide ligands: the
JAK-STAT pathway. Annu Rev Biochem 1995; 64:621-651.
Schindler C, Shuai K, Prezioso VR, Darnell jr JE. Interferon-dependent tyrosine
phosphorylation of a latent cytoplasmic transcription factor. Science
1992; 257:809-813.
Schmitt E J-P, Diaz T, Barros CM, de la Sota RL, Drost M, Fredriksson EW,
Staples CR, Thorner R, Thatcher WW. Differential response of the luteal
phase and fertility in cattle following ovulation of the first-wave follicle with
human chorionic gonadotropin or an agonist of gonadotropin-releasing
hormone. J Anim Sci 1996a; 74:1074-1083.
Schmitt E J-P, Diaz TC, Drost M, Thatcher WW. Use of a gonadotropin
releasing hormone agonist or human chorionic gonadotropin for timed
insemination in cattle. J Anim Sci 1996b; 74:1084-1091.
Schmitt E J-P, Drost M, Diaz T, Roomes C, Thatcher WW. Effect of
gonadotropin-releasing hormone agonist on follicle recruitment and
pregnancy rate in cattle. J Anim Sci 1996c; 74:154-161.
Short, RV. Implantation and the maternal recognition of pregnancy. In:
Wolstenholme GEW, OConnor M (eds), Foetal Autonomy. Ciba
foundation symposium. London: J and A Churchill Ltd; 1969:377-386.


45
of pregnancy, and not bIFN-x alone, probably also operate to inhibit PGF2a
production.
In an effort to pinpoint specific enzymes that bIFN-x altered in the PGF2a-
generating cascade, Xiao and others (1999) measured messenger ribonucleic
acid (mRNA) and protein expression for COX-2 in endometrial epithelial cells
treated with oxytocin and with oxytocin in combination with bIFN-t. Oxytocin
maximally stimulated COX-2 mRNA and protein from 3 to 24 hours compared to '
controls. Treatment with bIFN-x reduced this effect of oxytocin, and this was
consistent with a reduction in PGF2a secretion In medium. In contrast, Asselin
and coworkers (Asselin et al., 1997) found that bIFN-x actually stimulated
expression of COX-2, which would contradict the antiluteolytic role of bIFN-x.
However, they also reported that bIFN-x stimulated expression of an endometrial
prostaglandin E2-9-ketoreductase, which catalyzes the conversion of PGF2ainto
PGE2 (Asselin and Fortier, 1998). Since PGE2has been shown to have luteo-
protective actions (Pratt et al., 1977), they proposed a model whereby bIFN-x
actualiy stlmulates the PGF2a-generating machinery, but a conversion of PGF2a
to PGE2 at the end of the cascade would support an antiluteolytic effect of blFN-
x.
It is expected that in order to stimulate intracellular changes resulting in
decreased PGF2oproduction, bIFN-x needs to stimulate a receptor-mediated
mechanism of signal transduction. Such a mechanism should evoke intracellular


130
from epithelial cells (Danet-Desnoyers et al., 1994). Therefore, it is reasonable
to assume that blFN-x exerts most of its regulation on epithelial cells. However,
in WCE used for immunoprecipitation, proteins originated from epithelial cells
were diluted with proteins coming from the stroma, capillaries etc. It is possible
that STAT proteins are present in all cell types in the endometrium, but they
might become tyrosine phosphorylated preferentially in the epithelium. Due to
cellular heterogeneity of explants, immunoprecipitation of WCE collected a pool
of STATs from different cell types, and epithelial, tyrosine-phosphorylated STATs
are in too small of a proportion to be detected clearly. This would explain why
tyrosine phosphorylation was difficult to detect regardless of the abundant
presence of STATs. An alternative explanation is that tissue phosphatases
quickly de-phosphorylated STATs, which then could not be detected in
immunoblots. Although phosphatase inhibitors such as p-glycerophosphate and
Na3V04 were present in the homogenization buffer and homogenization was
performed on ice, it is possible that compartmentalized pools of phosphatases
originally present in the whole tissue were disrupted and an acute removal of
tyrosine phosphates took place. Whatever the reason, it became clear that a
different model of study would be necessary to evaluate critically the dynamics of
blFN-x-stimulated JAK-STAT pathway in the endometrium.
Experiment 2 tested an alternative system for study of blFN-x actions in
the endometrium, using scraped epithelial endometrial cells. Initial intention was
to use scraped cells for culture, to measure phosphorylation of STAT proteins in


68
as P8, was induced only in response to blFN-T, but not in response to IFN-a,
suggesting the possibility of blFN-x eliciting specific signal transduction and
protein synthesis. Moreover, P8 but not P16 secretion could be stimulated by
phorbol ester (Staggs et al., 1998). Amino acid analysis of the P8 revealed
identity with the alpha chemokine family: 92-100% identity with bovine bGCP-2
(Teixeira et al., 1997). Functions of bGCP-2 remain elusive, but it has been
suggested (Hansen et al., 1999) that being a chemokine, bGCP-2 may attract
conceptus cells to attachment sites in the endometrium. Also, bGCP-2 may
attract cells from the immune system, to release cytokines beneficial to embryonic
development. P16 was identified as a bovine ubiquitin-cross reactive protein
(Austin et al., 1996a,b). The bUCRP mRNA (Hansen et al., 1997) and protein
(Austin et al., 1996b) are induced by blFN-x, and sequence analysis of the
bUCRP gene revealed presence of a conserved ISRE in the promoter region,
indicating putative activation by blFN-x (Perry et al., 1997). Analysis of the
primary structure of bUCRP revealed presence of critical amino acids and
domains implicated in functions of ubiquitin, such as conjugating with other
proteins. However, bUCRP lacked residues required for targeting proteins to
proteasomal degradation (Austin et al., 1996a). Therefore, it was proposed that a
possible role for bUCRP was to modify uterine proteins during early pregnancy
(Hansen et al., 1999). In fact, Johnson and others (1998a) reported that specific
conjugates of bUCRP and endometrial cytosolic proteins were formed in


259
sheep endometrium and analyze synthesis of prostaglandins in relation to COX-
2 content. I failed to repeat their technique successfully. After scraping, 99% of
bovine endometrial cells were dead, as determined by trypan blue exclusion.
Therefore, use of this system for In vitro incubations was not possible with bovine
endometrium. However, the system was useful to generate data depicting levels
of COX-2 and PLA2 In endometrium epithelium just after slaughter. These data
led us to conclude that similar abundance of COX-2 and PLA2 in cows receiving
control or bIFN-x uterine infusions indicated that a reduction in PGF2a secretion
induced by PDBu may be due to bIFN-x regulation of their respective enzyme
activities. BEND cells provided a model adequate to measure cytosolic and
nuclear proteins related to the JAK-STAT pathway (Chapters 5 and 6) and to
measure PGF2cl secretion and expression of proteins related to the PGF2a
secretory machinery (Chapter 7). They originated from day 14 cyclic cows, but
how much of the original phenotype is maintained is questionable, and criticism
applicable to any cell culture system is also valid here. However, PGF2a
secretion regulation by both PDBu and bIFN-x are maintained. BEND cells do
not respond to oxytocin stimulation (Arnold, Badinga, Binelli and Thatcher,
unpublished observations) unless they are primed with steroids before oxytocin
challenge (Mirando, personal communication). Lack of responsiveness to
oxytocin is similar to non-responsiveness in explants from day 15 cyclic cows,
challenged with oxytocin (Arnold et al., 1999). Another similarity between
explants and BEND cells is that the two systems were responsive to PDBu


26
patterns of steroids. Gametes and embryos would tolerate mild perturbations in
the system and the reproductive processes would be carried out by internal, pre
determined programs, modestly influenced by the oviductal environment. The
oviduct would basically provide a physical substratum for events to occur. Leese
(1988) suggested that this possibility could be appropriately tested by trying to
culture embryos on an epithelium anatomically related to the oviduct, such as the
trachea (I.e., ciliated, secretory, containing active chloride Ion pump). To support
this first possibility, there is the fact that embryos can be matured and fertilized in
vitro in the absence oviductal cells, tissue or conditioned medium. Alternatively,
one could say that oviduct-gametes/embryos relationships have been optimized
in the course of evolution, to become a robust system, with little chance for
failure. Specific interactions would be required for success of reproductive
processes, including synthesis and secretion of oviductal proteins in a regional
and timely fashion. Moreover, such unique set of proteins would Interact with
gametes/embryos to maximize reproductive output. To test this last possibility,
secretory proteins in the oviduct would first need to be identified. Then,
removing specific proteins from the system with use of Immunoneutralizatlon,
knockouts, transgenics and anti-sense models for example, should provide
evidence for their importance. For example, addition of specific antibodies for a
hamster E2 -dependent oviduct protein prevents in vitro fertilization (Sakai et al,
1988).


179
Due to the relative low abundance of STAT proteins in cells, the
immunopreclpitatlon approach was chosen to concentrate STATs and facilitate
detection and analysis of STATs In immunoblots. The antl-human STAT
antibodies specifically immunoprecipltated and detected bands for bovine STATs
1, 2 and 3, based on their predicted molecular weight on immunoblots.
Therefore, all experiments were conducted using antl-human STAT antibodies. .
The initial objective of the present study was to determine a dose of blFN-
t to be used in the experiments. To my best knowledge, no precise
measurement of concentrations of blFN-x in the uterus have been taken.
Trophoblastic cells on the surface of the elongating embryo secrete blFN-x
locally, and blFN-x is probably readily taken up by epithelial cells in the
endometrium. Therefore, measurements of concentrations of blFN-x in uterine
fluid would probably be meaningless. One strategy to estimate physiological
doses of blFN-x is to examine the Kd for the IFN-t receptor. Based on the
calculated Kd for the ovine and blFN-x receptor in endometrium, the
physiological dose for blFN-x would be between .2 and 11.6 ng/ml (discussed in
Danet-Desnoyers et al., 1994), which is lower than the dose that elicited
maximum phosphorylation of STAT-1 (50 ng/ml). Compared to other
experiments utilizing blFN-x, the dose of 50 ng/ml that we chose to conduct
subsequent experiments Is low. For example, uterine infusions of up to 400
gg/day are necessary for extension of estrous cycle length In non-pregnant cows


