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Identification, characterization, and regulation of porcine oviductal plasminogen activator inhibitor-1 and functional analysis of oviductal-specific secretory glycoprotein

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Identification, characterization, and regulation of porcine oviductal plasminogen activator inhibitor-1 and functional analysis of oviductal-specific secretory glycoprotein
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Kouba, Andrew J., 1968-
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vii, 220 leaves : ill. ; 29 cm.

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Embryos ( jstor )
Fertilization ( jstor )
In vitro fertilization ( jstor )
Messenger RNA ( jstor )
Oocytes ( jstor )
Oviducts ( jstor )
Proteins ( jstor )
Spermatozoa ( jstor )
Swine ( jstor )
Zona pellucida ( jstor )
Animal Science thesis, Ph. D ( lcsh )
Dissertations, Academic -- Animal Science -- UF ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1999.
Bibliography:
Includes bibliographical references (leaves 193-219).
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Printout.
General Note:
Vita.
Statement of Responsibility:
by Andrew J. Kouba.

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IDENTIFICATION, CHARACTERIZATION, AND REGULATION OF PORCINE
OVIDUCTAL PLASMINOGEN ACTIVATOR INHIBITOR-1 AND FUNCTIONAL
ANALYSIS OF OVIDUCTAL-SPECIFIC SECRETARY GLYCOPROTEIN












By

ANDREW J. KOUBA


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
























This Dissertation is dedicated to Brenda and Jerry Kouba.














ACKNOWLEDGMENTS

First and foremost, I need to thank God for the accomplishment of this

dissertation. Many times when the path became difficult and the end seemed so far away,

His gentle hand would continue to push me forward. Throughout my graduate career,

His graciousness has provided me with countless blessings. My relationship with and

faith in God is the responsibility of two wonderful individuals to whom I owe a great deal

of gratitude. My parents, Brenda and Jerry Kouba, have been a constant source of

support, encouragement, and love. They were my first teachers and continue to play such

a role in my life. At a very young age, they encouraged me to make observations,

formulate questions, and test my hypothesis; hence, my parents were the first to teach me

the scientific method. I also owe a great deal of thanks to my siblings, Bonnie and Joann

Kouba, who have always been supportive of my endeavors.

Second, I would like to thank my mentor, Bill Buhi, for his advisement during my

time at the University of Florida. I am deeply grateful for his acceptance of me into his

laboratory and the time that was involved in my education. I will always treasure his

guidance and our numerous conversations on what it means to become a scientist. I also

need to thank members of my committee-Pete Hansen, Ken Drury, Frank Simmen, and

Bill Thatcher-for their assistance throughout my program. Their suggestions in the

design and analysis of my experiments are greatly appreciated and I look forward to

future collaborations with all of them.








Next, I would like to thank Idania Alvarez for the often difficult task of training

me in numerous aspects of protein biochemistry. During my graduate career, several

students, post-docs, and secretarial staff have aided, and I thank them for all their

assistance-Lalantha Abeydeera, Brant Burkhardt, Dan Arnold, Morgan Peltier, Lannett

Edwards, Michael Porter, Carrie Vance, Mary Ellen Hissem, and all those students who

heat-checked every morning with me. I also thank Billy Day and Maureen Goodenow

for allowing me to conduct experiments in their laboratories for the completion of this

dissertation. Lastly, heartfelt thanks go to all those friends who have constantly been

there for me and supported me throughout this journey you know who you are.
















TABLE OF CONTENTS


ACKN OW LEDGM ENTS ................................................ .....................iii

LIST OF TABLES... .................................... .................. ..... ....... .... .vii

LIST OF FIGURES .................. ....... ....... ... ...... .................. ... ...viii

ABSTRACT................... .. ............... ........ .. x

CHAPTERS

1 INTRODUCTION............................. ......... ... ............... 1

2 LITERATURE REVIEW

Biology of the Oviduct........... ............................... ..... ......... .............
Proteins, Growth Factors, and Cytokines of the Oviduct..............................19
Oviduct Secretions and Co-culture on Fertilization and Embryonic
Development....................... ................... .................. 26
Sperm-Oviduct Interactions........... ........................................ 29
Structure, Regulation, and Biological Actions of Plasminogen Activator
Inhibitor-1 ..................................... ................ ........ 31
Structure, Regulation, and Biological Actions of Porcine Oviductal-Specific
Secretory Glycoprotein....................................... ....... ............ 41

3 IDENTIFICATION AND LOCALIZATION OF PLASMINOGEN
ACTIVATOR INHIBITOR-1 (AI-1) IN THE PORCINE OVIDUCT

Introduction... ....... .................................... ....58
M materials and M ethods ................... ......... .......... .......... ... ... .........60
Results....................................... ............. .... ..... ........... 66
D discussion .......................... ....... ...............83









4 OVIDUCTAL PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1)
PROTEIN, mRNA, AND HORMONAL REGULATION

Introduction... ............. ... ... ........................... ...................... 89
Materials and Methods........... ............. ...... .. ...... ... ..............91
Results.......................... ... ........... ... ...... 98
D discussion .......1......... .............................................. .......109

5 PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) AND uPA ACTIVITY
IN THE OVIDUCT AND ASSOCIATION OF PAI-1 WITH THE
PREIMPLANTATION EMBRYO

Introduction............................................................. ............... 115
Materials and Methods...................... .............. ...... .... ......117
Results................... ...................................... 123
Discussion .......... .... ........... ...................... .................. 146

6 EFFECTS OF A PORCINE OVIDUCT-SPECIFIC GLYCOPROTEIN ON
FERTILIZATION, POLYSPERMY, AND EARLY EMBRYONIC
DEVELOPMENT IN VITRO

Introduction................... ............ ............................. ............. 151
Materials and Methods................... ................ .. ............ 153
R esults................... ............. .................. ......... .....162
Discussion........................................ ...... ............... 176

7 SUMMARY AND CONCLUSIONS............................................... 183


REFERENCES............................. .......................................... 193

BIOGRAPHICAL SKETCH...... ........... .................. .................220















LIST OF TABLES


Table Pag

6-1 Effect ofpOSP on fertilization parameters of pig oocytes matured and fertilized in
vitro..................... ................ ................ .. ............. 167

6-2 Effect ofpOSP on zona pellucida solubility of pig oocytes matured in vitro....170















LIST OF FIGURES


Figure ag

2-1 Pericellular activation cascade for plasminogen and matrix metalloproteinases...57

3-1 Representative fluorograph after 2D-SDS-PAGE separation of['H]-labeled
proteins (100,000 cpm) from explant culture medium conditioned by the three
segments of the oviduct .................. .................................72

3-2 Representative Sepharose CL-6B and Sephadex G-100 fractionations of [H]-
labeled proteins in isthmus-conditioned medium.............................74

3-3 Comparison of the identified N-terminal amino acid sequence of the 45,000 Mr
protein from isthmic-conditioned culture media to the N-terminal sequence of
porcine PAI-1 .............. ........ ............... .......... ........ ... ..... 75

3-4 Immunoprecipitation ofplasminogen activator inhibitor-1 from porcine isthmic-
conditioned culture media after fractionation on a heparin-agarose affinity
column and elution with 0.4 M NaCI..............................................76

3-5 Representative immunocytochemical localization of PAI-1 in the isthmus from
crossbred gilts on Day 0 (Panel A), Day 2 (Panel B) or Day 12 (Panel C) of
pregnancy............................... ...... .. .... ....................77

3-6 Representative immunocytochemical localization of PAl-1 in oviductal tissue
from Day 2 cyclic (Panel A and C) and Day 2 pregnant (Panel B and D)
crossbred gilts from the isthmus (Panel C and D) and ampulla (Panel A and
B )........................................................... ..... ...... 78

3-7 Immunogold labeling ofPAI-1 within oviductal tissue from a Day 0 non-pregnant
crossbred gilt ........... ... .. ................ ................. 80

3-8 Immunogold labeling of PAI-1 within oviductal tissue from a Day 9 pregnant
crossbred gilt ................................ ............ ..... ........ ........ 82

4-1 Densitometry ofPAI-l protein in fluorographs from 2D-SDS-PAGE analyses of
isthmic- and ampulla-conditioned medium (100,000 cpm) from Large White
and Meishan gilts during early pregnancy.......................................102








4-2 PAI-1 protein in oviduct flushes from crossbred gilts during early pregnancy... 103

4-3 Densitometry of PAI-I protein in fluorographs from 2D-SDS-PAGE analyses of
isthmic-conditioned medium (100,000 cpm [3H]-leucine) from OVX cross
bred gilts................ .... ..... ............ ..... ... ......... .... ..... 104

4-4 Dot-blot hybridization analyses of oviductal total RNA from cyclic crossbred
gilts................... ................. ................... .............. ........... 105

4-5 Dot-blot hybridization analyses of oviductal total RNA from the infundibulum,
ampulla, and isthmus of crossbred gilts...........................................107

4-6 Dot-blot hybridization analysis of whole oviductal total RNA from OVX steroid-
treated crossbred gilts.......................... .......... ...... .... ..........108

5-1 Representative fluorograph of denaturant-activated or latent PAI-1 incubated in
the presence ofuPA after non-reducing 1D-SDS-PAGE separation..........126

5-2 Oviductal PAI-1 inhibition ofuPA activity over time.............................128

5-3 Dose-dependent inactivation of uPA activity by activated PAI-1.................129

5-4 Inhibition ofuPA activity with activated PAI-1 (40 pg) 30 minutes after initiation
of the enzymatic reaction (cleavage of the chromogenic substrate, 2-44x, by
uPA)............................. .... ...... ................130

5-5 Plasminogen activator activity in oviduct flushes from early pregnant crossbred
gilts....................................................... .............. .............131

5-6 Dose-dependent inhibition ofuPA activity by amiloride.........................132

5-7 Inhibition ofoviductal flush PA activity by amiloride .............................133

5-8 Electron micrograph of a pig preovulatory follicular oocyte with surrounding
cumulus cells....................................... .............. .............. 135

5-9 Immunogold localization ofPAI-1 in association with pig preovulatory follicular
oocytes and cumulus cells......................... ..............................137

5-10 Immunogold localization of PAI-1 in association with oviductal oocytes........139

5-11 Immunogold localization of PAI-I in association with oviductal embryos (2-4
cell) and spermatozoa attached and embedded in the zona pellucida......... 141

5-12 Immunogold localization of PAI-1 in association with early uterine embryos (8-16
cell)................................................... ... ........ .......... ...... .... 143











5-13 Controls for immunogold localization of PAI-1 in association with follicular
oocytes, oviductal oocytes and embryos, and uterine embryos................145

6-1 Representative fluorograph of [3H]-leucine labeled proteins (500 pg) from whole
oviduct explant culture media (Day 0/1) subjected to heparin-agarose affinity
column chromatography and separated by 2D-SDS-PAGE..................166

6-2 Photograph ofpolyspermic porcine oocyte matured and fertilized without
exposure to pOSP ...... ..................................... ...................168

6-3 Effect of in vitro incubation of pOSP and anti-pOSP IgG on the polyspermy rate
of porcine oocytes matured and fertilized in vitro...............................169

6-4 Effect of in vitro incubation ofpOSP on sperm binding to porcine putative
zygotes.................. ..... ..... ............ ...................................... 171

6-5 Photograph of sperm binding to putative zygotes after pre-incubation and
fertilization in the presence of 0 pg/ml pOSP (A) or 100 pg/ml pOSP (B).. 173

6-6 Effect of in vitro incubation ofpOSP on cleavage rate using porcine oocytes
matured, fertilized and cultured in vitro........................................174

6-7 Effect of in vitro incubation of pOSP on blastocyst development using porcine
oocytes matured, fertilized and cultured in vitro. ............................175

7-1 Model for the de novo synthesis and secretion of proteins by the porcine
oviduct........... ............................. ........................... 192














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

IDENTIFICATION, CHARACTERIZATION, AND REGULATION OF PORCINE
OVIDUCTAL PLASMINOGEN ACTIVATOR INHIBITOR-1 AND FUNCTIONAL
ANALYSIS OF OVIDUCTAL-SPECIFIC SECRETARY GLYCOPROTEIN

by

Andrew J. Kouba

December 1999

Chairperson: William C. Buhi
Major Department: Animal Science

In mammals the oviduct de novo synthesizes and secretes numerous proteins that

may act to facilitate the processes of fertilization and early cleavage-stage embryonic

development. Several of these proteins have been shown to be steroid-modulated and

have cycle-specific expression. However, the majority of these proteins remain

unidentified and have only been characterized in a limited fashion by molecular weight

and isoelectric point. Functional data relating to these proteins is especially lacking. The

first objective was to identify and characterize the major de novo secretary product of the

isthmus. The second objective was the examine potential functions of the well-

characterized pOSP during in vitro fertilization and embryo culture.

The major de novo secretary product of the isthmus was identified as plasminogen

activator inhibitor-1 (PAI-1). It was demonstrated that this protein is localized to

oviductal epithelium and packaged into putative secretary granules for exocytosis. Upon








release into the oviductal lumen, PAI-I was shown to associate with oviductal oocytes,

embryos, and spermatozoa. Characterization of this protein revealed that (1) estrogen

inhibits mRNA and protein expression, (2) mRNA and protein is greatest during

fertilization and early embryo development, and (3) once activated, PAI-1 can form a

complex with uPA and inhibit its activity. In addition, PAI-1 levels are greatest when PA

activity was highest. These results suggest an important role for the regulation of the

plasminogen/plasmin system during fertilization and early embryonic development.

Functional analysis of pOSP revealed several potential actions of this protein

during fertilization. It was demonstrated that pOSP significantly reduced the incidence of

polyspermy in pig oocytes fertilized in vitro. After fertilization in the presence ofpOSP,

a reduction in the number of sperm cells attached to the zona pellucida was observed.

This may be a potential mechanism whereby pOSP reduces the number of sperm cells

that can attach and penetrate an egg. Pig oocytes fertilized in the presence of pOSP had

increased blastocyst development compared to the control. Therefore, the reduction in

the rate of polyspermic fertilization may have resulted in enhanced development to

blastocyst. The pOSP reduction in polyspermy could be inhibited by anti-pOSP

indicating that the decreased incidence of polyspermic fertilization was specific to pOSP.

The characterization and functional analysis of these two proteins, PAI-I and

pOSP, indicates an important relationship between their expression, association with the

oocyte or embryo, and potential actions during fertilization and development.















CHAPTER 1
INTRODUCTION


This dissertation will attempt to enlighten and convey to the reader the importance

of the mammalian oviduct as not only a conduit for the transport of gametes but also as

an important reproductive organ that contributes to the well-being and development of

the embryo. Numerous studies have been done indicating that a pregnancy can be

established without the presence of features and structures that are located within or are

specific to the oviduct. Perhaps the most pervasive example is that of in vitro fertilization

(IVF), in which an oocyte is matured, fertilized, and developed in vitro with subsequent

transplantation of the embryo directly into the uterus, resulting in a successful pregnancy.

Techniques such as IVF, which remove the oocyte and spermatozoa from contact with

the tubule, have generated much debate on the question of whether the mammalian

oviduct is an essential part of the female reproductive tract.

Over the past decade, numerous advances have been accomplished for embryonic

development in vitro, especially during oocyte maturation and fertilization. These

advances have been primarily due to careful formulation of culture medium and use of

co-culture with oviductal epithelial cells. Interpretation of these studies has led to two

prevalent hypotheses. One view is that the oviduct has a more passive role in terms of

establishing an environment with an optimum pH, temperature, osmotic pressure,

nutrients, and oxygen tension for fertilization and early cleavage-stage development. The

second is the oviduct has a more active role, investing energy into the expression and

I







2

synthesis of molecules, which may facilitate or regulate these events. Evidence to date

suggests that these two views are not mutually exclusive but are cooperative in the

formation of a microenvironment, which sustains fertilization and early cleavage-stage

development. Perhaps the importance of this reproductive organ has to be viewed with

respect to its importance in the establishment of a species population. An insightful

paragraph by Dr. Ronald H. F. Hunter in the book The Fallopian Tubes (1988) touches on

this theory:



For most biologists, successful reproduction needs to be considered in
population terms, and all features that contribute to the stability of a
breeding population, and thereby to perpetuation of the species, need to be
brought to mind. It is in this light that the many specific contributions of
the Fallopian tubes are best interpreted. Whilst individual morphological
and biochemical components may not be of overriding importance in
themselves for the establishment of single pregnancies, together they can
be viewed as contributing to the successful maintenance of a breeding
population.


Although embryonic development in vitro has recently seen significant advances,

scientists are still far from reaching a degree of development and viability similar to that

seen in vivo. In virtually every reported case, cultured embryos exhibit reduced cell

numbers, decreased viability with increasing duration of culture, and reduced

development to later stages when compared with their counterparts growing in vivo

[Bavister, 1988]. These observations indicate that some important characteristics of the

embryonic milieu are missing from the artificial culture environment. Peptide and

macromolecules are the oviductal constituents most suspected of having specific

embryotrophic actions, particularly de novo synthesized and secreted products of the

epithelium. The first objective was to identify an unknown major de novo secretary









product of the porcine isthmus. Sequence analysis revealed this protein to be porcine

plasminogen activator inhibitor-1 (PAI-I). Experiments were designed to characterize

this protein throughout the estrous cycle or early pregnancy and to evaluate its

association with the pre-implantation embryo. The second part of this dissertation

concerns the functional analyses of a well-characterized oviductal protein, porcine

oviductal secretary glycoprotein (pOSP), during fertilization and early embryonic

development. It may be that with the identification and functional analysis of specific

oviductal molecules, conditions for in vitro development of embryos may come to mimic

more closely that of the in vivo situation. This dissertation will present data that indicates

a more active role for this reproductive organ in the successful establishment of

pregnancy.















CHAPTER 2
LITERATURE REVIEW



Biology of the Oviduct

Historical Perspective

The first observations of the oviduct, date back to Aristotle (322-384 BC), who

noted a convoluted structure connecting the uterus to the ovary. However, a common

misinterpretation that perpetuated for several centuries was that the ducts transmitted

female semen to the urinary bladder where it was excreted. Accurate anatomical

descriptions were first made by Galen (AD 130-200), linking the ovaries to the uterus and

describing their termination in the uterine horns of bicornuate domestic animals.

However, his mistake was to perpetuate the concept that this tubule was a conduit for

female semen, which was filtered by the ovary from the bloodstream. This notion was

based upon his observation that the female Fallopian tubes were similar to the male

seminal vessels.

The idea of a tubule which transmitted female semen persisted for over 14

centuries. Descriptive anatomical drawings were not available until the excellent works

of Leonardo da Vinci (1452-1519) were published. Another anatomist, Andreas

Vesalius, extensively characterized the anatomy of the Fallopian tube in 1543 in a seven-

volume series known as De Humani Corporis Fabrica. His analyses of their function

echoed that of the past by supporting the hypothesis that these structures were responsible

4







5

for the transport of female semen. The critical structure of the oviduct and relationship of

this structure to the ovary and uterus were detailed by Fallopius, and due to his detailed

description of the tubule his name was lent to the structure (Fallopian tube). The

collected works of Harvey (1651), Van Home (1668), and de Graaf (1672) established

that female semen was a myth, but that instead an egg originates in the ovary (although

the follicle was misinterpreted as the egg), and that this egg is transported into the uterus

via the Fallopian tubes. It was not until significant advances in the technological

development of the microscope were made near the end of the 17-" century that a more

careful scrutiny of the oviduct and its structure, came about. For an excellent review on

the history of the oviduct see The Fallopian Tubes (1988), edited by Hunter and The

Mammalian Oviduct (1969), edited by Hafez and Blandau.

Prenatal Development and Macroscopic Anatomy of the Oviduct

The oviducts of adult mammals are specialized structures that originate during

organogenesis from the cranial region of the primitive Mullerian ducts [Price et al.,

1969]. Johannes Muller first described the Mullerian ducts in 1830, yet far less attention

has been directed toward the development of the oviduct in relation to the uterus.

Although the development of the oviduct prenatally differs widely among mammalian

species, several general statements can still be made. The oviduct undergoes

morphogenetic and histogenetic changes that distinguish it from the uterus which include

(1) coiling of the oviduct, (2) differences in size (diameter), (3) differentiation of

epithelium and muscle, (4) formation of mucosal folds, (5) development of the fimbriae,

and (6) development of the three segments infundibulum, ampulla, and isthmus [Price

et al., 1969]. For a detailed description of comparative development of the mammalian







6

oviduct, the reader is referred to The Mammalian Oviduct (1969), edited by Hafez and

Blandau.

The paired oviducts connect within the uterus at the uterine tubule junction (UTJ)

and link this structure to the ovary. The oviduct is convoluted in most species (except the

rabbit and man), and is supported by the mesosalpinx, which is a part of the broad

ligament [Nalbandov, 1969]. A specialized portion of the mammalian oviduct is often

referred to as the fimbriae infundibulum, which forms a fringed open funnel. In the pig,

which has open fimbriae, this is an intimate structure which encapsulates the ovary. In

species such as mouse and rat this structure forms a sac around the ovary called the bursa

ovarii. The fimbriae of the infundibulum are extremely motile and direct fluid movement

into the oviduct during the time of ovulation. In the pig, unilateral removal of the

infundibulum does not cause infertility as the contralateral fimbriae can redirect and

capture oocytes from the peritoneal cavity [Nalbandov, 1969]. The segment adjacent to

the infundibulum is referred to as the ampulla and has the largest diameter of the three

segments of the oviduct. The intricate folding of the mucous membrane is also

characteristic of this section. The diameter of the ampulla tapers down as it becomes the

isthmus which, in the pig, connects into the uterus at the tip of the uterine horn. The pig

oviduct averages 26-28 cm in length, with the isthmus comprising approximately 1/3 of

the overall length with the majority being made up of the ampulla.

The portion of the isthmus which enters the uterus is referred to as the uterotubal

junction (UTJ), and in most litter-bearing species such as the pig, it contains a high

density of folds and finger-like processes (villi) [Hafez and Black, 1969]. These

structures are arranged in such a fashion so as to prevent the passage of fluid and uterine







7

contents into the oviduct. Anderson (1927a) showed that in pigs, a greater amount of

pressure is required to force uterine fluid into the oviducts when eggs are present (Day 2-

4 of the estrous cycle) than during the luteal phase. This suggests that the UTJ maintains

the oviductal environment protecting it from contamination by secretions from the uterus.

As reviewed by Hunter (1988), the pig oviduct contains an outer layer of

longitudinal muscle and an inner layer of circular muscle. External to these muscle

layers is a well-vascularized serosa layer of tissue. The isthmus portion of the oviduct

has a greater amount of circular muscle, allowing for a greater contraction potential than

the other two segments. The circular muscle layers of the isthmus and ampullary-isthmic

junction are highly innervated with adrenergic nerve terminals originating in the ovarian

or hypogastric plexus. The innervation of the ampulla and infundibulum is very poor

compared to the isthmus, and is usually restricted to the walls of blood vessels. The high

density of circular muscle and adrenergic innervation of the isthmus indicates an

important role for this segment in the transport of gametes and embryos.

The lymphatic vessels of the pig oviduct follow the ovarian and uterine lymphatic

drainage and show a similar concentration in the isthmus as was described for the

sympathetic innervation of nerve bundles. Anderson's (1927b) study of the domestic pig,

revealed that cyclical variations could be noted in lymphatic development and that these

changes might be due to ovarian steroids. It has been suggested that the lymphatics

might be involved in transmission of embryonic information to either the ovary or uterus,

since the greatest amount of tubule resistance to the embryo is found within the isthmus.

The vascular supply to the oviduct is supported by the uterine and ovarian

arteries, and varies according to the estrous cycle or pregnancy. The capillary bed and







8

larger blood vessels of the infundibulum and ampulla become engorged with blood close

to the time of ovulation and are regulated by estrogen [Hunter, 1988]. This increase in

blood supply may also account for the increased tubule secretion and transudation in the

pig at estrus.

Microscopic Anatomy of the Oviduct

The architecture of the ampulla and isthmus is centered on an epithelium with

numerous tubal mucosal folds that show cyclical changes in morphology. The actual

space within the oviduct lumen is very negligible (unlike the lumen of a gland).

Therefore, gametes often remain in intimate contact with the epithelial lining as opposed

to floating free in an aqueous environment. This epithelial lining is composed of simple

columnar and modified cells of the goblet cell type, which are secretary in nature. There

are two distinct differentiated cell types within the oviduct, referred to as ciliated and

non-ciliated secretaryy), although this is not absolute as sometimes differentiating cells

display both cilia and secretary granules [Jansen and Bajpai, 1982]. Interspersed

throughout the ciliated cells are non-ciliated cells that contain electron-dense secretary

granules that can be identified throughout the cell but are most often found in the apical

region. These secretary granules exude their contents with greater frequency during

ovulation, contributing to the decline in epithelial height shortly thereafter [Hunter,

1988]. In most domestic species, epithelial cell height and secretary activity are greatest

at the time of ovulation.

Estrous Cycle of the Pig and Hormonal Regulation by Ovarian Steroids

The influence of ovarian steroid hormones on the oviductal environment has been

well documented in several species for ciliogenesis, secretion, contractility, and







9

morphology of the tubal mucosa and musculature [Hunter, 1988]. It is difficult to

discuss these processes in relation to the oviduct without first describing some aspects of

the pig estrous cycle and its changing hormonal environment. A prepubertal gilt becomes

sexually mature at 6-8 months of age and is described as polyestrous, which means a lack

of seasonality in its reproductive cycle. The average cycle length for post-pubescent gilts

is 21 days, with Day 0 being the onset of standing estrus. The average length of estrus is

2-3 days, with ovulation occurring 30-40 hours after the onset of estrus. A pig is a

polytoccous, or litter bearing, animal and ovulation occurs over a time period of 1 to 5

hours [Hunter, 1988]. Estrogen begins to rise several days prior to estrus (proestrus) and

returns to basal levels by Day 2 of the estrous cycle [Karlbom et al., 1982]. Progesterone

levels begin to rise by Day 2 of the estrous cycle in conjunction with development of the

corpus luteum, reaching a peak around Days 14-15 and quickly declining thereafter

[Karlbom et al., 1982].

Ovarian steroids are transported to the oviduct through the blood [Giorgi, 1980]

which then bind to specific cytosolic receptors and are transported to the nucleus [Chan

and O'Malley, 1976]. If steroid receptors are absent in the oviduct, the tissue becomes

refractory to the action of that hormone. This provides a unique model in which

oviductal function can be examined in intact and ovariectomized animals. Tissue

concentrations of estradiol and progesterone receptors in the porcine oviduct were first

examined by Stanchev et al. (1985). These researchers looked at both cytoplasmic and

nuclear receptors for estrogen (ER) and progesterone (PR) throughout the estrous cycle.

The ampulla showed cyclic variations in the concentration of cytoplasmic and nuclear

ER, increasing during proestrus, reaching a maximum concentration during standing







10

estrus and declining by Days 3-4 of the cycle. The ampulla also showed changes in

cytoplasmic and nuclear PR levels during the estrous cycle, with increasing

concentrations found from standing estrus through the luteal phase. The isthmus had a

much lower concentration of cytoplasmic and nuclear ER than the ampulla, and isthmic

cytoplasmic ER showed no cyclic variation. The isthmus and ampulla showed similar

levels of PRs and also had a similar expression of nuclear PR during the estrous cycle.

However, no changes were noted for levels of cytoplasmic PR in the isthmus during the

estrous cycle. These studies indicate that the isthmus has a constant low-level expression

of cytosolic receptors, while nuclear receptor concentrations vary according to the

hormonal state of the animal. The ampulla, which is believed to be more sensitized to the

effects of estrogen and progesterone than the infundibulum or isthmus, revealed cyclic

changes in both cytoplasmic and nuclear receptors during the estrous cycle. The higher

proliferative and secretary nature of the ampulla during the preovulatory and ovulatory

period may account for these differences in ER expression [Iritani et al., 1974].

Contractility and Ciliogenesis of the Oviduct

Oviductal muscular activity has been examined throughout the estrous cycle, and

although investigators are not in complete agreement, it appears that the most vigorous

contractions occur at about the time of estrus and ovulation. Observations done nearly 76

years ago in the pig revealed that the strength of oviductal muscular contractions was

related to the dimensions of the preovulatory follicles [Seckinger 1923, Wislocki and

Guttmacher 1924]. The larger the diameter of the follicle the stronger the contractions.

As reviewed by Boling (1969), the predominant movement is peristaltic, and

contractions occur in localized segments or loops rather than traveling long distances.







11

These contractions are primarily abovarian in direction. Kymographic records

radiographicc examination in which the range of involuntary movements are recorded) of

the pig oviduct revealed that the ampulla undergoes regular and mild contractions, while

those of the isthmus are more vigorous [Kok, 1926]. Decreased contractility has been

noted in the castrate rabbit although this activity did not become completely quiescent

[Greenwald, 1963]. Compared to intact animals, a delay in tubal egg transport was

observed in castrate animals. Greenwald (1967) showed that species differences are

observed in egg transport in response to exogenous estrogen. In the rat, single injections

of estrogen on Days 1, 2, and 3 of early pregnancy accelerate oviductal transport of ova

[Ortiz et al., 1979, 1991]. A recent study suggests that protein synthesis is required in rat

oviducts for the estrogen effect on oviductal transport to occur [Rios et al., 1997].

Several reports have indicated that estrogen and progesterone together regulate the

contractility and are summarized by Boling (1969). It was observed that the oviductal

musculature remains relatively quiet under the influence of estrogen, and begins to

contract more vigorously when estrogen is withdrawn. Likewise, tissue under the

influence of estrogen begins to contract vigorously shortly after an injection with

progesterone. These data indicate that perhaps estrogen has an inhibitory effect on

vigorous oviductal contractions. Much of these data appears contradictory, especially

since observations at or near estrus indicate this period as having the strongest

contractions. Contractions of oviductal musculature may be controlled by prostaglandins

of the F series. Prostaglandin F2a has been observed in both the Fallopian walls as well as

in oviduct fluid. Lippes (1979) found that PGF2. in human tubal fluid showed cyclical

variations and had higher pre-ovulatory than post-ovulatory levels. It may be that steroid







12

hormones and prostaglandins program the amplitude and frequency of contractions via

the regulation of calcium transport. However, information regarding control of oviductal

contractions is lacking.

Ciliogenesis refers to the growth and development of oviductal cilia throughout

the estrous cycle and pregnancy. Perhaps the greatest amount of information on this

subject is from investigations on the number and morphology of oviductal cilia

throughout the estrous and menstrual cycles and pregnancy. However, there are also

several studies using experimental procedures such as hypophysectomy and ovariectomy,

followed by steroid hormone replacement. As summarized by Brenner (1969),

overwhelming evidence suggests that estrogen stimulates the growth of ciliated cells and

progesterone antagonizes this effect. In the presence of prolonged progesterone, cells

become undifferentiated and difficult to clarify [Brenner et al., 1983]. Distinct

morphological changes have been noted in relation to a changing steroid hormone

environment in humans [Verhage et al., 1979] and rabbits [Brenner, 1969]. Although the

hormonal regulation of ciliogenesis is widely accepted, the number and character of

ciliated cells varies depending on the species studied, the portion of the oviduct sampled,

and the phase of the sexual cycle [Schaffer, 1908]. For example, in the domestic pig,

active ciliated cells have been seen at all stages of the cycle, without any significant

changes in their distribution, number, or activity [Snyder, 1923]. Only a change in cell

height was observed with a maximum occurring around the time of ovulation (Day 0-3)

and a minimum height about two weeks after ovulation. Observations by Buhi

(unpublished data) indicate that in the pig, gradients in the number of ciliated vs secretary

cells exist between the ampulla and isthmus. The pig isthmus contains about 80%







13

ciliated cells and 20% secretary cells (personal communication). During the luteal phase

of the pig, the distal portion of the ciliated cells become constricted from the rest of the

cell and are cut off in a process called deciliation [Flerko, 1954]. It has been proposed

that deciliated cells fill up with secretary granules and become active secretary cells.

Depleted secretary cells then become peg cells and are eventually lost at the end of the

cycle [Brenner, 1969]. As summarized by Hunter (1988), ciliary beat appears to be

regulated by circulating levels of ovarian hormones and is greatest at or near the time of

ovulation. In most mammals examined thus far, cilia of the ampulla beat in the direction

of the ampullary-isthmic junction [Blandau and Verdugo, 1976], suggesting a role for

cilia in the process of egg transport to the site of fertilization.

Fertilization and Early Embryogenesis in the Oviduct

In most mammals, new life begins in the oviduct through the union of germ cells

during fertilization. Wassarman (1999a) defines fertilization as the process of joining

two germ cells, egg and sperm, whereby the somatic chromosome number is restored and

the development of a new individual exhibiting characteristics of the species is initiated.

This process includes several ordered steps: final maturation of both male and female

gametes, sperm acrosome binding, acrosome reaction and penetration through the zona

pellucida, fusion of sperm and oocyte membranes, the corticol granule reaction, and

activation of the cell. During ovulation, fully-grown oocytes from antral follicles

undergo meiotic maturation to become oocytes that are now capable of interacting with

spermatozoa and being fertilized [Wassarman, 1999a]. Capacitation of spermatozoa

within the oviduct is viewed as the final phase of sperm maturation, conferring upon the

cells the ability to penetrate the egg investments [Hunter, 1995]. One consequence of







14

capacitation is an increased flagellar beat of spermatozoa, termed "hyperactivation,"

which is believed to assist in the penetration of the hamster zona pellucida [Yanagimachi,

1988]. A second consequence is membrane vesiculation due to point fusions on the

anterior portion of the sperm head, releasing proteolytic enzymes such as acrosin [Hunter,

1995]. The acrosome reaction has been suggested to be initiated by the binding of sperm

to the zona pellucida. It is generally accepted that one of the zona pellucida proteins,

ZP3, is the natural agonist for stimulation of the acrosome reaction in acrosome-intact

spermatozoa. Binding of spermatozoa to ZP3 activates G-proteins which stimulate a

change in intracellular pH and Ca++; however, the sperm receptors that activate these G-

proteins remain elusive [Wassarman, 1999b]. In the mouse, the zona matrix is composed

of three ZP glycoproteins, ZP 1-3. ZP3 and ZP2 dimers are located throughout the zona

pellucida and are crosslinked by ZPI to create a three-dimensional matrix [Wassarman,

1999b]. In the pig, rabbit, and non-human primates, ZP1 has been shown to be the

primary sperm receptor [Dunbar et al., 1998]. Although these proteins from different

species are conserved within each family, they exhibit distinct biological properties as the

mouse ZP3 is the primary sperm receptor.

Once sperm has penetrated the egg investments, its plasma membrane must bind

and fuse with the oocyte vitelline membrane. Fertilin-P, a sperm membrane-bound

protein, is thought to be the primary molecule responsible for binding of acrosome-

reacted sperm to the plasma membrane [Frayne and Hall, 1999]. This protein is a

member of the ADAM (disintegrin and metalloprotease domain) family of

transmembrane proteins. The disintegrin domain of fertilin-0 is believed to interact with

integrins on the oocyte plasma membrane, mediating the process of fusion. Recently, it







15

was shown that mice that are homozygous null for fertilin-P (fertilin-1 -/-) have a

significantly reduced fertilization rate [Wassarman, 1999b]. Shortly after spermatozoa-

oocyte membrane fusion, the quiescent egg "awakens" to initiate a series of biochemical

events known as egg activation. This activation leads to an explosive release of Ca"

from intracellular storage sites, a transient increase in intracellular pH, an irreversible

activation of oxidative pathways, lipid metabolism, and protein and DNA synthesis

[Yanagimachi, 1988]. This activation is believed to be initiated by paternal proteins

obtained during fusion. The massive release of intracellular Ca" stimulates a Ca"-

dependent exocytosis of corticol granules (CG) which have an important role in

modification of the egg envelope during fertilization. As reviewed by Yanagimachi

(1988), exocytosis in mouse eggs begins near the point of sperm-egg fusion and

propagates from this point in a wave-like fashion to the opposite side of the egg. The

CG, which contain hydrolytic enzymes and saccharide components, alter the physical and

chemical characteristics of the zona pellucida so that the zona becomes refractory to

sperm attachment and penetration. This occurs due to proteolytic modifications of the

sperm-binding ZP receptors. This process is referred to as the Zonaa reaction" and is a

key event for establishing a block to polyspermy in pigs [Hunter, 1991]. Once the sperm

nucleus is incorporated into the cytoplasm of the mature mouse egg (characterized by

ejection of the second polar body) there is a rapid breakdown of the nuclear envelope,

decondensation ofchromatin, and formation of the male pronucleus.

The formation of the female and male pronucleus and their subsequent migration

through the cytoplasm is due to microtubules and microfilaments of the cytosolic

framework [Yanagimachi, 1988]. Besides fertilization, the oviduct is also the site of







16

early cleavage-stage embryonic development. Compared to other domestic species, the

pig has a relatively short cleavage-stage development within the oviduct, entering the

uterus at about the 4-cell stage [Kopecny, 1989]. Cleavage is invariably asynchronous in

mammalian embryos cultured in vitro and development is retarded by about 2 h each cell

cycle, thus, the development to the blastocyst stage may be an entire day longer than

embryos in vivo [Pedersen, 1988]. This indicates that oviduct components may

contribute to the morphologic and metabolic regulation of the developing embryo,

leading to enhanced rates of development of in vivo compared to in vitro embryos.

Oviductal Fluid

Oviductal fluid which can be found within the tubal lumen throughout the

reproductive cycle varies in volume as well as composition and is most abundant when

gametes or embryos are present [Hunter, 1988]. Because tubal volume was found to be

greatest during estrus and near to or just after ovulation, it was suggested that circulating

ovarian steroids might regulate tubal fluid secretion. Bishop (1956) showed that after

ovariectomy in estrous rabbits, tubal fluid secretion dropped from 1.2 ml to 0.2 ml/24 hr

and that systemic injections of estradiol could restore fluid production to estrus volumes.

Comparable observations in tubal fluid accumulation have also been noted in the ewe,

cow, and pig [Hunter, 1988]. The pig was shown to have a fluid accumulation of 6.3

ml/24 hr during estrus falling to 2.1 ml/24 hr in the luteal phase [Iritani et al., 1974].

Another distinction seen between estrus and the luteal phase, is the direction of fluid

flow. Seminal experiments in tubal ligation revealed significant changes in tubal fluid

retention depending on the stage of the cycle. During estrus, the bulk of the fluid passes

from the ampulla into the peritoneal cavity generating a retrograde flow, however, by the







17

time embryos descend through the uterotubal junction, much of the reduced fluid volume

is being redirected toward the uterus [Hunter, 1988]. The restriction of tubal flow

towards the uterus at estrus, is thought to be primarily due to edema found within the

isthmic mucosa. Constriction of the isthmus is thought to be programmed by circulating

levels of estrogen and wanes as a result of elevating progesterone levels [Hunter,1988].

It is difficult to envision the relationship between tubal fluid flow and movement

of the gametes through the lumen. From the observations of Parker (1931), suggestions

have been made that a microflow system close to the epithelium and within the grooves

or channels of the surface may differ in direction from that of the bulk flow. This

microflow system may be intimately involved with the movement of spermatozoa or

oocytes through the oviductal lumen. Detailed chemical analyses of oviduct fluid has

revealed a complex mixture of constituents derived from plasma and the oviductal

epithelium including the oviduct-specific secretary macromolecules. The passage of

plasma constituents into the lumen has been referred to as transudationn.' Because

certain components in oviduct fluid are present at concentrations different from those in

plasma the term transudation was expanded to 'selective transudation' [Leese, 1988].

Therefore, the term 'secretion' has been applied to the formation and discharge of de novo

proteins from the oviductal epithelium. Protein concentration in oviductal fluid is 10-

15% that found in serum [Gandolfi, 1995]. The molecules and proteins involved are

transported across the epithelium within endocytic vesicles that travel from the

basolateral membrane to the apical surface where their contents are released into the

lumen [Parr and Parr, 1986]. The epithelial cell tight junctional complexes restrict

passage of proteins into the lumen between epithelial cells. During early pregnancy in







18

the pig, total recovered oviductal fluid protein was greatest on days 0-3 indicating a

cyclical variation in total protein [Buhi et al., 1998, Kouba unpublished]. Similar cyclic

changes in total protein have been observed during the human menstrual cycle [Lippes et

al., 1981].

Biosynthetic Activity

The mammalian oviduct is a highly synthetic and secretive tissue. The

biosynthetic capacity has been evaluated by measuring the incorporation of radiolabeled

precursor into secreted nondialyzeable macromolecules. Biosynthetic activity of the

entire oviduct was greatest during proestrus, estrus, and metestrus compared to other days

of the cycle in the pig [Buhi et al., 1989], sheep [Buhi et al., 1991] and cow [Malayer et

al., 1988]. Synthetic differences were not observed between mated/pregnant and cyclic

gilts, indicating that the presence of gametes or embryos does not affect the biosynthetic

activity. These findings are in contrast to that of the rabbit where pregnant animals were

shown to have decreased incorporation rates compared to non-pregnant animals [Roy et

al.,1972]. The greater activity during estrus suggests that ovarian steroid hormones may

regulate this function. In ovariectomized pigs [Buhi et al., 1992] or sheep [Buhi et al.,

1991], biosynthetic activity was greater with estrogen than with other steroid treatments.

Furthermore, pigs unilaterally ovariectomized showed no differences in biosynthetic

activity from either the contra- or ipsilateral oviduct containing an ovary with follicles or

corpus lutea. Therefore, circulating ovarian hormones produced by one ovary were

sufficient to control protein synthesis of both oviducts [Buhi et al., 1997].

A recent study showed that the presence of a persistent dominant follicle

decreased the synthetic activity of oviductal tissue in the cow [Binelli et al., 1999]. These







19

investigators suggest that prolonged exposure to high levels of estrogen may have caused

down-regulation of the estrogen receptor, thus suppressing biosynthetic activity.

Interestingly, fluctuations in the abundance of de novo synthesized proteins in animals

containing a persistent dominant follicle, compared to a fresh dominant follicle, were

observed. It was suggested by Binelli et al. (1999) that these fluctuations might be due to

a release of the inhibitory actions of estrogen on certain proteins. Of the three oviduct

segments, the ampulla had the greatest synthetic activity with estrogen and the lowest

with progesterone. Similar to the biosynthetic capacity of the entire oviduct, the capacity

of the ampulla was greatest on Days 0-4 of the estrous cycle, while the isthmus showed

no differences. Likewise, the ampulla was shown to have greater biosynthetic activity

than the isthmus regardless of day of the estrous cycle. Similar rates of activity were

found for the ampulla and infundibulum.

Proteins. Growth Factors, and Cvtokines of the Oviduct

Fluxes of molecules from the fluid bathing the serosal surface to those bathing the

mucosal surface are generated giving the oviductal epithelium its physiological

characteristics [Leese, 1988]. This flux includes water, electrolytes, metabolic non-

electrolytes, amino acids, steroids and proteins. The secretion of specific oviduct fluid

constituents will be discussed here in light of their possible contributions to gamete-

embryo interactions. The majority of this section will cover the identification of proteins

found within oviductal secretions, and the other constituents will be briefly highlighted.

Water is not thought to be transported into the oviductal lumen through an active process,

but rather in response to osmotic gradients established by the transport of ions [Leese,

1988]. Numerous ions have been identified in oviduct fluid including sodium, chloride,







20

potassium, bicarbonate, magnesium, calcium and inorganic phosphorous [Leese, 1988,

Ellington, 1991, Boatman, 1997]. As summarized in these reviews, electrolyte

concentrations in the oviduct vary considerably from those found in serum. Varying

electrolyte concentrations in culture medias during in vitro fertilization and embryo

culture have shown the importance of these constituents on fertilization and embryonic

development. The volume of literature on this topic is extensive and will not be covered

in this literature review. The presence of non-electrolytes in oviduct fluid has also been

reviewed [see Leese, 1988, Boatman, 1997]. Metabolic precursors found in oviduct fluid

include glucose, lactate, pyruvate and amino acids. Differences in concentrations of

these energy substrates in oviductal fluid and blood plasma of pigs during the peri-

ovulatory period have been observed [Nichol et al., 1992]. Several of these energy

substrates (pyruvate and lactate) are routinely used during in vitro fertilization. Two

amino acids, taurine and hypotaurine, have been shown to be present in oviductal fluid at

concentrations much greater than those found in serum [Guerin et al., 1995]. These

amino acids, as well as the converting enzyme that synthesizes hypotaurine from cysteine

intermediates, such as cysteine sulphinate decarboxylase [Guerin and Menezo, 1995],

have been localized to oviductal epithelial cells. Actions of taurine and hypotaurine

include; osmoregulation, calcium modulation, phospholipid interactions, membrane

protein receptor interactions, and antioxidation [Huxtable, 1992, Boatman, 1997].

Specific functions of these amino acids relative to the oviduct include; motility and

viability of sperm, progression through the cell block, improved development of zygotes

to the morulae or blastocyst stages, and increased cell numbers in vitro [Boatman, 1997].

The oviductal epithelium also produces another potent modulator of oxidative stress, the







21

antioxidant glutathione [McNutt-Scott and Harris, 1998]. Through addition of both

electrolyte and non-electrolyte energy substrates and careful formulation of culture

mediums to mimic oviductal concentrations of these constituents, improvements to in

vitro fertilization and development to blastocyst have been achieved. Therefore, the

relative contribution of these constituents to establishment of an appropriate oviductal

environment for fertilization and early embryonic development should be underscored.

Steroids present in the oviductal environment are not generally assumed to be

produced by the oviduct but arrive through serum transudation or from the ruptured

follicle and cumulus cells. However, one report suggests that the oviductal epithelium

contains the machinery for steroidogenesis [Ogunranti, 1992]. Many of the effects of

steroids on gamete function in the oviduct are mediated indirectly through the

vasculature, serosa, mucosa, and muscularis affecting protein secretion, gamete transport,

and fluid accumulation [Hunter, 1988, Hafez and Blandau, 1969]. Recent evidence

suggests that progesterone may have direct actions on the gametes, inducing

hyperactivation and the acrosome reaction in mammalian spermatozoa [Meizel, 1995,

Boatman, 1997]. Use of an anti-progesterone antibody in vivo showed decreased

cleavage rates and embryonic cell numbers [McRae, 1994]. The actions of progesterone

on spermatozoa are non-genomic in origin, and genomic actions of steroids on oviductal

gametes has not been reported. Similarly, there are no reported non-genomic actions of

steroids on metaphase II-oocytes through to the blastocyst stage [Boatman, 1997].

In the pig, analyses of oviductal fluid proteins reveals the presence of hundreds of

proteins, most of which originate from serum as transudate [Buhi et al., 1999]. The most

abundant serum proteins in oviductal fluid are albumin, heavy and light chains of







22

immunoglobulins and transferring. Similar to measurements of total protein in oviduct

fluid, electrophoretic analyses of serum proteins show cyclical variations in their relative

distribution [Buhi et al., 1999]. Additionally, several other proteins have been localized

to the oviductal epithelium and while these proteins have not been shown to be oviduct-

derived and secreted, deserve some mention. Numerous protease inhibitors and enzymes

have been detected including, but not limited to; alkaline phosphatase, amylase, lactate

dehydrogenase, diesterase, lysozyme, acid phosphatase, esterases, lipase, aminopeptidase,

glucosidase and galactosidase [Hunter, 1988]. These molecules may be important in

detoxifying the oviductal environment of substrates harmful to either gametes or

embryos. Recently, catalase was found in porcine, human, and bovine oviduct fluid

[Lapointe et al., 1998]. Catalase concentration was greatest near estrus, and could bind to

the acrosomal cap of spermatozoa. These investigators suggested that the ability of the

oviduct to maintain sperm viability was partially dependent on the ability of catalase to

protect sperm from oxidative damage.

Local countercurrent transfer of products from the ovary or the uterus to other

parts of the reproductive tract are well known. Molecules originating from these tissues

and found within oviduct epithelium or circulation include PGF2, and PGE2 [Harper,

1988], oxytocin [Schramm et al., 1986], and endothelin 1 [Rosselli et al., 1994]. In

addition, the oxytocin receptor has been identified in the oviduct of the ewe [Ayad et al.,

1990]. These molecules have been shown to be important stimulators of oviductal

muscular activity. Local distributions of prostaglandin, oxytocin, and endothelin-1 in the

bovine oviduct were shown to be cyclic and greater concentrations were detected at

estrus in association with elevated levels of oviductal estradiol [Wijayagunawardane et







23

al., 1997]. In the pig, LH/hCG receptors [Gawronska et al., 1999] and receptor mRNA

[Derecka et al., 1995] have been identified. It was observed that LH/hCG receptors were

greatest at the preovulatory stage of the estrous cycle, and caused relaxation of the

oviduct. It may be that LH in conjunction with oxytocin, endothelin 1 and prostaglandin

regulate gamete/embryo transfer.

Alpha-fetoprotein (AFP) has recently been found in human oviduct fluid and may

be regulated by progesterone [Lippes and Wagh, 1993]. AFP binds estrone and estradiol,

acting as a carrier molecule for this steroid and its metabolites in biological fluids. AFP

has also been shown to have immunosuppressive effects. Wagh and Lippes (1993)

showed that AFP binds to the sperm acrosomal cap and may act as an acrosome-

stabilizing factor, preventing a premature acrosome reaction. Other possible

immunosuppressing agents in the oviduct are mucins. MUC1, a transmembrane mucin

gene product, has been localized in the human Fallopian tube, and similar to mucins in

the cervix may act as a barrier to sperm and pathogens [Gipson et al., 1997]. MUC1 may

also be important for gamete and embryo transfer and prevention of adhesion to oviductal

epithelium.

A large number of peptide growth factors and cytokines in the mammalian

oviduct have been described [Kane et al., 1997, Buhi et al., 1997]. These factors likely

originate from both the oviductal epithelium and serum transudate. Growth factors and

cytokines that have been identified within oviductal fluid or the epithelium during

ovulation, fertilization, and early cleavage-stage development in domestic species

include; epidermal growth factor (EGF), heparin-binding EGF, transforming growth

factor (TGF)-ct,, insulin-like growth factor (IGF)-I and II, IGF binding proteins (1-4),







24

colony stimulating factor (CSF)-1, acidic and basic fibroblast growth factor, platelet

derived growth factor, granulocyte macrophage-CSF-1, interleukin-6 and the IGF-II

receptor [Gandolfi, 1995, Chegini, 1996, Buhi et al., 1997, Kane et al., 1997, Buhi et al.,

1999]. Several growth factor and cytokine mRNAs and proteins in the oviduct appear to

be modulated by steroids and temporally associated with elevated estrogen levels at or

near estrus. For examples of their presence and regulation during the estrous cycle the

reader is referred to the reviews listed above. Another interesting cytokine, leukemia

inhibitory factor (LIF) has been identified by the presence of mRNA and protein in the

bovine [Reinhart et al., 1998] and human [Keltz et al., 1996, Barmat et al., 1997] oviduct.

Reinhart et al. (1998) showed that estrogen but not progesterone, stimulated LIF

synthesis and that this stimulation was not receptor-mediated, suggesting a non-genomic

action for estrogen derivitized products. Retinol-binding protein (RBP) has been

localized in the porcine [Harey et al., 1994], equine [McDowell et al., 1993] and ovine

[Eberhardt et al., 1999] oviduct. Eberhardt et al. (1999a) showed that RBP mRNA and

protein synthesis are stimulated by estrogen and that levels of RBP mRNA are greater on

Day 1 of the estrous cycle than Days 5 or 10. In one experiment, embryos collected at

the 1- to 4-cell stage from retinol-treated superovulated pregnant ewes and cultured in

vitro for 7 days, showed a higher development to blastocyst (79%) than control animals

(5%). Retinoids may have beneficial effects during fertilization and early embryonic

development especially in embryonic morphogenesis, cell growth, and differentiation.

Oviduct-derived proteins have been identified in experiments using radiolabeled

precursors for protein synthesis during oviductal organ explant or epithelial cell cultures.

Electrophoretic characterization of de novo synthesized and secreted proteins by ID- and







25

2D-SDS-PAGE and fluorography have been performed in the pig, cow, ewe, human, and

baboon [Buhi et al.,1997]. However, very few of these proteins have been identified and

have only been characterized in a limited fashion by isoelectric point and molecular

weight. Two of these proteins, the estrogen-dependent oviduct secretary glycoprotein

(OSP) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), have been

extensively characterized [Buhi et al., 1997, 1999]. Pig OSP will be discussed in greater

detail later in the literature review. TIMP-1 is a major de novo synthesized and secreted

protein of the isthmic segment of the porcine oviduct and its mRNA and protein

expression are greatest on Day 2 of the estrous cycle or early pregnancy. TIMP-1 protein

production and release in the isthmus portion of the oviduct was shown to be greater with

estrogen treatment indicating potential regulation of this inhibitor by ovarian steroids.

This is supported by radioimmunoassay analyses of oviductal flush TIMP-1 protein,

which showed significantly greater levels at Day 0 than other days of the cycle. TIMP-I,

a serine protease inhibitor, has been shown to improve in vitro development of embryos

in both the cow [Satoh et al., 1994] and pig [Funahashi et al., 1997]. This evidence

suggests that TIMP-1, present in the isthmus at the time of fertilization and early

embryonic development, may have important regulatory actions on these processes.

Recently, serine protease inhibitors of the uterus have been shown to stimulate DNA

synthesis in a glandular endometrial epithelial cell line [Badinga et al., 1999], thereby

suggesting a mitogenic role for TIMP-I stimulation of embryonic development.

Four other de novo synthesized proteins, complement C3b, immunoglobulin A

heavy chain, the carboxy terminal region of preprocollagen, and clusterin, have been

identified in the pig oviduct by N-terminal amino acid microsequence analysis [Buhi et







26

al., 1998, Buhi et al., 1999b]. Similar to pOSP and TIMP-1, these four proteins appear to

show synthetic gradients within the oviduct. Of the four, only clusterin and

preprocollagen are found within the pig isthmus. Preliminary evidence indicates that

these proteins exhibit a cyclic secretion pattern and may be partially controlled by

ovarian steroids [Buhi, personal communication]. Potential functions of these four

proteins include immunoprotection, lipid transport, and tissue remodeling. Of the many

de novo synthesized proteins secreted by the porcine (>14) [Buhi et al., 1997] or bovine

(>30) [Malayer et al., 1988] oviducts, only seven have been positively identified (OSP,

TIMP-1, PAI-1, preprocollagen, immunoglobulin A, clusterin and complement C3b).

Oviduct Secretions and Co-culture on Fertilization and Embryonic Development

A massive amount of literature indicates that the oviduct creates a unique

environment for sustaining embryo development. Prior to modern day embryo culture

systems, embryos removed from the oviduct or produced as a result of in vitro

fertilization tended to slow down or block developmentally when maintained in culture

media [Bavister, 1988]. However, when embryos are transferred to the oviduct in vivo,

development could occur from 1-cell to the blastocyst. This beneficial effect of the

oviduct was shown not to be species-specific as bovine embryos could develop in the

sheep or rabbit oviduct. The ability of the oviduct to overcome the cell block to

development is mimicked in vitro by use of chemically defined media supplemented with

protein additives (example; serum or BSA) and a low oxygen tension. Experiments in

the cow, sheep, and mouse where embryos are transferred to oviducts in vivo or in vitro

indicate an important role for this reproductive organ in sustaining embryonic growth

[Bavister, 1988, Hosoi et al., 1995]. A reduced degree of embryo growth obtained in







27

culture media alone [Bowman and McLaren, 1970, Bavister, 1988] and growth

abnormalities following embryo transfer [Farin and Farin, 1995], have been noted for

embryos produced by in vitro fertilization.

If indeed the oviduct creates a unique environment that sustains embryo

development, supplementation of oviductal fluid or the use of oviductal epithelial cell

(OEC) co-cultures during in vitro embryo development should increase the

developmental capacity. Addition of oviduct secretions/fluid to culture medium has

either had no observable effect or has retarded embryo development when compared to

controls [Sirard et al., 1985, Eberhardt et al., 1994]. However, careful fractionation of

oviductal cell-conditioned medium and subsequent addition of these fractions during in

vitro fertilization or embryo culture has had more promising results [Minami et al., 1992,

Mermillod et al., 1993, Liu et al., 1995, Vansteenbrugge et al., 1996, Hill et al., 1996, Liu

et al., 1998]. Preliminary results by these investigators have characterized, by molecular

weight, the embryotrophic activities of oviductal cell-conditioned media. These activities

have both high (>10 kDa) and low (<10 kDa) molecular weight components. None of the

determined molecular weights correspond to OSP. Perhaps the greatest contribution of

oviductal secretion analysis to date has resulted from careful evaluation of its ionic

composition. The concentrations of oviductal electrolytes differs considerably from that

of blood sera and media formulated to resemble the ionic composition of oviductal fluid

show improved embryonic development [Bavister, 1988, Boatman, 1997]. This media,

referred to as synthetic oviduct fluid (SOF), was commonly used during the late 1980s

and early 1990s. However, its use is uncommon today, as laboratories are moving

towards using protein-free chemically-defined culture media for repeatability.







28

Perhaps the most pervasive example of the contribution of the oviduct to embryo

culture can be seen using OEC co-cultures. Seminal experiments by Gandolfi and Moor

(1987) showed that in vivo fertilized sheep 1-cell embryos had a markedly increased

development (8- to 16-cell stage) when co-cultured with OEC (86%) or fetal fibroblast

cells (93%) than in culture media alone (13%). Interestingly, embryos cultured for 6 days

in the presence of OEC had an increased development to blastocyst (46%) when

compared to the fetal fibroblast cell co-culture (5%). Likewise, embryo transfer

experiments, revealed that embryos cultured in the presence of OEC showed twice the

viability of embryos cultured with fetal fibroblast cells. Similar results have been shown

in cattle [Eyestone et al., 1987, Eyestone and First, 1989, Ellington et al., 1990]. As

suggested by Bavister (1988), morphological criteria alone are not sufficient evidence of

the quality of cultured embryos. Hosoi et al. (1995) designed experiments to identify the

developmental stage at which embryos could be influenced by the oviductal environment.

These investigators found that in both mice and cattle the oviductal influence on

blastocyst development occurs during the switch from maternal to embryonic genome

control. The largest contribution of OEC has been shown in humans. Human embryos

co-cultured with OEC have reduced fragmentation [Bongso et al., 1989, Yeung et al.,

1992, Morgan et al., 1995], better morphological characteristics [Morgan et al., 1995],

higher cleavage [Morgan et al., 1995], blastulation [Bongso et al., 1989, Wiemer et al.,

1993], hatching [Yeung et al., 1992] and pregnancy rates [Bongso et al., 1992]. The

beneficial effect of the OEC may include the removal of toxic components from the

medium and/or production of embryotrophic factors. The benefits of OEC co-cultures

are not restricted to the oocyte alone but may also act on spermatozoa. Human sperm co-







29

cultured with bovine OEC show decreased chromatin structural degeneration [Ellington

et al., 1998] and in the pig, a reduced incidence of polyspermy [Nagai and Moor, 1990,

Dubuc and Sirard, 1995]. In addition to OEC effects on enhancing early embryonic

development, OEC play an important role in sperm-oviduct interactions.

Sperm-Oviduct Interactions

In most domestic species, of the millions of sperm that are ejaculated during a

natural mating, only a few thousand reach the isthmus of the oviduct, and there most are

held in a "reservoir" [Suarez, 1998]. Of these thousand which enter the isthmus only a

few reach the ampulla which is the site of fertilization. This oviductal sperm reservoir

has been suggested to be involved in the prevention of polyspermic fertilization [Hunter,

1991], maintenance of sperm fertility between the onset of estrus and fertilization

[Pollard et al., 1991, Chian and Sirard 1994, Smith, 1998], capacitation [Chian et al.,

1995, Mahmoud and Parrish, 1996], and/or motility hyperactivation [Kervancioglu et al.,

1994]. The oviductal sperm reservoir is established by spermatozoa binding to oviductal

epithelial cells and has been observed in cattle, mice, hamsters, pigs, and horses [Suarez,

1998]. For most species studied thus far, spermatozoa bind to the apical membrane of

isthmic oviductal epithelial cells (OEC) and adhesion is specific to the rostral region of

the sperm head [Smith, 1998]. The narrowness of the isthmic oviductal lumen enhances

sperm entrapment within the isthmus and increases the amount of contact between sperm

and the mucosal surface. The binding moieties between spermatozoa and OEC are still

unknown. Nor is it completely understood how and when sperm are released from these

cells. Some evidence suggests that sperm binding and release may depend on the

physical state and motor functions of the sperm. Smith and Yanagimachi (1991) reported







30

that hamster sperm would not bind to OEC once capacitated or hyperactivated. Likewise,

hyperactivation was shown to be a primary factor for release of bound spermatozoa in the

mouse [DeMott and Suarez, 1992]. Therefore, binding and release of spermatozoa may

be due to changes in surface properties of the sperm head, which might lead to increased

flagellar beat amplitude and asymmetry. Binding of sperm to OEC may be due to a lectin

or carbohydrate present on sperm cells, as sperm attachment was inhibited by fetuin in

the hamster [DeMott et al., 1995] or fucose in the cow [Lefebvre et al., 1997]. Suarez

(1998) and Kervancioglu et al. (1994) suggest that epithelial secretions, initiated by

signals of impending ovulation, could enhance sperm capacitation, binding, and release.

Soluble oviductal factors have been shown to induce capacitation in bull sperm

[Parrish et al., 1989, Chian et al., 1995, Mahmoud and Parrish, 1996]. Heparin has been

shown to induce capacitation similar to oviduct fluid [Parrish et al, 1988] and is widely

used during in vitro fertilization of bovine oocytes. However, the intracellular signaling

conditions which induce capacitation (increased intracellular Ca' and pH) are not

equivalent between heparin and oviduct fluid [Parrish et al., 1994]. Recently, Fazeli et al.

(1999) examined induction of capacitation by porcine OEC. Similar to observations in

other species, capacitated boar spermatozoa do not bind to porcine OEC. These

investigators also found that unbound spermatozoa in co-culture with ampulla or isthmic

OEC were slowly capacitated over time. This observation was not noted for cells of

nonreproductive origin. Kervancioglu et al. (1994) reports similar observations between

human OEC and vero cells (kidney epithelium). This suggests that the induction of

capacitation may be specific to oviductal secretions. With boar spermatozoa, the slow

induction of capacitation in vitro by OEC [Fazeli et al., 1999] is similar to in vivo bovine







31

sperm capacitation which may occur for as long as 24 h [Parrish, 1989]. Besides

suggestions by Parrish (see above references) that a heparin-like glycosaminoglycan is

responsible for capacitation activity in oviduct fluid, other protein components have been

shown to induce capacitation, including the bovine estrus-associated glycoprotein

[McNutt and Killian, 1991, King et al., 1994]. Capacitation can also occur spontaneously

in vitro in a defined medium without the addition of biological fluids. This suggests that

the process is intrinsically modulated by sperm itself However, this does not rule out the

influence of positive/negative regulatory factors within the oviduct. It is generally

accepted that capacitation is induced by the removal of decapacitating factors,

specifically cholesterol from the sperm membrane [as reviewed by Visconti and Kopf,

1998 and Cross, 1998]. Cholesterol-binding proteins are present in follicular and

oviductal fluids [DeLamirande et al., 1997] and may be involved in cholesterol removal.

Structure. Regulation and Biological Actions of Plasminogen Activator Inhibitor (PAI)-I

Controlled and targeted proteolytic activity or inhibition of such activity is an

important process of any biological system integrating tissue remodeling, tissue

destruction, or cell migration. This extracellular proteolysis is tightly regulated by the

plasminogen activator (PA)/plasmin system and inhibitors. One of the regulatory

molecules, plasminogen activator inhibitor-I (PAI-1), regulates the biological activity of

PA by preventing conversion of plasminogen to the broad-spectrum enzyme, plasmin.

Plasmin is primarily responsible for the proteolytic degradation of fibrin as well as other

ECM proteins. Therefore, it is principally involved in fibrinolysis and breakdown of the

ECM and basement membranes. PAI-I belongs to a superfamily of "serpins" serinee

protease inhibitors), which represent about 10% of the total protein in blood plasma [Gils







32

and Declerck, 1998]. Of these serpins, al-proteinase represents about 70%. While PAI-1

has been traditionally thought of as a regulator of fibrinolysis, the plasminogen activator

cascade has been shown to be important in other biological systems requiring

extracellular proteolysis such as ovulation, mammary gland involution, blastocyst

implantation, and tumor metastasis [Ny et al., 1993]. Plasminogen biosynthesis occurs

primarily in the liver and is found in high concentrations within blood plasma (2 mM)

thereby providing an unlimited source of potential proteolytic activity [Ny et al., 1993].

Thus, the rate-limiting step in these reactions are synthesis and activation of PA. The

proteolytic cascade is very dynamic and must be considered a multi-component system.

This multi-level system allows for precise regulation and fine-tuning at discrete focal

areas requiring tissue remodeling, so that systemic proteolysis is controlled.

The primary component of this system, plasmin, is formed by proteolytic

cleavage of plasminogen. One of two specific plasminogen activators, tissue-type (tPA)

or urokinase (uPA), initiates the cleavage of plasminogen. Both belong to the serpin

superfamily, are immunologically distinct and encoded by different genes [Ny et al.,

1993]. PAI-1 binds to both tPA and uPA acting as a suicide substrate and thereby

inactivating their enzymatic activity. In addition to this function, uPA- and tPA-PAI-1

complexes can bind to the multifunctional clearance receptor, low-density lipoprotein

related-receptor protein/a2-macroglobulin receptor (LRP/o2-MR or Megalin) [Ny et al.,

1993]. These complexes are internalized by endocytosis and subsequently degraded.

This is another potential mechanism for PAI-I regulation of uPA and tPA proteolytic

activity. Urokinase PA also has a cell surface receptor (uPAR) along with plasminogen,

and binding of these molecules to cellular surfaces is thought to lead to generation of







33

plasmin, which coordinates extracellular matrix (ECM) remodeling and degradation.

Degradation of receptor-bound uPA is enhanced three- to four-fold following complex

formation with PAI-1 [Ny et al., 1993]. Plasmin is the proteolytic enzyme primarily

responsible for the degradation of fibrin (fibrinolysis), breakdown of ECM and basement

membranes as well as thrombolysis. While tPA generation of plasmin is linked to the

fibrinolytic activity, uPA generation of plasmin is associated with the ECM

remodeling/degradation [Andreasen et al., 1990]. Plasmin can also activate promatrix

metalloproteinases, procollagenase, and progelatinase. A diagram of the pericellular

activation cascade for plasminogen and matrix metalloproteinases can be found in Figure

2-1.

PAI-1, a single chain glycoprotein of 50 kDa, is synthesized in an active form,

which converts spontaneously to an inactive latent form that can be partially reactivated

by denaturing agents such as SDS, guanidinium hydrochloride, and urea [Hekman and

Loskutoff, 1985]. In plasma, PAI-1 occurs in a complex with an integrin, vitronectin,

resulting in a significant stabilization [Declerck et al., 1988, Mimuro and Loskutoff,

1989]. PAl-1 forms a stable equimolar complex with both uPA and tPA, presumably of a

covalent nature [Andreasen et al., 1990], and exists in at least three distinct

conformational forms; active, latent, and substrate-cleaved [Lawrence et al., 1997].

Active PAI-1 decays to the latent form with a half-life of 1-2 h at 370 C, unless stabilized

by vitronectin. Similar to other serpins, PAI-I has a tightly ordered tertiary structure

consisting of three P-pleated sheets, A, B and C, a-helices A through I and a reactive site

loop [Gils and Declerck, 1998]. The reactive site, comprising the "bait" peptide bond, is

located 30-40 amino acid residues from the carboxy-terminal end. Nucleotide







34

sequencing of the cDNA and amino acid sequencing revealed a mature protein of 379-

381 amino acids long with heterogeneity among species primarily in the amino terminus

[Andreasen et al., 1990]. This protein was also found to be a glycoprotein and shares a

high degree of similarity (91%) between species examined, cow, human, rat, rabbit, and

mouse. Recently, pig PAl-1 was cloned and sequenced from a cDNA library prepared

from cultured pig aortic cells [Bijnens et al., 1997]. The cDNA consists of a 131-bp 5'

untranslated sequence, a 1206-bp open reading frame encoding a 402-amino acid protein,

and a 1651-bp untranslated sequence. The preprotein is cleaved at amino acid position

23, resulting in release of the mature 379-amino acid protein, PAI-1.

Hormonal Regulation of PAl-1

PAl-1 is produced by a variety of cell lines and primary cell cultures. These in

vitro culture systems have served as important models for obtaining information about

the intracellular mechanism of action stimulated by various regulatory agents.

Investigations have resulted in the identification of several hormones and steroids that

control the synthesis and secretion of PAI-I. As reviewed by Andreasen et al. (1990),

PAI-1 synthesis and/or activity is stimulated by glucocorticoids, insulin, endotoxin,

interleukin-1, tumor necrosis factor-a, EGF, CSF-1, and phorbol esters. Inhibitors of

PAI-1 synthesis and/or activity are molecules that stimulate intracellular increases in

cAMP production such as follicle stimulating hormone (FSH) and leutinizing hormone

(LH). Other potential regulators of PAl-1 synthesis/activity are prolactin [Liu et al.,

1998] and prostaglandin F2a [Zhang et al., 1996].

In addition to regulation by the molecules described above, PAl-1 expression and

activity is regulated by ovarian steroids. Progesterone has been shown to significantly







35

increase PAl-1 synthesis in several endometrial cell lines [Schatz et al., 1994, Casslen et

al., 1992, Casslen et al., 1995, Miyauchi et al., 1995]. As reviewed by Schatz et al.

(1994), estrogen had either no effect or inhibited PAI-I synthesis. However, the effect of

progesterone on PAl-1 synthesis doubled in response to the presence of both estrogen and

progesterone. Furthermore, exogenous steroids elicit similar changes in expression of

PAI-1 mRNA as seen for PAI-I protein. Progesterone was shown to not interact with the

804-bp promoter region of the human endometrial PAI-1 gene, but rather increased the

stability of PAI-1 mRNA [Sandberg et al., 1997]. After menopause, hormone

replacement therapy may reduce the risk of coronary heart disease that is associated with

fibrinolytic activity. Women receiving estradiol were shown to have significantly lower

plasma levels of PAI-1 [Lindoff et al., 1996, Shahar et al., 1996], indicating that estrogen

has a significant role in inhibition of PA-1 expression. However, some investigators

suggest that estrogen might also increase PAl-1 synthesis [Sobel et al., 1995, Fujimoto et

al., 1996] which is contradictory to the majority of other findings to date.

Plasminogen Activator and its Inhibitor in the Uterus and Ovary

In man and rodents, endometrial stromal cells proliferate and differentiate into

decidual cells as the implanting embryo invades the endometrium in order to establish an

intimate contact with the maternal blood supply. Although the implanting embryo

produces matrix-degrading enzymes for implantation, ECM-degrading enzymes are also

produced in the human endometrium under the control of steroid hormones [Wang et al.,

1996]. In the human, an increase in tissue-factor (TF) and PAl-1, and inhibition oftPA,

uPA and matrix metalloproteinases (MMPs) are associated with progestin-induced

decidualization of estrogen-primed endometrial stromal cells in vivo and in vitro







36

[Lockwood and Schatz, 1996]. These important regulators of fibrinolysis, hemostasis,

and ECM turnover in the decidualized stromal and decidual cells suggest a mechanism to

explain the absence of hemorrhage during invasion of the endometrial vasculature by

trophoblasts. Progesterone withdrawal reduces TF and PAl-1, yet increases uPA, tPA

and MMPs thereby increasing hemorrhage, fibrinolysis, ECM degradation and vascular

injury characterizing menstruation [Lockwood and Schatz, 1996]. Trophoblast cells are

so highly invasive that they are called pseudomalignant and when grafted to ectopic sites,

invade uncontrollably [Axelrod, 1985]. The modified uterine tissue, decidua, is rendered

resistant to invasion by the well-controlled coordinate expression of protease inhibitors

such as PAI-1 [Waterhouse et al., 1993, Teesalu et al., 1996, Lala and Hamilton, 1996].

The PA system has also been shown to be an important regulator of ovulation. As

early as 1916, it was proposed that proteolytic activity was involved in degradation of the

follicle wall at the time of ovulation [Ny et al., 1993]. Levels of plasminogen in

follicular fluid are comparable to levels found in serum. Injection of agents which inhibit

PA and plasmin activity into the ovarian bursa suppress ovulation [Ny et al., 1993].

Combined uPA and tPA gene-deficient mice were also shown to have a 26% reduced

ovulation efficiency [Leonardsson et al., 1995]. The loss of an individual PA is

functionally compensated by the activity of the remaining PA, however the loss of both is

not obligatory for ovulation. As reviewed by Andreasen et al. (1990) and Ny et al.

(1993), ovulation-associated hormones, FSH and LH, are effective stimulators of PA

secretion from granulosa cells. In the preovulatory follicle, the LH surge stimulates a

cascade of proteolytic enzymes, including PA, plasmin, and MMPs. These enzymes

bring about the degradation of the perifollicular matrix and the decomposition of the







37

meshwork of collagen fibers which provide strength to the follicle wall [Tsafriri and

Reich, 1999]. Increased ovarian proteolytic activity is controlled by high levels of PAI-1

and TIMP-1 expression in theca cells from growing follicles, ensuring their development

by protection from enzymes diffusing from ovulatory follicles [Tsafriri and Reich, 1999].

FSH, LH, and activators of cAMP suppress PAl-1 synthesis and activity in granulosa

cells. However, one report in the rat indicates cell-specific expression of PAI-1 and

TIMP-1 mRNAs in the LH/hCG-stimulated ovary suggesting a co-expression of both

enzymes and their inhibitors during ovulation [Chun et al., 1992]. PAI-1 was also shown

to be a major secretary product of the corpus luteum and its expression is stimulated by

PGF2a [Smith et al., 1997]. These investigators suggest an important role for PAI-1

during ECM remodeling of the ovary following ovulation.