41
There is good evidence for pregnancy-induced suppression of luteolytic
stimulus in cattle relative to the attenuation of E2 effects. Pregnant cows have
reduced circulating concentrations of E2 (Pritchard et al., 1994), probably as a
result of reduced folliculogenesis (total production of follicles) and decreased
production of E2 per follicle (decreased aromatase activity; Thatcher et al., 1991).
Moreover, in day 18 pregnant cows, administration of E2 stimulates only a
modest increase in PGF2ci secretion, indicating that presence of the conceptus
attenuates E2 effects (Thatcher et al., 1984).
Regarding alterations on the P4-primed uterus, Arnold and others (1999)
incubated endometrial explants obtained from day 17 cyclic or day 17 pregnant
cows with intracellular stimulators of PGF2a synthesis, and measured
concentrations of PGF2osecreted into the culture medium. Melittin, PDBu and
calcium ionophore each stimulated release of PGF2a from explants of cyclic cows
compared to control treatment (medium alone). In contrast, all stimulators
mentioned above failed to induce release of PGF2a in explants originated from
pregnant cows. This indicated that pregnancy affected the intracellular PGF2a-
generating machinery to suppress its ability to stimulate PGF2a. Interpretation of
these data suggests that pregnancy may have inhibitory effects at each of the
steps stimulated by treatments, which include PKC (PDBu) and PLA2 (melittin,
ionophore). Alternatively, pregnancy may affect a distal, convergence point in
the pathway, for example, at the COX-2 level. Effects on the enzymatic
machinery can be to decrease expression and/or activity of PKC, PLA2and COX-


19
beating (towards the uterus) and oviduct muscular contractions (towards ovary)
maintain eggs in constant rotation, which is essential for fertilization and to
prevent oviduct implantation (Hafez, 1993a). Non-ciliated epithelial cells have
primarily a secretory function. They contain secretory granules at their apical
aspect, and these accumulate during the follicular phase of the estrous cycle,
and are released into the lumen after ovulation (Murray, 1992). Treatment of
ovariectomized sheep with E2 stimulates hypertrophy of secretory organelles and
accumulation of granules in non-ciliated cells of the AMP (Murray, 1995).
Oviductal secretions contribute to the formation of the oviductal fluid, discussed
next.
The oviduct fluid. Reproductive processes occurring in the oviduct are
exposed and subjected to regulatory influences of ingredients in the oviductal
fluid. Chemical analyses of the oviductal fluid indicated that it is a mixture of
constituents derived from the plasma, through selective transudation, plus
specific proteins synthesized and secreted by the oviductal epithelium (Leese,
1988). The major classes of components are water, gases (02), electrolytes (Ca,
Na, K, Cl), non-electrolytes (glucose, fructose, complex carbohydrates) and
proteins. Some proteins originate from serum (albumin, immunoglobulins) while
others are synthesized de novo in the oviduct [plasminogen activator inhibitor
(Kouba et al., 1997) and bovine oviductal glycoprotein (Boyce et al., 1990)].
Functions of oviductal fluid electrolytes and non-electrolytes are reviewed in
Leese (1988). De novo synthesized oviductal proteins may affect reproductive


28
controlling length of ovarian cycles (described above) and, as a consequence,
its own uterine cycle. The uterine cycle can be divided into a long progestational
phase and a short, estrogen-dominated phase (Hansel and Convey, 1983). At
the end of progestational phase, the uterus gains the ability to produce and
secrete PGF2(I, which acts to cause structural and functional demise of the CL
(McCracken et al., 1971). An immediate consequence of PGF2oactions is a
decrease in circulating concentrations of P4 (Nett et al., 1976). This initiates the
estrogen dominated period that lasts until the next ovulation and formation of
new CL. Controlling CL life span, the uterus controls the ovarian cycle. During
pregnancy, presence of the conceptus blocks luteolytlc mechanisms so that the
CL remains functional and the uterus remains In a progestational stage until
parturition (McCracken et al., 1984). Similar to oviducts, uterine morphology and
secretory activity are modulated by ovarian steroids, as discussed below.
Functional anatomy and morphology. The uterus is suspended in the
pelvis by the mesometrium, a caudal division of the broad ligament. In cows, the
uterus can be described anatomically in two continuous portions, the gestational
part of the uterus (consisting of uterine horns and uterine body), and the cervix.
Similar to the oviduct, the uterus is a tube-shaped organ, which contains a lumen
(Bartol, 1999). Histologically, a cross section of the uterus reveals an inner
mucosal layer, the endometrium, an adluminal layer of smooth musculature, the
myometrium and an outer, serous peritoneal coat of the uterus, the perimetrium
(Bartol, 1999). For the remaining of this discussion, I will focus on characteristics


Arnaldo, Aurora, Lola, Dinda, Dindo, Carmen, Maga, Guilherme, Paula, Neco,
Ana, Rico, Beto, Aldo, Ines, Janete, Maria Helena, Farjala (late), Lenita, Lucia,
Elisa, Leandro, Lucio, Elisa Kampf, Lolo Kampf, Claudia, Gabi e Yara. I Thank
them for their dedication, faith, love, patience, inspiration, support and
encouragement.
Most specially of all, with my whole heart I wish to thank my wife Nana, for
helping me to externalize the best of my being and for being side-by-side with
me on this long road.
VI


30
ovarian artery. The later is coiled about the surface of the uteroovarian vein
(Bartol, 1999). Countercurrent exchange of PGF2[twas demonstrated elegantly
by Knickerbocker and coworkers (1996). The authors sampled blood originating
from uterine branch of ovarian artery(UBOA) and facial artery (FA), and
measured changes In PGF2a concentrations in response to a challenge with E2.
There was a greater concentration of PGF2aln UBOA compared to FA, indicating
existence of local countercurrent exchange between uterine venous drainage
and ovarian artery.
The hvstotroph. Hystotroph is the secretions present within the uterine
lumen for nourishment of the developing conceptus (Roberts and First, 1983). A
broader definition should also include functions such as paracrine regulation of
conceptus physiology and development and protection of the conceptus from the
maternal immune system. Solymosi and Florn (1994) measured protein content
in uterine milk (i.e., hystotroph) of cows and determined that 73% of the dry
matter content was composed proteinaceous material. Information on nature of
proteins contained in uterine milk is limited in cattle. Electrophoretic analysis
reveled at least nine proteins, seven minor and 2 major, which were identified as
lactoferrin and acid phosphatase (Bazer and First, 1983). Lactoferrin may have
a bacteriostatic function in the uterine luminal environment. The acid
phosphatase has basic PI, which is similar to uteroferrin in pigs. Uteroferrin is
involved in iron transport to the conceptus (Roberts and Bazer, 1988), but
whether bovine acid phosphatase has the same role in cattle is unknown.


278
Knickerbocker JJ, Thatcher WW, Bazer FW, Drost M, Barron DH, Fincher KB,
Roberts RM. Proteins secreted by day-16 to -18 conceptuses extend
corpus luteum function in cows. J Reprod Frtil 1986; 77:381-391.
Korzus E, Nagaase H, Rydell R, Travis J. The mitogen-activated proteinkinase
and JAK-STAT signaling pathways are required for an oncostatin-
responsive element-mediated activation of matrix metalloproteinase 1
gene expression. J Biol Chem 1997; 272:1188-1196.
Kotwica J, Skarzynski D, Bogacki M, Melin P, Starostka B. The use of an
oxytocin antagonist to study the function of ovarian oxytocin during
luteolysis in cattle. Theriogenology 1997; 48:1287-1299.
Kouba AJ, Aberydeera LR, Alvarez IM, Day BN, Buhi WC. Effects of porcine
oviduct-specific glycoprotein (pOSP) on fertilization and polyspermy. Biol
Reprod 1999; 60 (suppl 1):228(Abstr.).
Kouba AJ, Alvarez IM, Buhi WC. Purification and characterization of of a 45 kD
protein synthesized de novo by oviductal tissue from early pregnant and
ovariectomized (OVX) steroid-treated gilts. Biol Reprod 1997; 56 (suppl
1):187.
Lafrance M, Goff AK. Effects of pregnancy on oxytocin-induced release of
prostaglandin F2n release in heifers. Biol Reprod 1985; 33:1113-1119.
Lafrance M, Goff AK. Effects of progesterone and oestradiol-17p on oxytocin-
induced release of prostaglandin F2a in heifers. J Reprod and Frtil 1988;
82:429-436.
Lamming GE, Darwash AO, Back HL. Corpus luteum function in dairy cows and
embryo mortality. J Reprod Frtil 1989; 37 (suppl):245-252.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 1970; 227:680-685.
Leaman DW, Leung S, Li X, Stark GR. Regulation of STAT-dependent
pathways by growth factors and cytokines. FASEB J 1996; 10:1578-
1588.
Lee EH, Rikihisa Y. Protein kinase A-mediated inhibition of gammainterferon-
induced tyrosine phosphorylation of Janus kinases and latent cytoplasmic
transcription factors in human monocytes by Ehrlichia chaffeensis. Infect
Immun 1998; 66:2514-2520.