PA in the Oviduct During Fertilization and Embryo Development

A significant amount of literature has been compiled on PA activity during

fertilization and its association with the preimplantation embryo. However, there are no

data on its physiological inhibitor, PAl-1, in the oviductal lumen or its association with

the early embryo. The importance of PAl-1 in the reproductive system is not fully

understood. PAI-1 gene-deficient mice are viable, fertile, and without abnormalities in

organogenesis and development [Carmeliet et al., 1993]. Other factors (PAI-2, 2-

antiplasmin, a2-macroglobulin, Cl-esterase inhibitor and ca-antitrypsin) known to reduce

plasminogen activation and/or plasmin activity, may compensate for the loss of PAl-1.

However, to the authors knowledge, these proteins have not been identified in the

oviduct. This portion of the discussion will concern data available on PA that may lead

to insights on PAI-1 function within the oviduct.







38

As early as 1968, PA was shown to be associated with the oviductal mucosa

[Tympanidis and Astrup, 1968]. PA activity has also been shown to be associated with

rat [Liedholm and Astedt, 1975] and mouse [Sherman, 1980] preimplantation embryos

from the 2-cell to blastocyst stage. Sherman (1980) showed that this activity was

associated with the zona pellucida, as denuded embryos have no PA activity. These

earlier studies concluded that zona-associated PA did not originate from the embryo but

from genital tract secretions. The observation that PA activity steadily decreased as

preimplantation development proceeded both in vivo and in vitro raises the possibility

that the zona pellucida contains residual activity from follicular or oviductal fluid

[Sherman, 1980].

PA activity has been associated with several important reproductive processes

including oocyte maturation, fertilization, and early embryogenesis. Pig cumulus-cell

oocyte complexes (COCs) have been shown to produce two PAs during in vitro

maturation [Kim and Menino, 1995]. These PAs were shown to be tPA and tPA-inhibitor

complex. Stimulators of PA activity in pig COCs, like cAMP and okadaic acid, were

found to inhibit oocyte maturation. Kim and Menino (1995) suggest that although PA

production is temporally associated with oocyte maturation, coordination of these two

processes is differentially regulated. The production of PA in rat COCs has also been

reported [Liu et al., 1986, 1987]. PA activity in rat [Salustri et al., 1985, Ny et al., 1987]

and pig [Kim and Menino, 1995] COCs is through a stimulation of protein kinase A and

C. Recently, cow COCs matured in vitro, were shown to contain mRNA for all elements

of a proteolytic cascade including uPA, PAl-1, MMP-1 and TIMP-1 [Bieser et al., 1997].

These investigators suggest a potential role for this extracellular proteolysis in cumulus







39

expansion during oocyte maturation. In the human, transcription of PAI-1 and PAI-2

genes have been described for cumulus cells and granulosa-luteal cells [Piquette et al.,

1993]. Similarly, cow COCs show production of PA during in vitro maturation and this

production was stimulated by EGF [Park et al., 1999]. This activity was shown to be

associated with uPA and was not present in denuded oocytes, indicating the importance

that cumulus cells play in its synthesis. For a current discussion on the production of PA

in COCs relative to the time of germinal vesicle breakdown and resumption of meiosis,

the reader is referred to Park et al. (1999).

As reviewed by Huarte et al. (1993) the PA/plasmin proteolytic cascade may have

a significant role during fertilization. Proteolytic enzymes participate in multiple phases

of mammalian fertilization including the acrosome reaction, sperm-binding to the zona

pellucida, zona pellucida penetration, and the zona reaction. Mouse gametes express

plasminogen-dependent proteolytic activities; ovulated eggs synthesize and secrete tPA

while ejaculated spermatozoa have uPA activity. Likewise, plasminogen was shown to

bind to both mouse spermatozoa and eggs, and the presence of plasminogen increased the

in vitro fertilization rate [Huarte et al., 1993] presumably due to increased plasmin

generation. In addition, antibodies, which inhibit the catalytic activity of plasmin, were

shown to decrease the fertilization rate. Exogenously added plasminogen was likely

converted to plasmin by zona pellucida-associated uPA or tPA activity. These findings

are supported by previous work indicating that a positive correlation existed between the

ability of oocytes fertilized in vitro and the PA activity of the corresponding follicular

fluid and granulosa cells [Deutinger et al., 1988, Milwidsky et al., 1989]. Zhang et al.

(1992) showed that tPA was released from rat oocytes as a result of oocyte activation and







40

suggested that tPA may act in the perivitelline space on the zona pellucida during

fertilization and/or activation. As described by Zhang et al. (1992), tPA may be a

component of corticol granules that are involved in the zona block to polyspermy. In

their study, activation-induced zona hardening (limited proteolysis of zona protein ZP2)

was prevented by a anti-tPA monoclonal antibody. Inadvertent activation under in vitro

conditions in mouse and human IVF systems often times leads to premature loss of

fertility in respective oocytes. Addition of leupeptin, a serine protease inhibitor, during

mouse oocyte calcium-ionophore activation reversed the decrease in the capacity of

oocytes to fertilize and develop in vitro [Tawia and Lopata, 1992]. The data support

evidence for PA activity in zona hardening and suggests a coordinate expression between

activator and inhibitor in regulating this process. Additionally, supplementation of

culture medium with proteases increased hatching rate of mouse embryos [Lee et al.,

1997], while addition of protease inhibitors to culture medium inhibited hatching in vitro

[Dabich, 1981, Yamazaki et al., 1985]. This suggests an important role for oviductal-

derived protease inhibitors in the prevention of premature hatching, prior to the correct

developmental stage within the uterus. The effect may be directed towards proteolysis of

the zona matrix. Zona of porcine oviductal oocytes and embryos were shown to be more

resistant to proteolytic digestion than either follicular oocytes or embryos recovered from

the uterus [Broermann et al., 1989], indicating an important role for protease inhibitors in

protecting the zona pellucida from degradative proteases.

Preimplantation mouse [Harvey et al., 1995, Zhang et al., 1996], rat [Zhang et al.,

1994], sheep [Menino et al., 1989, Bartlett and Menino, 1993] and cow [Dyk and

Menino, 1991, Berg and Menino, 1992] embryos were shown to have PA activity.







41

Kaaekuahiwi and Menino (1990), showed that as embryonic size and cell number

increase and development progresses, bovine embryos liberate more PA. Recently,

bovine embryonic PA was shown to induce changes in the electrophoretic protein profile

of the zona pellucida when embryos were incubated with exogenous plasminogen

[Cannon and Menino, 1998].

In addition to PA's association with maturation, fertilization and embryogenesis, it

might also have an important role in spermatozoa physiology. Urokinase PA is

synthesized in epithelial cells of the caudal epididymis, vas deferens, and seminal

vesicles [Huarte et al., 1987], while tPA was found in the prostate gland [Reese et al.,

1988]. In addition, Sertoli cells have been shown to secrete PA and PAI-1 [reviewed by

Gilabert et al., 1995]. The presence of uPA and tPA has been shown in ejaculated

spermatozoa of man and various animal species [Smokovitis et al., 1987]. These

investigators report a direct influence on sperm motility and suggest that the PA are

membrane bound. Recently, boar spermatozoa were shown to have uPA and tPA

associated with spermatozoal membranes and differences were detected in their

localization [Smokovitis et al., 1992]. The outer acrosomal membranes contained tPA

while the inner acrosomal membrane contained both tPA and uPA.

Structure. Regulation, and Biological Actions of Oviductal Secretory Glycoprotein (OSP)

In vitro Synthesis and Hormonal Regulation of OSP

Experiments by Oliphant and Ross (1982) in the rabbit describe the identification

and purification of three sulfated glycoproteins from oviduct fluid. This investigation

was one of the first studies utilizing a radiolabeled precursor, [35S], to evaluate

incorporation into macromolecular components of oviductal epithelial cells and oviductal







42

explant tissue in culture. Utilizing this technique, Oliphant and Ross (1982) reported the

de novo synthesis and secretion ofglycoproteins by oviductal epithelium. Subsequently,

numerous studies have been done in various species. Evaluations of stage-specific

proteins in the pig oviduct were first reported by Buhi et al. (1989). The de novo

synthesis of porcine oviductal proteins by cyclic and early pregnant tissues in explant

culture revealed that incorporation of [3H]-leucine into nondialyzeable macromolecules

was greatest on Days 0 and 2 and secretary activity was lowest on Days 10 to 15. The

ampulla was shown to have greater incorporation than the isthmus and increased rates of

incorporation were temporally associated with elevated estrogen at proestrus, estrus, and

metestrus [Buhi et al., 1989]. 1D-SDS-PAGE and fluorographic analysis of oviductal

culture medium revealed three proteins of 335 k, 115 k, and 85 k Mr, associated with

proestrus, estrus, and metestrus. High resolution 2D-SDS-PAGE further resolved the

isoelectric and molecular weight variants of these radiolabeled proteins initially detected

by 1D-SDS-PAGE [Buhi et al., 1990]. The major 115 k Mr band was resolved into two

major glycoproteins, one basic (100 k M,, pi > 8) and one acidic (100 k Mr, pl 4.5-5.5).

The 85 k Mr protein was resolved into a very acidic (pl< 4) protein of 75 to 85 k Mr.

These three proteins have been designated pOSP El (85 k Mr), pOSP E2 (100 k Mr;

acidic) and pOSP E3 (100 k Mr; basic). It was suggested by Buhi et al. (1990) that these

denatured and reduced subunits may be part of a higher molecular weight complex and

were related (pOSP E2 had the same N-terminal sequence as El and the N-terminal

sequence for E3 was identical to an internal sequence of E2). Incorporation of [35S]-

glucosamine indicates that these three estrus-associated proteins are glycosylated [Buhi et

al., 1990].







43

Oviduct-specific secretary proteins have been identified in human [Verhage,

1988, Buhi et al., 1989], baboon [Fazleabas and Verhage, 1986, Verhage and Fazleabas,

1988], cow [ Malayer et al., 1988, Boice et al., 1990], sheep [ Sutton et al., 1984, Sutton

et al., 1986, Gandolfi et al., 1989, Buhi et al., 1991, Murray, 1992], mouse [Kapur and

Johnson, 1985] rhesus monkey [Verhage et al., 1997], and hamster [Robitaille et al.,

1988, Abe et al., 1998]. Although isoelectric points are similar for the subunits examined

in other species, differences have been noted in molecular weights for these

glycoproteins. Studies on these glycoproteins have led to several key observations. First,

these glycoproteins are primarily secretary products of the ampulla, although their

synthesis can be seen in other segments at a much lower rate [Verhage and Fazleabas,

1988, Buhi et al., 1990, Boice et al., 1990, Gandolfi et al., 1991, Murray, 1992, and

O'Day-Bowman et al., 1995]. Second, the presence of these proteins only during estrus,

temporally elevated at estrus or the follicular stage in humans indicates that this protein is

regulated by estrogen. Therefore, the pattern (but not presence) of distribution for these

proteins is dependent on the presence or absence of specific ovarian steroids. In the pig,

treatment of ovariectomized (OVX) gilts with estrogen increased incorporation rate in the

ampulla and revealed that the oviduct-specific glycoproteins were estrogen-dependent

[Buhi et al., 1992]. This is supported by the observation that progesterone

antagonizes/abrogates the stimulatory effect of estrogen on their synthesis. Similar

observations on OSP steroid regulation using OVX animals have been made in sheep

[Buhi et al., 1991, Murray and DeSouza, 1995] and baboon [Verhage and Fazleabas,

1988]. Newborn golden hamsters (1.5 days) injected daily with estrogen or progesterone

showed that estrogen could induce hamster OSP synthesis in the undifferentiated







44

epithelial cells of neonates, while progesterone could not [Abe et al., 1998]. This family

of estrogen-dependent and oviduct-specific glycoproteins is the most abundant

radiolabeled secretary product of the oviduct, yet assigning specific functions to these

proteins has been elusive. Investigations on the localization of OSPs in the oviduct and

their association with the ovulated oocyte and early embryo, as detailed below, suggest

possible involvement during fertilization and early cleavage-stage embryonic

development.

Immunolocalization ofpOSP in the Oviduct. Oocvte. Spermatozoa and Early Embryo

Immunogold localization of pOSP in the pig has been evaluated at the cellular

level utilizing electron microscopy [Buhi et al., 1993]. These investigators found that

pOSP was localized to putative secretary granules in non-ciliated secretary cells of the

ampulla from both cyclic and OVX estrogen-treated gilts. This study was not extended

to evaluate distribution of pOSP in either the infundibulum or isthmus of the pig. Similar

observations of OSP localizing to secretary granules in the ampulla have been observed

in sheep [Murray, 1992, Gandolfi et al., 1991], cow [Boice et al., 1990], baboon [Verhage

et al., 1990, Verhage et al., 1989], rhesus monkey [Verhage et al., 1997], and human

[O'Day-Bowman et al., 1995, Rapisarda et al., 1993]. Studies done in the human and

baboon identified OSP in secretary granules of the isthmus as well as the ampulla,

indicating that this segment also synthesizes OSP. Evaluations of colloidal gold densities

in segments treated with estrogen, progesterone, or estrogen + progesterone have not

been performed. Therefore, it is unknown whether there are dynamic changes of OSP in

secretary granules relative to hormonal status, except for the observation with estrogen

alone, as detailed above. Hamster OSP of oviductal origin is associated with uterine







45

epithelial cells during the first 3 days of pregnancy, but is reduced by Day 4, and is

absent by Days 5 and 6 [Roux et al., 1997]. Because Days 5 and 6 correspond to the time

of implantation in the hamster, and levels of OSP remain constant in the oviduct, these

investigators suggest that OSP may be involved in uterine receptivity.

The macromolecular composition of the zona pellucida in the mammalian

oocyte/embryo is quite complex and three major glycoproteins have been identified each

containing about 15 isoelectric species [Dunbar, 1983]. Several studies have revealed

changes in the composition of the porcine zona pellucida during development from the

oocyte to the 4-cell embryo. Brown and Cheng (1986) observed that the zona pellucida

of the follicular oocyte to the early embryo acquired three glycoproteins from the oviduct

at estrus. These investigators observed two proteins of 250 k and 90 k Mr when the zona

pellucida was analyzed by 1D- and 2D-SDS-PAGE under non-reducing conditions.

When these proteins were electrophoresed under reducing conditions, subunits of 90 k,

79 k, and 69 k M, were detected. Similar differences in the macromolecular composition

of zona pellucida from pig oocytes, eggs, and zygotes have been observed by Hedrick et

al. (1987), although there is some discrepancy in the molecular weight of these proteins

when compared to those of Brown and Cheng. These are the first studies indicating that

proteins of oviductal origin associate with the zona pellucida and that their molecular

weight (80-90 k) corresponds to that of pOSP. The study by Buhi et al. (1993) supports

these earlier results and specifically indicates that oviductal pOSP associates with

unfertilized oviductal oocytes and early embryos. Pig OSP immunoreactivity was

observed through early development to Day 7 hatched porcine embryos but had

disappeared by Day 9 of pregnancy [Buhi et al., 1993]. In addition to association with the







46

zona pellucida, pOSP also associates with flocculent material in the perivitelline space

and with vitelline and blastomere membranes. Limited data supports the possibility that

the embryo itself endocytoses OSP during its early development. Hamster OSP has been

localized in coated pits, apical vesicles, mutivesicular bodies, and lysozome-like

structures [Kan and Roux, 1995]. The association of pOSP with the early developing

embryo suggests this protein may have an important role during fertilization and early

cleavage-stage embryonic development.

Localization of OSP to the zona pellucida of oviductal oocytes or early embryos

has been shown in the hamster [Kan et al., 1989, Kan and Roux, 1995, Abe and Oikawa,

1990, Leveille et al., 1987], cow [Wegner and Killian, 1991], sheep [Gandolfi et al.,

1991], rhesus monkey [Verhage et al., 1997], and baboon [Boice et al., 1990]. In the

mouse, OSP was not associated with the zona pellucida and was only localized within the

perivitelline space [Kapur and Johnson, 1985]. An interesting observation by Kan et al.

(1995) was that hamster OSP localized to the flocculent material in the perivitelline space

in oviductal oocytes only after fertilization. These investigators suggest that OSP

interacts with secretions of corticol granules after fertilization and may be involved in the

block to polyspermy. This hypothesis is supported by our work on polyspermy described

in Chapter 4.

While observations for an association of OSP with the oviductal oocyte or early

embryo are consistent across species studied thus far, a similar consensus does not exist

for localization of OSP to spermatozoa. In the hamster [Kimura et al., 1994, Boatman

and Magnoni, 1995] and cow [King and Killian, 1994, Abe et al., 1995], OSP has been

shown to associate with the spermatozoal membrane. However, a report in the human







47

[Reuter et al., 1994] failed to find an association of human OSP with human

spermatozoa.

Molecular Biology ofpOSP

Cloning and characterization of a full length cDNA for pOSP has been reported

by Buhi et al. (1996). A full length clone (2022-bp) was identified and sequenced,

yielding an open reading frame of 1581 bp that coded for a protein of 527 amino acids.

A putative signal sequence was identified (a.a. 1-21) and is highly conserved, having

100% homology among bovine, human, and ovine signal sequences. Sequence analysis

of pOSP revealed 3 consensus N-glycosylation sites as well as several potential O-

glycosylation sites, suggesting large amounts of carbohydrate addition. This would also

explain the differences between the predicted native molecular weight of 55,600 and

those observed by 2D-SDS-PAGE (El; 75 k, E2,3; 100 k M,). The pOSP cDNA

sequence revealed significant identities and similarity to oviductal secretary

glycoproteins (OSP) from a number of other species, especially in the 5' end of the open

reading frame. Sequence analysis of pOSP cDNA also revealed two potential

phosphorylation sites, a consensus heparin-binding sequence, a region similar to the

chitinase catalytic site, and a potential C-terminal region similar to a chitinase binding

domain. However, this protein does not have chitinase activity and is missing an

essential amino acid required for such activity. Observations have been made

establishing a structural similarity between OSPs and the chitinase protein family [Arias

et al., 1994, Sendai et al., 1995, Suzuki et al., 1995, Buhi et al., 1996]. Sendai et al.

(1995) has suggested that the chitinase-like structure in OSPs may be involved with

carbohydrate moieties of the oocyte or surface of the spermatozoa. Since chitinase binds







48

to chitin (poly-pl,4-N-GlcNAc) and hydrolyzes it, Suzuki et al. (1995) suggests that

OSPs may be a GlcNAc binding protein, which interacts with carbohydrate components,

located on the zona pellucida. OSPs have been cloned in several other species including

the baboon [Donnelly et al., 1991], human [Arias et al., 1994], hamster [Suzuki et al.,

1995], mouse [Sendai et al., 1995], cow [Sendai et al., 1994], sheep [DeSouza and

Murray, 1995] and rhesus monkey [Verhage et al., 1997]. These researchers also found

that a significant degree of homology exists among oviduct-specific secretary

glycoproteins of various mammalian species studied thus far. However, OSP cDNA

appears to lack homology to the frog oviduct-specific protein-1 gene found in Xenopus

oviduct cells [Donnelly et al., 1991]. Sequence analysis of the various species listed

above revealed several N- and O-glycosylation sites, which may reflect why differences

are observed between species in isoelectric point and molecular weight when evaluated

by 2D-SDS-PAGE. Identification of the polypeptide precursors and characterization of

their biosynthetic maturation within oviductal cells for hamster OSP has been examined

in detail and hypothesis on the polymorphism of these variants due to translational and

posttranslational events has been reviewed by Malette et al. (1995). Southern blot

analysis ofgenomic DNA has shown that there is a single copy of the hamster OSP gene

in the hamster genome and PCR amplification revealed that it is contained within a single

exon, excluding the possibility of alternative splicing [Paquette et al., 1995]. Although

these studies have not been done in other species to date, immunological and northern

blot analysis suggest that the family of OSPs detected by 1- and 2D-SDS-PAGE all

derive from a single gene. Hamster OSP has recently been referred to as a secretary

mucin, due to extensive O-glycosylation (> 50%) not seen in other species, tandemly







49

repeated amino acid motifs, and the presence of multiple alleles (Paquette et al., 1995].

These tandem repeats are present in rodents and humans but are not seen in ungulates,

raising interesting questions from an evolutionary viewpoint. A review of the various

sequences cloned revealed significant differences in the C-terminal region of the OSPs

and may reflect species differences in the OSP molecule. For instance, Sendai et al.

(1995) identified a unique seven-residue repeat sequence (21 repeats) in the C-terminal

side of mouse OSP which are not seen in cow, sheep, pig or baboon OSP.

Probes for pOSP mRNA detected only a single 2.25 kb message in oviductal

tissue and mRNA was present in all 3 segments of the oviduct. Levels of mRNA were

greatest in the ampulla indicating this segment as the primary site of its expression.

Northern blot analysis of numerous other tissues (heart, intestine, cervix, kidney,

endometrium, myometrium, spleen, lung, aorta, liver, or stomach) confirmed that

expression of this protein is specific to the porcine oviduct. Steady-state levels of pOSP

mRNA in the oviduct were greatest at estrus (Days 0,1) consistent with elevated estrogen,

decreased rapidly by Day 2, and remained low throughout diestrus. These data correlates

well with previous reports on pOSP protein synthesis [Buhi et al., 1990, Buhi et al.,

1992]. Levels of pOSP mRNA in oviducts of steroid-treated OVX gilts confirmed that

this protein's expression is up-regulated by estrogen and that progesterone antagonizes

this effect. Data from other species including human [Arias et al., 1994], baboon

[Donnelly et al., 1991], and bovine [Sendai et al., 1994] have also shown that the

message for this protein is greatest during the late follicular phase and in estradiol-

dominated oviducts.







50

A study in the hamster, utilizing in situ hybridization for detection of OSP

message, found evidence for OSP in both the ampulla and isthmus, although the signal

intensity was greatest in the ampulla [Komiya et al., 1996]. The distribution of OSP

message was different within the two sections, with the ampulla having signal detected in

both the perinuclear and basal regions, while the isthmus only had signal in the basal

compartment of epithelial cells. Komiya et al. (1996) also found that the message for

OSP is greatest when serum estradiol/progesterone levels are higher and that the mRNA

for OSP wanes with age and correlates with decreasing serum estradiol levels. However,

these investigators also showed that progesterone is required for hamster OSP gene

expression, unlike data from the baboon [Donnelly KM et al, 1991] and pig [Buhi et al.,

1996], which shows OSP strongly suppressed by progesterone. Another interesting

difference is that the hamster [Paquette et al., 1995] rabbit [Donnelly et al., 1991], and

mouse [Donnelly et al., 1991] showed a constant level of mRNA expression for OSP

throughout the estrous cycle which differs from that of other species observed thus far

where a strong estrogen-dependence is required for its expression. Contrary to findings

by Komiya et al. (1996), Murray and DeSouza (1995) found that transcripts encoding

sheep OSP were in the basal compartment and at the apical tips in fimbria and ampulla

epithelial cells at the free margins of mucosal folds. Because mRNA was localized to the

apical tips, these investigators suggested that mRNA encoding OSP was translated at a

unique cytoplasmic foci. Electron microscopy reveals that all the cytoplasmic machinery

required for protein synthesis is also located within the apical tips of oviductal epithelial

cells, which suggests a quick response time from the point of translation to release of

OSP into the lumen [Murray and DeSouza, 1995].







51

Cyclical, structural, and functional changes in the oviduct are thought to be

brought about primarily by the actions of estradiol and progesterone [Verhage and Jaffe,

1986], and numerous reports have been cited above showing regulation of OSPs and

pOSP by estrogen/progesterone levels. Recent studies have demonstrated that human

[Lie et al, 1993] and pig [Gawronska et al., 1999] oviducts contain luteinizing hormone

(LH)/human chorionic gonadotropin (hCG) receptors, suggesting that factors other than

circulating ovarian steroids may regulate oviductal function. One study by Sun et al.

(1997), revealed that culturing bovine oviductal epithelial cells with hCG resulted in a

time and dose-dependent increase of OSP protein and transcript. A nuclear transcription

run-on assay was used to determine that hCG did not increase the transcription rate of the

gene but rather "stabilized" its transcript [Sun et al., 1997]. These investigators suggested

that higher LH levels present during the periovulatory periods may act to stabilize the

message for OSP in the oviduct, thus leading to an increased secretion rate. Recently,

heterozygous mutant mice have been generated, which lack a large portion of the OSP

coding region [Sendai et al., 1999]. In the future, homozygous offspring may clarify the

role of this protein in vivo.

Biological Actions of OSPs

The observations that in all species examined so far, with the notable exception of

the mouse, OSPs associate with the zona pellucida, suggest an intimate role for these

estrogen-dependent proteins during fertilization and early cleavage-stage embryonic

development. However, the abundant information detailing a morphological association

of OSPs with spermatozoa, oocytes, or embryos does not correspond to a suitable amount

of functional information. Functional information regarding OSPs have been hindered by







52

difficulties in the protein purification process. In fact, no direct in vivo data on precise

functions of these proteins are available to date. However, several in vitro observations

of activities associated with the inclusion of OSP have led to some attractive hypotheses.

Macromolecules secreted by the hamster oviduct in the ampulla region have been

shown to facilitate penetration of sperm through the egg investments [Boatman et al.,

1994] and follicular eggs collected from the ampulla were shown to be more penetrable

and fertilizeable than follicular eggs collected from the ovarian bursa or unovulated

follicles [Boatman and Magnoni, 1995]. Similar increases in penetration rates have been

observed in the bovine [Martus et al., 1998] and were linked to the addition of

concentrated bovine OSP during in vitro fertilization (IVF). One conflicting report in the

hamster [Kimura et al., 1994] indicates that penetration rates were decreased in the

presence of OSP. These data, although partially conflicting, indicate that association of

OSP with the oocyte may have a functional role during fertilization. It is unknown

whether these observed effects were due to actions on spermatozoa, oocyte or both.

Martus et al. (1997) showed that exposure of bovine oocytes to bovine OSP during any

phase of the IVF process increased fertilization rates, while exposure of spermatozoa to

bovine OSP had no effect.

Several investigators suggest that OSPs induce capacitation [McNutt et al., 1992,

Anderson and Killian 1994] and/or the acrosome reaction. Therefore, an increase in the

number of capacitated or acrosome-reacted spermatozoa could possibly lead to the above

observations on increased penetration. However, direct evidence for the estrogen-

dependent OSP on these activities is lacking.







53

Another potential modulator for this increase in penetration may be due to actions

on spermatozoa movement characteristics. Abe et al. (1995) found that bovine OSP

effectively maintained the viability and motility of bovine spermatozoa relative to control

medium and that this activity was dose-dependent. The ability of bovine OSP to

maintain motility and viability may allow a greater proportion of spermatozoa to

penetrate the egg investments and subsequently fertilize the oocyte. The OSPs may also

have a role in gamete recognition and binding. In the hamster [Schmidt et al., 1997a,

1997b] and human [O'Day-Bowman et al., 1996], OSP was found to increase the number

of tightly bound sperm attached to the zona pellucida. This increase might also lead to

the increased penetration rates described above. These data are supported by the

observation that a redistribution in the localization of hamster OSP occurs depending on

the state of capacitation [Boatman and Magnoni, 1995].

Functional data on embryonic development are especially lacking, and

investigations to date are less than encouraging. No effects of bovine OSP

[Vansteenbrugge et al., 1997] or ovine OSP [Hill et al., 1997] were observed on bovine

embryonic development. Numerous investigations have shown the need for homologous

systems when studying this protein, therefore observations by Hill et al. (1997) may be

due to the heterologous system employed. Previous studies by Hill et al. (1996 a, b)

examining ovine OSP effects on ovine embryonic development demonstrated several

subtle but consistent results. The most significant of these being that a reduced

proportion of one-cell embryos underwent first cleavage, but no overall decrease on the

proportion of blastocysts that formed. These investigators suggest that this may reflect a

selection mechanism occurring in vivo. Bovine OSP has been found to increase the







54

number of bovine zygotes that cleaved compared to controls, but by Day 7 of embryonic

development, no difference could be detected in the number of blastocysts that formed

[Martus et al., 1997]. However these investigators did observe an increased number of

blastocysts on Day 6 of in vitro culture. This may suggest that bovine OSP, while not

increasing the number of blastocysts, increases their rate of development. However, a

slight loss in the number of blastocysts between Day 6 and Day 7 was shown and might

indicate that the concentration of bovine OSP tested or length of exposure (>6 days) is

detrimental to subsequent development. This increased rate of development may be due

to an unknown stimulation on the expression of maternal or embryonic genes and

increased protein synthesis. One study indicates that in vivo fertilized pig embryos (one-

or two-cell stage), cultured in vitro in the presence of one subunit (97 k Mr) of semi-

purified pOSP, showed increased rates of incorporation of methionine into protein at the

four-cell stage [Wallenhaupt et al., 1996]. The data described above indicate that pOSP

may facilitate or enhance fertilization and early cleavage-stage embryonic development.

This review thus leaves a number of unanswered questions which need to be

addressed. The first is; what are the unidentified de novo synthesized and secreted

macromolecules of the oviduct? The second is; for those proteins which have been

identified and characterized, what is the specific functions) in relation to reproductive

processes in the oviduct; union of gametes, fertilization, and early cleavage-stage

embryonic development. Chapters 3, 4, and 5 describe the identification,

characterization, and regulation of PAI- in the oviduct. The objectives of these three

chapters include; 1) localization of PAI-I within the oviduct, 2) hormonal regulation of

oviductal PAI-1 by ovarian steroids, 3) expression of PA-1 mRNA and protein during







55

early pregnancy, 4) evaluation of PAI-1 and uPA activity in the oviduct during early

pregnancy, and 5) localization of PAI-1 on oviductal oocytes and embryos. Chapter 6

will examine the functionality of pOSP during in vitro fertilization and embryo culture.

Effects of pOSP on the fertilization rate, polyspermy rate, and development to blastocyst

will be examined. Results of these studies will begin to define the roles of oviductal PAI-

1 and pOSP during fertilization and embryonic development.

















Figure 2-1. Pericellular activation cascade for plasminogen and matrix metalloproteinase.

uPA, urokinase plasminogen activator; uPA-R, urokinase plasminogen activator receptor;
PAI-1, plasminogen activator inhibitor-1; a2 AP, alpha-two anti-plasmin inhibitor; TIMP,
tissue inhibitor of matrix metalloproteinase; MMP, matrix metalloproteinase. Proteolytic
enzymes and inhibitors (outlined in blue) involved in the regulation of fibrinolysis and
extracellular matrix remodeling/degradation.















cell
tPAl-1
TIMPns


pla II noTel s IIi















CHAPTER
IDENTIFICATION AND LOCALIZATION OF PLASMINOGEN ACTIVATOR
INHIBITOR-1 (PAI-1) IN THE PORCINE OVIDUCT


Introduction

The porcine oviduct provides an important microenvironment for final maturation

of gametes, fertilization, and early cleavage-stage embryonic development. In part to

provide an effective environment for these reproductive processes, numerous proteins

derived from serum as a transudate or from oviductal epithelium contribute to the

composition of oviductal luminal fluid [Feigelson and Kay, 1972, Sutton et al., 1984,

Buhi et al., 1997]. Oviductal luminal fluid and its protein composition vary during the

estrous cycle and early pregnancy, and maximal levels of oviductal fluid appear to

coincide with elevated estrogen [Hunter, 1988]. When estrogen is maximal, Days 0 and 1

of the estrous cycle or early pregnancy, biosynthetic activity in the oviduct is greatest

[Buhi et al., 1989]. The infundibulum and ampulla, regardless of day of the estrous cycle

or early pregnancy, have a biosynthetic activity significantly greater (2-3 times) than that

of the isthmus [Buhi et al., 1997]. This activity, measured in explant culture-conditioned

media, reflects the de novo synthesis and secretion of secretary proteins from these three

segments.

The relative importance of oviductal-derived proteins to fertilization and early

embryonic development is not known, nor have many of these proteins been identified.

Several studies have shown the importance of utilizing the oviduct or its constituents for

58







59

enhancing embryo development in vitro [Archibong et al., 1989, White et al., 1989, Liu

et al., 1998]. In the pig, glycoproteins derived from oviductal fluid have been shown to

associate with the zona pellucida of oocytes after ovulation, at fertilization and during

early embryonic development [Brown and Cheng, 1986, Hedrick et al., 1987, Buhi et al.,

1993]. It has been suggested that these proteins may be facilitating the beneficial effects

of oviductal epithelial cell co-cultures on embryo development in vitro [Buhi et al.,

1997].

The pig oviduct has been shown to synthesize and secrete de novo 14 major

proteins into explant culture medium [Buhi et al., 1990]. The majority of these proteins

have been described electrophoretically by isoelectric point and relative molecular mass.

Two of these proteins have been identified and characterized, the porcine oviduct-specific

secretary glycoprotein (pOSP) [Buhi et al., 1992] and tissue inhibitor of matrix

metalloproteinase-1 (TIMP-1) [Buhi et al., 1996]. Of the numerous proteins yet to be

identified, one protein with an apparent molecular weight of 45,000, appeared to be

synthesized and distributed similar to TIMP-1. Like TIMP-1, this protein was shown to

incorporate 3H-glucosamine, be composed of 5-6 isoelectric species, and be synthesized

primarily by the isthmus [Buhi et al., 1990]. Thus, with characteristics similar to

oviductal TIMP-1, it was suggested that the 45,000 Mr protein may also be a protease

inhibitor.

This study was designed to further identify and characterize the 45,000 M, de

novo synthesized protein of the porcine oviduct and to better understand specific protein

contributions of each oviductal segment relative to ovulation, gamete transport,

fertilization and early cleavage-stage embryonic development. With identification of the







60

unknown 45,000 M, protein as porcine plasminogen activator inhibitor (PAI)-1

(described here in Chapter 3), studies were designed to examine its synthesis within the

infundibulum, ampulla, and isthmus, and to evaluate its distribution throughout the

oviduct. While PAI-1 has been localized in the uterus and ovary from a variety of

species and potential mechanisms examined, no studies to our knowledge have been done

to assess PAI-1 within the oviduct.

Materials and Methods

Materials

Acrylamide, N,N' diallyltartardiamide, urea, Nonidet P-40, and sodium dodecyl

sulfate were purchased from Gallard-Schlesinger (Carle Place, NY); X-Omat AR film

and photography reagents were a product of Eastman Kodak Co. (Rochester, NY); amino

acids and protein standards were purchased from Sigma-Aldrich (St. Louis, MO);

ampholines were from Pharmacia-Biotech (Piscataway, NJ); all other supplies and

reagents for gel electrophoresis were purchased from Bio-Rad Laboratories (Richmond,

CA) or Fisher Scientific (Orlando, FL). All medium and culture supplies were purchased

from Life Technologies (Grand Island, NY). L-[4,5-3H]leucine (specific activity, 120

Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Immobilin-P (PVDF)

membranes were purchased from Millipore Corporation (Bedford, MA); Vectastain ABC

Elite kit was obtained from Vector Laboratories (Burlingame, CA). Affinity-purified

goat anti-human PAI-1 was purchased from American Diagnostica (Greenwich, CT);

goat IgG and normal rabbit serum (NRS) were purchased from Sigma-Aldrich. All other

reagents, including column chromatography supplies, were products from Sigma, Fisher,

Life Technologies or Pharmacia-Biotech.









Tissue Collection. Explant Culture, and Electrophoresis

Florida crossbred (Yorkshire x Duroc x Hampshire) or European Large White

gilts were observed daily for behavioral estrus in the presence of an intact boar. After the

completion of at least two estrous cycles, animals for pregnancy studies were bred on the

first day of standing estrus, designated as Day 0, and 24 h later (except for gilts assigned

to Day 0). Pregnant or cyclic gilts were taken to the abattoir on Days 0, 2, and 12 for

slaughter. Oviducts were collected aseptically and separated by gross dissection into the

three functional segments, which were then cultured as previously described [Buhi et al.,

1989]. For culture, 0.5 g of tissue was incubated in leucine-deficient (modified) Eagle's

minimum essential medium (MEM) containing 100 PCi [3H]-leucine for 24 h at 390 C in

a defined atmosphere. Conditioned culture media was then separated from tissue and

frozen at -200 C until analyzed by 2D-SDS-PAGE and fluorography or utilized for

purification of the 45,000 Mr protein.

In order to evaluate synthesis of the 45,000 Mr protein within the three segments,

oviductal tissue was collected from Day 2 pregnant Large White gilts and cultured as

described above. Culture media was dialyzed (12,000 molecular weight cut-off) against

10 mM Tris-HC1 buffer (pH 7.6), containing 0.15 M NaCI and 0.02% (w/v) NaN3,

followed by dialysis against dH20 (2 changes, 4L each, 24 h each) at 40 C. Total protein

content of dialyzed culture media was measured by the Bio-Rad microassay (according to

manufacturer's instructions) and radiolabeled proteins measured by liquid scintillation

spectrophotometry. Samples representing each oviductal segment, containing 100,000

cpm, were lyophilized, solubilized in Laemmli's buffer [Laemmli, 1970] and analyzed







62

by 2D-SDS-PAGE and subjected to fluorography as described previously [Buhi et al.,

1995]. All X-ray films were exposed for 14 days at -800 C and developed.

Protein Fractionation

Explant culture media conditioned by isthmic tissue on Days 0, 2, and 12 of early

pregnancy, were pooled and subjected to gel filtration chromatography on a Sepharose

CL-6B column as described previously [Buhi et al., 1990] with some modifications. The

Sepharose CL-6B column (1.8 x 92 cm) was equilibrated in column buffer [10 mM Tris-

HCI, 0.4 M NaCI, 0.02% (w/v) NaN3; pH 7.5] at 40 C. Culture media (7 ml) centrifuged

at 2,200 x g for 10 minutes at 40 C to remove particulate material, was added slowly to

the column. After collection of the void volume, elution profiles were generated by

collecting 2 ml fractions, measuring protein (absorption at 280 nm) and determining

radioactivity in each fraction. Chromatographic peaks corresponding to the elution of

ovalbumin (45,000 Mr standard) were pooled from individual column runs.

Immunoglobulins were removed from pooled fractions by incubation with Protein A-

Sepharose beads in 10 mM phosphate-buffered saline (PBS), pH 8.0, overnight at 40 C.

Beads were then separated from the supernatant by centrifugation at 1,700 X g for 10

minutes at 40 C, washed 3x in PBS, and pooled supernatants dialyzed against dH20 (2

changes, 4 L each, 24 h each, 40 C). The dialyzed sample was analyzed for protein

content and radioactivity, as indicated above, and lyophilized. Lyophilized samples were

then resuspended in column buffer (lml/15 mg protein) and further fractionated on a

Sephadex G-100 column (1.5 x 75 cm) at 40 C. The Sephadex G-100 column was

calibrated previously with Blue Dextran, apotransferrin, ovalbumin and cytochrome C as

molecular weight markers. Elution profiles were generated using methodology







63

established for the Sepharose CL-6B column. The pooled chromatographic peaks from

individual column runs corresponding to the 45,000 Mr standard were utilized for

subsequent Western blotting and N-terminal amino acid sequence analysis. Purification

at each step was examined by 2D-SDS-PAGE and fluorography [Buhi et al., 1990].

Western Blotting and Sequence Analysis

The pooled chromatographic peaks containing the 45,000 Mr protein were

separated by Tris-tricine 2D-SDS-PAGE [Schagger and von Jagow, 1987]. Proteins were

then transferred by semi-dry electrophoresis (Milli-Blot SDE system, Millipore Corp,

Bedford, CA) to a PVDF membrane as described previously [Buhi et al., 1995] with

some modifications. Following separation of proteins by Tris-tricine 2D-SDS-PAGE, the

gel was rinsed in three changes of dH20 for 5 min each, then equilibrated in 25 mM Tris-

HCI buffer (pH 9.4) containing 2% (w/v) SDS, for 30 min. The gel was washed in three

changes of dH20 for 5 min, and incubated in two changes of cathode C buffer (25 mM

Tris-HCl, 5.25 g/L norleucine, 10% (v/v) methanol; pH 9.4) for 10 min at room

temperature. After assembly of the TransUnit sandwich, proteins were transferred for 1 h

under constant current (2.5 mA per cm2). Subsequently, the membrane was washed

(dH20, 5 min), and stained in 0.1% (w/v) Coomassie blue R-250 in 50% (v/v) methanol

for 1 min. The membrane was destined (50% methanol, 5 min) with constant rocking

and rinsed in three changes of dH20. The PVDF membrane was allowed to air dry,

proteins corresponding to the 45,000 Mr protein were excised, and subjected to N-

terminal amino acid microsequencing at the Interdisciplinary Center for Biotechnology

Research (ICBR) facility using a 470A gas phase protein sequencer (Applied

Biosystems) with an on-line analytical HPLC system. The peptide sequence was







64

analyzed with the National Center for Biotechnology Information (NCBI) BLAST

program [Altschul et al., 1990].

After identification of the 45,000 M, protein as porcine PAI-1 (see Results), a

one-step partial-purification method was employed using heparin-agarose affinity column

chromatography. PAI-1 has been shown previously to quantitatively bind to heparin-

Sepharose and elute with increasing concentrations of NaCI [Ehrlich et al., 1991].

Isthmic-conditioned culture media (Day 12 pregnant and Day 1 cyclic) were pooled,

centrifuged as described above, diluted (1:3) in 20 mM Tris-HCI (pH 7.6, 40 C)

containing 0.02% (w/v) NaN3, and slowly loaded onto a heparin-agarose column (2.5 x

8.2 cm) at 40 C. PAI-1 was eluted utilizing stepwise increments of NaCl (0.1-3.0 M), and

the protein pooled and dialyzed against dH20 (two changes, 24 h each, 4L each, 40 C).

Protein content was determined as above, and aliquots containing 0.5 mg of protein

lyophilized and used for immunoprecipitation. Fractions were analyzed for the presence

of PAI- by 2D-SDS-PAGE and fluorographic analysis.

Immunoprecipitation

PAI-1, semi-purified by heparin-agarose affinity column chromatography, was

immunoprecipitated with a polyclonal rabbit anti-hPAI-1 antiserum (kindly provided by

Dr. Schleef, Scripps Institute, La Jolla, CA). Lyophilized protein samples (0.5 mg) were

solubilized in 900 Il of NET buffer [50 mM Tris-HC1, 0.15 M NaCI, 0.1% (v/v) Nonidet

P-40, 1 mM EDTA, 0.25% (w/v) gelatin, and 0.02% (w/v) NaN3; pH 7.5 at 250 C].

Protein A-Sepharose beads were equilibrated in NET buffer and 100 Al of swollen beads

were incubated with 100 pl of either undiluted PAI-1 antiserum or NRS and 300 ul of

NET buffer for 1 h at 250 C. After incubation, Protein A-Sepharose beads were pulse-







65

centrifuged (Beckman, microcentrifuge) for 30 sec and washed in three changes of NET

buffer (0.5 ml). Semi-purified PAI-I protein (100-200 pl) was then incubated with NRS

or rabbit anti-hPAI-1 antibody-coated beads (200 il), respectively, for 2 h at 250 C with

constant rotation. Complexes were pelleted by centrifugation and washed as above.

Proteins conjugated to the Protein A-Sepharose beads were solubilized in Laemmeli

buffer [Laemmeli, 1970], boiled for 3 min, separated on a 10% (w/v) 1D-SDS-PAGE gel

and subjected to fluorography [Buhi et al., 1995]. Heparin-agrarose fractionated proteins,

which were not used for immunoprecipitation, were removed from solution using

standard acetone precipitation procedures [Harlow and Lane, 1988] and solubilized in

Laemmeli buffer as above for positive radiolabeled PAI-I identification.

Immunocvtochemistry

A polyclonal affinity-purified goat anti-hPAI-1 was used to immunolocalize PAI-

1 in porcine oviductal tissues from cyclic and early pregnant animals. To compare

distribution of PAI-1 in cyclic and early pregnant animals, infundibulum, ampulla and

isthmic tissues were collected on Days 0, 2, and 12, (n=3 animals/day) and

immunocytochemistry performed as described [Chegini et al., 1992]. Tissues were cut

into 5 mm portions, fixed in Bouin's solution, embedded in paraffin, sectioned (0.5 pm),

and mounted on precoated glass slides. A goat Vectastain ABC Elite kit (Vector

Laboratories, Burlingame, CA) was used according to the manufacturer's instructions.

Controls included use of an affinity-purified goat IgG and the absence of primary

antibody. Goat anti-hPAI-1 and goat IgG were used at a dilution of 1:10 in PBS (pH

7.4).









Immunogold Electron Microscopy (EM)

Oviductal tissue from the three segments of Day 0 non-pregnant and Day 9

pregnant crossbred gilts, were fixed for 1 h in PBS, pH 7.4, containing 0.5% (v/v)

glutaraldehyde, 4% (v/v) paraformaldehyde at 40 C. The two days selected were times of

elevated estrogen (Day 0) or progesterone (Day 9) production. After fixation and rinsing

in PBS, tissues were dehydrated in graded ethanol series and embedded in Unicryl

(British BioCell International, UK ) under UV light at -100 C for 2 days. Thin sections

(0.5 mm) were cut and collected on Formvar-coated 100 mesh nickel grids, and PAI-I

antigen detected by immunogold labeling. The polyclonal rabbit anti-human PAI-I and

preimmune rabbit sera, diluted 1:1000 in a high salt Tween buffer (0.02 M Tris-HCI, 0.5

M NaCI, 1% [v/v] Tween 20, pH 7.2) supplemented with 1% (w/v) ovalbumin, were

incubated overnight with grids in a humid chamber at 40 C. Sections were then

incubated with a secondary antibody (goat anti-rabbit IgG, 1:30 dilution in PBS)

conjugated to 18 nm colloidal gold (Jackson ImmunoResearch Laboratories, Inc, West

Grove, PA) for 1 h at room temperature. Sections were then post-stained with 2% (w/v)

uranyl acetate and Reynolds lead citrate. Grids were examined on a Hitachi H-7000

transmission electron microscope (Hitachi Scientific Instruments, Danbury, CT). Digital

micrographs were taken on a Gatan BioScan/Digital Micrograph 2.5 (Gatan Inc,

Pleasanton, CA).

Results

Electrophoretic Analysis

Representative 2D-SDS-PAGE and fluorographic analyses of explant culture

media from the infundibulum, ampulla, and isthmus containing radiolabeled de novo







67

synthesized proteins are shown in Figure 3-1. The 45,000 Mr protein, found in all three

segments, is seen as the major radiolabeled protein from the isthmic portion of the

oviduct (Figure 3-1C). The level of protein expression appears to be reduced in the

ampulla (Figure 3-1B) and nearly absent in the infundibulum (Figure 3-1A). Thus, the

45,000 Mr protein appears to have a specific spatial synthesis and release in the oviduct.

This specific spatial expression is similar to that reported for the matrix metalloproteinase

inhibitor, TIMP-1, in the oviduct [Buhi et al., 1996] (Figure 3-1).

Purification, Blotting, and N-terminal Sequencing

With identification of the 45,000 Mr protein as the major radiolabeled protein of

the isthmus, the next objective was to purify this protein for N-terminal sequencing. A

two-step gel-filtration chromatographic procedure was developed in order to separate and

enrich this protein. Elution profiles for protein and radioactivity from Sepharose CL-6B

and Sephadex G-100 columns are shown in Figure 3-2A and 3-2B, respectively.

Fractionation of explant culture media on these two columns, each showed two peaks of

radioactivity. The broad second peak in both graphs, contained the greatest amount of

radioactivity and included the 45,000 Mr protein. A representative fluorograph of the

45,000 Mr protein after fractionation on a Sephadex G-100 column is shown in Figure 3-

2C. This protein appears to contain at least 5 isoelectric species, of which 3 species,

including an acidic and basic species, were submitted for N-terminal amino acid

sequence analysis. A search of protein, RNA and DNA data banks indicated that the

derived N-terminal amino acid sequence of 26 amino acids, identical for all three

isoelectric species, was 96% identical (100% similar) to porcine PAI-1 (Figure 3-3). This

sequence (1-26) corresponded to amino acid positions 20-46 of mature PAI-1 protein,







68

indicating removal of the hydrophobic leader peptide prior to release from the cell

[Bijnens et al., 1997].

Immunoprecipitation

To confirm that the 45,000 Mr protein identified by N-terminal amino acid

microsequencing was PAl-1, an anti-hPAI-1 serum was used for immunoprecipitation of

this protein from isthmic culture media after fractionation by heparin-agarose affinity

column chromatography. Fractionated culture media containing de novo synthesized

radiolabeled proteins were treated with either Protein A-complexed rabbit anti-hPAI-1

serum or Protein A-complexed NRS. Bound proteins were solubilized and examined by

1D-SDS-PAGE and fluorography (Figure 3-4A and 3-4B). As shown in Figure 3-4B,

anti-hPAI-1 serum specifically recognized and precipitated radiolabeled PAl-1, while

NRS did not. These results indicate that this antibody does not cross-react with other

radiolabeled proteins present within the fractionated culture media. Western Immunoblot

analysis showed that the anti-hPAI-1 antisera cross-reacted with only 2-3 minor

unlabeled proteins, possibly oftransudate origin. The primary protein recognized by this

antibody on the Western Immunoblot was the PAI-1 family (5-6 isoelectric species) (data

not shown).

Immunocytochemistry

With identification of PA-1, the next objective was to examine its distribution

throughout the oviduct on Days 0, 2, and 12 of the estrous cycle or early pregnancy.

Immunoreactive PAl-1 was detected in all three segments of the oviduct regardless of

day of cycle examined and no differences in staining intensity could be detected between

days (infundibulum not shown). Representative data of the immunocytochemical







69

localization in the isthmus on these days is shown in Figure 3-5. No difference in

staining intensity could be detected (subjective visualization of three representative

animals) between pregnant and cyclic tissues within the three segments (Figure 3-6).

PAI-1 was localized primarily within the oviductal epithelium, while only background

staining could be identified within muscle and stroma tissue (Data not shown for muscle

and stroma). Here, PAI-1 appeared to be heavily concentrated at the apical region of the

epithelium (Figure 3-5C, arrow) with little staining found in the basal region. However,

PAI-I immunoreactivity was also associated within cells lining blood vessels in the

stroma. Immunocytochemistry pictures shown are representative of 3 animals/day.

Because staining intensity varied between each animal examined, subjective

comparisons on the level of staining intensity between pregnant and cyclic gilts were not

made.

Electron Microscopy

Since immunocytochemistry was not able to resolve PAI-1 association with

specific cellular structures or organelles within the epithelium, an examination of PAI-1

distribution within the epithelium was performed using electron microscopic

immunogold localization. PAI-I was found primarily in epithelium of isthmic non-

ciliated secretary cells and appeared to be concentrated in putative secretary granules at

or near the apical border of cells in both Day 0 non-pregnant and Day 9 pregnant animals

(Figures 3-7A and 3-8A). In addition, this protein was also found to be associated with

the isthmic luminal epithelial border and cilia (Figures 3-7B and 3-8B). Localization of

PAI-1 within ampullary epithelium (both ciliated and non-ciliated) was negligible (Figure

3-8C). Isthmic tissue incubated with preimmune sera (control) showed no labeling







70

within secretary granules or at the epithelial border (Figure 3-8D). As detailed in the

immunoprecipitation results, the antibody employed showed no potential cross-reactions

with other proteins found within oviduct-coniditioned culture medium.

















Figure 3-1. Representative fluorograph after 2D-SDS-PAGE separation of [HI-labeled
proteins (100,000 cpm) from explant culture media conditioned by the three segments of
the oviduct.

Tissue was collected from the A) infundibulum, B) ampulla or C) isthmus of Large
White X Landrace gilts on Day 2 of pregnancy. The 45,000 Mr protein is indicated by an
arrow in each panel. TIMP-1 is marked by an arrowhead only in panel C. Molecular
weight markers (x 103) are indicated and the pH gradient runs from left (pH 8) to right
(pH 4).







72




















s. ? r *i
iT -

U -


4* .

















Figure 3-2. Representative Sepharose CL-6B and Sephadex G-100 fractionations of
[3H]-labeled proteins in isthmus-conditioned medium.

Fractions (2 ml) collected from the CL-6B column elution (Panel A) were counted for
radioactivity and measured for protein (see Methods). In Panel A, tubes 58-75 (peak 2,
bracket) containing the 45,000 M, protein, were pooled, treated with Protein A beads to
remove immunoglobulins and further factionated on a G-100 column (Panel B). In panel
B, tubes 48-60 (peak 2, bracket), containing the 45,000 Mr protein were pooled, and
separated by Tris-tricine 2D-SDS-PAGE for electroblotting. Arrows in panel A and B
reflect elution point of 45,000 Mr ovalbumin molecular weight standard. Panel C, shows
a representative fluorograph of the 45,000 Mr protein after chromatographic separation by
Tris-tricine 2D-SDS-PAGE. Three isoelectric species (marked with arrows) were
excised and subjected to N-terminal amino acid sequence analysis. Molecular weight
markers (x103) are indicated and the pH gradient runs from left (pH 8) to right (pH 4).












1 20 00 ,n


.0W






o 10 s 30 40 5a so 70 0









oI -ieo
a 10 20 0 40 so 1 7




o tio a so
vms--------------- --, R







Fraion anuir

C
97
66





29' *r 0
~ ~ 10 M M W C t *
















20.... 25 .... 30 ... 35 .. 40 .... 45 .
Porcine
EGSAS SHHQS LAARL ATDFG VKVFR QV PAI-1

EGSAS SHHQS LAARL ATDFG VKXFR QV 45,000 M,
isthmic protein
1.. 5 .. .. 10 .. 15 .. .. 20 .. 25 .



Figure 3-3. Comparison of the identified N-terminal amino acid sequence of the 45,000
Mr protein from isthmic-conditioned culture media to the N-terminal sequence of porcine
PAI-1.

Proteins were fractionated by size-exclusion chromatography using a Sepharose CL-6B
and Sephadex G-100 column, separated by Tris-tricine 2D-SDS-PAGE, transferred by
electroblotting to PVDF membranes and subjected to N-terminal amino acid micro-
sequence analysis. X (residue 22) indicates an undetermined amino acid residue. A 96%
sequence identity was found to porcine plasminogen activator inhibitor-i (Accession #
1870170).






-----.- 76











2 3

I
















1 2 3




Figure 3-4. Immunoprecipitation of plasminogen activator inhibitor-1 from porcine
isthmic-conditioned culture media after fractionation on a heparin-agarose affinity
column and elution with 0.4 M NaCI.

A representative one-dimensional SDS-polyacrylamide gel (Panel A) containing
Coomasie blue-stained proteins from either a non-precipitated sample (lane 1), proteins
precipitated using a rabbit anti-human PAI-1 (lane 2) or normal rabbit sera (lane 3).
Although not visible on the gel, the arrow indicates the apparent location of PAI-I
protein. Panel B shows a fluorograph generated from the same gel in Panel A. Lanes are
as indicated above. PAI-1 found in semi-purified culture media, was specifically
precipitated by antibody (lane 2), but not normal rabbit serum (lane 3).










rr


Figure 3-5. Representative immunocytochemical localization of PAI-1 in the isthmus
from crossbred gilts on Day 0 (Panel A), Day 2 (Panel B) or Day 12 (Panel C) of
pregnancy.

The control is shown in Panel D. Arrow indicates intense staining of PA-1 within the
apical region ofisthmic epithelium. X 20.







































Figure 3-6. Representative immunocytochemical localization of PAI-1 in oviductal
tissue from Day 2 cyclic (Panel A and C) and Day 2 pregnant (Panel B and D) crossbred
gilts from the isthmus (Panel C and D) and ampulla (Panel A and B).

The infundibulum is not shown. Arrow indicates intense staining of PAI-1 within the
apical region of the epithelium. X 20.

















Figure 3-7. Immunogold labeling of PAI-1 within oviductal tissue from a Day 0 non-
pregnant crossbred gilt.

Gold particles are seen in secretary granules of isthmic non-ciliated cells (Panel A,
arrow) as well as associated with the epithelial luminal border and cilia (Panel B, arrow).
X 20,000.























Figure 3-8. Immunogold labeling of PAl-I within oviductal tissue from a Day 9 pregnant
crossbred gilt.

Gold particles can be seen clustered within putative secretary granules of isthmic non-
ciliated cells (Panel A, arrow) and associated with the epithelial luminal border and cilia
(Panel B, arrow). Gold particles were not detected in secretary granules of the ampulla
although some scattered particles were associated with cilia (Panel C). Immunogold
staining was not detected in the isthmus treated with preimmune serum (Panel D, arrow).
X 20,000.





82









; .,, f-.



P o. ,


^' ,, ;. \.y. %' % ,,
'* j, ." '.^"" .' t-" "

.. -,- -.,- :'.-,',.

,A, ". "' .'." '.,'IL ''Yd










Discussion

The mammalian oviduct secretes numerous de novo synthesized proteins into the

oviductal lumen, some of which are present before ovulation, or during fertilization and

early cleavage-stage embryonic development. In order to characterize the 14 major de

novo proteins secreted by the porcine oviduct, explant tissue (infundibulum, ampulla, and

isthmus), was incubated with [3H]-leucine, and synthesized and secreted proteins

separated by 2D-SDS-PAGE, detected by fluorography, and characterized by molecular

weight and isoelectric points [Buhi et al., 1990]. Two of these proteins have been

extensively examined, while others remain unidentified [Buhi et al., 1997], including a

45,000 M, protein synthesized primarily by the isthmic portion of the oviduct. Sequence

analyses revealed that this isthmic protein was porcine plasminogen activator inhibitor-1.

Recently, an unidentified 45,000 M, protein found in bovine oviductal fluid was shown to

associate with the zona pellucida of bovine oocytes [Staros and Killian, 1998] and may

be the protein under investigation.

PA-1, a member of the serpin family of serine protease inhibitors, is the primary

inhibitor ofurokinase plasminogen activator (uPA) and tissue-type plasminogen activator

(tPA). It is a glycoprotein consisting of 379 amino acids with an apparent molecular

mass of 45,000 [Andreasen et al., 1990]. Among the serpins, PAI-1 is secreted in an

active form that rapidly converts to an inactive latent form that can be reactivated by

phospholipids and a variety of denaturants [Hekman and Loskutoff, 1985]. The

inhibitory activity of PAl-I, however, is stabilized by vitronectin in either serum or

extracellular matrix (ECM), thus preventing transformation to the inactive latent form

[Declerck et al., 1988]. Both tPA and uPA initiate proteolysis by converting plasminogen







84

to the broad-specificity enzyme plasmin. This extracellular protease is reported to be

involved in the remodeling of ECM, fibrinolysis, cell migration, and tumor metastasis

[Andreasen et al., 1990]. Plasmin can also activate pro-matrix metalloproteinases

(MMPs) thereby regulating the pericellular activation cascade leading to ECM

degradation [Murphy et al., 1992]. Complex control of this activation cascade is

regulated by PAI-1 and tissue inhibitors of matrix metalloproteinases (TIMPs).

Immunoprecipitation confirmed the presence of PAI-1 which in the oviduct,

appeared to consist of at least five or more isoelectric species as shown by fluorographic

analysis, N-terminal amino acid sequencing and immunoblot analysis (data not shown for

immunoblot). The presence of these isoforms suggests that newly synthesized and

secreted PAI-1 was post-translationaly modified. One modification was characterized as

glycosylation with incorporation of ['H]-glucosamine into PAI-1 during tissue explant

culture [Buhi et al., 1990]. Localization and synthesis of PAI-1 appears to mimic a

matrix metalloproteinase inhibitor from the oviduct, TIMP-1 [Buhi et al., 1996]. Both

TIMP-1 and PAI-1 show a similar spatial expression by the oviduct segments, with a

greater expression in the isthmus relative to either the ampulla or infundibulum. This

would suggest an important function relative to its spatial expression. The isthmus

portion of the oviduct has been described as a spermatozoa reservoir where sperm

undergo capacitation and hyperactivation [Suarez, 1998]. In the pig, the ampulla-isthmic

junction is the location of fertilization and early cleavage-stage embryonic development

[Buhi et al., 1997]. This protein may therefore facilitate or regulate these important

reproductive events that occur within or near the isthmus. However, PAI-1 may act in

other segments of the oviduct as well, due to retrograde movement of oviductal fluid into







85

the peritoneal cavity during estrus [Hunter, 1988]. Localization of PAI-I in the

infundibulum and ampulla epithelium and protein secretion by explant tissues, suggests

that these segments may also contribute to luminal PAl-1, although synthesis of PAI-1 in

these segments appears to be very low.

Fibrin deposits have been located on the tubal mucosa of the oviduct, which could

possibly interact with the ECM components of the zona pellucida and prevent tubal

transport [Liedholm and Astedt, 1975]. Liedholm and Astedt (1975), observed

fibrinolytic activity associated with the unfertilized ovum in rats and suggested that this

activity may be involved with the prevention of adhesion to fibrin deposits that may

hinder gamete transport. This activity was also shown to be associated with spermatozoa.

Prevention of cellular adhesion to the oviductal mucosa and fibrin deposits might also be

regulated by uPA and tPA. The developing embryo may produce uPA/tPA in response to

signals from the surrounding oviductal environment. Fibrin has been shown to increase

expression of uPA mRNA and protein in 3.5 day old uterine embryos of the mouse

[Zhang et al., 1996]. Both mouse and rat preimplantation embryos have been shown to

have tPA activity [Zhang et al., 1992, Carroll et al., 1993] and uPA activity [Zhang et al.,

1994, Harvey et al., 1995]. Expression of this proteolytic activity, or the respective

mRNA, was found to be developmental- and stage-specific. While the fibrinolytic

activity of the ovum and proteinase expression by the embryo may facilitate their

transport through the oviduct, these molecules are potent modulators of their immediate

environment with respect to ECM remodeling. PAl-1, an important regulator of both

fibrinolysis and plasminogen activators, may act as a stabilizing or counter-regulatory

factor for maintaining ECM integrity of the oviductal epithelium. Therefore, production







86

of PAI-1 by the oviduct might act to prevent premature nidation of the preimplantation

embryo. While the pig has noninvasive placentation, the trophoblast of the pig has

invasive potential as shown by its transfer to ectopic sites [Samuel, 1971]. Part of this

invasive potential may be due to fibrinolytic and plasminogen activator activity of the

embryo. Liedholm and Astedt (1975) suggested that the fibrinolytic activity of the ovum

may be depressed by an inhibitor of plasminogen activation. Plasmin-induced proteolysis

has been shown to be important for implantation in invasive species such as the mouse

[Sappino et al., 1989, Zhang et al., 1996]. However, in the pig, an endometrial inhibitor

of plasmin production has been suggested to protect the endometrium from the

blastocyst-induced proteolysis during placentation [Fazleabas et al., 1982]. PAI-1

secretion within the oviduct, may therefore have a similar function to that of the

endometrial plasmin inhibitor.

Immunolocalization results indicated that PAI-I is localized within the oviductal

epithelium and is heavily concentrated near the apical membrane, suggesting secretion

into the lumen. Immunogold EM of the isthmus, revealed that PAI-1 was located in

putative secretary granules within the lumen and associated with cilia. While PAI-1 was

localized to the ampulla using immunocytochemistry, immunogold electron microscopy

was unable to detect its presence. This may reflect a difference in specificity and

dilutions of two different antibodies used, difference in tissue sections examined between

the two different procedures and 2D-SDS-PAGE and fluorographic analyses, which

suggest that PAI-1 protein secretion is low in the ampulla. Both immunocytochemistry

and fluorographic analysis have shown that PAI-1 is localized in the ampulla and

infundibulum, however, the inability to locate immunoreactivity utilizing electron







87

microscopy may be that protein synthesis of this molecule is very low in these segments

and is present in secretary granules in very small amounts. PAI-1 was found in secretary

granules from isthmic tissue exposed to either a high estrogen (Day 0 cyclic) or high

progesterone (Day 9 pregnant) environment indicating that this protein is synthesized

throughout the estrous cycle or early pregnancy. Evidence from our laboratory

(unpublished) indicates that PAI-1 secretion may vary during the estrous cycle or early

pregnancy and its synthesis might be controlled by ovarian steroids. Reports on PAI-1

regulation in endometrial stromal and decidual cell cultures suggest that this protein is

up-regulated by progesterone, while estrogen antagonizes this effect [Schatz and

Lockwood, 1993]. The oviduct along with the uterus, is a major target for ovarian

steroids and steroid-modulated and cycle-specific changes have been noted for two other

de novo synthesized products of the epithelium, pOSP and TIMP-1 [Buhi et al., 1997].

Because the zona pellucida can be subject to proteolytic degradation, oviductal

PAI-1 may also protect the integrity of the zona pellucida from embryonic or oviductal

plasminogen activator activity. To our knowledge, PAI-I mRNA or activity has not been

examined in the early cleavage-stage embryo or in the oviduct. The porcine zona

pellucida of oviductal oocytes or embryos was found to be more resistant to proteolytic

degradation than that of either follicular oocytes or embryos collected from the uterine

environment [Broermann et al., 1989], suggesting a potential interaction of oviductal

protease inhibitors with the oocyte/embryo. Changes in resistance of the zona pellucida

to proteases may be dependent upon addition of glycoproteins/inhibitors obtained during

transit through the oviduct. Thus, an oviduct-specific factor may act to protect the zona

pellucida and embryo from degradation by proteolytic enzymes. Proteases are present in







88

oviductal flush and include the plasminogen activators and matrix metalloproteinases

(Kouba AJ, unpublished). PAI-1 working together with TIMP-I, may act to tightly

regulate this proteolytic activity. Plasminogen, the natural substrate for uPA and tPA, is

found in many extracellular fluids including seminal plasma [Kobayashi et al., 1992] and

follicular fluid [Beers, 1975], and may be enriched in oviductal fluid at or near the time

of fertilization. Estrogens have been shown to stimulate the uptake of plasminogen from

plasma by the mouse uterus [Finlay et al., 1983] and a similar function may occur in the

oviduct during estrus. Plasminogen has been shown to bind to mouse spermatozoa,

oocytes, and cumulus cells, which enhanced the local generation of plasmin [Huarte et

al., 1993] and this binding allows for PA-induced proteolysis to discrete focal areas.

Huarte et al. (1993) also showed that addition of plasminogen or antibodies to plasmin

during in vitro fertilization could increase and decrease, respectively, the fertilization

rate. Therefore, PAI-1 within the oviduct may inhibit oocyte or embryonic generation of

plasmin from plasminogen, due to their inherent tPA and uPA activity, thus maintaining

integrity of the zona pellucida while not affecting fertilization.

Further objectives will be to evaluate the biological role of PAI-1 within the

oviduct. PAl-1 may act in a autocrine/paracrine fashion to prevent proteolytic

degradation of ovulated oocytes or early embryos in the oviduct or uterus, prevent

premature hatching, regulate ECM remodeling of the oviduct or early embryo, inhibit

embryonic invasion of the oviductal lining, and promote embryonic development.




Full Text
IDENTIFICATION, CHARACTERIZATION, AND REGULATION OF PORCINE
OVIDUCT AL PLASMINOGEN ACTIVATOR INHIBITOR-1 AND FUNCTIONAL
ANALYSIS OF OVIDUCTAL-SPECIFIC SECRETORY GLYCOPROTEIN
By
ANDREW J. KOUBA
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

This Dissertation is dedicated to Brenda and Jerry Kouba

ACKNOWLEDGMENTS
First and foremost, I need to thank God for the accomplishment of this
dissertation. Many times when the path became difficult and the end seemed so far away,
His gentle hand would continue to push me forward. Throughout my graduate career,
His graciousness has provided me with countless blessings. My relationship with and
faith in God is the responsibility of two wonderful individuals to whom I owe a great deal
of gratitude. My parents, Brenda and Jerry Kouba, have been a constant source of
support, encouragement, and love. They were my first teachers and continue to play such
a role in my life. At a very young age, they encouraged me to make observations,
formulate questions, and test my hypothesis; hence, my parents were the first to teach me
the scientific method. I also owe a great deal of thanks to my siblings, Bonnie and Joann
Kouba, who have always been supportive of my endeavors.
Second, I would like to thank my mentor, Bill Buhi, for his advisement during my
time at the University of Florida. I am deeply grateful for his acceptance of me into his
laboratory and the time that was involved in my education. I will always treasure his
guidance and our numerous conversations on what it means to become a scientist. I also
need to thank members of my committee-Pete Hansen, Ken Drury, Frank Simmen, and
Bill Thatcher-for their assistance throughout my program. Their suggestions in the
design and analysis of my experiments are greatly appreciated and I look forward to
future collaborations with all of them.
iii

Next, I would like to thank Idania Alvarez for the often difficult task of training
me in numerous aspects of protein biochemistry. During my graduate career, several
students, post-docs, and secretarial staff have aided, and I thank them for all their
assistance-Lalantha Abeydeera, Brant Burkhardt, Dan Arnold, Morgan Peltier, Lannett
Edwards, Michael Porter, Carrie Vance, Mary Ellen Hissem, and all those students who
heat-checked every morning with me. I also thank Billy Day and Maureen Goodenow
for allowing me to conduct experiments in their laboratories for the completion of this
dissertation. Lastly, heartfelt thanks go to all those friends who have constantly been
there for me and supported me throughout this journey - you know who you are.
IV

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT xi
CHAPTERS
1 INTRODUCTION 1
2 LITERATURE REVIEW
Biology of the Oviduct 4
Proteins, Growth Factors, and Cytokines of the Oviduct 19
Oviduct Secretions and Co-culture on Fertilization and Embryonic
Development 26
Sperm-Oviduct Interactions 29
Structure, Regulation, and Biological Actions of Plasminogen Activator
Inhibitor-1 31
Structure, Regulation, and Biological Actions of Porcine Oviductal-Specific
Secretory Glycoprotein 41
3 IDENTIFICATION AND LOCALIZATION OF PLASMINOGEN
ACTIVATOR INHIBITOR-1 (PAI-1) IN THE PORCINE OVIDUCT
Introduction 58
Materials and Methods 60
Results 66
Discussion 83
v

4 OVIDUCTAL PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1)
PROTEIN, mRNA, AND HORMONAL REGULATION
Introduction 89
Materials and Methods 91
Results 98
Discussion 109
5 PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) AND uPA ACTIVITY
IN THE OVIDUCT AND ASSOCIATION OF PAI-1 WITH THE
PREIMPLANTATION EMBRYO
Introduction 115
Materials and Methods 117
Results 123
Discussion 146
6 EFFECTS OF A PORCINE OVIDUCT-SPECIFIC GLYCOPROTEIN ON
FERTILIZATION, POLYSPERMY, AND EARLY EMBRYONIC
DEVELOPMENT IN VITRO
Introduction 151
Materials and Methods 153
Results 162
Discussion 176
7 SUMMARY AND CONCLUSIONS 183
REFERENCES 193
BIOGRAPHICAL SKETCH 220
vi

LIST OF TABLES
Table Page
6-1 Effect of pOSP on fertilization parameters of pig oocytes matured and fertilized in
vitro 167
6-2 Effect of pOSP on zona pellucida solubility of pig oocytes matured in vitro.. . . 170
vii

LIST OF FIGURES
Figure Page
2-1 Pericellular activation cascade for plasminogen and matrix metalloproteinases.57
3-1 Representative fluorograph after 2D-SDS-PAGE separation of [3H]-labeled
proteins (100,000 cpm) from explant culture medium conditioned by the three
segments of the oviduct 72
3-2 Representative Sepharose CL-6B and Sephadex G-100 fractionations of [3H]-
labeled proteins in isthmus-conditioned medium 74
3-3 Comparison of the identified N-terminal amino acid sequence of the 45,000 Mr
protein from isthmic-conditioned culture media to the N-terminal sequence of
porcine PAI-1 75
3-4 Immunoprecipitation of plasminogen activator inhibitor-1 from porcine isthmic-
conditioned culture media after fractionation on a heparin-agarose affinity
column and elution with 0.4 M NaCl 76
3-5 Representative immunocytochemical localization of PAI-1 in the isthmus from
crossbred gilts on Day 0 (Panel A), Day 2 (Panel B) or Day 12 (Panel C) of
pregnancy 77
3-6 Representative immunocytochemical localization of PAI-1 in oviductal tissue
from Day 2 cyclic (Panel A and C) and Day 2 pregnant (Panel B and D)
crossbred gilts from the isthmus (Panel C and D) and ampulla (Panel A and
B) 78
3-7 Immunogold labeling of PAI-1 within oviductal tissue from a Day 0 non-pregnant
crossbred gilt 80
3-8 Immunogold labeling of PAI-1 within oviductal tissue from a Day 9 pregnant
crossbred gilt 82
4-1 Densitometry of PAI-1 protein in fluorographs from 2D-SDS-PAGE analyses of
isthmic- and ampulla-conditioned medium (100,000 cpm) from Large White
and Meishan gilts during early pregnancy 102
viii

4-2 PAI-1 protein in oviduct flushes from crossbred gilts during early pregnancy. .103
4-3 Densitometry of PAI-1 protein in fluorographs from 2D-SDS-PAGE analyses of
isthmic-conditioned medium (100,000 cpm [3H]-leucine) from OVX cross
bred gilts 104
4-4 Dot-blot hybridization analyses of oviductal total RNA from cyclic crossbred
gilts 105
4-5 Dot-blot hybridization analyses of oviductal total RNA from the infundibulum,
ampulla, and isthmus of crossbred gilts 107
4-6 Dot-blot hybridization analysis of whole oviductal total RNA from OVX steroid-
treated crossbred gilts 108
5-1 Representative fluorograph of denaturant-activated or latent PAI-1 incubated in
the presence of uPA after non-reducing 1D-SDS-PAGE separation 126
5-2 Oviductal PAI-1 inhibition of uPA activity over time 128
5-3 Dose-dependent inactivation of uPA activity by activated PAI-1 129
5-4 Inhibition of uPA activity with activated PAI-1 (40 |tg) 30 minutes after initiation
of the enzymatic reaction (cleavage of the chromogenic substrate, 2-44x, by
uPA) 130
5-5 Plasminogen activator activity in oviduct flushes from early pregnant crossbred
gilts 131
5-6 Dose-dependent inhibition of uPA activity by amiloride 132
5-7 Inhibition of oviductal flush PA activity by amiloride 133
5-8 Electron micrograph of a pig preovulatory follicular oocyte with surrounding
cumulus cells 135
5-9 Immunogold localization of PAI-1 in association with pig preovulatory follicular
oocytes and cumulus cells 137
5-10 Immunogold localization of PAI-1 in association with oviductal oocytes 139
5-11 Immunogold localization of PAI-1 in association with oviductal embryos (2-4
cell) and spermatozoa attached and embedded in the zona pellucida 141
5-12 Immunogold localization of PAI-1 in association with early uterine embryos (8-16
cell) 143
IX

5-13 Controls for immunogold localization of PAI-1 in association with follicular
oocytes, oviductal oocytes and embryos, and uterine embryos 145
6-1 Representative fluorograph of [3H]-leucine labeled proteins (500 pg) from whole
oviduct explant culture media (Day 0/1) subjected to heparin-agarose affinity
column chromatography and separated by 2D-SDS-PAGE 166
6-2 Photograph of polyspermic porcine oocyte matured and fertilized without
exposure to pOSP 168
6-3 Effect of in vitro incubation of pOSP and anti-pOSP IgG on the polyspermy rate
of porcine oocytes matured and fertilized in vitro 169
6-4 Effect of in vitro incubation of pOSP on sperm binding to porcine putative
zygotes 171
6-5 Photograph of sperm binding to putative zygotes after pre-incubation and
fertilization in the presence of 0 pg/ml pOSP (A) or 100 pg/ml pOSP (B)..173
6-6 Effect of in vitro incubation of pOSP on cleavage rate using porcine oocytes
matured, fertilized and cultured in vitro 174
6-7 Effect of in vitro incubation of pOSP on blastocyst development using porcine
oocytes matured, fertilized and cultured in vitro 175
7-1 Model for the de novo synthesis and secretion of proteins by the porcine
oviduct 192
x

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
IDENTIFICATION, CHARACTERIZATION, AND REGULATION OF PORCINE
OVIDUCTAL PLASMINOGEN ACTIVATOR INHIBITOR-1 AND FUNCTIONAL
ANALYSIS OF OVIDUCTAL-SPECIFIC SECRETORY GLYCOPROTEIN
by
Andrew J. Kouba
December 1999
Chairperson: William C. Buhi
Major Department: Animal Science
In mammals the oviduct de novo synthesizes and secretes numerous proteins that
may act to facilitate the processes of fertilization and early cleavage-stage embryonic
development. Several of these proteins have been shown to be steroid-modulated and
have cycle-specific expression. However, the majority of these proteins remain
unidentified and have only been characterized in a limited fashion by molecular weight
and isoelectric point. Functional data relating to these proteins is especially lacking. The
first objective was to identify and characterize the major de novo secretory product of the
isthmus. The second objective was the examine potential functions of the well-
characterized pOSP during in vitro fertilization and embryo culture.
The major de novo secretory product of the isthmus was identified as plasminogen
activator inhibitor-1 (PAI-1). It was demonstrated that this protein is localized to
oviductal epithelium and packaged into putative secretory granules for exocytosis. Upon
YÍ

release into the oviductal lumen, PAI-1 was shown to associate with oviductal oocytes,
embryos, and spermatozoa. Characterization of this protein revealed that (1) estrogen
inhibits mRNA and protein expression, (2) mRNA and protein is greatest during
fertilization and early embryo development, and (3) once activated, PAI-1 can form a
complex with uPA and inhibit its activity. In addition, PAI-1 levels are greatest when PA
activity was highest. These results suggest an important role for the regulation of the
plasminogen/plasmin system during fertilization and early embryonic development.
Functional analysis of pOSP revealed several potential actions of this protein
during fertilization. It was demonstrated that pOSP significantly reduced the incidence of
polyspermy in pig oocytes fertilized in vitro. After fertilization in the presence of pOSP,
a reduction in the number of sperm cells attached to the zona pellucida was observed.
This may be a potential mechanism whereby pOSP reduces the number of sperm cells
that can attach and penetrate an egg. Pig oocytes fertilized in the presence of pOSP had
increased blastocyst development compared to the control. Therefore, the reduction in
the rate of polyspermic fertilization may have resulted in enhanced development to
blastocyst. The pOSP reduction in polyspermy could be inhibited by anti-pOSP
indicating that the decreased incidence of polyspermic fertilization was specific to pOSP.
The characterization and functional analysis of these two proteins, PAI-1 and
pOSP, indicates an important relationship between their expression, association with the
oocyte or embryo, and potential actions during fertilization and development.