64
the autophosphorylation domains were also effective in phosphorylating STATs.
This indicates that an alternative mechanism, where docking through SH2 domain
of STATs is not required for phosphorylation, is in place for EGF-induced STAT
phosphorylation.
The obligatory intracellular bacterium of macrophages, Ehrlichia
chaffeensis, blocked tyrosine phosphorylation of STAT-1, JAK-1 and JAK-2 in
response to IFN-y within 30 minutes of infection (Lee and Rikihisa, 1998). Also, .
PKA activity was increased 25 fold after infection. Inhibitors of PKA activity
partially abrogated the E chaffeensis-induced inhibition of STAT-1 tyrosine
phosphorylation, suggesting negative regulation of the JAK-STAT pathway by the
PKA-dependent mechanisms.
Another interesting theme is the occurrence of synergistic effects as a
result of coactivation of cellular pathways involving the JAK-STAT system. For
example, cooperation of interferon-y and tumor necrosis factor (TNF) during
inflammatory responses is a result of cooperation between STAT-1 and the
transcription factor NF-icp. Synergistic expression of several genes involved in
the inflammatory process was contingent on presence of both transcription
factors (Ohmori et al., 1997). Stimulation by oncostatin M (OSM) induces
expression of matrix metalloproteinases (MMPs). Analysis of the regulatory
region of MMP-1 gene revealed presence of an AP-1 site as well as a STAT
binding element. Korzus and coworkers (1997) reported enhancement of MMP


254
Nucleus
o
1*
-O
IFTM-inducible gene
OO
p
PGFsynthesis-related gene
t
j
IFN-T-regulated protein
IFN-r-induced protein
PKC-regulated protein
PKC-induced protein


257
phosphorylation and activity of the serine kinase PI-3' kinase (Pfeffer et al.,
1997). This enzyme has been implicated in stimulation of transcriptional
activation of COX-2, through activation of PKC activity. Interferons a, p and y
(actually a type II interferon, but that also acts through activation of the JAK-
STAT pathway) induce synthesis and activation of PLA2 (Wu et al., 1994), and
PLA2 was shown to be required for maximum formation of ISGF-3 complex (Flati
et al., 1996). Finally, Interferon p stimulated PLC activity in human lymphocytes,
and this effect was independent of ISGF-3 formation (Miscia et al., 1997).
Collectively, these data suggest that either the endometrium has a unique
responsiveness to interferons or blFN-i modulates the PGF2asecretion
machinery differently than other types of interferons. The second hypothesis is
less attractive, since early experiments demonstrated that interferon a is able to
extend interestrus interval and CL lifespan in dairy cows (Plante et al., 1989).
A question that was asked at the outset of experiments to determine
presence and function of JAK-STAT pathway in the endometrium was what is
the appropriate experimental model to use (i.e., primary cells, explants etc).
Criteria for choice were physiological relevance of a system and convenience of
a system for performing intensive, frequent experiments. The system chosen
initially was the use of explants, collected from day 15 cyclic cows (Chapter 3).
Explant culture is attractive in that structural arrangement of tissue is maintained,
allowing critical cell-cell communication to be maintained. Moreover, tissues
were collected from specific stages of the estrous cycle, which provided a clear


178
Embryonic Trophoblastic Cell~)
Endometrial Epithelial Cell
Nucleus
flSREl filElr*
IFN-regulated genes
-IRF-1, IRF-2 (+)
-UCRP (+)
-Estradiol receptor (-)
-Oxytocin receptor (-)
Figure 5-23. hypothetical model for the blFN-i-stimulated JAK-STAT pathway of signal
transduction in endometrial epithelial cells. Trophoblastic cells on the conceptus secrete bIFN-t
into the uterine lumen, and blFN-r interacts with its receptor in the apical aspect of endometrial
epithelial cells. Binding of blFN-r elicits tyrosine phsphorylation, homo- and hetero-dimer
formation of pre-existing, unphosphorylated STAT proteins. Dimers of STATs translocate to the
nucleus where they bind to specific cis-activating elements [sis-inducible element (SIE) and
interferon-stimulus response element (ISRE)] present in the regulatory region of interferon-
regulated genes to stimulate their transcription. It is hypothesized that proteins induced by blFN-
1, such as ubiquitin cross-reactive protein (UCRP), interferon regulatory factor (IRF)-1 and IRF-2
may act to suppress synthesis of PGF2from endometrial cells. Also, the JAK-STAT pathway
may suppress synthesis of proteins involved the PGF2asynthesis machinery, such as receptors
for oxytocin and estradiol.
The BEND cells used In our studies are a spontaneously immortalized cell
line, derived from endometrial epithelial cells of cows on day 14 of the estrous
cycle (Austin et al., 1996a). Similarly to endometrial explants, BEND cells
secrete both UCRP and GCP-2 In response to blFN-x (Austin et al., 1996b;
Teixelra et al., 1997; Johnson et al., 1998a; Staggs et al., 1998), suggesting that
they are an adequate model for studying IFN-stimulated signal transduction.


158
a
blFN-r, 50ng/ml
Figure 5-9. Immunoblotting analysis of STAT-3 immunoprecipitated from whole cell
extracts from BEND cells treated with blFN-r for increasing intervals of time.
a) Representative enhanced chemiluminescence (ECL) exposure of tyrosine
phosphorylation of STAT-3; b) Representative ECL exposure of abundance of STAT-3.
nucleus was only detected In one experiment, where it increased by 15 minutes
and remained elevated for up to 120 minutes (Figure 5-13).
STAT-3. Similar to STATs 1 and 2, there was an increase in abundance
of STAT-3 that lasted from 3 to 120 minutes in CE (Figure 5-14). In the nucleus,
blFN-x increased abundance of STAT-3, which lasted from 8 to 60 minutes
(Figure 5-15). Tyrosine phosphorylation of STAT-3 increased gradually to reach
maximum levels at 15 minutes in the cytosol (Figure 5-16) and at 30 minutes In
the nucleus (Figure 5-17). Levels of phosphorylation decreased abruptly after 60
and 120 minutes exposure to blFN-x.


59
molecule with the carboxy-terminal portion of DNA-bound p48 stabilizes ISGF-3
(Horvath et al., 1996). Vickenmeier and coworkers (1996) reported direct binding
of recombinant, tyrosine phosphorylated STAT-1:1 dimers to tandem DNA
sequences. STAT-2 also forms homodimers, but requires p48 for strong
transactivation of transcription (Bluyssen and Levy, 1997). However, interactions
with DNA were not stable. Addition of STAT-1 increased the affinity and altered
sequence selectivity of p48-DNA interactions. In this scenario, ISGF-3 assembly
involves p48 functioning as an adaptor protein to recruit STAT-1 and STAT-2 to
an ISRE, STAT-2 contributes with potent transactivation but is unable to directly
contact DNA, while STAT-1 stabilizes the complex by contacting DNA directly.
Alternatively to transcription-induction through ISRE binding, interferons also
induce genes like IRF-1 which lack ISREs. Such genes are induced through
sequences named Inverted Repeats, present in their promoters (Haque and
Williams, 1994).
JAK-STAT pathway regulation
As in other tyrosine-phosphorylation-induced signaling systems, biological
responses resulting from activation of the JAK-STAT pathway are transient (Shuai
et al., 1992). Although the pathway of activation via the JAK-STAT pathway is
well established, few molecules have been identified that switch the signal off
(Starr and Hilton, 1999). Intuitively, one would predict that regulation of a tyrosine
phosphorylation pathway could occur through the actions of phosphatases, to
inactivate phosphotyrosil groups on receptors, JAKS and STATs, and proteases,


3 PERSISTENT DOMINANT FOLLICLE ALTERS PATTERN OF
OVIDUCTAL SECRETORY PROTEINS FROM COWS AT ESTRUS . 76
Introduction 76
Materials and Methods 79
Materials 79
Preparation of Medium 80-
Animals and Treatments 80
Tissue Culture 82
Two-Dimensional Electrophoresis 83
Densitometry 83
Hormone Assays 84
Statistical Analysis 85
Results 85
Ultrasonography and Hormone Measurements 85
Incorporation Rate 88
Fluorography and Densitometry 88
Discussion 95
4 EFFECTS OF BOVINE INTERFERON-TAU ON THE JAK-STAT SIGNAL
TRANSDUCTION PATHWAY AND SYNTHESIS OF PROTEINS IN
BOVINE ENDOMETRIUM AND ON THE MECHANISM OF
GENERATION OF PROSTAGLANDIN F2a IN ENDOMETRIAL
EPITHELIAL CELLS 103
Introduction 103'
Materials and Methods 105
Materials 105
Experiment 1 107
Experiment 2 114
Statistical Analysis 117
Results 117
Experiment 1 117
Experiment 2 121
Discussion 123
5 BOVINE INTERFERON-TAU STIMULATES THE JAK-STAT PATHWAY
IN BOVINE ENDOMETRIAL EPITHELIAL CELLS 133
Introduction 133
Materials and Methods 135
Materials 135
viii


98
Therefore, it was expected that the sustained high E2/low P milieu of PDF cows
and the increasing E2 and P4 milieu of FDF cows would differentially modulate
protein synthesis and secretion in the oviduct.
Analysis of the biosynthetic activity (incorporation rates) indicated that
presence of a PDF decreased the synthetic activity of oviductal tissues.
However, the overall increased incorporation of label into synthesized and
secreted macromolecules for FDF cows compared to PDF cows in all oviductal
regions appears to be in variance with the similar abundance of specific proteins
for both PDF and FDF cows (Tables 1 and 2). Several explanations for this
dichotomy are possible. Higher E2 and lower P4 concentrations associated with
presence of a PDF over an extended period of time, apparently had a
suppressive effect on overall biosynthetic activity of the different oviductal
regions. Prolonged exposure to high levels of E2 in PDF cows may have caused
down-regulation of E2 receptor, which would explain the suppression in
biosynthetic activity of oviducts from PDF cows compared to FDF cows.
However, synthesis and secretion of proteins that are actually inhibited by E2
could be stimulated as a result of down-regulation of E2 receptors. Studies in the
hen (Kawashima et al., 1996), mouse (Fuentealba, 1988), pig (Stanchev et al.,
1985) and primates (Slayden and Brenner., 1994) indicate that absolute and
relative amounts of receptors for E2 and P4 vary in the oviduct, during the estrous
cycle and pregnancy. Moreover, oviductal functions such as velocity of egg
transport (Fuentealba et al., 1988) and oviductal epithelial cell proliferation


88
(estrus). For PDF cows, E2 remained at approximately 15 pg/ml from day 9 until
day 19, After PGF2a injection on day 7, P4 concentrations decreased for both
groups between day 7 and day 11. After day 11, P4 increased in association
with development of a new CL in FDF cows (3/3) while concentrations of P4
remained low for PDF cows. After CIDR removal and PGF2a injection on day 16,
P4 concentrations decreased for FDF and PDF cows.
Incorporation Rate
Incorporation rate of radiolabel into protein can be used as a measure of
the protein biosynthetic activity of tissues (i.e., amount of 3H-leu incorporated
into newly synthesized and secreted macromolecules). There was a significant
(p<0.05) treatment by region interaction (Figure 3-4). The FDF increased
incorporation rate of 3H-leu into proteins for all oviductal regions (treatment
effect; p<0.01). However, stimulation was not significant in the 1ST (INF and
AMP vs. 1ST by treatment contrast p<0.01). No side or side by treatment effects
were detected (p>0.1).
Fluoroaraphv and Densitometry
The pattern of proteins secreted by explants of INF, AMP and 1ST, as
resolved by two-dimensional SDS-PAGE, are shown in representative
fluorographs in Figure 3-5. Proteins analyzed were designated P1 to P20 based
on their location in the fluorograph following a clockwise pattern starting in the