CHAPTER 1
INTRODUCTION
This dissertation will attempt to enlighten and convey to the reader the importance
of the mammalian oviduct as not only a conduit for the transport of gametes but also as
an important reproductive organ that contributes to the well-being and development of
the embryo. Numerous studies have been done indicating that a pregnancy can be
established without the presence of features and structures that are located within or are
specific to the oviduct. Perhaps the most pervasive example is that of in vitro fertilization
(IVF), in which an oocyte is matured, fertilized, and developed in vitro with subsequent
transplantation of the embryo directly into the uterus, resulting in a successful pregnancy.
Techniques such as IVF, which remove the oocyte and spermatozoa from contact with
the tubule, have generated much debate on the question of whether the mammalian
oviduct is an essential part of the female reproductive tract.
Over the past decade, numerous advances have been accomplished for embryonic
development in vitro, especially during oocyte maturation and fertilization. These
advances have been primarily due to careful formulation of culture medium and use of
co-culture with oviductal epithelial cells. Interpretation of these studies has led to two
prevalent hypotheses. One view is that the oviduct has a more passive role in terms of
establishing an environment with an optimum pH, temperature, osmotic pressure,
nutrients, and oxygen tension for fertilization and early cleavage-stage development. The
second is the oviduct has a more active role, investing energy into the expression and
1

2
synthesis of molecules, which may facilitate or regulate these events. Evidence to date
suggests that these two views are not mutually exclusive but are cooperative in the
formation of a microenvironment, which sustains fertilization and early cleavage-stage
development. Perhaps the importance of this reproductive organ has to be viewed with
respect to its importance in the establishment of a species population. An insightful
paragraph by Dr. Ronald H. F. Hunter in the book The Fallopian Tubes (1988) touches on
this theory:
For most biologists, successful reproduction needs to be considered in
population terms, and all features that contribute to the stability of a
breeding population, and thereby to perpetuation of the species, need to be
brought to mind. It is in this light that the many specific contributions of
the Fallopian tubes are best interpreted. Whilst individual morphological
and biochemical components may not be of overriding importance in
themselves for the establishment of single pregnancies, together they can
be viewed as contributing to the successful maintenance of a breeding
population.
Although embryonic development in vitro has recently seen significant advances,
scientists are still far from reaching a degree of development and viability similar to that
seen in vivo. In virtually every reported case, cultured embryos exhibit reduced cell
numbers, decreased viability with increasing duration of culture, and reduced
development to later stages when compared with their counterparts growing in vivo
[Bavister, 1988], These observations indicate that some important characteristics of the
embryonic milieu are missing from the artificial culture environment. Peptide and
macromolecules are the oviductal constituents most suspected of having specific
embryotrophic actions, particularly de novo synthesized and secreted products of the
epithelium. The first objective was to identify an unknown major de novo secretory

3
product of the porcine isthmus. Sequence analysis revealed this protein to be porcine
plasminogen activator inhibitor-1 (PAI-1). Experiments were designed to characterize
this protein throughout the estrous cycle or early pregnancy and to evaluate its
association with the pre-implantation embryo. The second part of this dissertation
concerns the functional analyses of a well-characterized oviductal protein, porcine
oviductal secretory glycoprotein (pOSP), during fertilization and early embryonic
development. It may be that with the identification and functional analysis of specific
oviductal molecules, conditions for in vitro development of embryos may come to mimic
more closely that of the in vivo situation. This dissertation will present data that indicates
a more active role for this reproductive organ in the successful establishment of
pregnancy.

CHAPTER 2
LITERATURE REVIEW
Biology of the Oviduct
Historical Perspective
The first observations of the oviduct, date back to Aristotle (322-384 BC), who
noted a convoluted structure connecting the uterus to the ovary. However, a common
misinterpretation that perpetuated for several centuries was that the ducts transmitted
female semen to the urinary bladder where it was excreted. Accurate anatomical
descriptions were first made by Galen (AD 130-200), linking the ovaries to the uterus and
describing their termination in the uterine horns of bicomuate domestic animals.
However, his mistake was to perpetuate the concept that this tubule was a conduit for
female semen, which was filtered by the ovary from the bloodstream. This notion was
based upon his observation that the female Fallopian tubes were similar to the male
seminal vessels.
The idea of a tubule which transmitted female semen persisted for over 14
centuries. Descriptive anatomical drawings were not available until the excellent works
of Leonardo da Vinci (1452-1519) were published. Another anatomist, Andreas
Vesalius, extensively characterized the anatomy of the Fallopian tube in 1543 in a seven-
volume series known as De Humani Corporis Fabrica. His analyses of their function
echoed that of the past by supporting the hypothesis that these structures were responsible
4

5
for the transport of female semen. The critical structure of the oviduct and relationship of
this structure to the ovary and uterus were detailed by Fallopius, and due to his detailed
description of the tubule his name was lent to the structure (Fallopian tube). The
collected works of Harvey (1651), Van Home (1668), and de Graaf (1672) established
that female semen was a myth, but that instead an egg originates in the ovary (although
the follicle was misinterpreted as the egg), and that this egg is transported into the uterus
via the Fallopian tubes. It was not until significant advances in the technological
development of the microscope were made near the end of the 17- century that a more
careful scrutiny of the oviduct and its structure, came about. For an excellent review on
the history of the oviduct see The Fallopian Tubes (1988), edited by Hunter and The
Mammalian Oviduct (1969), edited by Hafez and Blandau.
Prenatal Development and Macroscopic Anatomy of the Oviduct
The oviducts of adult mammals are specialized structures that originate during
organogenesis from the cranial region of the primitive Mullerian ducts [Price et al.,
1969], Johannes Muller first described the Mullerian ducts in 1830, yet far less attention
has been directed toward the development of the oviduct in relation to the uterus.
Although the development of the oviduct prenatally differs widely among mammalian
species, several general statements can still be made. The oviduct undergoes
morphogenetic and histogenetic changes that distinguish it from the uterus which include
(1) coiling of the oviduct, (2) differences in size (diameter), (3) differentiation of
epithelium and muscle, (4) formation of mucosal folds, (5) development of the fimbriae,
and (6) development of the three segments - infundibulum, ampulla, and isthmus [Price
et al., 1969], For a detailed description of comparative development of the mammalian

6
oviduct, the reader is referred to The Mammalian Oviduct (1969), edited by Hafez and
Blandau.
The paired oviducts connect within the uterus at the uterine tubule junction (UTJ)
and link this structure to the ovary. The oviduct is convoluted in most species (except the
rabbit and man), and is supported by the mesosalpinx, which is a part of the broad
ligament [Nalbandov, 1969], A specialized portion of the mammalian oviduct is often
referred to as the fimbriae infundibulum, which forms a fringed open funnel. In the pig,
which has open fimbriae, this is an intimate structure which encapsulates the ovary. In
species such as mouse and rat this structure forms a sac around the ovary called the bursa
ovarii. The fimbriae of the infundibulum are extremely motile and direct fluid movement
into the oviduct during the time of ovulation. In the pig, unilateral removal of the
infundibulum does not cause infert'lity as the contralateral fimbriae can redirect and
capture oocytes from the peritoneal cavity [Nalbandov, 1969], The segment adjacent to
the infundibulum is referred to as the ampulla and has the largest diameter of the three
segments of the oviduct. The intricate folding of the mucous membrane is also
characteristic of this section. The diameter of the ampulla tapers down as it becomes the
isthmus which, in the pig, connects into the uterus at the tip of the uterine horn. The pig
oviduct averages 26-28 cm in length, with the isthmus comprising approximately 1/3 of
the overall length with the majority being made up of the ampulla.
The portion of the isthmus which enters the uterus is referred to as the uterotubal
junction (UTI), and in most litter-bearing species such as the pig, it contains a high
density of folds and finger-like processes (villi) [Hafez and Black, 1969], These
structures are arranged in such a fashion so as to prevent the passage of fluid and uterine

7
contents into the oviduct. Anderson (1927a) showed that in pigs, a greater amount of
pressure is required to force uterine fluid into the oviducts when eggs are present (Day 2-
4 of the estrous cycle) than during the luteal phase. This suggests that the UTJ maintains
the oviductal environment protecting it from contamination by secretions from the uterus.
As reviewed by Hunter (1988), the pig oviduct contains an outer layer of
longitudinal muscle and an inner layer of circular muscle. External to these muscle
layers is a well-vascularized serosa layer of tissue. The isthmus portion of the oviduct
has a greater amount of circular muscle, allowing for a greater contraction potential than
the other two segments. The circular muscle layers of the isthmus and ampullary-isthmic
junction are highly innervated with adrenergic nerve terminals originating in the ovarian
or hypogastric plexus. The innervation of the ampulla and infundibulum is very poor
compared to the isthmus, and is usually restricted to the walls of blood vessels. The high
density of circular muscle and adrenergic innervation of the isthmus indicates an
important role for this segment in the transport of gametes and embryos.
The lymphatic vessels of the pig oviduct follow the ovarian and uterine lymphatic
drainage and show a similar concentration in the isthmus as was described for the
sympathetic innervation of nerve bundles. Anderson's (1927b) study of the domestic pig,
revealed that cyclical variations could be noted in lymphatic development and that these
changes might be due to ovarian steroids. It has been suggested that the lymphatics
might be involved in transmission of embryonic information to either the ovary or uterus,
since the greatest amount of tubule resistance to the embryo is found within the isthmus.
The vascular supply to the oviduct is supported by the uterine and ovarian
arteries, and varies according to the estrous cycle or pregnancy. The capillary bed and

8
larger blood vessels of the infundibulum and ampulla become engorged with blood close
to the time of ovulation and are regulated by estrogen [Hunter, 1988], This increase in
blood supply may also account for the increased tubule secretion and transudation in the
pig at estrus.
Microsconic Anatomy of the Oviduct
The architecture of the ampulla and isthmus is centered on an epithelium with
numerous tubal mucosal folds that show cyclical changes in morphology. The actual
space within the oviduct lumen is very negligible (unlike the lumen of a gland).
Therefore, gametes often remain in intimate contact with the epithelial lining as opposed
to floating free in an aqueous environment. This epithelial lining is composed of simple
columnar and modified cells of the goblet cell type, which are secretory in nature. There
are two distinct differentiated cell types within the oviduct, referred to as ciliated and
non-ciliated (secretory), although this is not absolute as sometimes differentiating cells
display both cilia and secretory granules [Jansen and Bajpai, 1982], Interspersed
throughout the ciliated cells are non-ciliated cells that contain electron-dense secretory
granules that can be identified throughout the cell but are most often found in the apical
region. These secretory granules exude their contents with greater frequency during
ovulation, contributing to the decline in epithelial height shortly thereafter [Hunter,
1988], In most domestic species, epithelial cell height and secretory activity are greatest
at the time of ovulation.
Estrous Cycle of the Pie and Hormonal Regulation bv Ovarian Steroids
The influence of ovarian steroid hormones on the oviductal environment has been
well documented in several species for ciliogenesis, secretion, contractility, and

9
morphology of the tubal mucosa and musculature [Hunter, 1988], It ¡s difficult to
discuss these processes in relation to the oviduct without first describing some aspects of
the pig estrous cycle and its changing hormonal environment. A prepubertal gilt becomes
sexually mature at 6-8 months of age and is described as polyestrous, which means a lack
of seasonality in its reproductive cycle. The average cycle length for post-pubescent gilts
is 21 days, with Day 0 being the onset of standing estrus. The average length of estrus is
2-3 days, with ovulation occurring 30-40 hours after the onset of estrus. A pig is a
polytoccous, or litter bearing, animal and ovulation occurs over a time period of 1 to 5
hours [Hunter, 1988], Estrogen begins to rise several days prior to estrus (proestrus) and
returns to basal levels by Day 2 of the estrous cycle [Karlbom et al., 1982], Progesterone
levels begin to rise by Day 2 of the estrous cycle in conjunction with development of the
corpus luteum, reaching a peak around Days 14-15 and quickly declining thereafter
[Karlbom et al., 1982],
Ovarian steroids are transported to the oviduct through the blood [Giorgi, 1980]
which then bind to specific cytosolic receptors and are transported to the nucleus [Chan
and O’Malley, 1976], If steroid receptors are absent in the oviduct, the tissue becomes
refractory to the action of that hormone. This provides a unique model in which
oviductal fiinction can be examined in intact and ovariectomized animals. Tissue
concentrations of estradiol and progesterone receptors in the porcine oviduct were first
examined by Stanchev et al. (1985). These researchers looked at both cytoplasmic and
nuclear receptors for estrogen (ER) and progesterone (PR) throughout the estrous cycle.
The ampulla showed cyclic variations in the concentration of cytoplasmic and nuclear
ER, increasing during proestrus, reaching a maximum concentration during standing

10
estrus and declining by Days 3-4 of the cycle. The ampulla also showed changes in
cytoplasmic and nuclear PR levels during the estrous cycle, with increasing
concentrations found from standing estrus through the luteal phase. The isthmus had a
much lower concentration of cytoplasmic and nuclear ER than the ampulla, and isthmic
cytoplasmic ER showed no cyclic variation. The isthmus and ampulla showed similar
levels of PRs and also had a similar expression of nuclear PR during the estrous cycle.
However, no changes were noted for levels of cytoplasmic PR in the isthmus during the
estrous cycle. These studies indicate that the isthmus has a constant low-level expression
of cytosolic receptors, while nuclear receptor concentrations vary according to the
hormonal state of the animal. The ampulla, which is believed to be more sensitized to the
effects of estrogen and progesterone than the infundibulum or isthmus, revealed cyclic
changes in both cytoplasmic and nuclear receptors during the estrous cycle. The higher
proliferative and secretory nature of the ampulla during the preovulatory and ovulatory
period may account for these differences in ER expression [Iritani et al., 1974],
Contractility and Ciliogenesis of the Oviduct
Oviductal muscular activity has been examined throughout the estrous cycle, and
although investigators are not in complete agreement, it appears that the most vigorous
contractions occur at about the time of estrus and ovulation. Observations done nearly 76
years ago in the pig revealed that the strength of oviductal muscular contractions was
related to the dimensions of the preovulatory follicles [Seckinger 1923, Wislocki and
Guttmacher 1924], The larger the diameter of the follicle the stronger the contractions.
As reviewed by Boling (1969), the predominant movement is peristaltic, and
contractions occur in localized segments or loops rather than traveling long distances.

11
These contractions are primarily abovarian in direction. Kymographic records
(radiographic examination in which the range of involuntary movements are recorded) of
the pig oviduct revealed that the ampulla undergoes regular and mild contractions, while
those of the isthmus are more vigorous [Kok, 1926], Decreased contractility has been
noted in the castrate rabbit although this activity did not become completely quiescent
[Greenwald, 1963], Compared to intact animals, a delay in tubal egg transport was
observed in castrate animals. Greenwald (1967) showed that species differences are
observed in egg transport in response to exogenous estrogen. In the rat, single injections
of estrogen on Days 1, 2, and 3 of early pregnancy accelerate oviductal transport of ova
[Ortiz et al., 1979, 1991], A recent study suggests that protein synthesis is required in rat
oviducts for the estrogen effect on oviductal transport to occur [Rios et al., 1997],
Several reports have indicated that estrogen and progesterone together regulate the
contractility and are summarized by Boling (1969). It was observed that the oviductal
musculature remains relatively quiet under the influence of estrogen, and begins to
contract more vigorously when estrogen is withdrawn. Likewise, tissue under the
influence of estrogen begins to contract vigorously shortly after an injection with
progesterone. These data indicate that perhaps estrogen has an inhibitory effect on
vigorous oviductal contractions. Much of these data appears contradictory, especially
since observations at or near estrus indicate this period as having the strongest
contractions. Contractions of oviductal musculature may be controlled by prostaglandins
of the F series. Prostaglandin F20, has been observed in both the Fallopian walls as well as
in oviduct fluid. Lippes (1979) found that PGF2a in human tubal fluid showed cyclical
variations and had higher pre-ovulatory than post-ovulatory levels. It may be that steroid

12
hormones and prostaglandins program the amplitude and frequency of contractions via
the regulation of calcium transport. However, information regarding control of oviductal
contractions is lacking.
Ciliogenesis refers to the growth and development of oviductal cilia throughout
the estrous cycle and pregnancy. Perhaps the greatest amount of information on this
subject is from investigations on the number and morphology of oviductal cilia
throughout the estrous and menstrual cycles and pregnancy. However, there are also
several studies using experimental procedures such as hypophysectomy and ovariectomy,
followed by steroid hormone replacement. As summarized by Brenner (1969),
overwhelming evidence suggests that estrogen stimulates the growth of ciliated cells and
progesterone antagonizes this effect. In the presence of prolonged progesterone, cells
become undifferentiated and difficult to clarify [Brenner et al., 1983], Distinct
morphological changes have been noted in relation to a changing steroid hormone
environment in humans [Verhage et al., 1979] and rabbits [Brenner, 1969], Although the
hormonal regulation of ciliogenesis is widely accepted, the number and character of
ciliated cells varies depending on the species studied, the portion of the oviduct sampled,
and the phase of the sexual cycle [Schaffer, 1908], For example, in the domestic pig,
active ciliated cells have been seen at all stages of the cycle, without any significant
changes in their distribution, number, or activity [Snyder, 1923], Only a change in cell
height was observed with a maximum occurring around the time of ovulation (Day 0-3)
and a minimum height about two weeks after ovulation. Observations by Buhi
(unpublished data) indicate that in the pig, gradients in the number of ciliated vs secretory
cells exist between the ampulla and isthmus. The pig isthmus contains about 80%

13
ciliated cells and 20% secretory cells (personal communication). During the luteal phase
of the pig, the distal portion of the ciliated cells become constricted from the rest of the
cell and are cut off in a process called deciliation [Flerko, 1954], It has been proposed
that deciliated cells fill up with secretory granules and become active secretory cells.
Depleted secretory cells then become peg cells and are eventually lost at the end of the
cycle [Brenner, 1969], As summarized by Hunter (1988), ciliary beat appears to be
regulated by circulating levels of ovarian hormones and is greatest at or near the time of
ovulation. In most mammals examined thus far, cilia of the ampulla beat in the direction
of the ampullary-isthmic junction [Blandau and Verdugo, 1976], suggesting a role for
cilia in the process of egg transport to the site of fertilization.
Fertilization and Early Embrvogenesis in the Oviduct
In most mammals, new life begins in the oviduct through the union of germ cells
during fertilization. Wassarman (1999a) defines fertilization as the process of joining
two germ cells, egg and sperm, whereby the somatic chromosome number is restored and
the development of a new individual exhibiting characteristics of the species is initiated.
This process includes several ordered steps: final maturation of both male and female
gametes, sperm acrosome binding, acrosome reaction and penetration through the zona
pellucida, fusion of sperm and oocyte membranes, the corticol granule reaction, and
activation of the cell. During ovulation, fijlly-grown oocytes from antral follicles
undergo meiotic maturation to become oocytes that are now capable of interacting with
spermatozoa and being fertilized [Wassarman, 1999a]. Capacitation of spermatozoa
within the oviduct is viewed as the final phase of sperm maturation, conferring upon the
cells the ability to penetrate the egg investments [Hunter, 1995], One consequence of

14
capacitation is an increased flagellar beat of spermatozoa, termed "hyperactivation,”
which is believed to assist in the penetration of the hamster zona pellucida [Yanagimachi,
1988], A second consequence is membrane vesiculation due to point fusions on the
anterior portion of the sperm head, releasing proteolytic enzymes such as acrosin [Hunter,
1995], The acrosome reaction has been suggested to be initiated by the binding of sperm
to the zona pellucida. It is generally accepted that one of the zona pellucida proteins,
ZP3, is the natural agonist for stimulation of the acrosome reaction in acrosome-intact
spermatozoa. Binding of spermatozoa to ZP3 activates G-proteins which stimulate a
change in intracellular pH and Ca++; however, the sperm receptors that activate these G-
proteins remain elusive [Wassarman, 1999b], In the mouse, the zona matrix is composed
of three ZP glycoproteins, ZP 1-3. ZP3 and ZP2 dimers are located throughout the zona
pellucida and are crosslinked by ZP1 to create a three-dimensional matrix [Wassarman,
1999b], In the pig, rabbit, and non-human primates, ZP1 has been shown to be the
primary sperm receptor [Dunbar et al., 1998], Although these proteins from different
species are conserved within each family, they exhibit distinct biological properties as the
mouse ZP3 is the primary sperm receptor.
Once sperm has penetrated the egg investments, its plasma membrane must bind
and fuse with the oocyte vitelline membrane. Fertilin-P, a sperm membrane-bound
protein, is thought to be the primary molecule responsible for binding of acrosome-
reacted sperm to the plasma membrane [Frayne and Hall, 1999], This protein is a
member of the ADAM (disintegrin and metalloprotease domain) family of
transmembrane proteins. The disintegrin domain of fertilin-p is believed to interact with
integrins on the oocyte plasma membrane, mediating the process of fusion. Recently, it

15
was shown that mice that are homozygous null for fertilin-(3 (fertilin-ft -/-) have a
significantly reduced fertilization rate [Wassarman, 1999b], Shortly after spermatozoa-
oocyte membrane fusion, the quiescent egg "awakens" to initiate a series of biochemical
events known as egg activation. This activation leads to an explosive release of Ca^
from intracellular storage sites, a transient increase in intracellular pH, an irreversible
activation of oxidative pathways, lipid metabolism, and protein and DNA synthesis
[Yanagimachi, 1988], This activation is believed to be initiated by paternal proteins
obtained during fusion. The massive release of intracellular Ca++ stimulates a Ca*4-
dependent exocytosis of corticol granules (CG) which have an important role in
modification of the egg envelope during fertilization. As reviewed by Yanagimachi
(1988), exocytosis in mouse eggs begins near the point of sperm-egg fusion and
propagates from this point in a wave-like fashion to the opposite side of the egg. The
CG, which contain hydrolytic enzymes and saccharide components, alter the physical and
chemical characteristics of the zona pellucida so that the zona becomes refractory to
sperm attachment and penetration. This occurs due to proteolytic modifications of the
sperm-binding ZP receptors. This process is referred to as the "zona reaction" and is a
key event for establishing a block to polyspermy in pigs [Hunter, 1991], Once the sperm
nucleus is incorporated into the cytoplasm of the mature mouse egg (characterized by
ejection of the second polar body) there is a rapid breakdown of the nuclear envelope,
decondensation of chromatin, and formation of the male pronucleus.
The formation of the female and male pronucleus and their subsequent migration
through the cytoplasm is due to microtubules and microfilaments of the cytosolic
framework [Yanagimachi, 1988], Besides fertilization, the oviduct is also the site of

16
early cleavage-stage embryonic development. Compared to other domestic species, the
pig has a relatively short cleavage-stage development within the oviduct, entering the
uterus at about the 4-cell stage [Kopecny, 1989], Cleavage is invariably asynchronous in
mammalian embryos cultured in vitro and development is retarded by about 2 h each cell
cycle, thus, the development to the blastocyst stage may be an entire day longer than
embryos in vivo [Pedersen, 1988], This indicates that oviduct components may
contribute to the morphologic and metabolic regulation of the developing embryo,
leading to enhanced rates of development of in vivo compared to in vitro embryos.
Oviductal Fluid
Oviductal fluid which can be found within the tubal lumen throughout the
reproductive cycle varies in volume as well as composition and is most abundant when
gametes or embryos are present [Hunter, 1988], Because tubal volume was found to be
greatest during estrus and near to or just after ovulation, it was suggested that circulating
ovarian steroids might regulate tubal fluid secretion. Bishop (1956) showed that after
ovariectomy in estrous rabbits, tubal fluid secretion dropped from 1.2 ml to 0.2 ml/24 hr
and that systemic injections of estradiol could restore fluid production to estrus volumes.
Comparable observations in tubal fluid accumulation have also been noted in the ewe,
cow, and pig [Hunter, 1988], The pig was shown to have a fluid accumulation of 6.3
ml/24 hr during estrus falling to 2.1 ml/24 hr in the luteal phase [Iritani et al., 1974],
Another distinction seen between estrus and the luteal phase, is the direction of fluid
flow. Seminal experiments in tubal ligation revealed significant changes in tubal fluid
retention depending on the stage of the cycle. During estrus, the bulk of the fluid passes
from the ampulla into the peritoneal cavity generating a retrograde flow, however, by the

17
time embryos descend through the uterotubal junction, much of the reduced fluid volume
is being redirected toward the uterus [Hunter, 1988], The restriction of tubal flow
towards the uterus at estrus, is thought to be primarily due to edema found within the
isthmic mucosa. Constriction of the isthmus is thought to be programmed by circulating
levels of estrogen and wanes as a result of elevating progesterone levels [Hunter,1988],
It is difficult to envision the relationship between tubal fluid flow and movement
of the gametes through the lumen. From the observations of Parker (1931), suggestions
have been made that a microflow system close to the epithelium and within the grooves
or channels of the surface may differ in direction from that of the bulk flow. This
microflow system may be intimately involved with the movement of spermatozoa or
oocytes through the oviductal lumen. Detailed chemical analyses of oviduct fluid has
revealed a complex mixture of constituents derived from plasma and the oviductal
epithelium including the oviduct-specific secretory macromolecules. The passage of
plasma constituents into the lumen has been referred to as ‘transudation.’ Because
certain components in oviduct fluid are present at concentrations different from those in
plasma the term transudation was expanded to 'selective transudation' [Leese, 1988],
Therefore, the term 'secretion' has been applied to the formation and discharge of de novo
proteins from the oviductal epithelium. Protein concentration in oviductal fluid is 10-
15% that found in serum [Gandolfi, 1995], The molecules and proteins involved are
transported across the epithelium within endocytic vesicles that travel from the
basolateral membrane to the apical surface where their contents are released into the
lumen [Parr and Parr, 1986], The epithelial cell tight junctional complexes restrict
passage of proteins into the lumen between epithelial cells. During early pregnancy in

18
the pig, total recovered oviductal fluid protein was greatest on days 0-3 indicating a
cyclical variation in total protein [Buhi et al., 1998, Kouba unpublished]. Similar cyclic
changes in total protein have been observed during the human menstrual cycle [Lippes et
al., 1981],
Biosynthetic Activity
The mammalian oviduct is a highly synthetic and secretive tissue. The
biosynthetic capacity has been evaluated by measuring the incorporation of radiolabeled
precursor into secreted nondialyzeable macromolecules. Biosynthetic activity of the
entire oviduct was greatest during proestrus, estrus, and metestrus compared to other days
of the cycle in the pig [Buhi et al., 1989], sheep [Buhi et al., 1991] and cow [Malayer et
al., 1988], Synthetic differences were not observed between mated/pregnant and cyclic
gilts, indicating that the presence of gametes or embryos does not affect the biosynthetic
activity. These findings are in contrast to that of the rabbit where pregnant animals were
shown to have decreased incorporation rates compared to non-pregnant animals [Roy et
al.,1972]. The greater activity during estrus suggests that ovarian steroid hormones may
regulate this function. In ovariectomized pigs [Buhi et al., 1992] or sheep [Buhi et al.,
1991], biosynthetic activity was greater with estrogen than with other steroid treatments.
Furthermore, pigs unilaterally ovariectomized showed no differences in biosynthetic
activity from either the contra- or ipsilateral oviduct containing an ovary with follicles or
corpus lútea. Therefore, circulating ovarian hormones produced by one ovary were
sufficient to control protein synthesis of both oviducts [Buhi et al, 1997],
A recent study showed that the presence of a persistent dominant follicle
decreased the synthetic activity of oviductal tissue in the cow [Binelli et al., 1999], These

19
investigators suggest that prolonged exposure to high levels of estrogen may have caused
down-regulation of the estrogen receptor, thus suppressing biosynthetic activity.
Interestingly, fluctuations in the abundance of de novo synthesized proteins in animals
containing a persistent dominant follicle, compared to a fresh dominant follicle, were
observed. It was suggested by Binelli et al. (1999) that these fluctuations might be due to
a release of the inhibitory actions of estrogen on certain proteins. Of the three oviduct
segments, the ampulla had the greatest synthetic activity with estrogen and the lowest
with progesterone. Similar to the biosynthetic capacity of the entire oviduct, the capacity
of the ampulla was greatest on Days 0-4 of the estrous cycle, while the isthmus showed
no differences. Likewise, the ampulla was shown to have greater biosynthetic activity
than the isthmus regardless of day of the estrous cycle. Similar rates of activity were
found for the ampulla and infundibulum.
Proteins. Growth Factors, and Cytokines of the Oviduct
Fluxes of molecules from the fluid bathing the serosal surface to those bathing the
mucosal surface are generated giving the oviductal epithelium its physiological
characteristics [Leese, 1988], This flux includes water, electrolytes, metabolic non¬
electrolytes, amino acids, steroids and proteins. The secretion of specific oviduct fluid
constituents will be discussed here in light of their possible contributions to gamete-
embryo interactions. The majority of this section will cover the identification of proteins
found within oviductal secretions, and the other constituents will be briefly highlighted.
Water is not thought to be transported into the oviductal lumen through an active process,
but rather in response to osmotic gradients established by the transport of ions [Leese,
1988], Numerous ions have been identified in oviduct fluid including sodium, chloride,

20
potassium, bicarbonate, magnesium, calcium and inorganic phosphorous [Leese, 1988,
Ellington, 1991, Boatman, 1997], As summarized in these reviews, electrolyte
concentrations in the oviduct vary considerably from those found in serum. Varying
electrolyte concentrations in culture medias during in vitro fertilization and embryo
culture have shown the importance of these constituents on fertilization and embryonic
development. The volume of literature on this topic is extensive and will not be covered
in this literature review. The presence of non-electrolytes in oviduct fluid has also been
reviewed [see Leese, 1988, Boatman, 1997], Metabolic precursors found in oviduct fluid
include glucose, lactate, pyruvate and amino acids. Differences in concentrations of
these energy substrates in oviductal fluid and blood plasma of pigs during the peri-
ovulatory period have been observed [Nichol et al., 1992], Several of these energy
substrates (pyruvate and lactate) are routinely used during in vitro fertilization. Two
amino acids, taurine and hypotaurine, have been shown to be present in oviductal fluid at
concentrations much greater than those found in serum [Guerin et al., 1995], These
amino acids, as well as the converting enzyme that synthesizes hypotaurine from cysteine
intermediates, such as cysteine sulphinate decarboxylase [Guerin and Menezo, 1995],
have been localized to oviductal epithelial cells. Actions of taurine and hypotaurine
include; osmoregulation, calcium modulation, phospholipid interactions, membrane
protein receptor interactions, and antioxidation [Huxtable, 1992, Boatman, 1997],
Specific functions of these amino acids relative to the oviduct include; motility and
viability of sperm, progression through the cell block, improved development of zygotes
to the morulae or blastocyst stages, and increased cell numbers in vitro [Boatman, 1997],
The oviductal epithelium also produces another potent modulator of oxidative stress, the

21
antioxidant glutathione [McNutt-Scott and Harris, 1998], Through addition of both
electrolyte and non-electrolyte energy substrates and careful formulation of culture
mediums to mimic oviductal concentrations of these constituents, improvements to in
vitro fertilization and development to blastocyst have been achieved. Therefore, the
relative contribution of these constituents to establishment of an appropriate oviductal
environment for fertilization and early embryonic development should be underscored.
Steroids present in the oviductal environment are not generally assumed to be
produced by the oviduct but arrive through serum transudation or from the ruptured
follicle and cumulus cells. However, one report suggests that the oviductal epithelium
contains the machinery for steroidogenesis [Ogunranti, 1992], Many of the effects of
steroids on gamete function in the oviduct are mediated indirectly through the
vasculature, serosa, mucosa, and muscularis affecting protein secretion, gamete transport,
and fluid accumulation [Hunter, 1988, Hafez and Blandau, 1969], Recent evidence
suggests that progesterone may have direct actions on the gametes, inducing
hyperactivation and the acrosome reaction in mammalian spermatozoa [Meizel, 1995,
Boatman, 1997], Use of an anti-progesterone antibody in vivo showed decreased
cleavage rates and embryonic cell numbers [McRae, 1994], The actions of progesterone
on spermatozoa are non-genomic in origin, and genomic actions of steroids on oviductal
gametes has not been reported. Similarly, there are no reported non-genomic actions of
steroids on metaphase II-oocytes through to the blastocyst stage [Boatman, 1997],
In the pig, analyses of oviductal fluid proteins reveals the presence of hundreds of
proteins, most of which originate from serum as transudate [Buhi et al., 1999], The most
abundant serum proteins in oviductal fluid are albumin, heavy and light chains of

22
immunoglobulins and transferrin. Similar to measurements of total protein in oviduct
fluid, electrophoretic analyses of serum proteins show cyclical variations in their relative
distribution [Buhi et al., 1999], Additionally, several other proteins have been localized
to the oviductal epithelium and while these proteins have not been shown to be oviduct-
derived and secreted, deserve some mention. Numerous protease inhibitors and enzymes
have been detected including, but not limited to; alkaline phosphatase, amylase, lactate
dehydrogenase, diesterase, lysozyme, acid phosphatase, esterases, lipase, aminopeptidase,
glucosidase and galactosidase [Hunter, 1988], These molecules may be important in
detoxifying the oviductal environment of substrates harmful to either gametes or
embryos. Recently, catalase was found in porcine, human, and bovine oviduct fluid
[Lapointe et al., 1998], Catalase concentration was greatest near estrus, and could bind to
the acrosomal cap of spermatozoa. These investigators suggested that the ability of the
oviduct to maintain sperm viability was partially dependent on the ability of catalase to
protect sperm from oxidative damage.
Local countercurrent transfer of products from the ovary or the uterus to other
parts of the reproductive tract are well known. Molecules originating from these tissues
and found within oviduct epithelium or circulation include PGF2a and PGEz [Harper,
1988], oxytocin [Schramm et al., 1986], and endothelin 1 [Rosselli et al., 1994], In
addition, the oxytocin receptor has been identified in the oviduct of the ewe [Ayad et al.,
1990], These molecules have been shown to be important stimulators of oviductal
muscular activity. Local distributions of prostaglandin, oxytocin, and endothelin-1 in the
bovine oviduct were shown to be cyclic and greater concentrations were detected at
estrus in association with elevated levels of oviductal estradiol [Wijayagunawardane et

23
al., 1997], In the pig, LH/hCG receptors [Gawronska et al., 1999] and receptor mRNA
[Derecka et al., 1995] have been identified. It was observed that LH/hCG receptors were
greatest at the preovulatory stage of the estrous cycle, and caused relaxation of the
oviduct. It may be that LH in conjunction with oxytocin, endothelin 1 and prostaglandin
regulate gamete/embryo transfer.
Alpha-fetoprotein (AFP) has recently been found in human oviduct fluid and may
be regulated by progesterone [Lippes and Wagh, 1993], AFP binds estrone and estradiol,
acting as a carrier molecule for this steroid and its metabolites in biological fluids. AFP
has also been shown to have immunosuppressive effects. Wagh and Lippes (1993)
showed that AFP binds to the sperm acrosomal cap and may act as an acrosome-
stabilizing factor, preventing a premature acrosome reaction. Other possible
immunosuppressing agents in the oviduct are mucins. MUC1, a transmembrane mucin
gene product, has been localized in the human Fallopian tube, and similar to mucins in
the cervix may act as a barrier to sperm and pathogens [Gipson et al., 1997], MUC1 may
also be important for gamete and embryo transfer and prevention of adhesion to oviductal
epithelium.
A large number of peptide growth factors and cytokines in the mammalian
oviduct have been described [Kane et al., 1997, Buhi et al., 1997], These factors likely
originate from both the oviductal epithelium and serum transudate. Growth factors and
cytokines that have been identified within oviductal fluid or the epithelium during
ovulation, fertilization, and early cleavage-stage development in domestic species
include; epidermal growth factor (EGF), heparin-binding EGF, transforming growth
factor (TGF)-a,p, insulin-like growth factor (IGF)-I and II, IGF binding proteins (1-4),

24
colony stimulating factor (CSF)-l, acidic and basic fibroblast growth factor, platelet
derived growth factor, granulocyte macrophage-CSF-1, interleukin-6 and the IGF-II
receptor [Gandolfi, 1995, Chegini, 1996, Buhi et al., 1997, Kane et al., 1997, Buhi et al.,
1999], Several growth factor and cytokine mRNAs and proteins in the oviduct appear to
be modulated by steroids and temporally associated with elevated estrogen levels at or
near estrus. For examples of their presence and regulation during the estrous cycle the
reader is referred to the reviews listed above. Another interesting cytokine, leukemia
inhibitory factor (LIF) has been identified by the presence of mRNA and protein in the
bovine [Reinhart et al., 1998] and human [Keltz et al., 1996, Barmat et al., 1997] oviduct.
Reinhart et al. (1998) showed that estrogen but not progesterone, stimulated LIF
synthesis and that this stimulation was not receptor-mediated, suggesting a non-genomic
action for estrogen derivitized products. Retinol-binding protein (RBP) has been
localized in the porcine [Harney et al., 1994], equine [McDowell et al., 1993] and ovine
[Eberhardt et al., 1999] oviduct. Eberhardt et al. (1999a) showed that RBP mRNA and
protein synthesis are stimulated by estrogen and that levels of RBP mRNA are greater on
Day 1 of the estrous cycle than Days 5 or 10. In one experiment, embryos collected at
the 1- to 4-cell stage from retinol-treated superovulated pregnant ewes and cultured in
vitro for 7 days, showed a higher development to blastocyst (79%) than control animals
(5%). Retinoids may have beneficial effects during fertilization and early embryonic
development especially in embryonic morphogenesis, cell growth, and differentiation.
Oviduct-derived proteins have been identified in experiments using radiolabeled
precursors for protein synthesis during oviductal organ explant or epithelial cell cultures.
Electrophoretic characterization of tie novo synthesized and secreted proteins by ID- and

25
2D-SDS-PAGE and fluorography have been performed in the pig, cow, ewe, human, and
baboon [Buhi et al.,1997]. However, very few of these proteins have been identified and
have only been characterized in a limited fashion by isoelectric point and molecular
weight. Two of these proteins, the estrogen-dependent oviduct secretory glycoprotein
(OSP) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), have been
extensively characterized [Buhi et al., 1997, 1999], Pig OSP will be discussed in greater
detail later in the literature review. TIMP-1 is a major de novo synthesized and secreted
protein of the isthmic segment of the porcine oviduct and its mRNA and protein
expression are greatest on Day 2 of the estrous cycle or early pregnancy. TIMP-1 protein
production and release in the isthmus portion of the oviduct was shown to be greater with
estrogen treatment indicating potential regulation of this inhibitor by ovarian steroids.
This is supported by radioimmunoassay analyses of oviductal flush TIMP-1 protein,
which showed significantly greater levels at Day 0 than other days of the cycle. TIMP-1,
a serine protease inhibitor, has been shown to improve in vitro development of embryos
in both the cow [Satoh et al., 1994] and pig [Funahashi et al., 1997], This evidence
suggests that TIMP-1, present in the isthmus at the time of fertilization and early
embryonic development, may have important regulatory actions on these processes.
Recently, serine protease inhibitors of the uterus have been shown to stimulate DNA
synthesis in a glandular endometrial epithelial cell line [Badinga et al., 1999], thereby
suggesting a mitogenic role for TIMP-1 stimulation of embryonic development.
Four other de novo synthesized proteins, complement C3b, immunoglobulin A
heavy chain, the carboxy terminal region of preprocollagen, and clusterin, have been
identified in the pig oviduct by N-terminal amino acid microsequence analysis [Buhi et

26
al., 1998, Buhi et al., 1999b]. Similar to pOSP and TIMP-1, these four proteins appear to
show synthetic gradients within the oviduct. Of the four, only clusterin and
preprocollagen are found within the pig isthmus. Preliminary evidence indicates that
these proteins exhibit a cyclic secretion pattern and may be partially controlled by
ovarian steroids [Buhi, personal communication]. Potential functions of these four
proteins include immunoprotection, lipid transport, and tissue remodeling. Of the many
de novo synthesized proteins secreted by the porcine (>14) [Buhi et al., 1997] or bovine
(>30) [Malayer et al., 1988] oviducts, only seven have been positively identified (OSP,
TIMP-1, PAI-1, preprocollagen, immunoglobulin A, clusterin and complement C3b).
Oviduct Secretions and Co-culture on Fertilization and Embryonic Development
A massive amount of literature indicates that the oviduct creates a unique
environment for sustaining embryo development. Prior to modem day embryo culture
systems, embryos removed from the oviduct or produced as a result of in vitro
fertilization tended to slow down or block developmentally when maintained in culture
media [Bavister, 1988], However, when embryos are transferred to the oviduct in vivo,
development could occur from 1-cell to the blastocyst. This beneficial effect of the
oviduct was shown not to be species-specific as bovine embryos could develop in the
sheep or rabbit oviduct. The ability of the oviduct to overcome the cell block to
development is mimicked in vitro by use of chemically defined media supplemented with
protein additives (example; serum or BSA) and a low oxygen tension. Experiments in
the cow, sheep, and mouse where embryos are transferred to oviducts in vivo or in vitro
indicate an important role for this reproductive organ in sustaining embryonic growth
[Bavister, 1988, Hosoi et al., 1995], A reduced degree of embryo growth obtained in

27
culture media alone [Bowman and McLaren, 1970, Bavister, 1988] and growth
abnormalities following embryo transfer [Farin and Farm, 1995], have been noted for
embryos produced by in vitro fertilization.
If indeed the oviduct creates a unique environment that sustains embryo
development, supplementation of oviductal fluid or the use of oviductal epithelial cell
(OEC) co-cultures during in vitro embryo development should increase the
developmental capacity. Addition of oviduct secretions/fluid to culture medium has
either had no observable effect or has retarded embryo development when compared to
controls [Sirard et al., 1985, Eberhardt et al., 1994], However, careful fractionation of
oviductal cell-conditioned medium and subsequent addition of these fractions during in
vitro fertilization or embryo culture has had more promising results [Minami et al., 1992,
Mermillod et al., 1993, Liu et al., 1995, Vansteenbrugge et al., 1996, Hill et al., 1996, Liu
et al., 1998], Preliminary results by these investigators have characterized, by molecular
weight, the embryotrophic activities of oviductal cell-conditioned media. These activities
have both high (>10 kDa) and low (<10 kDa) molecular weight components. None of the
determined molecular weights correspond to OSP. Perhaps the greatest contribution of
oviductal secretion analysis to date has resulted from careful evaluation of its ionic
composition. The concentrations of oviductal electrolytes differs considerably from that
of blood sera and media formulated to resemble the ionic composition of oviductal fluid
show improved embryonic development [Bavister, 1988, Boatman, 1997], This media,
referred to as synthetic oviduct fluid (SOF), was commonly used during the late 1980s
and early 1990s. However, its use is uncommon today, as laboratories are moving
towards using protein-free chemically-defined culture media for repeatability.

28
Perhaps the most pervasive example of the contribution of the oviduct to embryo
culture can be seen using OEC co-cultures. Seminal experiments by Gandolfi and Moor
(1987) showed that in vivo fertilized sheep 1-cell embryos had a markedly increased
development (8- to 16-cell stage) when co-cultured with OEC (86%) or fetal fibroblast
cells (93%) than in culture media alone (13%). Interestingly, embryos cultured for 6 days
in the presence of OEC had an increased development to blastocyst (46%) when
compared to the fetal fibroblast cell co-culture (5%). Likewise, embryo transfer
experiments, revealed that embryos cultured in the presence of OEC showed twice the
viability of embryos cultured with fetal fibroblast cells. Similar results have been shown
in cattle [Eyestone et al., 1987, Eyestone and First, 1989, Ellington et al., 1990], As
suggested by Bavister (1988), morphological criteria alone are not sufficient evidence of
the quality of cultured embryos. Hosoi et al. (1995) designed experiments to identify the
developmental stage at which embryos could be influenced by the oviductal environment.
These investigators found that in both mice and cattle the oviductal influence on
blastocyst development occurs during the switch from maternal to embryonic genome
control. The largest contribution of OEC has been shown in humans. Human embryos
co-cultured with OEC have reduced fragmentation [Bongso et al., 1989, Yeung et al.,
1992, Morgan et al., 1995], better morphological characteristics [Morgan et al., 1995],
higher cleavage [Morgan et al., 1995], blastulation [Bongso et al., 1989, Wiemer et al.,
1993], hatching [Yeung et al., 1992] and pregnancy rates [Bongso et al., 1992], The
beneficial effect of the OEC may include the removal of toxic components from the
medium and/or production of embryotrophic factors. The benefits of OEC co-cultures
are not restricted to the oocyte alone but may also act on spermatozoa. Human sperm co-

29
cultured with bovine OEC show decreased chromatin structural degeneration [Ellington
et al., 1998] and in the pig, a reduced incidence of polyspermy [Nagai and Moor, 1990,
Dubuc and Sirard, 1995], In addition to OEC effects on enhancing early embryonic
development, OEC play an important role in sperm-oviduct interactions.
Sperm-Oviduct Interactions
In most domestic species, of the millions of sperm that are ejaculated during a
natural mating, only a few thousand reach the isthmus of the oviduct, and there most are
held in a "reservoir" [Suarez, 1998], Of these thousand which enter the isthmus only a
few reach the ampulla which is the site of fertilization. This oviductal sperm reservoir
has been suggested to be involved in the prevention of polyspermic fertilization [Hunter,
1991], maintenance of sperm fertility between the onset of estrus and fertilization
[Pollard et al., 1991, Chian and Sirard 1994, Smith, 1998], capacitation [Chian et al.,
1995, Mahmoud and Parrish, 1996], and/or motility hyperactivation [Kervancioglu et al.,
1994], The oviductal sperm reservoir is established by spermatozoa binding to oviductal
epithelial cells and has been observed in cattle, mice, hamsters, pigs, and horses [Suarez,
1998], For most species studied thus far, spermatozoa bind to the apical membrane of
isthmic oviductal epithelial cells (OEC) and adhesion is specific to the rostral region of
the sperm head [Smith, 1998], The narrowness of the isthmic oviductal lumen enhances
sperm entrapment within the isthmus and increases the amount of contact between sperm
and the mucosal surface. The binding moieties between spermatozoa and OEC are still
unknown. Nor is it completely understood how and when sperm are released from these
cells. Some evidence suggests that sperm binding and release may depend on the
physical state and motor functions of the sperm. Smith and Yanagimachi (1991) reported

30
that hamster sperm would not bind to OEC once capacitated or hyperactivated. Likewise,
hyperactivation was shown to be a primary factor for release of bound spermatozoa in the
mouse [DeMott and Suarez, 1992], Therefore, binding and release of spermatozoa may
be due to changes in surface properties of the sperm head, which might lead to increased
flagellar beat amplitude and asymmetry. Binding of sperm to OEC may be due to a lectin
or carbohydrate present on sperm cells, as sperm attachment was inhibited by fetuin in
the hamster [DeMott et al., 1995] or fucose in the cow [Lefebvre et ah, 1997], Suarez
(1998) and Kervancioglu et ah (1994) suggest that epithelial secretions, initiated by
signals of impending ovulation, could enhance sperm capacitation, binding, and release.
Soluble oviductal factors have been shown to induce capacitation in bull sperm
[Parrish et ah, 1989, Chian et al., 1995, Mahmoud and Parrish, 1996], Heparin has been
shown to induce capacitation similar to oviduct fluid [Parrish et al, 1988] and is widely
used during in vitro fertilization of bovine oocytes. However, the intracellular signaling
conditions which induce capacitation (increased intracellular Ca+ and pH) are not
equivalent between heparin and oviduct fluid [Parrish et ah, 1994], Recently, Fazeli et al.
(1999) examined induction of capacitation by porcine OEC. Similar to observations in
other species, capacitated boar spermatozoa do not bind to porcine OEC. These
investigators also found that unbound spermatozoa in co-culture with ampulla or isthmic
OEC were slowly capacitated over time. This observation was not noted for cells of
nonreproductive origin. Kervancioglu et ah (1994) reports similar observations between
human OEC and vero cells (kidney epithelium). This suggests that the induction of
capacitation may be specific to oviductal secretions. With boar spermatozoa, the slow
induction of capacitation in vitro by OEC [Fazeli et ah, 1999] is similar to in vivo bovine

31
sperm capacitation which may occur for as long as 24 h [Parrish, 1989], Besides
suggestions by Parrish (see above references) that a heparin-like glycosaminoglycan is
responsible for capacitation activity in oviduct fluid, other protein components have been
shown to induce capacitation, including the bovine estrus-associated glycoprotein
[McNutt and Killian, 1991, King et al., 1994], Capacitation can also occur spontaneously
in vitro in a defined medium without the addition of biological fluids. This suggests that
the process is intrinsically modulated by sperm itself. However, this does not rule out the
influence of positive/negative regulatory factors within the oviduct. It is generally
accepted that capacitation is induced by the removal of decapacitating factors,
specifically cholesterol from the sperm membrane [as reviewed by Visconti and Kopf,
1998 and Cross, 1998], Cholesterol-binding proteins are present in follicular and
oviductal fluids [DeLamirande et al., 1997] and may be involved in cholesterol removal.
Structure. Regulation and Biological Actions of Plasminogen Activator Inhibitor fPAD-1
Controlled and targeted proteolytic activity or inhibition of such activity is an
important process of any biological system integrating tissue remodeling, tissue
destruction, or cell migration. This extracellular proteolysis is tightly regulated by the
plasminogen activator (PA)/plasmin system and inhibitors. One of the regulatory
molecules, plasminogen activator inhibitor-1 (PAI-1), regulates the biological activity of
PA by preventing conversion of plasminogen to the broad-spectrum enzyme, plasmin.
Plasmin is primarily responsible for the proteolytic degradation of fibrin as well as other
ECM proteins. Therefore, it is principally involved in fibrinolysis and breakdown of the
ECM and basement membranes. PAI-1 belongs to a superfamily of "serpins" (serine
protease inhibitors), which represent about 10% of the total protein in blood plasma [Gils

32
andDeclerck, 1998], Of these serpins, ai-proteinase represents about 70%. While PAI-1
has been traditionally thought of as a regulator of fibrinolysis, the plasminogen activator
cascade has been shown to be important in other biological systems requiring
extracellular proteolysis such as ovulation, mammary gland involution, blastocyst
implantation, and tumor metastasis [Ny et al., 1993], Plasminogen biosynthesis occurs
primarily in the liver and is found in high concentrations within blood plasma (2 mM)
thereby providing an unlimited source of potential proteolytic activity [Ny et al., 1993],
Thus, the rate-limiting step in these reactions are synthesis and activation of PA. The
proteolytic cascade is very dynamic and must be considered a multi-component system.
This multi-level system allows for precise regulation and fine-tuning at discrete focal
areas requiring tissue remodeling, so that systemic proteolysis is controlled.
The primary component of this system, plasmin, is formed by proteolytic
cleavage of plasminogen. One of two specific plasminogen activators, tissue-type (tPA)
or urokinase (uPA), initiates the cleavage of plasminogen. Both belong to the serpin
superfamily, are immunologically distinct and encoded by different genes [Ny et al.,
1993], PAI-1 binds to both tPA and uPA acting as a suicide substrate and thereby
inactivating their enzymatic activity. In addition to this function, uPA- and tPA-PAI-1
complexes can bind to the multifunctional clearance receptor, low-density lipoprotein
related-receptor protein/ci2-macroglobul¡n receptor (LRP/ct2-MR or Megalin) [Ny et al.,
1993], These complexes are internalized by endocytosis and subsequently degraded.
This is another potential mechanism for PAI-1 regulation of uPA and tPA proteolytic
activity. Urokinase PA also has a cell surface receptor (uPAR) along with plasminogen,
and binding of these molecules to cellular surfaces is thought to lead to generation of

33
plasmin, which coordinates extracellular matrix (ECM) remodeling and degradation.
Degradation of receptor-bound uPA is enhanced three- to four-fold following complex
formation with PAI-1 [Ny et al., 1993], Plasmin is the proteolytic enzyme primarily
responsible for the degradation of fibrin (fibrinolysis), breakdown of ECM and basement
membranes as well as thrombolysis. While tPA generation of plasmin is linked to the
fibrinolytic activity, uPA generation of plasmin is associated with the ECM
remodeling/degradation [Andreasen et al., 1990], Plasmin can also activate promatrix
metalloproteinases, procollagenase, and progelatinase. A diagram of the pericellular
activation cascade for plasminogen and matrix metalloproteinases can be found in Figure
2-1.
PAI-1, a single chain glycoprotein of 50 kDa, is synthesized in an active form,
which converts spontaneously to an inactive latent form that can be partially reactivated
by denaturing agents such as SDS, guanidinium hydrochloride, and urea [Hekman and
Loskutoff, 1985], In plasma, PAI-1 occurs in a complex with an integrin, vitronectin,
resulting in a significant stabilization [Declerck et al., 1988, Mimuro and Loskutoff,
1989], PAI-1 forms a stable equimolar complex with both uPA and tPA, presumably of a
covalent nature [Andreasen et al., 1990], and exists in at least three distinct
conformational forms; active, latent, and substrate-cleaved [Lawrence et al., 1997],
Active PAI-1 decays to the latent form with a half-life of 1-2 h at 37° C, unless stabilized
by vitronectin. Similar to other serpins, PAI-1 has a tightly ordered tertiary structure
consisting of three P-pleated sheets, A, B and C, a-helices A through I and a reactive site
loop [Gils and Declerck, 1998], The reactive site, comprising the "bait" peptide bond, is
located 30-40 amino acid residues from the carboxy-terminal end. Nucleotide

34
sequencing of the cDNA and amino acid sequencing revealed a mature protein of 379-
381 amino acids long with heterogeneity among species primarily in the amino terminus
[Andreasen et al., 1990], This protein was also found to be a glycoprotein and shares a
high degree of similarity (91%) between species examined, cow, human, rat, rabbit, and
mouse. Recently, pig PAI-1 was cloned and sequenced from a cDNA library prepared
from cultured pig aortic cells [Bijnens et ah, 1997], The cDNA consists of a 131-bp 5'
untranslated sequence, a 1206-bp open reading frame encoding a 402-amino acid protein,
and a 1651-bp untranslated sequence. The preprotein is cleaved at amino acid position
23, resulting in release of the mature 379-amino acid protein, PAI-1.
Hormonal Regulation of PAI-1
PAI-1 is produced by a variety of cell lines and primary cell cultures. These in
vitro culture systems have served as important models for obtaining information about
the intracellular mechanism of action stimulated by various regulatory agents.
Investigations have resulted in the identification of several hormones and steroids that
control the synthesis and secretion of PAI-1. As reviewed by Andreasen et ah (1990),
PAI-1 synthesis and/or activity is stimulated by glucocorticoids, insulin, endotoxin,
interleukin-1, tumor necrosis factor-a, EGF, CSF-1, and phorbol esters. Inhibitors of
PAI-1 synthesis and/or activity are molecules that stimulate intracellular increases in
cAMP production such as follicle stimulating hormone (FSH) and Ieutinizing hormone
(LH). Other potential regulators of PAI-1 synthesis/activity are prolactin [Liu et ah,
1998] and prostaglandin Fía [Zhang et ah, 1996],
In addition to regulation by the molecules described above, PAI-1 expression and
activity is regulated by ovarian steroids. Progesterone has been shown to significantly

35
increase PAI-1 synthesis in several endometrial cell lines [Schatz et al., 1994, Casslen et
al., 1992, Casslen et al., 1995, Miyauchi et al., 1995], As reviewed by Schatz et al.
(1994), estrogen had either no effect or inhibited PAI-1 synthesis. However, the effect of
progesterone on PAI-1 synthesis doubled in response to the presence of both estrogen and
progesterone. Furthermore, exogenous steroids elicit similar changes in expression of
PAI-1 mRNA as seen for PAI-1 protein. Progesterone was shown to not interact with the
804-bp promoter region of the human endometrial PAI-1 gene, but rather increased the
stability of PAI-1 mRNA [Sandberg et al., 1997], After menopause, hormone
replacement therapy may reduce the risk of coronary heart disease that is associated with
fibrinolytic activity. Women receiving estradiol were shown to have significantly lower
plasma levels of PAI-1 [Lindoff et al., 1996, Shahar et al., 1996], indicating that estrogen
has a significant role in inhibition of PAI-1 expression. However, some investigators
suggest that estrogen might also increase PAI-1 synthesis [Sobel et al., 1995, Fujimoto et
al., 1996] which is contradictory to the majority of other findings to date.
Plasminogen Activator and its Inhibitor in the Uterus and Ovary
In man and rodents, endometrial stromal cells proliferate and differentiate into
decidual cells as the implanting embryo invades the endometrium in order to establish an
intimate contact with the maternal blood supply. Although the implanting embryo
produces matrix-degrading enzymes for implantation, ECM-degrading enzymes are also
produced in the human endometrium under the control of steroid hormones [Wang et al.,
1996], In the human, an increase in tissue-factor (TF) and PAI-1, and inhibition of tPA,
uPA and matrix metalloproteinases (MMPs) are associated with progestin-induced
decidualization of estrogen-primed endometrial stromal cells in vivo and in vitro

36
[Lockwood and Schatz, 1996], These important regulators of fibrinolysis, hemostasis,
and ECM turnover in the decidualized stromal and decidual cells suggest a mechanism to
explain the absence of hemorrhage during invasion of the endometrial vasculature by
trophoblasts. Progesterone withdrawal reduces TF and PAI-1, yet increases uPA, tPA
and MMPs thereby increasing hemorrhage, fibrinolysis, ECM degradation and vascular
injury characterizing menstruation [Lockwood and Schatz, 1996], Trophoblast cells are
so highly invasive that they are called pseudomalignant and when grafted to ectopic sites,
invade uncontrollably [Axelrod, 1985], The modified uterine tissue, decidua, is rendered
resistant to invasion by the well-controlled coordinate expression of protease inhibitors
such as PAI-1 [Waterhouse et al., 1993, Teesalu et al., 1996, Lala and Hamilton, 1996],
The PA system has also been shown to be an important regulator of ovulation. As
early as 1916, it was proposed that proteolytic activity was involved in degradation of the
follicle wall at the time of ovulation [Ny et al., 1993], Levels of plasminogen in
follicular fluid are comparable to levels found in serum. Injection of agents which inhibit
PA and plasmin activity into the ovarian bursa suppress ovulation [Ny et al., 1993],
Combined uPA and tPA gene-deficient mice were also shown to have a 26% reduced
ovulation efficiency [Leonardsson et al., 1995], The loss of an individual PA is
functionally compensated by the activity of the remaining PA, however the loss of both is
not obligatory for ovulation. As reviewed by Andreasen et al. (1990) and Ny et al.
(1993), ovulation-associated hormones, FSH and LH, are effective stimulators of PA
secretion from granulosa cells. In the preovulatory follicle, the LH surge stimulates a
cascade of proteolytic enzymes, including PA, plasmin, and MMPs. These enzymes
bring about the degradation of the perifollicular matrix and the decomposition of the

37
meshwork of collagen fibers which provide strength to the follicle wall [Tsafriri and
Reich, 1999], Increased ovarian proteolytic activity is controlled by high levels of PAI-1
and TIMP-1 expression in theca cells from growing follicles, ensuring their development
by protection from enzymes diffusing from ovulatory follicles [Tsafriri and Reich, 1999],
FSH, LH, and activators of cAMP suppress PAI-1 synthesis and activity in granulosa
cells. However, one report in the rat indicates cell-specific expression of PAI-1 and
TIMP-1 mRNAs in the LH/hCG-stimulated ovary suggesting a co-expression of both
enzymes and their inhibitors during ovulation [Chun et al., 1992], PAI-1 was also shown
to be a major secretory product of the corpus luteum and its expression is stimulated by
PGF2a [Smith et al., 1997], These investigators suggest an important role for PAI-1
during ECM remodeling of the ovary following ovulation.
PA in the Oviduct During Fertilization and Embryo Development
A significant amount of literature has been compiled on PA activity during
fertilization and its association with the preimplantation embryo. However, there are no
data on its physiological inhibitor, PAI-1, in the oviductal lumen or its association with
the early embryo. The importance of PAI-1 in the reproductive system is not fully
understood. PAI-1 gene-deficient mice are viable, fertile, and without abnormalities in
organogenesis and development [Carmeliet et al., 1993], Other factors (PAI-2, a2-
antiplasmin, 012-macroglobulin, Ci-esterase inhibitor and aj-antitrypsin) known to reduce
plasminogen activation and/or plasmin activity, may compensate for the loss of PAI-1.
However, to the authors knowledge, these proteins have not been identified in the
oviduct. This portion of the discussion will concern data available on PA that may lead
to insights on PAI-1 function within the oviduct.

38
As early as 1968, PA was shown to be associated with the oviductal mucosa
[Tympanidis and Astrup, 1968], PA activity has also been shown to be associated with
rat [Liedholm and Astedt, 1975] and mouse [Sherman, 1980] preimplantation embryos
from the 2-cell to blastocyst stage. Sherman (1980) showed that this activity was
associated with the zona pellucida, as denuded embryos have no PA activity. These
earlier studies concluded that zona-associated PA did not originate from the embryo but
from genital tract secretions. The observation that PA activity steadily decreased as
preimplantation development proceeded both in vivo and in vitro raises the possibility
that the zona pellucida contains residual activity from follicular or oviductal fluid
[Sherman, 1980],
PA activity has been associated with several important reproductive processes
including oocyte maturation, fertilization, and early embryogenesis. Pig cumulus-cell
oocyte complexes (COCs) have been shown to produce two PAs during in vitro
maturation [Kim and Menino, 1995], These PAs were shown to be tPA and tPA-inhibitor
complex. Stimulators of PA activity in pig COCs, like cAMP and okadaic acid, were
found to inhibit oocyte maturation. Kim and Menino (1995) suggest that although PA
production is temporally associated with oocyte maturation, coordination of these two
processes is differentially regulated. The production of PA in rat COCs has also been
reported [Liu et al., 1986, 1987], PA activity in rat [Salustri et al., 1985, Ny et al., 1987]
and pig [Kim and Menino, 1995] COCs is through a stimulation of protein kinase A and
C. Recently, cow COCs matured in vitro, were shown to contain mRNA for all elements
of a proteolytic cascade including uPA, PAI-1, MMP-1 and TIMP-1 [Bieser et al., 1997],
These investigators suggest a potential role for this extracellular proteolysis in cumulus

39
expansion during oocyte maturation. In the human, transcription of PAI-1 and PAI-2
genes have been described for cumulus cells and granulosa-luteal cells [Piquette et al.,
1993], Similarly, cow COCs show production of PA during in vitro maturation and this
production was stimulated by EGF [Park et ah, 1999], This activity was shown to be
associated with uPA and was not present in denuded oocytes, indicating the importance
that cumulus cells play in its synthesis. For a current discussion on the production of PA
in COCs relative to the time of germinal vesicle breakdown and resumption of meiosis,
the reader is referred to Park et ah (1999).
As reviewed by Huarte et ah (1993) the PA/plasmin proteolytic cascade may have
a significant role during fertilization. Proteolytic enzymes participate in multiple phases
of mammalian fertilization including the acrosome reaction, sperm-binding to the zona
pellucida, zona pellucida penetration, and the zona reaction. Mouse gametes express
plasminogen-dependent proteolytic activities; ovulated eggs synthesize and secrete tPA
while ejaculated spermatozoa have uPA activity. Likewise, plasminogen was shown to
bind to both mouse spermatozoa and eggs, and the presence of plasminogen increased the
in vitro fertilization rate [Huarte et ah, 1993] presumably due to increased plasmin
generation. In addition, antibodies, which inhibit the catalytic activity of plasmin, were
shown to decrease the fertilization rate. Exogenously added plasminogen was likely
converted to plasmin by zona pellucida-associated uPA or tPA activity. These findings
are supported by previous work indicating that a positive correlation existed between the
ability of oocytes fertilized in vitro and the PA activity of the corresponding follicular
fluid and granulosa cells [Deutinger et ah, 1988, Milwidsky et ah, 1989], Zhang et al.
(1992) showed that tPA was released from rat oocytes as a result of oocyte activation and

40
suggested that tPA may act in the perivitelline space on the zona pellucida during
fertilization and/or activation. As described by Zhang et al. (1992), tPA may be a
component of corticol granules that are involved in the zona block to polyspermy. In
their study, activation-induced zona hardening (limited proteolysis of zona protein ZP2)
was prevented by a anti-tPA monoclonal antibody. Inadvertent activation under in vitro
conditions in mouse and human IVF systems often times leads to premature loss of
fertility in respective oocytes. Addition of leupeptin, a serine protease inhibitor, during
mouse oocyte calcium-ionophore activation reversed the decrease in the capacity of
oocytes to fertilize and develop in vitro [Tawia and Lopata, 1992], The data support
evidence for PA activity in zona hardening and suggests a coordinate expression between
activator and inhibitor in regulating this process. Additionally, supplementation of
culture medium with proteases increased hatching rate of mouse embryos [Lee et al.,
1997], while addition of protease inhibitors to culture medium inhibited hatching in vitro
[Dabich, 1981, Yamazaki et al., 1985], This suggests an important role for oviductal-
derived protease inhibitors in the prevention of premature hatching, prior to the correct
developmental stage within the uterus. The effect may be directed towards proteolysis of
the zona matrix. Zona of porcine oviductal oocytes and embryos were shown to be more
resistant to proteolytic digestion than either follicular oocytes or embryos recovered from
the uterus [Broermann et al., 1989], indicating an important role for protease inhibitors in
protecting the zona pellucida from degradative proteases.
Preimplantation mouse [Harvey et al., 1995, Zhang et al., 1996], rat [Zhang et al.,
1994], sheep [Menino et al., 1989, Bartlett and Menino, 1993] and cow [Dyk and
Menino, 1991, Berg and Menino, 1992] embryos were shown to have PA activity.