185
levels by 120 minutes. This Indicates that homo and heterodimers of STATs
were formed in the cytoplasm, migrated to the nucleus and returned to the
cytoplasm.
Association of STATs was noted even without exposure to blFN-i, both in
NE and CE, Indicating a hormone-independent basal level of association of
STATs. In support to our findings, Stancato and coworkers (1996) showed that
STAT heterodimers (1:2 and 1:3) exist in the cytosol prior to cytokine stimulation.
There was a faster rate of Increase In abundance of STAT-2 In the nucleus
compared to STAT-1, associated with STAT-3. This is in conflict with data from
Horvath and Darnell (unpublished observations cited in Darnell, 1997) which
suggest weak binding of STAT dimers 2:3, compared to 1:2 or 1:3. However,
Ghlslain and Fish (1996) reported formation of STAT 2:3 complex after
stimulation of U266 cells with Interferon-a. Moreover, after maximum abundance
at 15 minutes, there was a sharp decrease of STAT-2 in NE, while STAT-1 was
still associated with STAT-3 In high amounts by 30 minutes. It Is proposed that
the dynamics of association with STAT-3 is different for STATs 1 and 2.
Furthermore, due to the apparently greater abundance of STAT-1 in cells
compared to STAT-2, one should expect greater formation of STAT 1:3
complexes, rather that 2:3. Since the opposite is the case, bIFN-t apparently
preferentially induced formation of STAT 2:3 complexes.
The present series of experiments supports the concept that blFN-x is
able to activate the JAK-STAT pathway of signal transduction in bovine


147
immunoprecipitated with anti-STAT-1, -2 and -3 antibodies simultaneously (1 pg
each).
Coimmunoprecipitation of STATs
Association of proteins in complexes may be detected by
coimmunoprecipitation methodology. To verify whether blFN-t induces
formation of complexes of STATs 1 and 2 with STAT-3, eight plates of BEND
cells were assigned randomly to be treated with 50 ng/ml of bIFN-T for 1, 3, 8,
15, 30, 60 or 120 minutes or nothing (0 minutesYcontrol treatment). Cytosolic
extracts and NE were obtained from each plate and immunoprecipitated with
anti-STAT-3. Immunoprecipitated proteins were analyzed by immunoblots for
STAT-1 and -2, phosphotyrosine and STAT-3, in that order. In a separate
experiment, designed to test whether blFN-x induces formation of complexes of
STAT-3 with STATs 1 and 2, CE and NE were immunoprecipitated with STATs 1
and 2 and proteins were analyzed by immunoblotting for STAT-3,
phosphotyrosine and STATs 1 and 2, in that sequence.
Densitometric Analysis
Abundance (i.e., amount of chemiluminescence signal associated with a
given protein present in the sample and detected with an antibody specific for
that protein) and phosphorylation (i.e., amount of chemiluminescence signal
associated with phosphorylation of a given protein present in the sample and


280
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Time Response to bIFN-x
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Binelli M, Diaz T, Flansen TR, Thatcher VWV. Bovine interferon-tau induces
phosphorylation and nuclear translocation of STAT-1, -2 and -3 in
endometrial epithelial cells. Biol Reprod 1998; 58 (Suppl. 1):214 (Abstr.).
Binelli M, Hampton J, Buhi WC, Thatcher WW. Persistent dominant follicle alters,
pattern of oviductal secretory proteins from cows at estrus. Biol Reprod
1999; 61:127-134.
Binelli M, Subramaniam PS, Thatcher WW. Bovine interferon-tau promotes
transient phosphorylation of STAT-1 protein in endometrium. Biol Reprod
.1996; 54(Suppl. 1):169 (Abstr.).
Bluyssen AR, Durbin JE, Levy DE. ISGF3 gamma p48, a specificity switch for
interferon activated transcription factors. Cytokine Growth Factor Rev
1996; 7:11-17.
Bluyssen AR, Levy DE. STAT-2 is a transcriptional activator that requires
sequence-specific-contacts provided by STAT 1 and p48 forstabel
interaction with DNA. J Biol Chem 1997; 272:4600-4605.
Boatman EB, Magnoni GE. Identification of a sperm penetration factor in the
oviduct of the golden hamster. Biol Reprod 1995; 52:199-207.


CHAPTER 4
EFFECTS OF BOVINE INTERFERON-TAU ON THE JAK-STAT SIGNAL
TRANSDUCTION PATHWAY AND SYNTHESIS OF PROTEINS IN BOVINE
ENDOMETRIUM AND ON THE MECHANISM OF GENERATION OF
PROSTAGLANDIN F2a IN ENDOMETRIAL EPITHELIAL CELLS
Introduction
Bovine (b) conceptuses secrete copious amounts of the glycoprotein
interferon-tau (IFN-x) around day 17 of pregnancy (Farln et al., 1990). Bovine
IFN-x suppresses luteolytic pulses of prostaglandin F2a (PGF2cl) to rescue the
maternal corpus luteum (CL) from luteolysls (Thatcher et al., 1997). Therefore,
blFN-x is considered to be a mediator to maintain pregnancy. This Chapter
describes experiments designed to examine two separate aspects of blFN-x
effects in the bovine endometrium, namely (1) signal transduction and protein
synthesis and (2) effects on the PGF2n-synthesizing machinery.
Trophoblasts secrete blFN-x into the uterine lumen and blFN-x binds to
receptors on the surface of endometrial cells (Hansen et al., 1989; Li and
Roberts, 1994). Binding of blFN-x elicits intracellular events that stimulate
synthesis and secretion of specific proteins in bovine endometrial explants
(Nalvar et al, 1995) and decreases secretion of PGF2a by endometrial cells
103


248
maintenance, in which timely exchange of signals between maternal and
conceptus units is required for maintenance of pregnancy (Thatcher et al.,
1994b; 1997). Such exchange of signals reduces uterine PGF2a secretion
(Helmer et al., 1989a; Meyer et al., 1995; Arnold et al., 1999), decreases
follicular development (Thatcher et al., 1991) and may support conceptus growth
through uterine growth factor production (de Moraes and Hansen, 1997; Paula-
Lopes et al., 1998; 1999). These more complex, more highly interactive sets of
interactions offer greater opportunity for failure, what could explain the greater
percentage of embryo losses during the maternal recognition of pregnancy
window associated with CL maintenance at approximately 17 days after estrus.
The fact that a major regulation of oviductal function in exerted through
the actions of ovarian steroids, alterations in the endogenous patterns of E2 and
P4 secretions may have negative implications on fertility. Chapter 3 illustrates
modulation of oviductal secretory protein patterns by altered follicular function
and steroid secretion. The distinct steroidal milieus conditioned by fresh vs.
persistent dominant follicles caused both region and side specific changes in
abundance of specific oviductal proteins. Such changes may be associated with
the decreased fertility observed in cattle submitted to estrous synchronization
protocols that cause formation of persistent dominant follicles (Savio et al.,
1993b; Cooperative Regional Research Project, NE-161, 1996). Mechanistically,
there could have been changes in critical proteins associated with reproductive
processes in the oviduct, such as fertilization and early embryo development.


66
culture medium. PLA, was associated with JAK-1, and inhibitors of PLA2 activity
prevented formation of active ISGF3 transcription complexes. However, such
inhibition did not block binding of activated STAT-1 to inverted repeat sequences,
such as present in the regulatory region of IRF-1. Moreover, treatment of cells
with interferon-a stimulates tyrosine phosphorylation of PLA2. The authors argue
for a structural role of PLA2, which may be required for correct assembly of the
ISGF3 transcription complex.
The JAK-STAT pathway in bovine endometrium
In addition to the bIFN-x receptor data mentioned above, very little has
been done on elucidation of the signal transduction system activated by blFN-x in
the endometrium. In fact, one of the main objectives of this dissertation was to
provide evidence of existence, as well as details on the bIFN-x -activated,
endometrial JAK-STAT pathway.
Spencer and others (1998) conducted two studies to detect induction of
interferon-stimulated transcription factors, IRF-1 and IRF-2. Both factors were
absentJn cyclic ewes and present in pregnant ewes (days 11 and 13, cyclic and
days 13,15 and 17, pregnant). In cyclic ewes with ligated uterine horns,
unilateral infusion ovine IFN-x induced expression of IRF-1 and IRF-2 but not the
uterine horn receiving a BSA infusion. Since expression of these factors is
contingent on a functional JAK-STAT pathway, these data support existence of
such a pathway in the endometrium. Bathgate and coworkers (1998) also


272
Fu X-Y, Schindler C, Improta T, Aebersold R, Darnell jr JE. The proteins of
ISGF-3, the Interferon transcriptional activator, define a gene family
involved in signal transduction. Proc Natl Acad Sci USA 1992; 89:7840-
7843.
Fu XY, Zhang JJ. Transcription factor p91 interacts with the epidermal growth
factor receptor and mediates activation of the c-fos gene promoter. Cell
1993; 74:1135-1145.
Fuchs A-R, Fields MJ. Parturition, non-human mammals. In: Knobil E, Neil JD
(eds.), Encyclopedia of Reproduction, vol. 3. New York: Academic Press;
1999: 703-716.
Fuentealba B, Nieto M, Croxatto HB. Estrogen and progesterone receptors in
the oviduct during egg transport in cyclic and pregnant rats. Biol Reprod
1988; 39:751-757.
Gandolfi F, Brevini TAL, Richardson L, Brown CR, Moor RM. Characterization of
proteins secreted by sheep oviductal epithelial cells and their function in
embryonic development. Development 1989: 106:303-312.
Geisert RD, Fox TC, Morgan GL, Wells ME, Wettmann RP, Zavy MT. Survival of
bovine embryos transferred to progesterone-treated asynchronous
recipients. J Reprod Frtil 1991; 92:475-482
Gerhartz C, Heesel B, Sasse J, Hemmann U, Landgraf C, Schneider-Mergener
J, Horn F, Heinrich PC, Graeve L. Differential activation of acute phase
response factor/STAT3 and STAT1 via the cytoplasmic domain of the
interleukin 6 signal transducer gp130. I. Definition of a novel
phosphotyrosine motif mediating STAT1 activation. J Biol Chem 1996;
271:12991-12998.
Ghislain JJ, Fish EN. Application of genomic DNA affinity chromatography
identifies multiple interferon-alpha-regulated Stat2 complexes. J Biol
Chem 1996; 271:12408-12413.
Godkin JD, Bazer FW, Moffatt J, Sessions F, Roberts RM. Purification and
properties of a major, low molecular weight protein released by the
trophoblast sheep blastocycts at day 13-21. J Reprod Frtil 1982; 65:141-
150.
Gorlich D, Mattaj JW. Nucleocytoplasmic transport. Science 1996; 271:1513-
1518.