41
Kaaekuahiwi and Menino (1990), showed that as embryonic size and cell number
increase and development progresses, bovine embryos liberate more PA. Recently,
bovine embryonic PA was shown to induce changes in the electrophoretic protein profile
of the zona pellucida when embryos were incubated with exogenous plasminogen
[Cannon and Menino, 1998],
In addition to PA's association with maturation, fertilization and embryogenesis, it
might also have an important role in spermatozoa physiology. Urokinase PA is
synthesized in epithelial cells of the caudal epididymis, vas deferens, and seminal
vesicles [Huarte et al., 1987], while tPA was found in the prostate gland [Reese et al.,
1988], In addition, Sertoli cells have been shown to secrete PA and PAI-1 [reviewed by
Gilabert et al., 1995], The presence of uPA and tPA has been shown in ejaculated
spermatozoa of man and various animal species [Smokovitis et al., 1987], These
investigators report a direct influence on sperm motility and suggest that the PA are
membrane bound. Recently, boar spermatozoa were shown to have uPA and tPA
associated with spermatozoal membranes and differences were detected in their
localization [Smokovitis et al., 1992], The outer acrosomal membranes contained tPA
while the inner acrosomal membrane contained both tPA and uPA.
Structure. Regulation, and Biological Actions of Oviductal Secretory Glycoprotein ÍOSP1
In vitro Synthesis and Hormonal Regulation of OSP
Experiments by Oliphant and Ross (1982) in the rabbit describe the identification
and purification of three sulfated glycoproteins from oviduct fluid. This investigation
was one of the first studies utilizing a radiolabeled precursor, [35S], to evaluate
incorporation into macromolecular components of oviductal epithelial cells and oviductal

42
explant tissue in culture. Utilizing this technique, Oliphant and Ross (1982) reported the
de novo synthesis and secretion of glycoproteins by oviductal epithelium. Subsequently,
numerous studies have been done in various species. Evaluations of stage-specific
proteins in the pig oviduct were first reported by Buhi et al. (1989). The de novo
synthesis of porcine oviductal proteins by cyclic and early pregnant tissues in explant
culture revealed that incorporation of [3H]-leucine into nondialyzeable macromolecules
was greatest on Days 0 and 2 and secretory activity was lowest on Days 10 to 15. The
ampulla was shown to have greater incorporation than the isthmus and increased rates of
incorporation were temporally associated with elevated estrogen at proestrus, estrus, and
metestrus [Buhi et al., 1989], 1D-SDS-PAGE and fluorographic analysis of oviductal
culture medium revealed three proteins of 335 k, 115 k, and 85 k Mr, associated with
proestrus, estrus, and metestrus. High resolution 2D-SDS-PAGE further resolved the
isoelectric and molecular weight variants of these radiolabeled proteins initially detected
by 1D-SDS-PAGE [Buhi et al., 1990], The major 115 k Mr band was resolved into two
major glycoproteins, one basic (100 k Mr, pi > 8) and one acidic (100 k Mr, pi 4.5-S.5).
The 85 k Mr protein was resolved into a very acidic (pl< 4) protein of 75 to 85 k Mr.
These three proteins have been designated pOSP El (85 k M,), pOSP E2 (100 k Mr;
acidic) and pOSP E3 (100 k Mr; basic). It was suggested by Buhi et al. (1990) that these
denatured and reduced subunits may be part of a higher molecular weight complex and
were related (pOSP E2 had the same N-terminal sequence as El and the N-terminal
sequence for E3 was identical to an internal sequence of E2). Incorporation of [35S]-
glucosamine indicates that these three estrus-associated proteins are glycosylated [Buhi et
al., 1990],

43
Oviduct-specific secretory proteins have been identified in human [Verhage,
1988, Buhi et ah, 1989], baboon [Fazleabas and Verhage, 1986, Verhage and Fazleabas,
1988], cow [ Malayer et al., 1988, Boice et al., 1990], sheep [ Sutton et al., 1984, Sutton
et al,, 1986, Gandolfi et al., 1989, Buhi et al,, 1991, Murray, 1992], mouse [Kapur and
Johnson, 1985] rhesus monkey [Verhage et al., 1997], and hamster [Robitaille et al.,
1988, Abe et al., 1998], Although isoelectric points are similar for the subunits examined
in other species, differences have been noted in molecular weights for these
glycoproteins. Studies on these glycoproteins have led to several key observations. First,
these glycoproteins are primarily secretory products of the ampulla, although their
synthesis can be seen in other segments at a much lower rate [Verhage and Fazleabas,
1988, Buhi et al., 1990, Boice et al., 1990, Gandolfi et al., 1991, Murray, 1992, and
O'Day-Bowman et al., 1995], Second, the presence of these proteins only during estrus,
temporally elevated at estrus or the follicular stage in humans indicates that this protein is
regulated by estrogen. Therefore, the pattern (but not presence) of distribution for these
proteins is dependent on the presence or absence of specific ovarian steroids. In the pig,
treatment of ovariectomized (OVX) gilts with estrogen increased incorporation rate in the
ampulla and revealed that the oviduct-specific glycoproteins were estrogen-dependent
[Buhi et al., 1992], This is supported by the observation that progesterone
antagonizes/abrogates the stimulatory effect of estrogen on their synthesis. Similar
observations on OSP steroid regulation using OVX animals have been made in sheep
[Buhi et al., 1991, Murray and DeSouza, 1995] and baboon [Verhage and Fazleabas,
1988], Newborn golden hamsters (1.5 days) injected daily with estrogen or progesterone
showed that estrogen could induce hamster OSP synthesis in the undifferentiated

44
epithelial cells of neonates, while progesterone could not [Abe et al., 1998], This family
of estrogen-dependent and oviduct-specific glycoproteins is the most abundant
radiolabeled secretory product of the oviduct, yet assigning specific functions to these
proteins has been elusive. Investigations on the localization of OSPs in the oviduct and
their association with the ovulated oocyte and early embryo, as detailed below, suggest
possible involvement during fertilization and early cleavage-stage embryonic
development.
Immunolocalization of pOSP in the Oviduct. Oocvte. Spermatozoa and Early Embryo
Immunogold localization of pOSP in the pig has been evaluated at the cellular
level utilizing electron microscopy [Buhi et al., 1993], These investigators found that
pOSP was localized to putative secretory granules in non-ciliated secretory cells of the
ampulla from both cyclic and OVX estrogen-treated gilts. This study was not extended
to evaluate distribution of pOSP in either the infundibulum or isthmus of the pig. Similar
observations of OSP localizing to secretory granules in the ampulla have been observed
in sheep [Murray, 1992, Gandolfi et al., 1991], cow [Boice et al., 1990], baboon [Verhage
et al., 1990, Verhage et al., 1989], rhesus monkey [Verhage et al., 1997], and human
[O'Day-Bowman et al., 1995, Rapisarda et al., 1993], Studies done in the human and
baboon identified OSP in secretory granules of the isthmus as well as the ampulla,
indicating that this segment also synthesizes OSP. Evaluations of colloidal gold densities
in segments treated with estrogen, progesterone, or estrogen + progesterone have not
been performed. Therefore, it is unknown whether there are dynamic changes of OSP in
secretory granules relative to hormonal status, except for the observation with estrogen
alone, as detailed above. Hamster OSP of oviductal origin is associated with uterine

45
epithelial cells during the first 3 days of pregnancy, but is reduced by Day 4, and is
absent by Days 5 and 6 [Roux et al., 1997], Because Days 5 and 6 correspond to the time
of implantation in the hamster, and levels of OSP remain constant in the oviduct, these
investigators suggest that OSP may be involved in uterine receptivity.
The macromolecular composition of the zona pellucida in the mammalian
oocyte/embryo is quite complex and three major glycoproteins have been identified each
containing about 15 isoelectric species [Dunbar, 1983], Several studies have revealed
changes in the composition of the porcine zona pellucida during development from the
oocyte to the 4-cell embryo. Brown and Cheng (1986) observed that the zona pellucida
of the follicular oocyte to the early embryo acquired three glycoproteins from the oviduct
at estrus. These investigators observed two proteins of 250 k and 90 k Mr when the zona
pellucida was analyzed by ID- and 2D-SDS-PAGE under non-reducing conditions.
When these proteins were electrophoresed under reducing conditions, subunits of 90 k,
79 k, and 69 k M, were detected. Similar differences in the macromolecular composition
of zona pellucida from pig oocytes, eggs, and zygotes have been observed by Hedrick et
al. (1987), although there is some discrepancy in the molecular weight of these proteins
when compared to those of Brown and Cheng. These are the first studies indicating that
proteins of oviductal origin associate with the zona pellucida and that their molecular
weight (80-90 k) corresponds to that of pOSP. The study by Buhi et al. (1993) supports
these earlier results and specifically indicates that oviductal pOSP associates with
unfertilized oviductal oocytes and early embryos. Pig OSP immunoreactivity was
observed through early development to Day 7 hatched porcine embryos but had
disappeared by Day 9 of pregnancy [Buhi et al., 1993], In addition to association with the

46
zona pellucida, pOSP also associates with flocculent material in the perivitelline space
and with vitelline and blastomere membranes. Limited data supports the possibility that
the embryo itself endocytoses OSP during its early development. Hamster OSP has been
localized in coated pits, apical vesicles, mutivesicular bodies, and lysozome-like
structures [Kan and Roux, 1995], The association of pOSP with the early developing
embryo suggests this protein may have an important role during fertilization and early
cleavage-stage embryonic development.
Localization of OSP to the zona pellucida of oviductal oocytes or early embryos
has been shown in the hamster [Kan et al., 1989, Kan and Roux, 1995, Abe and Oikawa,
1990, Leveille et al., 1987], cow [Wegner and Killian, 1991], sheep [Gandolfi et al.,
1991], rhesus monkey [Verhage et al., 1997], and baboon [Boice et al., 1990], In the
mouse, OSP was not associated with the zona pellucida and was only localized within the
perivitelline space [Kapur and Johnson, 1985], An interesting observation by Kan et al.
(1995) was that hamster OSP localized to the flocculent material in the perivitelline space
in oviductal oocytes only after fertilization. These investigators suggest that OSP
interacts with secretions of corticol granules after fertilization and may be involved in the
block to polyspermy. This hypothesis is supported by our work on polyspermy described
in Chapter 4
While observations for an association of OSP with the oviductal oocyte or early
embryo are consistent across species studied thus far, a similar consensus does not exist
for localization of OSP to spermatozoa. In the hamster [Kimura et al., 1994, Boatman
and Magnoni, 1995] and cow [King and Killian, 1994, Abe et al., 1995], OSP has been
shown to associate with the spermatozoal membrane. However, a report in the human

47
[Reuter et al., 1994] failed to find an association of human OSP with human
spermatozoa.
Molecular Biology of pOSP
Cloning and characterization of a full length cDNA for pOSP has been reported
by Buhi et al. (1996). A full length clone (2022-bp) was identified and sequenced,
yielding an open reading frame of 1581 bp that coded for a protein of 527 amino acids.
A putative signal sequence was identified (a.a. 1-21) and is highly conserved, having
100% homology among bovine, human, and ovine signal sequences. Sequence analysis
of pOSP revealed 3 consensus N-glycosylation sites as well as several potential O-
glycosylation sites, suggesting large amounts of carbohydrate addition. This would also
explain the differences between the predicted native molecular weight of 55,600 and
those observed by 2D-SDS-PAGE (El; 75 k, E2,3; 100 k Mr). The pOSP cDNA
sequence revealed significant identities and similarity to oviductal secretory
glycoproteins (OSP) from a number of other species, especially in the 5' end of the open
reading frame. Sequence analysis of pOSP cDNA also revealed two potential
phosphorylation sites, a consensus heparin-binding sequence, a region similar to the
chitinase catalytic site, and a potential C-terminal region similar to a chitinase binding
domain. However, this protein does not have chitinase activity and is missing an
essential amino acid required for such activity. Observations have been made
establishing a structural similarity between OSPs and the chitinase protein family [Arias
et al., 1994, Sendai et al., 1995, Suzuki et al., 1995, Buhi et al., 1996], Sendai et al.
(1995) has suggested that the chitinase-like structure in OSPs may be involved with
carbohydrate moieties of the oocyte or surface of the spermatozoa. Since chitinase binds

48
to chitin (poly-pl,4-N-GlcNAc) and hydrolyzes it, Suzuki et al. (1995) suggests that
OSPs may be a GlcNAc binding protein, which interacts with carbohydrate components,
located on the zona pellucida. OSPs have been cloned in several other species including
the baboon [Donnelly et al., 1991], human [Arias et al., 1994], hamster [Suzuki et al.,
1995], mouse [Sendai et al., 1995], cow [Sendai et al., 1994], sheep [DeSouza and
Murray, 1995] and rhesus monkey [Verhage et al., 1997], These researchers also found
that a significant degree of homology exists among oviduct-specific secretory
glycoproteins of various mammalian species studied thus far. However, OSP cDNA
appears to lack homology to the frog oviduct-specific protein-1 gene found in Xenopus
oviduct cells [Donnelly et al., 1991], Sequence analysis of the various species listed
above revealed several N- and O-glycosylation sites, which may reflect why differences
are observed between species in isoelectric point and molecular weight when evaluated
by 2D-SDS-PAGE. Identification of the polypeptide precursors and characterization of
their biosynthetic maturation within oviductal cells for hamster OSP has been examined
in detail and hypothesis on the polymorphism of these variants due to translational and
posttranslational events has been reviewed by Malette et al. (1995). Southern blot
analysis of genomic DNA has shown that there is a single copy of the hamster OSP gene
in the hamster genome and PCR amplification revealed that it is contained within a single
exon, excluding the possibility of alternative splicing [Paquette et al., 1995], Although
these studies have not been done in other species to date, immunological and northern
blot analysis suggest that the family of OSPs detected by 1- and 2D-SDS-PAGE all
derive from a single gene. Hamster OSP has recently been referred to as a secretory
mucin, due to extensive O-glycosylation (> 50%) not seen in other species, tandemly

49
repeated amino acid motifs, and the presence of multiple alleles (Paquette et al., 1995],
These tandem repeats are present in rodents and humans but are not seen in ungulates,
raising interesting questions from an evolutionary viewpoint. A review of the various
sequences cloned revealed significant differences in the C-terminal region of the OSPs
and may reflect species differences in the OSP molecule. For instance, Sendai et al.
(1995) identified a unique seven-residue repeat sequence (21 repeats) in the C-terminal
side of mouse OSP which are not seen in cow, sheep, pig or baboon OSP.
Probes for pOSP mRNA detected only a single 2.25 kb message in oviductal
tissue and mRNA was present in all 3 segments of the oviduct. Levels of mRNA were
greatest in the ampulla indicating this segment as the primary site of its expression.
Northern blot analysis of numerous other tissues (heart, intestine, cervix, kidney,
endometrium, myometrium, spleen, lung, aorta, liver, or stomach) confirmed that
expression of this protein is specific to the porcine oviduct. Steady-state levels of pOSP
mRNA in the oviduct were greatest at estrus (Days 0,1) consistent with elevated estrogen,
decreased rapidly by Day 2, and remained low throughout diestrus. These data correlates
well with previous reports on pOSP protein synthesis [Buhi et al., 1990, Buhi et al.,
1992], Levels of pOSP mRNA in oviducts of steroid-treated OVX gilts confirmed that
this protein's expression is up-regulated by estrogen and that progesterone antagonizes
this effect. Data from other species including human [Arias et al., 1994], baboon
[Donnelly et al., 1991], and bovine [Sendai et al., 1994] have also shown that the
message for this protein is greatest during the late follicular phase and in estradiol-
dominated oviducts.

50
A study in the hamster, utilizing in situ hybridization for detection of OSP
message, found evidence for OSP in both the ampulla and isthmus, although the signal
intensity was greatest in the ampulla [Komiya et ai., 1996], The distribution of OSP
message was different within the two sections, with the ampulla having signal detected in
both the perinuclear and basal regions, while the isthmus only had signal in the basal
compartment of epithelial cells. Komiya et al. (1996) also found that the message for
OSP is greatest when serum estradiol/progesterone levels are higher and that the mRNA
for OSP wanes with age and correlates with decreasing serum estradiol levels. However,
these investigators also showed that progesterone is required for hamster OSP gene
expression, unlike data from the baboon [Donnelly KM et al, 1991] and pig [Buhi et al.,
1996], which shows OSP strongly suppressed by progesterone. Another interesting
difference is that the hamster [Paquette et al., 1995] rabbit [Donnelly et al., 1991], and
mouse [Donnelly et al., 1991] showed a constant level of mRNA expression for OSP
throughout the estrous cycle which differs from that of other species observed thus far
where a strong estrogen-dependence is required for its expression. Contrary to findings
by Komiya et al. (1996), Murray and DeSouza (1995) found that transcripts encoding
sheep OSP were in the basal compartment and at the apical tips in fimbria and ampulla
epithelial cells at the free margins of mucosal folds. Because mRNA was localized to the
apical tips, these investigators suggested that mRNA encoding OSP was translated at a
unique cytoplasmic foci. Electron microscopy reveals that all the cytoplasmic machinery
required for protein synthesis is also located within the apical tips of oviductal epithelial
cells, which suggests a quick response time from the point of translation to release of
OSP into the lumen [Murray and DeSouza, 1995],

51
Cyclical, structural, and functional changes in the oviduct are thought to be
brought about primarily by the actions of estradiol and progesterone [Verhage and Jaffe,
1986], and numerous reports have been cited above showing regulation of OSPs and
pOSP by estrogen/progesterone levels. Recent studies have demonstrated that human
[Lie et al, 1993] and pig [Gawronska et al., 1999] oviducts contain luteinizing hormone
(LH)/human chorionic gonadotropin (hCG) receptors, suggesting that factors other than
circulating ovarian steroids may regulate oviductal function. One study by Sun et al.
(1997), revealed that culturing bovine oviductal epithelial cells with hCG resulted in a
time and dose-dependent increase of OSP protein and transcript. A nuclear transcription
run-on assay was used to determine that hCG did not increase the transcription rate of the
gene but rather "stabilized" its transcript [Sun et al., 1997], These investigators suggested
that higher LH levels present during the periovulatory periods may act to stabilize the
message for OSP in the oviduct, thus leading to an increased secretion rate. Recently,
heterozygous mutant mice have been generated, which lack a large portion of the OSP
coding region [Sendai et al., 1999], In the future, homozygous offspring may clarify the
role of this protein in vivo.
Biological Actions of OSPs
The observations that in all species examined so far, with the notable exception of
the mouse, OSPs associate with the zona pellucida, suggest an intimate role for these
estrogen-dependent proteins during fertilization and early cleavage-stage embryonic
development. However, the abundant information detailing a morphological association
of OSPs with spermatozoa, oocytes, or embryos does not correspond to a suitable amount
of functional information. Functional information regarding OSPs have been hindered by

52
difficulties in the protein purification process. In fact, no direct in vivo data on precise
functions of these proteins are available to date. However, several in vitro observations
of activities associated with the inclusion of OSP have led to some attractive hypotheses.
Macromolecules secreted by the hamster oviduct in the ampulla region have been
shown to facilitate penetration of sperm through the egg investments [Boatman et al.,
1994] and follicular eggs collected from the ampulla were shown to be more penetrable
and fertilizeable than follicular eggs collected from the ovarian bursa or unovulated
follicles [Boatman and Magnoni, 1995], Similar increases in penetration rates have been
observed in the bovine [Martus et al., 199S] and were linked to the addition of
concentrated bovine OSP during in vitro fertilization (IVF). One conflicting report in the
hamster [Kimura et al., 1994] indicates that penetration rates were decreased in the
presence of OSP. These data, although partially conflicting, indicate that association of
OSP with the oocyte may have a functional role during fertilization. It is unknown
whether these observed effects were due to actions on spermatozoa, oocyte or both.
Martus et al. (1997) showed that exposure of bovine oocytes to bovine OSP during any
phase of the IVF process increased fertilization rates, while exposure of spermatozoa to
bovine OSP had no effect.
Several investigators suggest that OSPs induce capacitation [McNutt et al., 1992,
Anderson and Killian 1994] and/or the acrosome reaction. Therefore, an increase in the
number of capacitated or acrosome-reacted spermatozoa could possibly lead to the above
observations on increased penetration. However, direct evidence for the estrogen-
dependent OSP on these activities is lacking.

53
Another potential modulator for this Increase in penetration may be due to actions
on spermatozoa movement characteristics. Abe et al. (1995) found that bovine OSP
effectively maintained the viability and motility of bovine spermatozoa relative to control
medium and that this activity was dose-dependent. The ability of bovine OSP to
maintain motility and viability may allow a greater proportion of spermatozoa to
penetrate the egg investments and subsequently fertilize the oocyte. The OSPs may also
have a role in gamete recognition and binding. In the hamster [Schmidt et al., 1997a,
1997b] and human [O'Day-Bowman et al., 1996], OSP was found to increase the number
of tightly bound sperm attached to the zona pellucida. This increase might also lead to
the increased penetration rates described above. These data are supported by the
observation that a redistribution in the localization of hamster OSP occurs depending on
the state of capacitation [Boatman and Magnoni, 1995],
Functional data on embryonic development are especially lacking, and
investigations to date are less than encouraging. No effects of bovine OSP
[Vansteenbrugge et al., 1997] or ovine OSP [Hill et al., 1997] were observed on bovine
embryonic development. Numerous investigations have shown the need for homologous
systems when studying this protein, therefore observations by Hill et al. (1997) may be
due to the heterologous system employed. Previous studies by Hill et al. (1996 a, b)
examining ovine OSP effects on ovine embryonic development demonstrated several
subtle but consistent results. The most significant of these being that a reduced
proportion of one-cell embryos underwent first cleavage, but no overall decrease on the
proportion of blastocysts that formed. These investigators suggest that this may reflect a
selection mechanism occurring in vivo. Bovine OSP has been found to increase the

54
number of bovine zygotes that cleaved compared to controls, but by Day 7 of embryonic
development, no difference could be detected in the number of blastocysts that formed
[Martus et al., 1997], However these investigators did observe an increased number of
blastocysts on Day 6 of in vitro culture. This may suggest that bovine OSP, while not
increasing the number of blastocysts, increases their rate of development. However, a
slight loss in the number of blastocysts between Day 6 and Day 7 was shown and might
indicate that the concentration of bovine OSP tested or length of exposure (>6 days) is
detrimental to subsequent development. This increased rate of development may be due
to an unknown stimulation on the expression of maternal or embryonic genes and
increased protein synthesis. One study indicates that in vivo fertilized pig embryos (one-
or two-cell stage), cultured in vitro in the presence of one subunit (97 k Mr) of semi-
purified pOSP, showed increased rates of incorporation of methionine into protein at the
four-cell stage [Wallenhaupt et al., 1996], The data described above indicate that pOSP
may facilitate or enhance fertilization and early cleavage-stage embryonic development.
This review thus leaves a number of unanswered questions which need to be
addressed. The first is; what are the unidentified de novo synthesized and secreted
macromolecules of the oviduct? The second is; for those proteins which have been
identified and characterized, what is the specific function(s) in relation to reproductive
processes in the oviduct; union of gametes, fertilization, and early cleavage-stage
embryonic development. Chapters 3, 4, and 5 describe the identification,
characterization, and regulation of PAI-1 in the oviduct. The objectives of these three
chapters include; 1) localization of PAI-1 within the oviduct, 2) hormonal regulation of
oviductal PAI-1 by ovarian steroids, 3) expression of PAI-1 mRNA and protein during

55
early pregnancy, 4) evaluation of PAI-1 and uPA activity in the oviduct during early
pregnancy, and 5) localization of PAI-1 on oviductal oocytes and embryos. Chapter 6
will examine the functionality of pOSP during in vitro fertilization and embryo culture.
Effects of pOSP on the fertilization rate, polyspermy rate, and development to blastocyst
will be examined. Results of these studies will begin to define the roles of oviductal PAI-
1 and pOSP during fertilization and embryonic development.

Figure 2-1. Pericellular activation cascade for plasminogen and matrix metalloproteinase.
uPA, urokinase plasminogen activator; uPA-R, urokinase plasminogen activator receptor;
PAI-1, plasminogen activator inhibitor-1; 012 AP, alpha-two anti-plasmin inhibitor; TIMP,
tissue inhibitor of matrix metalloproteinase; MMP, matrix metalloproteinase. Proteolytic
enzymes and inhibitors (outlined in blue) involved in the regulation of fibrinolysis and
extracellular matrix remodeling/degradation.

Pericellular activation cascade for
plasminogen and matrixmetalloproteinases

CHAPTER 3
IDENTIFICATION AND LOCALIZATION OF PLASMINOGEN ACTIVATOR
INHIBITOR-1 (PAI-1) IN THE PORCINE OVIDUCT
Introduction
The porcine oviduct provides an important microenvironment for final maturation
of gametes, fertilization, and early cleavage-stage embryonic development. In part to
provide an effective environment for these reproductive processes, numerous proteins
derived from serum as a transudate or from oviductal epithelium contribute to the
composition of oviductal luminal fluid [Feigelson and Kay, 1972, Sutton et al., 1984,
Buhi et al., 1997], Oviductal luminal fluid and its protein composition vary during the
estrous cycle and early pregnancy, and maximal levels of oviductal fluid appear to
coincide with elevated estrogen [Hunter, 1988], When estrogen is maximal, Days 0 and 1
of the estrous cycle or early pregnancy, biosynthetic activity in the oviduct is greatest
[Buhi et al., 1989], The infundibulum and ampulla, regardless of day of the estrous cycle
or early pregnancy, have a biosynthetic activity significantly greater (2-3 times) than that
of the isthmus [Buhi et al., 1997], This activity, measured in explant culture-conditioned
media, reflects the de novo synthesis and secretion of secretory proteins from these three
segments.
The relative importance of oviductal-derived proteins to fertilization and early
embryonic development is not known, nor have many of these proteins been identified.
Several studies have shown the importance of utilizing the oviduct or its constituents for
58

59
enhancing embryo development in vitro [Archibong et al., 1989, White et al., 1989, Liu
et al., 1998], In the pig, glycoproteins derived from oviductal fluid have been shown to
associate with the zona pellucida of oocytes after ovulation, at fertilization and during
early embryonic development [Brown and Cheng, 1986, Hedrick et al., 1987, Buhi et al.,
1993], It has been suggested that these proteins may be facilitating the beneficial effects
of oviductal epithelial cell co-cultures on embryo development in vitro [Buhi et al.,
1997],
The pig oviduct has been shown to synthesize and secrete de novo 14 major
proteins into explant culture medium [Buhi et al., 1990], The majority of these proteins
have been described electrophoretically by isoelectric point and relative molecular mass.
Two of these proteins have been identified and characterized, the porcine oviduct-specific
secretory glycoprotein (pOSP) [Buhi et al., 1992] and tissue inhibitor of matrix
metalloproteinase-1 (TIMP-1) [Buhi et al., 1996], Of the numerous proteins yet to be
identified, one protein with an apparent molecular weight of 45,000, appeared to be
synthesized and distributed similar to TIMP-1. Like TIMP-1, this protein was shown to
incorporate 3H-glucosamine, be composed of 5-6 isoelectric species, and be synthesized
primarily by the isthmus [Buhi et al, 1990], Thus, with characteristics similar to
oviductal TIMP-1, it was suggested that the 45,000 Mr protein may also be a protease
inhibitor.
This study was designed to further identify and characterize the 45,000 Mr de
novo synthesized protein of the porcine oviduct and to better understand specific protein
contributions of each oviductal segment relative to ovulation, gamete transport,
fertilization and early cleavage-stage embryonic development. With identification of the

60
unknown 45,000 M, protein as porcine plasminogen activator inhibitor (PAI)-l
(described here in Chapter 3), studies were designed to examine its synthesis within the
infundibulum, ampulla, and isthmus, and to evaluate its distribution throughout the
oviduct. While PAI-1 has been localized in the uterus and ovary from a variety of
species and potential mechanisms examined, no studies to our knowledge have been done
to assess PAI-1 within the oviduct.
Materials and Methods
Materials
Acrylamide, N,N' diallyltartardiamide, urea, Nonidet P-40, and sodium dodecyl
sulfate were purchased from Gallard-Schlesinger (Carle Place, NY); X-Omat AR film
and photography reagents were a product of Eastman Kodak Co. (Rochester, NY); amino
acids and protein standards were purchased from Sigma-Aldrich (St. Louis, MO);
ampholines were from Pharmacia-Biotech (Piscataway, NJ); all other supplies and
reagents for gel electrophoresis were purchased from Bio-Rad Laboratories (Richmond,
CA) or Fisher Scientific (Orlando, FL). All medium and culture supplies were purchased
from Life Technologies (Grand Island, NY). L-[4,5-3H]leucine (specific activity, 120
Ci/mmol) was obtained from Amersham (Arlington Heights, IL). Immobilin-P (PVDF)
membranes were purchased from Millipore Corporation (Bedford, MA); Vectastain ABC
Elite kit was obtained from Vector Laboratories (Burlingame, CA). Affinity-purified
goat anti-human PAI-1 was purchased from American Diagnostica (Greenwich, CT);
goat IgG and normal rabbit serum (NRS) were purchased from Sigma-Aldrich. All other
reagents, including column chromatography supplies, were products from Sigma, Fisher,
Life Technologies or Pharmacia-Biotech.

61
Tissue Collection. Explant Culture, and Electrophoresis
Florida crossbred (Yorkshire x Duroc x Hampshire) or European Large White
gilts were observed daily for behavioral estrus in the presence of an intact boar. After the
completion of at least two estrous cycles, animals for pregnancy studies were bred on the
first day of standing estrus, designated as Day 0, and 24 h later (except for gilts assigned
to Day 0). Pregnant or cyclic gilts were taken to the abattoir on Days 0, 2, and 12 for
slaughter. Oviducts were collected aseptically and separated by gross dissection into the
three functional segments, which were then cultured as previously described [Buhi et al.,
1989], For culture, 0.5 g of tissue was incubated in leucine-deficient (modified) Eagle's
minimum essential medium (MEM) containing 100 pCi [3H]-leucine for 24 h at 39° C in
a defined atmosphere. Conditioned culture media was then separated from tissue and
frozen at -20° C until analyzed by 2D-SDS-PAGE and fluorography or utilized for
purification of the 45,000 M, protein.
In order to evaluate synthesis of the 45,000 Mr protein within the three segments,
oviductal tissue was collected from Day 2 pregnant Large White gilts and cultured as
described above. Culture media was dialyzed (12,000 molecular weight cut-off) against
10 mM Tris-HCl buffer (pH 7.6), containing 0.15 M NaCl and 0.02% (w/v) NaNi,
followed by dialysis against dH20 (2 changes, 4L each, 24 h each) at 4° C. Total protein
content of dialyzed culture media was measured by the Bio-Rad microassay (according to
manufacturer's instructions) and radiolabeled proteins measured by liquid scintillation
spectrophotometry. Samples representing each oviductal segment, containing 100,000
cpm, were lyophilized, solubilized in Laemmli’s buffer [Laemmli, 1970] and analyzed

62
by 2D-SDS-PAGE and subjected to fluorography as described previously [Buhi et al.,
1995], All X-ray films were exposed for 14 days at -80° C and developed.
Protein Fractionation
Explant culture media conditioned by isthmic tissue on Days 0, 2, and 12 of early
pregnancy, were pooled and subjected to gel filtration chromatography on a Sepharose
CL-6B column as described previously [Buhi et al., 1990] with some modifications. The
Sepharose CL-6B column (1.8 x 92 cm) was equilibrated in column buffer [10 mM Tris-
HC1, 0.4 M NaCl, 0.02% (w/v) NaN3; pH 7.5] at 4° C. Culture media (7 ml) centrifuged
at 2,200 x g for 10 minutes at 4° C to remove particulate material, was added slowly to
the column. After collection of the void volume, elution profiles were generated by
collecting 2 ml fractions, measuring protein (absorption at 280 nm) and determining
radioactivity in each fraction. Chromatographic peaks corresponding to the elution of
ovalbumin (45,000 M, standard) were pooled from individual column runs.
Immunoglobulins were removed from pooled fractions by incubation with Protein A-
Sepharose beads in 10 mM phosphate-buffered saline (PBS), pH 8.0, overnight at 4° C.
Beads were then separated from the supernatant by centrifugation at 1,700 X g for 10
minutes at 4° C, washed 3x in PBS, and pooled supernatants dialyzed against dH20 (2
changes, 4 L each, 24 h each, 4° C). The dialyzed sample was analyzed for protein
content and radioactivity, as indicated above, and lyophilized. Lyophilized samples were
then resuspended in column buffer (lml/15 mg protein) and further fractionated on a
Sephadex G-100 column (1.5 x 75 cm) at 4° C. The Sephadex G-100 column was
calibrated previously with Blue Dextran, apotransferrin, ovalbumin and cytochrome C as
molecular weight markers. Elution profiles were generated using methodology

63
established for the Sepharose CL-6B column. The pooled chromatographic peaks from
individual column runs corresponding to the 45,000 M, standard were utilized for
subsequent Western blotting and N-terminal amino acid sequence analysis. Purification
at each step was examined by 2D-SDS-PAGE and fluorography [Buhi et al., 1990],
Western Blotting and Sequence Analysis
The pooled chromatographic peaks containing the 45,000 Mr protein were
separated by Tris-tricine 2D-SDS-PAGE [Schagger and von Jagow, 1987], Proteins were
then transferred by semi-dry electrophoresis (Milli-Blot SDE system, Millipore Corp,
Bedford, CA) to a PVDF membrane as described previously [Buhi et al., 1995] with
some modifications. Following separation of proteins by Tris-tricine 2D-SDS-PAGE, the
gel was rinsed in three changes of dH20 for 5 min each, then equilibrated in 25 mM Tris-
HC1 buffer (pH 9.4) containing 2% (w/v) SDS, for 30 min. The gel was washed in three
changes of dH20 for 5 min, and incubated in two changes of cathode C buffer (25 mM
Tris-HCl, 5.25 g/L norleucine, 10% (v/v) methanol; pH 9.4) for 10 min at room
temperature. After assembly of the TransUnit sandwich, proteins were transferred for 1 h
under constant current (2.5 mA per cm2). Subsequently, the membrane was washed
(dH20, 5 min), and stained in 0.1% (w/v) Coomassie blue R-250 in 50% (v/v) methanol
for 1 min. The membrane was destained (50% methanol, 5 min) with constant rocking
and rinsed in three changes of dH20. The PVDF membrane was allowed to air dry,
proteins corresponding to the 45,000 M, protein were excised, and subjected to N-
terminal amino acid microsequencing at the Interdisciplinary Center for Biotechnology
Research (ICBR) facility using a 470A gas phase protein sequencer (Applied
Biosystems) with an on-line analytical HPLC system. The peptide sequence was

64
analyzed with the National Center for Biotechnology Information (NCBI) BLAST
program [Altschul et al., 1990],
After identification of the 45,000 M, protein as porcine PAI-1 (see Results), a
one-step partial-purification method was employed using heparin-agarose affinity column
chromatography. PAI-1 has been shown previously to quantitatively bind to heparin-
Sepharose and elute with increasing concentrations of NaCI [Ehrlich et al., 1991],
Isthmic-conditioned culture media (Day 12 pregnant and Day 1 cyclic) were pooled,
centrifuged as described above, diluted (1:3) in 20 mM Tris-HCl (pH 7.6, 4° C)
containing 0.02% (w/v) NaN3, and slowly loaded onto a heparin-agarose column (2.5 x
8.2 cm) at 4° C. PAI-1 was eluted utilizing stepwise increments ofNaCl (0.1-3.0 M), and
the protein pooled and dialyzed against dHíO (two changes, 24 h each, 4L each, 4° C).
Protein content was determined as above, and aliquots containing 0.5 mg of protein
lyophilized and used for immunoprecipitation. Fractions were analyzed for the presence
of PAI-1 by 2D-SDS-PAGE and fluorographic analysis.
Immunoprecipitation
PAI-1, semi-purified by heparin-agarose affinity column chromatography, was
immunoprecipitated with a polyclonal rabbit anti-hPAI-1 antiserum (kindly provided by
Dr. Schleef, Scripps Institute, La Jolla, CA). Lyophilized protein samples (0.5 mg) were
solubilized in 900 pi of NET buffer [50 mM Tris-HCl, 0.15 M NaCI, 0.1% (v/v) Nonidet
P-40, 1 mM EDTA 0.25% (w/v) gelatin, and 0.02% (w/v) NaN3; pH 7.5 at 25° C],
Protein A-Sepharose beads were equilibrated in NET buffer and 100 pi of swollen beads
were incubated with 100 pi of either undiluted PAI-1 antiserum or NRS and 300 ul of
NET buffer for 1 h at 25° C. After incubation, Protein A-Sepharose beads were pulse-

65
centrifuged (Beckman, microcentrifuge) for 30 sec and washed in three changes of NET
buffer (0,5 ml). Semi-purified PAI-1 protein (100-200 pi) was then incubated with NRS
or rabbit anti-hPAI-1 antibody-coated beads (200 pi), respectively, for 2 h at 25° C with
constant rotation. Complexes were pelleted by centrifugation and washed as above.
Proteins conjugated to the Protein A-Sepharose beads were solubilized in Laemmeli
buffer [Laemmeli, 1970], boiled for 3 min, separated on a 10% (w/v) 1D-SDS-PAGE gel
and subjected to fluorography [Buhi et al., 1995], Heparin-agrarose fractionated proteins,
which were not used for immunoprecipitation, were removed from solution using
standard acetone precipitation procedures [Harlow and Lane, 1988] and solubilized in
Laemmeli buffer as above for positive radiolabeled PAI-1 identification.
Immunocvtochemistrv
A polyclonal affinity-purified goat anti-hPAI-1 was used to immunolocalize PAI-
1 in porcine oviductal tissues from cyclic and early pregnant animals. To compare
distribution of PAI-1 in cyclic and early pregnant animals, infundibulum, ampulla and
isthmic tissues were collected on Days 0, 2, and 12, (n=3 animals/day) and
immunocytochemistry performed as described [Chegini et al., 1992], Tissues were cut
into 5 mm portions, fixed in Bouin's solution, embedded in paraffin, sectioned (0.5 pm),
and mounted on precoated glass slides. A goat Vectastain ABC Elite kit (Vector
Laboratories, Burlingame, CA) was used according to the manufacturer’s instructions.
Controls included use of an affinity-purified goat IgG and the absence of primary
antibody. Goat anti-hPAI-1 and goat IgG were used at a dilution of 1:10 in PBS (pH
7.4).