75
Objectives of This Dissertation
1) To study the distribution pattern of oviductal secretory proteins secreted
by cows bearing persistent or fresh dominant follicle;
2) To examine the signal transduction system stimulated by blFN-t in
endometrium;
3) To characterize the effects of blFN-x on PGF2 production by BEND
cells.


255
accumulation. Using this system it was determined that PDBu (probably through
stimulation of PKC) stimulates both protein expression and enzymatic activity of
PLA, and COX-2 in BEND cells. Moreover, blFN-x was able to regulate PGF2a
secretion also through modulation of protein expression and enzymatic activity of
either or both enzymes. Perhaps regulation of PLA, activity is a critical feature of
bIFN-x-mediated PGF2ainhibition. In the experiment reported by Arnold and co- .
authors (1999), explants were obtained from cows that received either blFN-x or
control protein (bovine serum albumen) infusions in tero. Explants were treated
with intracellular stimulators of PGF2a secretion, and PGF2tlwas measured in
culture medium. In vivo treatment with blFN-x failed to inhibit PGF2o secretion
stimulated by melittin, a PLA2 activity stimulator. If inhibition of PLA2 activity is
essential for inhibition of PGF2asecretion, it is possible that treatment with
melittin overcame blFN-x-induced inhibition, resulting in PGF2oproduction. It is
important to note that when endometrium from pregnant vs. cyclic cows were
tested for PGF2secretion in response to the melittin, pregnancy effectively
blocked melittin induced PGF2asecretion (Arnold et al., 1999). This supports the
notion that blFN-x needs to act in concert with other embryonic factors to fully
suppress luteolytic mechanisms. Identification of such factors warrants
investigation. Studies by Graf and co-authors (1999) and Asselin and co-authors
(1997) indicate that regulation of PLA2 activity in vivo is a preferred hypothesis
compared to regulation of gene expression. Abundance of PLA2 mRNA and


136.
200pg/ml) and anti-STAT-3 (C-20; catalog number SC-482, 200pg/ml) antibodies
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-
phosphotyrosine (PY-20, 1 mg/ml) antibody was from Transduction Laboratories
(Lexington, KY). Enhanced Chemiluminescence kit (Renaissance Western
Blot Chemiluminescence Reagent Plus) was from NEN Life Science Products
(Boston, MA).
Cell Culture and Cell Extracts
BEND cells (Austin et al., 1996b; Staggs et al., 1998) were grown to
confluence in culture medium (40% Hams F-12, 40% MEM, 10 ml AbAm/l, 200
U insulin/I, 0.0343g D-valine/L, 10% fetal bovine serum, 10% horse serum;
complete culture medium) in cell culture flasks at 37 C in a C02 incubator
(Sanyo, model MCO 17AI, Sanyo Electric Co., Ltd, Japan) under a humidified
atmosphere containing 95% 02 and 5% C02. Upon reaching confluence cells
were collected from culture flasks and were used (1) for experiments, (2) plated
on new culture flasks to maintain a stock of growing cells for subsequent
experiments and/or (3) frozen for maintaining a frozen stock. To collect cells
from culture flasks, medium was discarded and 10 ml of trypsin solution were
added to the flask and placed in the incubator for 5 minutes. Gentle tapping of '
corners of the flask was used to dislodged cells. Five ml of complete culture
medium were added to the flask to stop trypsin reaction, and all contents from
the flask were transferred to a 15 ml conical polypropylene tube. Cells were


58
Interferon-directed gene activation
After translocation to the nuclear compartment, STAT complexes can act
as transcription factors, to direct expression of interferon-induced genes. The
best studied transcription activation complex containing STAT dimers is called
interferon-stimulated gene factor 3 (ISGF-3), which is composed of a STAT 1:2
dimer and a nuclear DNA binding protein, p48 (Darnell et al.t 1994; Bluyssen et
al., 1996). ISGF-3 was first identified in electrophoretic mobility shift assays as a
complex induced by interferon treatment. It was formed independent of protein
synthesis, and was found to bind to consensus sequences on the regulatory
region of interferon-stimulated genes (Kessler et al., 1988). Consensus
sequences are known as interferon-stimulus response elements (ISREs).
Williams and Haque (1997) present a summary of sequences of ISREs of known
interferon-induced genes. A second interferon-induced transcription-activation
complex also was identified and named ISGF-2 (Kessler et al., 1988). Such a
complex is formed contingent on protein synthesis, presents different pattern of
migration in mobility shift assays and was later identified as the transcription
factor interferon regulatory factor 1 (IRF-1; Parrington et al., 1993). Interestingly,
IRF-1 and p48 are from the same family of proteins and can bind to the same
promoter elements (i.e., ISREs) in the regulatory region of interferon-stimulated
genes (Kessler et al., 1988; Parrington et al., 1993). The p48 and STAT 1:2
dimer do not associate in a stable manner to form the ISGF-3 complex in the
absence of DNA. However, contacts of amino acids 150 to 250 in the STAT-1


256
protein did not change during the estrous cycle in sheep (Graf et al 1999) and
blFN-x failed to affect PLA2 gene expression in endometrial epithelial cells
(Asselin et al., 1997). Similarly, Boos (1998) monitored changes in
immunoreactive COX enzymes (both 1 and 2) in endometrial biopsies obtained
from a given animal at days 1,8, 15 and 19 of the estrous cycle (n=10). There
was not a remarkable variation in levels of COX throughout the estrous cycle,
also supporting the concept that modulation of activity of COX is associated with
PGF2aduring luteolysis.
Another intriguing possibility for regulation of PGF2asecretion by blFN-x
involves fine regulation of PKC activity. The fact that blFN-x inhibited PDBu-
stimulated PGF2asecretion and PDBu stimulates PKC activity supports the
concept of blFN-x inhibition of PKC. Since bovine GCP-2 is induced by blFN-x
(Staggs et al., 1998) and also is stimulated by phorbol esters, part of the blFN-x
effects could be exerted through PKC. Finally, melittin not only stimulates PLA2
activity, but also inhibits PKC activity (Gravitt et al., 1994). There could be fine
regulation of PKC subtypes to yield differential responses modulated by melittin,
PDBu and blFN-x.
The endometrium presents a interesting model for studying alternative
modes of action of type I interferons. The required down regulation of the PGF2o
generation system by blFN-x is in variance with effects of other type I interferons .
in other cell systems. For example, interferon-p stimulates tyrosine


218
In Experiment 2 treatment contrasts were (c1) control vs PDBu and blFN-x
+ PDBu and (c2) PDBu vs. blFN-x + PDBu. Contrasts c1 and c2 were calculated-
using dish within treatment as the error term. Time contrasts were 0 vs. 3, 6, 9,
and 12 (c3), 3 vs. 6, 9 and 12 (c4), 6 vs. 9 and 12 (c5) and 9 vs. 12 (c6).
Treatment by time interactions were obtained by multiplying c1 and c3, c1 and
c4, c1 and c5, c1 and c6, c2 and c3, c2 and c4, c2 and c5, c2 and c6.
In Experiment 3, treatment contrasts were the same as for Experiment 2
but time contrasts were 1 vs. 2, 3, 4, 5 and 6 (c7), 2 vs, 3, 4, 5 and 6 (c8), 3 vs.
4, 5 and 6 (c9), 4 vs 5 and 6 (c10), 5 vs. 6 (c11). Treatment by time interactions
were obtained by sequentially multiplying c1 and c2 by c7, c8, c9, c10 and c11,
as exemplified for Experiment 2.
In Experiment 4, treatment contrasts were control vs PDBu, PDBu + blFN-
t and PDBu + blFN-x 3h (c12), PDBu vs PDBu + blFN-x and PDBu + blFN-x -3h
(c13), PDBu + blFN-x vs. PDBu + blFN-x-3h (c14). Contrasts c12, c13 and c14
were calculated using dish within treatment as the error term. Time contrasts
were 0 vs 1, 2, 3, 4, 5 and 6 (c15), c7, c8, c9, c10 and c11. Treatment by time
interactions were obtained by sequentially multiplying c12, c13 and c14 by c15,
c7, c8, c9, c10 and c11, as exemplified for Experiment 2. In Experiment 4, a
separate analysis was performed in which only PDBu and PDBu + blFN-x-3h
treatments were included. This analysis was conducted on untransformed data,
since significant heterogeneity of variance was not detected. The PGF2a
secretion data were further analyzed by homogeneity of regression. This


131
response to bIFN-x in vitro. However, there was a 99% mortality rate (as
measured by trypan blue exclusion), and the system was considered inadequate
for such purpose. This was in contrast to Charpigny and others (1999), which
were able to measure conversion of labeled arachidonic acid into several
prostaglandin classes using this system with ovine endometrium. A second
purpose was to compare the abundance of PLA2and COX-2 in cells obtained
from cows previously treated with control protein (BSA) or bIFN-x in vivo. Since
PDBu-stimulated PGF2aproduction was attenuated in explants from these same
cows treated with bIFN-x in vivo (Arnold et a!., 1999), it was hypothesized that a
lower abundance of PLA2 and COX-2 would explain the PGF2 results. However,
similar abundance of both enzymes indicates that bIFN-x regulation of PGF2ois
exerted through regulation of cellular activity of these enzymes.
In conclusion, the experiments reported in this Chapter indicated that
bIFN-x stimulated synthesis of endometrial proteins which may correspond to the
previously described bGCP-2, bUCRP and Mx proteins. Such proteins may
have specific roles on the process of maternal recognition of pregnancy,
however, such roles have not been confirmed. In addition, bIFN-x stimulates a
functional JAK-STAT pathway in the endometrium. Yet, an alternative in vitro
model of study will be required for further understanding of details of the blFN-x-
induced signal transduction in the endometrium and its relationship with the
antiluteolytic actions of bIFN-x. Scraped endometrial epithelial cells proved not


191
mg/ml in water) and 5 pi of phenol saturated in 10 mM Tris 1 mM EDTA pH 8
buffer (TE) were added. The mixture was loaded onto a Sephadex-G10
chromatographic column. The chromatographic column was prepared to purify
fractions containing radiolabeled probe from the remaining reaction mixture. A
disposable borosilicate Pasteur pipette (5.75") was secured in a ring stand with a
clamp and used as a column. Autoclaved glass wool was packed at the bottom
of the column and moistened with TE buffer. A slurry of Sephadex G-10 beads
(50 % beads:50%TE buffer, prepared previously and kept at 4C) was added to
the column until beads were packed to approximately 5 mm above the ridge of
the column. The TE buffer was added continuously to the top of the column to
prevent beads from drying. When the labeling mixture was ready, the column
was allowed to run dry, and reaction mixture was loaded immediately onto the
column. Then, while adding TE buffer to the column in 100 pi volumes, 100 pi
fractions were collected into microcentrifuge tubes. Fractions were monitored for
radioactivity using a portable geiger counter and the three most active fractions
were pooled. Five pi of tRNA solution, 30 pi of 8 M ammonium acetate (kept at -
20 C) and 700 pi 100% ethanol (Kept at -20 C) were added to the combined
fractions. This entire mixture was hand mixed and incubated at -80 C for 2
hours. Then, mixture was thawed, centrifuged at 12000 x g for 15 minutes,
supernatant carefully aspirated and discarded. Pellet (not visible) was washed in
200 pi 70% ethanol (kept at -20 C), centrifuged at 12000 x g for 5 minutes,
supernatant removed and discarded. Remaining ethanol was removed by


38
al., 1994). As discussed beforehand, PGF2(xgalns access to the ovary through a
counter-current mechanism. Binding of PGF2c,to receptors in the CL stimulates
release of luteal oxytocin that in turn binds oxytocin receptors in the
endometrium to elicit further release of PGF2c,, characterizing a positive feedback
loop.
Arnold and coauthors (1999) demonstrated that a responsive uterus is not
necessarily a oxytocin-responsive uterus. They Incubated endometrial explants
obtained from day 17 cyclic cows with oxytocin or with intra-cellular stimulators of
the PGF2c,-generating cascade described above. They showed that despite
oxytocin failure to stimulate PGF2a secretion, the stimulator of PKC activity,
phorbol 12, 13 dibutyrate (PDBu), and the stimulators of PLA2 activity, calcium
ionophore and melittin, were able to induce PGF2ct release acutely. This supports
the notion that alternative ligands to oxytocin can play a role on pulsatile
secretion of PGF2cl.
A final comment on the role of P4 -priming of the uterus for pulsatile PGF2a
secretion, relates to P4 ability to induce accumulation of lipid droplets in bovine
uterine epithelial cells (Brinsfield and Flawk, 1973). In mice, such lipid droplets
contain phospholipids (Silvia et al., 1991), which are substrate for PLA2 and
source of AA, as mentioned above. Progesterone also Induces synthesis of
COX-2 (Raw et al., 1988).
Pregnant uterus. In cattle, the vast majority of embryos are found in the
uterine horn ipsilateral to the ovary where ovulation occurred, indicating a readily


247
There is a much higher percentage of embryonic losses during the
window of maternal recognition of pregnancy associated with CL maintenance,
compared to during the time gametes/embryos are in the oviduct (5 to 10% vs.
30% respectively, Henricks et al., 1971; Diskln and Sreenan, 1980). This could
be attributed simply to the fact that the oocyte/embryos only remain in the
oviduct for 72 to 84 hours (Betteridge and Flechon, 1988) vs. 14 days from when
they first arrive to the uterus to the time of maternal recognition of pregnancy
associated with maintenance of CL. However, I would like to postulate that one
main reason for this discrepancy in embryonic losses is associated to the degree
of maternal-embryonic interactions occurring in these two periods. In other
words, while in the oviduct, there is less exchange of signals occurring
(associated with less mortality), it is the opposite in uterus. Although critical
studies have not being performed, there is little evidence for embryonic
modulation of oviductal function. There is strong evidence for a role of the
oviductal environment to affect processes igamete maturation and transport,
fertilization and early embryo development (Gandolfi et al., 1989; Parrish et al.,
1989; Anderson and Killian, 1994; Boatman and Magnoni, 1995; Hill etal., 1996;
Buhi et al., 1997;), but such processes appear to be vastly regulated through the
action of ovarian steroids (Buhi etal., 1991; Murray, 1993), and role of the
embryo is negligible. So, although there is communication occurring, it is largely
unidirectional. This is in complete contrast with processes occurring in the
uterus during maternal recognition of pregnancy associated with the time of CL


249
The fact that pregnancy (Arnold et al., 1999) and blFN-x (Meyer et al.,
1995) are able to modulate PGF2c, secretion from the uterus prompted our
laboratory to study the nature of the signal transduction system stimulated by
blFN-x in the endometrium to exert such an effect. Since bIFN-t is a type-l
interferon, we hypothesized that blFN-i activated the classical JAK-STAT
pathway and formulated our working hypothesis: binding of blFN-x to
endometrial receptors activates the JAK-STAT pathway and induces synthesis of
bIFN-x-induced proteins, which function to downregulate the PGF2a synthetic
machinery. Data from Chapters 4, 5 and 6 confirmed presence of a functional
JAK-STAT pathway in the endometrium (Figure 5-23). Intracellular actions of
extracellularly added blFN-x suggest the presence of a functional blFN-x
receptor. Immunoblots confirmed presence, homo- and heterodimer formation
and nuclear translocation of STAT proteins. Tyrosine phosphorylation of STAT
proteins indicated presence of JAK kinases. Electrophoretic mobility shift assays
confirmed biological activity of nuclear localized STATs, which bound to specific
DNA sequences present in the regulatory region of interferon-induced genes.
Finally, blFN-x induced synthesis of intracellular, secretory and nuclear (IRF-1)
proteins in endometrial cells and explants. Flowever, data presented in this
dissertation does not provide a direct link between activation of JAK-STAT
pathway and blFN-x -induced suppression of PGF2a. However, several
possibilities can be considered after examination of results (Chapters 4, 5 and 6)


23
Regulation of Reproductive Processes Occurring in the Oviduct
As summarized by Hafez (1993b) and discussed by Harper (1982) and
Anderson (1991), transport of unfertilized and fertilized eggs and sperm in the
oviduct is regulated by four primary forces: (1) frequency and force of
contractions of the oviductal musculature, influenced by endocrine and neural
mechanisms; (2) direction and intensity of beating of cilia, which conditions
movement of oviductal fluids; (3) secretory activity of non-ciliated cells, which is
dependent on the E2/P4 ratio; and (4) hydrodynamic properties of luminal fluids.
Changes in these factors are modulated by concentrations of ovarian steroids.
The outcome of these activities is efficient transport of gametes and embryos
and fertilization. Next, I will emphasize the concerted actions of these factors for
the mechanisms of egg pick up and fertilization.
Egg pick up. At the time of ovulation, there is a noticeable increase in
frequency and amplitude of contractions in the smooth musculature supporting
the oviduct. Contractions of the mesotubarium superior and mesosalpinx draw
the oviduct in a crescent shape and slide the fimbriae over the surface of the
ovary. The fringe-like folds in the INF contract rhythmically to repeatedly touch
the ovarian surface (Hafez, 1993a). This pattern of movements constitutes an
efficient mechanism to pick up ovulated oocytes. Moreover, maximum density of
ciliated cells In the oviduct occur In the INF. During ovulation, the strokes of cilia
in the fimbriated portion of the oviduct are synchronized to propel the oocyte


46
second messengers to ultimately regulate molecules involved in the generation
of PGF2a. Such regulation could involve synthesis of proteins inhibitory to the
PGF2 the cell to suppress PGF2c, stimulatory actions. There is limited information on
the nature of IFN-t receptors. Knickerbocker and Niswender (1989) measured
numbers of unoccupied binding sites for IFN-t in endometrium of cyclic and
pregnant sheep. Number of unoccupied binding sites decreased for both cyclic
and pregnant ewes from day 4 to day 12. Then it increased for cyclic animals,
but was still decreased for pregnant ewes, indicating that blFN-x binding sites
were possibly being occupied by conceptus-secreted IFN-t. Interestingly, affinity
for binding sites increased after day 12 for pregnant ewes but decreased for
cyclic ewes.
Hansen and coauthors (1989) reported use of cross-linking experiments
to characterize association of iodinated ovine IFN-t to membrane peptides.
They identified binding of IFN-t to both 100 and 70 kD membrane polypeptides.
Comparison of binding kinetics of IFN-t with IFN-a in this experiment suggested
existence of different receptors for these two ligands. However, Li and Roberts
(1994) showed a reciprocal displacement of IFN-t and IFN-a from bovine
endometrial cell membranes, suggesting that binding sites for these two
molecules were the same. Recently, Han and Roberts (1998) reported cloning
and characterization of receptors for IFN-t in cattle endometrium. Sequences of'


186
endometrial epithelial cells. Complexes of phosphorylated STATs may act as
transcription factors to activate transcription of early response genes. Nuclear
extracts obtained from BEND cells stimulated with blFN-t have factors able to
bind ISREs In mobility shift assays, and treatment with bIFN-x Induces synthesis
of the interferon regulatory factor 1 (IRF-1; Chapter 6). This suggests that
activated STATs are functional and can stimulate gene transcription. Moreover,
treatment of BEND cells with bIFN-x Inhibits the phorbol ester-induced
stimulation of PGF2a (Chapter 7). Our working hypothesis is that blFN-x-
stlmulated proteins act on endometrial epithelial cells to decrease secretion of
PGF2o (Figures 2-3 and 5-23).
One way to confirm that blFN-x-induced phosphorylated STAT complexes
present in the nucleus are biologically active is to test the ability of proteins in
nuclear extracts to bind to specific cis-actlng response elements in
electrophoretic mobility shift assays. Chapter 6 tests this concept and also
examines whether bIFN-x has the ability to stimulate IRF-1 in BEND cells.


18
oviduct can be divided into three functional regions: the funnel shaped
abdominal opening near the ovary INF, which terminates in the fringe-like
fimbriae; the more distal dilated AMP and the 1ST, the narrow proximal portion of
the oviduct, connecting to the uterus (Hafez, 1993a). The oviduct can be simply
described as a muscular tube with a mucosal lining. There are two muscle
coats: an external longitudinal and an internal circular coat (Leese, 1988).
Thickness of the musculature increases from the ovarian to the uterine end of
the oviduct. Muscular contractions function to mix oviductal contents, aid sperm
transport, help dendate the egg, promote fertilization and regulate egg
transport. Patterns of oviduct muscular contractions vary with the stage of the
estrous cycle, indicating hormonal regulation of this process. Before ovulation,
contractions are gentle, but become more vigorous at ovulation. Muscular
contractions in the ovarian direction are more common than in the uterine
direction (Hafez, 1993a). The oviductal mucosa possesses characteristic folds,
with high, branched folds in the AMP and decreasing heights towards the 1ST to
become low ridges. The mucosa consists of one layer of columnar epithelial
cells, underlined by a submucosa containing smooth muscle fibers and
connective tissue. The oviductal epithelium contains both ciliated and non-
ciliated, secretory cells. Ciliated cells are most abundant in the INF and least in
the 1ST. Rate of cilia beating is affected by levels of ovarian hormones, with
maximal activity occurring at the periovulatory period. Cilia beating is
synchronized and toward the uterus. The opposite direction of coordinated cilia


39
"attachable" embryo, which only migrates minimally within the uterus (Flood,
1991). Embryos undergo rapid morphological changes In the first 3 weeks of
pregnancy. After a series of cellular divisions, formation and hatching of
blastocysts (day 9-10 after ovulation; Betterldge and Flechon, 1988),
conceptuses start to elongate on day 12 (Betteridge et al., 1980), to occupy the
whole length of the uterine horn ipsilateral to the CL by day 17 and to reach the
tip of the contralateral horn on day 21 (Kastellc et al., 1988; Flood, 1991). The
first intimate connection between the conceptus and the uterus occurs between
days 18 and 20 of pregnancy, when numerous papillae penetrate the openings
of uterine glands (Guillomot et al., 1981). There Is intimate contact of maternal
and embryonic tissues starting with apposition of apical cell membranes of
aligned epithelia from both units. Actual adhesion begins around day 22 and is
completed on day 27 after insemination. Adhesion Is characterized by
interdigitatlon of embryonic and maternal microvilli (Flood, 1991). The next
series of events Include development of placentomes and growth of placental
tissues.
Maternal recognition of pregnancy associated with CL maintenance
As mentioned previously, the turning point in the uterine cycle Is the
commitment to either luteolysls or pregnancy. In cattle, commitment to
pregnancy Is only accomplished If adequate signaling exists between maternal
and embryonic units. Maternal recognition of pregnancy has been defined as
the process by which the periattachment conceptus signals Its presence to the


263
in vitro versus in vivo techniques is lacking, but would be crucial to understand
long term effects of the In vitro technique. In summary, it is still open to debate
the validity of rescuing pregnancies destined to failure. However, with current
intensive management systems of high performance animals (e.g., lactatlng
dairy cow) and decreased reproductive performance, modifications of
endogenous mechanisms to improve embryonic survival that optimize maternal-
conceptus interactions may improve reproductive performance, propagate
genetically superior animals and further enhance food production for human
consumption. Furthermore, such knowledge may be utilized to regulate
reproductive rates in both animals and humans.


182
receptor chains (Ihle et al., 1995; Figure 5-23). Phosphorylation of the receptor
attracts unphosphorylated STAT proteins, present in the cytosol, to come in
contact with the receptor, where STATs become phosphorylated on tyrosine
residues (Greenlund et al., 1994). Phosphorylated STATs form homo- and
heterodimers and translocate to the nucleus (Shuai et al., 1994; Schindler and
Darnell, 1995). In the nucleus, STAT complexes associate with DNA-binding
proteins, such as p48, to form a transcription activation complex which binds to
specific response elements on IFN-regulated genes (Darnell et al., 1994;
Schindler and Darnell, 1995). Response elements can be for example the
interferon-stimulus response element (ISRE, Levy et al., 1988) and the sis-
inducible element (SIE, Sadowsky et al., 1993). Our working hypothesis is that
proteins synthesized in response to bIFN-T act to decrease secretion of PGF2a.
If blFN-x-induced or -repressed protein synthesis is a result of activation of the
classical JAK-STAT pathway, translocation of tyrosine -phosphorylated STAT
proteins to the nucleus is expected. Data in the present report support this
possibility.
Generally, blFN-x induced an increase in abundance of STAT proteins in
the nucleus over time, which reached a plateau and then decreased. Except for
numerical differences for STAT-2, this occurred without reciprocal changes in
abundance of STATs in CE. This could indicate that only a small fraction of
STATs present in the cytosol actually migrated to the nucleus and the
immunoblot technique used was not sensitive enough to detect a decrease of


288
Subramaniam PS, Khan SA, Pontzer CH, Johnson H. Differential recognition of
the type I interferon receptor by interferons t and a is responsible for their
disparate cytotoxicities. Proc Natl Acad Sci USA 1995; 92:12270-12274.
Sun T, Lei ZM, Rao CV. A novel regulation of the oviductal glycoprotein gene
expression by luteinizing hormone in bovine tubal epithelial ceils. Moll
Cell Endocrinol 1997; 131:97-108.
Tanabe TY, Cassida LE. The nature of reproductive failure of cows of low
fertility. J Dairy Sci 1949; 32:237-246.
Teixeira MG, Austin KJ, Perry DJ, Dooley VD, Johnson GA, Francis BR, Hansen
TR. Bovine granulocyte chemotactic protein-2 is secreted by the
endometrium in response to interferon-tau (IFN-x). Endocrine 1997; 6:31-
37.
Thatcher WW. Ruminants. In: Knobil E, Neill J (eds), Encyclopedia of
reproduction, vol4. San Diego: Academic Press; 1999:302-311.
Thatcher WW, Binelli M, Burke J, Staples CR, Ambrose JD, Coelho S.
Antiluteolytic signals between the conceptus and the endometrium.
Theriogenology 1997; 47:131-140.
Thatcher WW, de la Sota RL, Schmitt E J-P, Diaz TC, Badinga L, Simmen FA,
Staples CR, Drost M. Control and management of ovarian follicles in
cattle to optimize fertility. Reprod Frtil Dev 1996; 8:203-217.
Thatcher WW, Driancourt MA, Terqui M, Badinga L. Dynamics of ovarian
follicular development in cattle following hysterectomy and during early
pregnancy. Domest Anim Endocrinol 1991;8:223-234.
Thatcher WW, Hansen PJ. Systems to alter embryo survival. In Van Horn HH,
Wilcox CJ (eds), Large dairy herd management. Champaign: Am Dairy
Sci Assoc; 1992:164-175.
Thatcher WW, Macmillan KL, Hansen PJ, BazerFW. Embryonic losses: cause
and prevention. In: Fields MJ, Sands RS (eds.), Factors affecting calf
crop. Boca Raton: CRC Press Inc; 1994a: 135-153.
Thatcher WW, Meyer MD, Danet-Desnoyers G. Maternal recognition of
pregnancy. J Reprod Frtil 1995 (suppl);49:15-28.


infundibulum, ampulla and isthmus from oviducts ipsilateral and contralateral to
CL of cows bearing PDF or FDF were examined by two-dimensional
fluorography. Presence of PDF altered distribution of secretory proteins in a
side- and region-specific manner. Changes in the oviductal environment may
contribute to decreased fertility of cows bearing a PDF. Conceptus-produced
bIFN-T suppresses endometrial PGF2ct pulses in vivo, and is required for
maintenance of pregnancy. The hypothesis was that bIFN-x stimulated
synthesis of endometrial proteins through the jak kinases (JAK)-signal
transducer and activator of transcription (STAT) pathway of signal transduction.
Presence of and bIFN-x-induced tyrosine phosphorylation of STAT proteins were
demonstrated via immunoprecipitation (IP) and immunoblotting (IB) techniques,
while bIFN-x-induced secretory proteins were measured by fluorography in
endometrial explants obtained from day 15 cyclic cows. BEND cells were used
for remaining experiments. Presence of STATs, tyrosine-phosphorylation, dimer
complex formation and nuclear translocation were measured through IP, co-IP
and IB. Binding of activated STAT complexes to cis-acting elements present in
the regulatory region of interferon-inducible genes was determined using
electrophoretic mobility shift assays. Bovine IFN-x stimulated synthesis of
interferon-regulatory factor-1 (IRF-1) in BEND cells as determined by IB. The
bIFN-x regulates synthesis of phorbol 12,13 dibutyrate (PDBu)-induced PGF2o,,
as measured through radioimmunoassays (RIA). Regulation was associated
XII


33
(Kindahl et al., 1976). It was determined that for most of the estrous cycle, basal
secretion of PGFM ranged from 25 to 70 pg/ml In one heifer and from 60-100
pg/ml In a second heifer. However, around the time of luteolysis, four peaks of
about 500 pg/ml and four peaks of about 250 pg/ml were observed for the first
and second heifers, respectively (Kindahl et al., 1976). Despite the clear among
animal variability in this small experiment, there was an evident decrease in P4
concentrations, from ~5 ng/ml to less than 1 ng/ml, within 24 hours after the first
PGFM peak for both heifers.
Pulsatile release of PGF- Generation of PGF2a pulses requires presence
of a stimulatory signal and a responsive uterus. A responsive uterus contains
receptors for the stimulatory signal, functional intracellular pathways to transduce
the stimulus into a secretory pulse and adequate amounts of substrate for PGF2ol
synthesis.
In cattle, nature of the stimulatory signal for production of luteolytic pulses
of PGF2o remains unclear. It has been accepted generally that oxytocin is the
major stimulator of PGF2o, secretion in cattle. Armstrong and Hansel (1959)
demonstrated that exogenous oxytocin caused luteolysis in heifers. Moreover,
injections of oxytocin increased concentrations of PGF2ain the uterine vein
(Milvae and Hansel, 1980) and increased concentrations of PGFM in peripheral
circulation (Lafrance and Goff, 1985) in cows. However, while oxytocin is able to
stimulate PGF2cl secretion in these experiments, it remains unclear whether
oxytocin is in fact required for the process of luteolysis. In a recent report,


118
a
COW
2215
5354
5497
blFN-r, 50 ng/ml
-
+
-
+
-
+
28
19- J
1 9
1 2
b
control Interferon
-r 6
o
P19 P12
Figure 4-1. Fluorographic analysis of newly synthesized and secreted
proteins from endometrial explants incubated for 24 h in presence (+) or
absence (-) of blFN-t.
a) Representative fluorograph of one dimensional SDS-PAGE
analysis of secretory proteins (arrows indicate differentially expressed
proteins P19 and P12; b) Densitometric analysis of abundance of P19 and
P12 (least squares means SEM).


81
each cow for 7 days. One day prior to CIDR removal, cows received an injection
Pre-Treatment Period
Treatment Period
GnRH
(+/-)
I
J
CIDR
CIDR
I
0 6
I I
7 0 5
n
7
I
9
I
16 18
Estrus
Estrus
Slaughter
Ultrasonography, Blood Collection
Figure 3-1. Experimental protocol (see text).
of prostaglandin-F2a (PGF2a Lutalyse, 25 mg) to regress the CL. To aid with
estrus detection, tail heads were painted (Impervo) and chalked (All-weather
Paintstick). Cows were observed twice daily for signs of estrus, and paint scores
were assigned (Macmillan et al., 1988). The day of standing estrus was
designated experimental day 0. During the treatment period, ovaries were
examined by transrectal ultrasonography using an Aloka echo camera model
SSD 500 linear array ultrasound scanner equipped with a 7.5MHz transducer
(Aloka Co., Japan). From Days 5 to 18, follicles and CL were measured daily
and sizes recorded. In addition, blood samples were collected in heparinized
evacuated tubes (Vacutainers, Becton Dickson Vacutainer System USA,


8
physiology must be changed to suppress this pulsatile release of PGF2afor the
conceptus to survive. The conceptus-secreted interferon-x (IFN-x) interacts with
the uterine tissue to decrease production of PGF2a, thereby allowing for
maintenance of CL and consequent sustained elevated P4 concentrations.
The process of luteolysis includes the action of follicular estrogen on a P4-
primed uterus, which is capable of secreting PGF2a. Driancourt and coworkers
(1991) formulated the hypothesis that one possible aspect of the antiluteolytic
mechanism induced by conceptus could involve attenuation in development of
follicles on the ovary adjacent to the pregnant uterine horn. They determined
that the number of follicles greater that 7 mm was reduced in the ovary
containing the CL after day 22 of pregnancy. Moreover, size of the largest
follicle was greater on the ovary contralateral to the pregnant uterine horn. An
additional study comparing follicular development in pregnant versus
hysterectomized cows indicated that products of pregnancy, either secreted
directly or induced by the conceptus, decreased intraovarian follicular
development in a local manner (Thatcher et al., 1991). This could enhance
embryonic survival by attenuating luteolytic mechanisms (Thatcher et al., 1994b).
The examples above illustrate the common theme of maternal-gametic
and maternal-embryonic interactions, and their occurrences throughout the
reproductive cycle. Failure of appropriate communication between maternal and
embryonic units can lead to disruption of the reproductive cycle and termination
of pregnancy. Next I will examine the issue of embryonic mortality in cattle. In


21
found with intact animals, where a greater incorporation rate was found in the
periestrus stage of the estrous cycle.
It is important to keep in mind that functional regions within the oviduct
have specific roles probably associated with particular arrays of secretory
products. Thus, it is expected that different steroid environments (e g., estrous
cycle vs. pregnancy) have distinct effects on each oviductal region,
characterizing a biosynthetic gradient of proteins across regions. For example,
in studies with bulls (Anderson and Killian, 1994), it has been demonstrated that
culture medium conditioned by 1ST tissue at estrus capacitated more sperm than
did medium conditioned by AMP. This increase was abolished by heating the
conditioned medium and inactivating proteins before incubation with sperm.
Staros and Killian (1998) showed that four unidentified oviductal proteins and a
P1-like protein (Boice et al., 1990; Binelli et al., 1999; Chapter 3) from non-luteal
oviductal fluid would associate with the zona pellucida, suggesting a modulation
of sperm/egg binding or embryonic development by oviduct-derived proteins.
Biosynthetic protein gradients have been reported in the pig and sheep (Buhi et
al., 1992; Buhi et al., 1996; 28, DeSouza and Murray, 1995; Murray, 1993).
Moreover, DeSouza and Murray (1995) reported differential secretion of a
chitinase-like protein, similar to P1 in response to steroid treatments in sheep,
while Buhi et al. (1996) showed differential expression POSP mRNA among
oviductal regions in pigs.


72
conducive for embryonic attachment, and should bear intracellular mechanisms
to receive and transduce antiluteolytic signals from the conceptus that ultimately
inhibit the default, PGF2a-secretory pathway of the uterus. Thatcher and Hansen-
(1992) reported that day 17 conceptuses varied in size from 15 to 250 mm.
Since inhibition of PGF2Js probably dependent on total amount of blFN-r
secreted and on area of endometrium occupied by the conceptus, smaller
conceptuses would have already a smaller chance of survival. Environmental
effects such as heat stress (discussed above) decrease conceptus development
and apparently compromises ability of the conceptus to secrete bIFN-t, leading
to failure in pregnancy recognition. There is also evidence for a role of the
uterus to stimulate secretion of bIFN-t by conceptus. Hernandez-Ledezma and
coworkers (1992) cultured IVF (in vitro fertilization)-produced embryos to
blastocyst stage and either continued in vitro culture or transferred conceptuses
to synchronized recipient cows. Embryos were recovered 4 days later, placed in
culture dishes and secretion of blFN-x was quantified. Secretion of blFN-x was
highly stimulated by exposure to the uterine environment, indicating that optimal
production of the antiluteolytic signal by the conceptus is not solely determined
by the conceptus. Stojkovic and coworkers (1999) reported that bovine embryos
derived by embryo flushing and in vitro production produced more bIFN-t in long
term culture than embryos derived from nuclear transfer or embryo splitting Such
differences may contribute to lower pregnancy rates following embryo transfer to


25
musculature contractions (Ellington, 1991; Hafez 1993b). Within the 1ST, sperm
undergo hyperactivation, which is required for final sperm transport, completion
of sperm capacitaron and the acrosome reaction. Eventually, spermatozoa
become exposed to ampullary fluid, detach from the 1ST epithelium and continue
migration towards the site of fertilization. The control of concerted, opposite
direction-movement of sperm and eggs at similar times in the oviduct is
intriguing. Perhaps the isthmoampullary junction acts to retain oocytes in the
ampulla, while spermatozoa are allowed to enter the 1ST (Anderson, 1991). Low
doses of estrogen cause "tube locking, retaining ova at the isthmoampullary
junction, while larger doses promote quick movement through the Isthmus and to
the uterus (Hawk, 1988).
Oviductal Function and Reproductive Failure in Cattle
The fact that oviductal function is regulated In the multi-factorial,
integrated fashion described above could lead one to hypothesize that
perturbations in the system could easily lead to reproductive failure. However, in
normal cattle, embryonic losses occurring during the time when the embryo is in
the oviduct are small, relative to other phases, as described above. This could
be interpreted at least in two ways. First, one could say that the oviduct plays
only a passive role on the processes of gamete transport, fertilization and early
embryonic development. In this view, the oviduct would keep default modes of
function (i.e., similar in presence or absence of an embryo), modulated by


221
a
o 78 ^mi ; i 4*-" -cox-2
T \\
* 39.5
1 2 3 4 5 6 7 8
b
Figure 7-2. Experiment 1. Immunoblotting analysis of COX-2 in whole cell extracts from BEND
cells treated with medium alone (control), blFN-x (50 ng/ml; bIFN-tau), phorbol 12,13 dibutyrate
(100ng/ml; PDBu) or blFN-x and PDBu for 24 hours.
a) Enhanced chemiluminescence (ECL) exposure of abundance of COX-2 (control:
lanes 1 and 2; PDBu: lanes 3 and 4: blFN-t lanes 5 and 6; blFN-x and PDBu: lanes 7 and 8);
b) Least squares means and SE of abundance of COX-2 arbitrary densitometric units (ADU).


135
Materials and Methods
Materials
Reagents and materials utilized for preparation of cell extracts,
immunoprecipitation and immunoblotting are the same as described in Chapter
4, except where noted. Monoclonal anti-vimentin clone V9 antibody (catalog
number V6630; 6.2 mg/ml lgG1), monoclonal anti-pan cytokeratin antibody
(catalog number C2562; 45.6 mg total protein/ml), Hams F-12 (catalog number
N6760), MEM (catalog number M7395), antibiotic-antimycotic solution (AbAm;
catalog number A9909), insulin (catalog number I5500), D-valine (catalog
number V1255), horse serum (catalog number H1138) and trypsin solution were
from Sigma Chemical Co.(St. Luis, MO). Fetal bovine serum was acquired from
Atlanta Biologicals (catalog number S11550; Norcross, GA). Fifteen ml
propylene tubes and 100 x 16 mm polypropylene centrifuge tube were from
Fisher Scientific (Pittsburgh, PA). Tissue culture dishes were from Corning
Glass Works (Corning, NY). Recombinant blFN-x (dissolved in 20 mM Tris-HCI,
1 mM EDTA, pH 8.0 to 200 pg/ml; 1.08 x 107 units of antiviral activity) was a
generous gift from Dr. Michael Roberts (University of Missouri). Cell scraper and
cell culture flasks (175 cm2, polystyrene with vented cap; catalog number
83.1812.002) were from Sarstedt, Inc. (Newton, NC). Anti-STAT-1 (E-23;
catalog number SC-346, 200pg/ml), anti-STAT-2 (C-20; catalog number SC-476,


83
functional region were cultured (Buhi et al., 1990) in LEU-deficient minimal
essential medium supplemented with 3H-LEU in the ratio of 100 mg tissue/3 mL
medium/20 mCi 3H-LEU for 24 hours at 37C in a controlled atmosphere of
N2:02:C02 (50%:47.5%:2.5% by volume). For AMP and INF, 500 mg of tissue
were cultured per dish, while for 1ST variable amounts of tissue (between 140
and 290 mg) were used.
Two-Dimensional Electrophoresis
After 24 hours incubation, conditioned media were dialyzed extensively
(MW cut-off 3500) againstTris buffered saline (10 mM Tris, 150 mM NaCI) pH
7.6 (two changes of 4 liters each/24 hours) and then dialyzed against deionized
water (two changes of four li