66
Immunoeold Electron Microscopy (EMJ
Oviductal tissue from the three segments of Day 0 non-pregnant and Day 9
pregnant crossbred gilts, were fixed for 1 h in PBS, pH 7.4, containing 0.5% (v/v)
glutaraldehyde, 4% (v/v) paraformaldehyde at 4° C. The two days selected were times of
elevated estrogen (Day 0) or progesterone (Day 9) production. After fixation and rinsing
in PBS, tissues were dehydrated in graded ethanol series and embedded in Unicryl
(British BioCell International, UK ) under UV light at -10° C for 2 days. Thin sections
(0.5 mm) were cut and collected on Formvar-coated 100 mesh nickel grids, and PAI-1
antigen detected by immunogold labeling. The polyclonal rabbit anti-human PAI-1 and
preimmune rabbit sera, diluted 1:1000 in a high salt Tween buffer (0.02 M Tris-HCl, 0.5
M NaCl, 1% [v/v] Tween 20, pH 7.2) supplemented with 1% (w/v) ovalbumin, were
incubated overnight with grids in a humid chamber at 4° C. Sections were then
incubated with a secondary antibody (goat anti-rabbit IgG, 1:30 dilution in PBS)
conjugated to 18 nm colloidal gold (Jackson ImmunoResearch Laboratories, Inc, West
Grove, PA) for 1 h at room temperature. Sections were then post-stained with 2% (w/v)
uranyl acetate and Reynolds lead citrate. Grids were examined on a Hitachi H-7000
transmission electron microscope (Hitachi Scientific Instruments, Danbury, CT). Digital
micrographs were taken on a Gatan BioScan/Digital Micrograph 2.5 (Gatan Inc,
Pleasanton, CA).
Results
Electrophoretic Analysis
Representative 2D-SDS-PAGE and fluorographic analyses of explant culture
media from the infundibulum, ampulla, and isthmus containing radiolabeled de novo

67
synthesized proteins are shown in Figure 3-1. The 45,000 Mr protein, found in all three
segments, is seen as the major radiolabeled protein from the isthmic portion of the
oviduct (Figure 3-1C). The level of protein expression appears to be reduced in the
ampulla (Figure 3-1B) and nearly absent in the infundibulum (Figure 3-1A). Thus, the
45,000 Mr protein appears to have a specific spatial synthesis and release in the oviduct.
This specific spatial expression is similar to that reported for the matrix metalloproteinase
inhibitor, TIMP-1, in the oviduct [Buhi et al., 1996] (Figure 3-1).
Purification. Blotting, and N-terminal Sequencing
With identification of the 45,000 M, protein as the major radiolabeled protein of
the isthmus, the next objective was to purify this protein for N-terminal sequencing. A
two-step gel-filtration chromatographic procedure was developed in order to separate and
enrich this protein. Elution profiles for protein and radioactivity from Sepharose CL-6B
and Sephadex G-100 columns are shown in Figure 3-2A and 3-2B, respectively.
Fractionation of explant culture media on these two columns, each showed two peaks of
radioactivity. The broad second peak in both graphs, contained the greatest amount of
radioactivity and included the 45,000 Mr protein. A representative fluorograph of the
45,000 Mr protein after fractionation on a Sephadex G-100 column is shown in Figure 3-
2C. This protein appears to contain at least 5 isoelectric species, of which 3 species,
including an acidic and basic species, were submitted for N-terminal amino acid
sequence analysis. A search of protein, RNA and DNA data banks indicated that the
derived N-terminal amino acid sequence of 26 amino acids, identical for all three
isoelectric species, was 96% identical (100% similar) to porcine PAI-1 (Figure 3-3). This
sequence (1-26) corresponded to amino acid positions 20-46 of mature PAI-1 protein,

68
indicating removal of the hydrophobic leader peptide prior to release from the cell
[Bijnens et al., 1997],
Immunoprecinitation
To confirm that the 45,000 Mr protein identified by N-terminal amino acid
microsequencing was PAI-1, an anti-hPAI-1 serum was used for immunoprecipitation of
this protein from isthmic culture media after fractionation by heparin-agarose affinity
column chromatography. Fractionated culture media containing de novo synthesized
radiolabeled proteins were treated with either Protein A-complexed rabbit anti-hPAI-1
serum or Protein A-complexed NRS. Bound proteins were solubilized and examined by
1D-SDS-PAGE and fluorography (Figure 3-4A and 3-4B). As shown in Figure 3-4B,
anti-hPAI-1 serum specifically recognized and precipitated radiolabeled PAI-1, while
NRS did not. These results indicate that this antibody does not cross-react with other
radiolabeled proteins present within the fractionated culture media. Western Immunoblot
analysis showed that the anti-hPAI-1 antisera cross-reacted with only 2-3 minor
unlabeled proteins, possibly of transudate origin. The primary protein recognized by this
antibody on the Western Immunoblot was the PAI-1 family (5-6 isoelectric species) (data
not shown).
Immunocvtochemistrv
With identification of PAI-1, the next objective was to examine its distribution
throughout the oviduct on Days 0, 2, and 12 of the estrous cycle or early pregnancy.
Immunoreactive PAI-1 was detected in all three segments of the oviduct regardless of
day of cycle examined and no differences in staining intensity could be detected between
days (infundibulum not shown). Representative data of the immunocytochemical

69
localization in the isthmus on these days is shown in Figure 3-5. No difference in
staining intensity could be detected (subjective visualization of three representative
animals) between pregnant and cyclic tissues within the three segments (Figure 3-6).
PAI-1 was localized primarily within the oviductal epithelium, while only background
staining could be identified within muscle and stroma tissue (Data not shown for muscle
and stroma). Here, PAI-1 appeared to be heavily concentrated at the apical region of the
epithelium (Figure 3-5C, arrow) with little staining found in the basal region. However,
PAI-1 immunoreactivity was also associated within cells lining blood vessels in the
stroma. Immunocytochemistry pictures shown are representative of 3 animals/day.
Because staining intensitys varied between each animal examined, subjective
comparisons on the level of staining intensity between pregnant and cyclic gilts were not
made.
Electron Microscopy
Since immunocytochemistry was not able to resolve PAI-1 association with
specific cellular structures or organelles within the epithelium, an examination of PAI-1
distribution within the epithelium was performed using electron microscopic
immunogold localization. PAI-1 was found primarily in epithelium of isthmic non-
ciliated secretory cells and appeared to be concentrated in putative secretory granules at
or near the apical border of cells in both Day 0 non-pregnant and Day 9 pregnant animals
(Figures 3-7A and 3-8A). In addition, this protein was also found to be associated with
the isthmic luminal epithelial border and cilia (Figures 3-7B and 3-8B). Localization of
PAI-1 within ampullary epithelium (both ciliated and non-ciliated) was negligible (Figure
3-8C). Isthmic tissue incubated with preimmune sera (control) showed no labeling

70
within secretory granules or at the epithelial border (Figure 3-8D). As detailed in the
immunoprecipitation results, the antibody employed showed no potential cross-reactions
with other proteins found within oviduct-coniditioned culture medium.

Figure 3-1. Representative fluorograph after 2D-SDS-PAGE separation of [3H]-labeled
proteins (100,000 cpm) from explant culture media conditioned by the three segments of
the oviduct.
Tissue was collected from the A) infundibulum, B) ampulla or C) isthmus of Large
White X Landrace gilts on Day 2 of pregnancy. The 45,000 M, protein is indicated by an
arrow in each panel. TIMP-1 is marked by an arrowhead only in panel C. Molecular
weight markers (x 103) are indicated and the pH gradient runs from left (pH 8) to right
(pH 4).

72

Figure 3-2. Representative Sepharose CL-6B and Sephadex G-100 fractionations of
[3H]-labeled proteins in isthmus-conditioned medium.
Fractions (2 ml) collected from the CL-6B column elution (Panel A) were counted for
radioactivity and measured for protein (see Methods). In Panel A, tubes 58-75 (peak 2,
bracket) containing the 45,000 M, protein, were pooled, treated with Protein A beads to
remove immunoglobulins and further factionated on a G-100 column (Panel B). In panel
B, tubes 48-60 (peak 2, bracket), containing the 45,000 Mr protein were pooled, and
separated by Tris-tricine 2D-SDS-PAGE for electroblotting. Arrows in panel A and B
reflect elution point of 45,000 M, ovalbumin molecular weight standard. Panel C, shows
a representative fluorograph of the 45,000 M, protein after chromatographic separation by
Tris-tricine 2D-SDS-PAGE. Three isoelectric species (marked with arrows) were
excised and subjected to N-terminal amino acid sequence analysis. Molecular weight
markers (xlO3) are indicated and the pH gradient runs from left (pH 8) to right (pH 4).

Radíoacáivtty (cpm/100 ul) — Radioactivity (cpmrtQOui)
zv*

75
20. .
25 . .
30 . ...
35 . .
40 ... .
45 .
Porcine
EGSAS
SHHQS
LAARL
ATDFG
VKVFR
QV
PAI-1
EGSAS
SHHQS
LAARL
ATDFG
VKXFR
QV
45,000 Mf
isthmic protein
i . . . .
5 . . . .
10 . ...
15 . . . .
20 . . . .
25 .
Figure 3-3. Comparison of the identified N-terminal amino acid sequence of the 45,000
Mr protein from isthmic-conditioned culture media to the N-terminal sequence of porcine
PAI-1.
Proteins were fractionated by size-exclusion chromatography using a Sepharose CL-6B
and Sephadex G-100 column, separated by Tris-tricine 2D-SDS-PAGE, transferred by
electroblotting to PVDF membranes and subjected to N-terminal amino acid micro¬
sequence analysis. X (residue 22) indicates an undetermined amino acid residue. A 96%
sequence identity was found to porcine plasminogen activator inhibitor-1 (Accession #
1870170).

1
«M
2
3
76
A
1 2 3
Figure 3-4. Immunoprecipitation of plasminogen activator inhibitor-1 from porcine
isthmic-conditioned culture media after fractionation on a heparin-agarose affinity
column and elution with 0.4 M NaCl.
A representative one-dimensional SDS-polyacrylamide gel (Panel A) containing
Coomasie blue-stained proteins from either a non-precipitated sample (lane 1), proteins
precipitated using a rabbit anti-human PAI-1 (lane 2) or normal rabbit sera (lane 3).
Although not visible on the gel, the arrow indicates the apparent location of PAI-1
protein. Panel B shows a fluorograph generated from the same gel in Panel A. Lanes are
as indicated above. PAI-1 found in semi-purified culture media, was specifically
precipitated by antibody (lane 2), but not normal rabbit serum (lane 3).

Figure 3-5. Representative immunocytochemical localization of PAI-1 in the isthmus
from crossbred gilts on Day 0 (Panel A), Day 2 (Panel B) or Day 12 (Panel C) of
pregnancy.
The control is shown in Panel D. Arrow indicates intense staining of PAI-1 within the
apical region of isthmic epithelium. X 20.

78
Figure 3-6. Representative immunocytochemical localization of PAI-1 in oviductal
tissue from Day 2 cyclic (Panel A and C) and Day 2 pregnant (Panel B and D) crossbred
gilts from the isthmus (Panel C and D) and ampulla (Panel A and B).
The infundibulum is not shown. Arrow indicates intense staining of PAI-1 within the
apical region of the epithelium. X 20.

Figure 3-7. Immunogold labeling of PAI-1 within oviductal tissue from a Day 0 non-
pregnant crossbred gilt.
Gold particles are seen in secretory granules of isthmic non-ciliated cells (Panel A,
arrow) as well as associated with the epithelial luminal border and cilia (Panel B, arrow).
X 20,000.

80

Figure 3-8. Immunogold labeling of PAI-1 within oviductal tissue from a Day 9 pregnant
crossbred gilt.
Gold particles can be seen clustered within putative secretory granules of isthmic non-
ciliated cells (Panel A, arrow) and associated with the epithelial luminal border and cilia
(Panel B, arrow). Gold particles were not detected in secretory granules of the ampulla
although some scattered particles were associated with cilia (Panel C). Immunogold
staining was not detected in the isthmus treated with preimmune serum (Panel D, arrow).
X 20,000.

82

83
Discussion
The mammalian oviduct secretes numerous de novo synthesized proteins into the
oviductal lumen, some of which are present before ovulation, or during fertilization and
early cleavage-stage embryonic development. In order to characterize the 14 major de
novo proteins secreted by the porcine oviduct, explant tissue (infundibulum, ampulla, and
isthmus), was incubated with [’llj-leucine, and synthesized and secreted proteins
separated by 2D-SDS-PAGE, detected by fluorography, and characterized by molecular
weight and isoelectric points [Buhi et al., 1990], Two of these proteins have been
extensively examined, while others remain unidentified [Buhi et al., 1997], including a
45,000 M, protein synthesized primarily by the isthmic portion of the oviduct. Sequence
analyses revealed that this isthmic protein was porcine plasminogen activator inhibitor-1.
Recently, an unidentified 45,000 Mr protein found in bovine oviductal fluid was shown to
associate with the zona pellucida of bovine oocytes [Staros and Killian, 1998] and may
be the protein under investigation.
PAI-1, a member of the serpin family of serine protease inhibitors, is the primary
inhibitor of urokinase plasminogen activator (uPA) and tissue-type plasminogen activator
(tPA). It is a glycoprotein consisting of 379 amino acids with an apparent molecular
mass of 45,000 [Andreasen et al., 1990], Among the serpins, PAI-1 is secreted in an
active form that rapidly converts to an inactive latent form that can be reactivated by
phospholipids and a variety of denaturants [Hekman and Loskutoff, 1985], The
inhibitory activity of PAI-1, however, is stabilized by vitronectin in either serum or
extracellular matrix (ECM), thus preventing transformation to the inactive latent form
[Declerck et al., 1988], Both tPA and uPA initiate proteolysis by converting plasminogen

84
to the broad-specificity enzyme plasmin. This extracellular protease is reported to be
involved in the remodeling of ECM, fibrinolysis, cell migration, and tumor metastasis
[Andreasen et al., 1990], Plasmin can also activate pro-matrix metalloproteinases
(MMPs) thereby regulating the pericellular activation cascade leading to ECM
degradation [Murphy et al., 1992], Complex control of this activation cascade is
regulated by PAI-1 and tissue inhibitors of matrix metalloproteinases (TIMPs).
Immunoprecipitation confirmed the presence of PAI-1 which in the oviduct,
appeared to consist of at least five or more isoelectric species as shown by fluorographic
analysis, N-terminal amino acid sequencing and immunoblot analysis (data not shown for
immunoblot). The presence of these isoforms suggests that newly synthesized and
secreted PAI-1 was post-translationaly modified. One modification was characterized as
glycosylation with incorporation of [3H]-glucosamine into PAI-1 during tissue explant
culture [Buhi et al., 1990], Localization and synthesis of PAI-1 appears to mimic a
matrix metalloproteinase inhibitor from the oviduct, TIMP-1 [Buhi et al., 1996], Both
TIMP-1 and PAI-1 show a similar spatial expression by the oviduct segments, with a
greater expression in the isthmus relative to either the ampulla or infundibulum. This
would suggest an important function relative to its spatial expression. The isthmus
portion of the oviduct has been described as a spermatozoa reservoir where sperm
undergo capacitation and hyperactivation [Suarez, 1998], In the pig, the ampulla-isthmic
junction is the location of fertilization and early cleavage-stage embryonic development
[Buhi et al., 1997], This protein may therefore facilitate or regulate these important
reproductive events that occur within or near the isthmus. However, PAI-1 may act in
other segments of the oviduct as well, due to retrograde movement of oviductal fluid into

85
the peritoneal cavity during estrus [Hunter, 1988]. Localization of PAI-1 in the
infundibulum and ampulla epithelium and protein secretion by explant tissues, suggests
that these segments may also contribute to luminal PAI-1, although synthesis of PAI-1 in
these segments appears to be very low.
Fibrin deposits have been located on the tubal mucosa of the oviduct, which could
possibly interact with the ECM components of the zona pellucida and prevent tubal
transport [Liedholm and Astedt, 1975], Liedholm and Astedt (1975), observed
fibrinolytic activity associated with the unfertilized ovum in rats and suggested that this
activity may be involved with the prevention of adhesion to fibrin deposits that may
hinder gamete transport. This activity was also shown to be associated with spermatozoa.
Prevention of cellular adhesion to the oviductal mucosa and fibrin deposits might also be
regulated by uPA and tPA. The developing embryo may produce uPA/tPA in response to
signals from the surrounding oviductal environment. Fibrin has been shown to increase
expression of uPA mRNA and protein in 3.5 day old uterine embryos of the mouse
[Zhang et al., 1996], Both mouse and rat preimplantation embryos have been shown to
have tPA activity [Zhang et al., 1992, Carroll et al., 1993] and uPA activity [Zhang et al.,
1994, Harvey et al., 1995], Expression of this proteolytic activity, or the respective
mRNA, was found to be developmental- and stage-specific. While the fibrinolytic
activity of the ovum and proteinase expression by the embryo may facilitate their
transport through the oviduct, these molecules are potent modulators of their immediate
environment with respect to ECM remodeling. PAI-1, an important regulator of both
fibrinolysis and plasminogen activators, may act as a stabilizing or counter-regulatory
factor for maintaining ECM integrity of the oviductal epithelium. Therefore, production

86
of PAI-1 by the oviduct might act to prevent premature nidation of the preimplantation
embryo. While the pig has noninvasive placentation, the trophoblast of the pig has
invasive potential as shown by its transfer to ectopic sites [Samuel, 1971], Part of this
invasive potential may be due to fibrinolytic and plasminogen activator activity of the
embryo. Liedholm and Astedt (1975) suggested that the fibrinolytic activity of the ovum
may be depressed by an inhibitor of plasminogen activation. Plasmin-induced proteolysis
has been shown to be important for implantation in invasive species such as the mouse
[Sappino et al., 1989, Zhang et al., 1996], However, in the pig, an endometrial inhibitor
of plasmin production has been suggested to protect the endometrium from the
blastocyst-induced proteolysis during placentation [Fazleabas et al., 1982], PAI-1
secretion within the oviduct, may therefore have a similar function to that of the
endometrial plasmin inhibitor.
Immunolocalization results indicated that PAI-1 is localized within the oviductal
epithelium and is heavily concentrated near the apical membrane, suggesting secretion
into the lumen. Immunogold EM of the isthmus, revealed that PAI-1 was located in
putative secretory granules within the lumen and associated with cilia. While PAI-1 was
localized to the ampulla using immunocytochemistry, immunogold electron microscopy
was unable to detect its presence. This may reflect a difference in specificity and
dilutions of two different antibodies used, difference in tissue sections examined between
the two different procedures and 2D-SDS-PAGE and fluorographic analyses, which
suggest that PAI-1 protein secretion is low in the ampulla. Both immunocytochemistry
and fluorographic analysis have shown that PAI-1 is localized in the ampulla and
infundibulum, however, the inability to locate immunoreactivity utilizing electron

87
microscopy may be that protein synthesis of this molecule is very low in these segments
and is present in secretory granules in very small amounts. PAI-1 was found in secretory
granules from isthmic tissue exposed to either a high estrogen (Day 0 cyclic) or high
progesterone (Day 9 pregnant) environment indicating that this protein is synthesized
throughout the estrous cycle or early pregnancy. Evidence from our laboratory
(unpublished) indicates that PAI-1 secretion may vary during the estrous cycle or early
pregnancy and its synthesis might be controlled by ovarian steroids. Reports on PAI-1
regulation in endometrial stromal and decidual cell cultures suggest that this protein is
up-regulated by progesterone, while estrogen antagonizes this effect [Schatz and
Lockwood, 1993], The oviduct along with the uterus, is a major target for ovarian
steroids and steroid-modulated and cycle-specific changes have been noted for two other
de novo synthesized products of the epithelium, pOSP and TIMP-1 [Buhi et al., 1997],
Because the zona pellucida can be subject to proteolytic degradation, oviductal
PAI-1 may also protect the integrity of the zona pellucida from embryonic or oviductal
plasminogen activator activity. To our knowledge, PAI-1 mRNA or activity has not been
examined in the early cleavage-stage embryo or in the oviduct. The porcine zona
pellucida of oviductal oocytes or embryos was found to be more resistant to proteolytic
degradation than that of either follicular oocytes or embryos collected from the uterine
environment [Broermann et al., 1989], suggesting a potential interaction of oviductal
protease inhibitors with the oocyte/embryo. Changes in resistance of the zona pellucida
to proteases may be dependent upon addition of glycoproteins/inhibitors obtained during
transit through the oviduct. Thus, an oviduct-specific factor may act to protect the zona
pellucida and embryo from degradation by proteolytic enzymes. Proteases are present in

88
oviductal flush and include the plasminogen activators and matrix metalloproteinases
(Kouba AJ, unpublished). PAI-1 working together with TIMP-1, may act to tightly
regulate this proteolytic activity. Plasminogen, the natural substrate for uPA and tPA, is
found in many extracellular fluids including seminal plasma [Kobayashi et al., 1992] and
follicular fluid [Beers, 1975], and may be enriched in oviductal fluid at or near the time
of fertilization. Estrogens have been shown to stimulate the uptake of plasminogen from
plasma by the mouse uterus [Finlay et al., 1983] and a similar function may occur in the
oviduct during estrus. Plasminogen has been shown to bind to mouse spermatozoa,
oocytes, and cumulus cells, which enhanced the local generation of plasmin [Huarte et
al., 1993] and this binding allows for PA-induced proteolysis to discrete focal areas.
Huarte et al. (1993) also showed that addition of plasminogen or antibodies to plasmin
during in vitro fertilization could increase and decrease, respectively, the fertilization
rate. Therefore, PAI-1 within the oviduct may inhibit oocyte or embryonic generation of
plasmin from plasminogen, due to their inherent tPA and uPA activity, thus maintaining
integrity of the zona pellucida while not affecting fertilization.
Further objectives will be to evaluate the biological role of PAI-1 within the
oviduct. PAI-1 may act in a autocrine/paracrine fashion to prevent proteolytic
degradation of ovulated oocytes or early embryos in the oviduct or uterus, prevent
premature hatching, regulate ECM remodeling of the oviduct or early embryo, inhibit
embryonic invasion of the oviductal lining, and promote embryonic development.

CHAPTER 4
OVIDUCT AL PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) PROTEIN,
mRNA, AND HORMONAL REGULATION
Introduction
The mammalian oviduct is host to both male and female gametes as well as the
early cleavage-stage embryo. Oviductal fluid provides an important medium that
facilitates gamete union/fertilization and nurtures the resultant zygote and developing
embryo. Oviduct fluid is also a potential source of signal molecules such as growth
factors, protease inhibitors, and oviduct-specific secretory glycoproteins [Buhi et al.,
1997], Several proteins have been shown to be synthesized de novo and secreted into
culture medium from oviductal explant cultures [Buhi et al., 1990], Two of these
proteins have been identified as TIMP-1 [Buhi et al., 1996] and PAI-1 [refer to Chapter
3], These protease inhibitors have been described as tightly regulating an activation
cascade of proteases which leads to degradation and/or remodeling of the extracellular
matrix (ECM). Since gametes or early cleavage-stage embryos are in intimate contact
with the oviductal epithelium, a means is provided for cell-cell interaction within their
immediate environment [Hunter, 1988], Buhi et al. (1996) and Kouba et al. (Chapter 3)
have proposed that the protease inhibitors, PAI-1 and TIMP-1, provide protection against
enzymatic degradation and prevent adhesion and invasion of the early cleavage-stage
embryos within the porcine oviduct.
89

90
Experiments in mice have shown that the blastocyst can easily implant in a wide
variety of sites within the body upon transplantation [Kirby, 1963], yet the oviduct in
non-primates is extraordinarily resistant to tubal implantation [Bronson and McLaren,
1970], The early cleavage-stage embryo has been shown to produce both urokinase
plasminogen activator (uPA) [Zhang et al., 1994, Harvey et al., 1995] and tissue-type
plasminogen activator (tPA) [Carroll et al., 1993, Zhang et al., 1992], both of which can
lead to the generation of plasmin, a broad specificity enzyme that can initiate remodeling
and degradation of the ECM. Although the embryo is in an environment containing
proteases such as uPA and tPA, as well as their substrate plasminogen [Huarte et al.,
1993], ovulated oviductal oocytes are extremely resistant to proteolytic degradation
[Broermann et al., 1989], and oviducts are strongly resistant to adhesion and implantation
[Jansen, 1984], PAI-1 and TIMP-1 have been suggested to play a role in these
phenomenon [Buhi et al., 1996, Kouba et al., Chapter 3], The inhibitors PAI-1 and
TIMP-1, are major de novo synthesized and secreted proteins of the isthmic portion of
the oviduct, the location of early cleavage-stage embryonic development [Buhi et al.,
1996, Kouba et al., Chapter 3], Further, PAI-1 protein was found localized within
secretory granules of the isthmic epithelium and associated with isthmic cilia [refer to
Chapter 3],
Another oviductal protein which has been characterized is the porcine oviduct-
specific glycoprotein (pOSP), an estrogen-regulated protein synthesized primarily by the
ampulla and infundibulum [Buhi et al., 1996], Immunogold electron microscopy labeling
showed that pOSP associates with the zona pellucida, perivitelline space and vitelline or
blastomere membranes of ovulated oocytes and early embryos, respectively [Buhi et al.,

91
1993], Protein, mRNA, and hormonal regulation of TIMP-1 and pOSP in the oviduct has
been evaluated throughout the estrous cycle and early pregnancy [Buhi et al., 1996],
Elucidation of spatial and temporal secretion of PAI-1 is significant to our understanding
of how the cleavage-stage embryo may be protected in a protease rich environment and
provide resistance to implantation.
The specific objectives of this study were: 1) to examine de novo synthesis and
secretion of radiolabeled PAI-1 during early pregnancy in two breeds of pigs, the
standard European Large White and the highly prolific Chinese Meishan, 2) to determine
PAI-1 levels in oviduct flushes from early pregnant crossbred gilts, 3) to evaluate levels
of PAI-1 mRNA in crossbred gilts during the estrous cycle, early pregnancy, and after
ovariectomy (OVX) and steroid-hormone replacement therapy and 4) to determine
specific spatial differences in expression of PAI-1 mRNA and protein between different
segments of the oviduct.
Materials and Methods
Materials
Acrylamide, N,N' diallyltartardiamide, urea, Nonidet P-40, and Sodium dodecyl
sulfate were from Gallard-Schlesinger (Carle Place, NY); X-Omat AR film and
photography reagents were a product of Eastman Kodak Co. (Rochester, NY); leucine
and protein standards were acquired from Sigma-Aldrich (St. Louis, MO); ampholines
were from Pharmacia-Biotech (Piscataway, NI); all other supplies and reagents for gel
electrophoresis were procured from either Bio-Rad Laboratories (Richmond, CA) or
Fisher Scientific (Orlando, FL); and all medium and culture supplies were purchased
from Life Technologies (Grand Island, NY). L-[4,5-3H]leucine (specific activity, 120

92
Ci/mmol) was obtained from Amersham (Arlington Heights, IL). RNA hybridization
was performed using the enhanced chemiluminescent kit (ECLâ„¢) purchased from
Amersham Pharmacia-Biotech (Piscataway, NJ)
Tissue Collection
Estrous cycle Sexually mature crossbred gilts (Yorkshire X Duroc X
Hampshire) were observed daily for behavioral estrus for at least two estrous cycles in
the presence of an intact boar. The first day of standing estrus was designated Day 0 and
animals were taken to the abattoir for slaughter on Days 0, 1, 2, 4, 8, 10, 12, 15, and 18 of
the estrous cycle (n=3/day). After exsanguination, reproductive tracts were collected
aseptically and the oviduct separated by gross dissection into the infundibulum, ampulla,
and isthmus. Tissues from one oviduct per animal were frozen immediately in liquid
nitrogen and stored at -80° C for subsequent RNA extraction. The second oviduct was
utilized for explant tissue culture as described below.
Ovariectomy Crossbred gilts to be treated with various steroids (n=12) were
bilaterally ovariectomized (OVX) on the fourth day following a natural estrus and
randomly assigned to one of four treatment groups. Gilts were injected (i.m.) daily for 11
consecutive days (Buhi et al., 1992) and treated with the following steroid regimens.
Treatment 1 received vehicle (2 ml), corn oil and ethanol (9:1, v/v; n=3); treatment 2
received 100 pg estradiol valerate (EV; n=3); treatment 3 received 200 mg progesterone
(P4 ) (n=3); treatment 4 received 200 mg P4 + 100 pg EV. OVX gilts were anesthetized
24 h after the last treatment, and subjected to surgery as described below. The oviduct
was flushed as described below, oviductal segments cultured, and/or tissue snap-frozen
for RNA analysis.

93
Pregnancy In order to evaluate PAI-1 message during early pregnancy, crossbred
gilts were mated with boars at the onset of estrus and again 24 h later to ensure fertile
mating (except animals assigned to Day 0 of pregnancy). Tissues were obtained by
surgery (ovariohysterectomy) as detailed below or gilts were taken to the abattoir for
slaughter on Days 0, 2, and 12 of early pregnancy (n=3/day). Tissue was collected
aseptically, separated into the functional segments and frozen in liquid nitrogen as
detailed above. Samples were stored at -80° C for subsequent RNA extraction.
Differences in protein secretion of PAI-1 were compared in two breeds of pigs, European
Large White and Chinese Meishan. Gilts were observed for behavioral estrus for four
estrous cycles, and on Day 0 of the fifth cycle, gilts were bred with a boar of the
respective breed as described above. Animals were taken to slaughter and tissues were
collected aseptically on Days 0, 2, and 5 of early pregnancy (n=3/day) and cultured as
tissue explants as detailed below.
Oviductal flush Oviducts from crossbred pregnant gilts (Days 0, 1, 2, 5, 8, 10,
and 12) were flushed after surgery (as detailed below) or sacrifice at the local abbatoir.
Reproductive tracts were immediately flushed from the fimbria-infundibulum through the
isthmus with 5 ml of (modified) Eagles minimum essential medium (MEM) using a 10
ml disposable syringe and 20-guage needle. Flushed material was collected into a sterile
15 ml conical tube and stored at -20° C. Animal-use protocols were approved by the
University of Florida Institutional Animal Care and Use Committee.
Surgery (ovariectomy and ovariohysterectomy! Anesthesia was induced with a
combination of Telzol (Fort Dodge, IA; 2.2 mg/kg) and Xylazine (Fort Dodge; 2.2
mg/kg) administered intramuscularly. Following induction of anesthesia, the anesthetic

94
plane was maintained via inhalation of halothane (Halocarbon Laboratories, River edge,
NJ)-oxygen mixture. Gilts were placed in dorsal recumbancy and the abdomen was
prepared for midventral laparotomy. The ovaries and/or uterus were excised by ligation
of the ovarian pedicles, followed by the broad ligament and the uterine body, cranial to
the cervix. The incision was closed and gilts allowed to recover for 24 h.
Explant Culture. Electrophoresis. Fluorography, and Densitometry
Oviductal functional segments (infundibulum, ampulla, isthmus) from early
pregnant (Days 0, 2, and 5) Large White or Meishan gilts and steroid-treated OVX
crossbred gilts were cultured as previously described (Buhi et al., 1990). Oviducts from
pregnant pigs were opened longitudinally and washed in several volumes of (modified)
Eagle's minimum essential medium (MEM). Tissue segments were cut into 1- to 3-mm3
sections and explants (500 mg) were cultured in 15 ml of leucine-deficient MEM
containing 100 pCi of [3H]-leucine on a rocking platform at 39° C under an atmosphere
of 50% N2: 47.5% O2: 2.5% CO2 (v:v:v). After 24 h of culture, media was aspirated, and
frozen at -20° C until analyzed by 2D-SDS-PAGE and fluorography. Prior to
electrophoresis, culture medium was dialyzed against 10 mM Tris-HCl buffer (pH 7.6),
containing 0.15 M NaCl and 0.02% (w/v) NaN3, followed by dialysis against dH20 (2
changes, 4 L each, 24 h each 4° C). Total protein content of dialyzed culture media was
measured by the Bio-Rad microassay (according to manufacture's instructions) and
radioactivity measured by liquid scintillation spectrophotometry. Dialyzed culture media
(100,000 cpm) from early pregnant and OVX tissues, were lyophilized, solubilized and
proteins separated by 2D-SDS-PAGE and subjected to fluorography as previously
described [Buhi et al., 1995], Ampulla and isthmic fluorographs were exposed for 7 days

95
at -80° C and developed, while infundibulum fluorographs required a longer exposure (14
days). Densitometry of fluorographs were measured on a Alphalmager 2,000 and
Multilmage Light Cabinet (Alpha Innotech Co., San Leandro, CA) according to
manufacturer's specifications. Data were adjusted for wet weight of tissue and volume of
culture media and are expressed as densitometric units/culture/gram of tissue.
Oviductal PAI-1 During Early Pregnancy
PAI-1 was measured in oviduct flushes collected during early pregnancy (Days 0,
1, 2, 5, 8, 10 and 12) using the Immulyse PAI-1 ELISA kit (Biopool International,
Ventura, CA). The antibody combination measures active and latent PAI-1, as well as
that complexed as tPA/PAI-1 or uPA/PAI-1. Briefly, oviduct flushes were pooled by
day, dialyzed, measured for protein content, and lyophilized as described. Lyophilized
samples were resuspended in ELISA buffer and 200 pg of sample from each day were
added to individual wells of a 96-well plate. The ELISA was run according to
manufacturer's instructions. After termination of the reaction, the absorbance (450 nm)
was recorded and PAI-1 expressed as pg of PAI-1 per pg of oviductal protein. All
samples were run in duplicate and PAI-1 concentrations were determined from the
standard curve supplied with the ELISA kit (range 0-50 ng/ml; detection limit 0.9 ng/ml).
RNA Isolation and Analysis
Total cellular RNA was isolated from snap-frozen (-80° C) oviductal tissue
(cyclic, pregnant, and OVX) with TRIzol (Life Technologies, Grand Island, NY)
according to the manufacturer's instructions. After isolation, total RNA was resuspended
in water, and purity and concentration determined by spectrophotometric analysis. For
dot blot analyses, 5 pg of total RNA was blotted onto nylon membranes using a vacuum

96
manifold apparatus (Schleicher and Schuell, Keene, NH) and UV cross-linked (1 minute
at 1200 mJ/cm2) to the membrane. A porcine PAI-1 cDNA insert in the pCR 2.1 cloning
vector (Invitrogen, Carlsbad, CA) was obtained courtesy of Dr. Paul Declerck, Leuven,
Belgium (Bijnens, et al., 1997). Plasmid DNA was digested with Ncol restriction
enzyme to generate a cDNA probe of 1143 base pairs in length. The insert was gel-
purified using the QIA Quick Gel Extraction Kit (Santa Clarita, CA) according to
manufacturer's specifications. Sequence of the PAI-1 probe was confirmed using the
ABI 373 Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer,
Folster City, CA). The gel-purified porcine PAI-1 cDNA insert was labelled and
hybridized using the ECL1 M direct nucleic acid labelling and detection system according
to manufacturer's specifications. Blots were hybridized overnight in a hybridization
incubator (Robbins Scientific, Model 1000, Sunny Vale, CA) at 42° C, exposed to X-ray
film (Fujifilm, Stamford, CT) for 2 h at 4° C and developed. Hybridization signals were
quantified by densitometry using Sigma Gel Scan (Jantel Corporation, San Rafael, CA)
and mean background value subtracted. All membranes were reprobed following the
chemiluminescent reaction and inactivation of the enzyme label, using a random-primed
cDNA corresponding to the coding region of porcine 18S rRNA (courtesy of Dr. Frank
Simmen, University of Florida) to confirm equal loading of RNA and mean background
value subtracted. The positive control included total RNA (5 pg) taken from the ovarian
corpus luteum [Smith et al., 1997], while the negative control was 5 pg of yeast total
RNA. The porcine PAI-1 cDNA was characterized previously by Dr. Paul Declerck and
was shown to specifically recognize only PAI-1. Northern analysis of the RNA was not
done for PAI-1.

97
Statistical Analyses
All densitometric values obtained from X-ray image analyses were subjected to
ANOVA using the General Linear Models procedure of the Statistical Analysis System
(SAS Institute Inc., Cary NC, 1988). Values with a p<0.1 were considered significant.
The model for PAI-1 protein levels in oviductal segments of early pregnant gilts included
main effects of day, breed, segment and all higher level interactions. Data are expressed
as least-squares means ± SEM. PAI-1 protein secretion in OVX steroid-hormone
replacement animals was evaluated using a 2 x 2 factorial design. Densitometric values
were subjected to transformation due to heterogeneity of variability and are expressed as
the mean+ SEM. The relationship between PAI-1 levels in oviduct flushes (expressed as
pg/pg oviduct protein) and days of early pregnancy were evaluated by regression
analysis.
The model for PAI-1 mRNA in the oviduct during the estrous cycle or early
pregnancy included the main effects of day, segment, and day by segment where
appropriate. A similar 2x2 factorial design was used to analyze PAI-1 mRNA in OVX
steroid-hormone replacement animals. RNA data are presented as least-squares means +
SEM and have been adjusted for differences in RNA loading by a covariate analysis with
18S rRNA hybridization values. A set of pre-planned orthogonal contrasts were used to
evaluate differences for PAI-1 mRNA levels among days of the estrous cycle or early
pregnancy. Contrasts shown in Figure 4-4 include comparing Day 2 + Day 12 against
other days of the cycle in the isthmus (Panel A) and Day 1 + Day 2 against other days of
the cycle in the whole oviduct (Panel B). Contrasts shown in Figure 4-5 included

98
comparing Day 2 vs. Day 12 and Day 2 + Day 12 vs. Day 0 in each segment
(infundibulum, ampulla, and isthmus) for both cyclic and pregnant gilts.
Results
De Novo Synthesis and Secretion of PAI-1 During Early Pregnancy
To evaluate PAI-1 synthesis during early pregnancy, oviductal tissue from the
three functional segments of Large White and Meishan pigs, were placed into explant
culture and media analyzed by 2D-SDS-PAGE and fluorography. Densitometric analysis
of fluorographs revealed changes in PAI-1 secretion patterns during early pregnancy. A
greater secretion of PAI-1 (p<0.01) was found in the isthmus than in the ampulla portion
of the oviduct regardless of day of pregnancy or breed (Figure 4-1). An effect of segment
(p<0.01) and day (p<0.1) were detected for PAI-1 protein, however no differences were
detected for breed. Interactions for breed by segment (p<0.1), breed by day (p<0.01) and
breed by segment by day (p<0.1) were detected. The Large White had a greater
expression of PAI-1 protein in the isthmus on Day 2 of early pregnancy compared to
other days examined, while the Meishan had greater amounts on Day 0. Since
infundibulum PAI-1 protein was undetectable in fluorographs exposed for 7 days at -80°
C, exposure for 14 days was required. The ampulla and isthmic fluorographs could not
be measured in a direct comparison with the infundibulum since 14 days resulted in an
over exposure of fluorographs from these segments. However, no differences were
detected for infundibulum P AI-1 levels between breeds and days of early pregnancy (data
not shown).

99
PAI-1 in Oviduct Flushes During Early Pregnancy
PAI-1 was undetectable by ELISA in individual samples. Therefore, flushings
from animals, collected and pooled according to day of early pregnancy, were
concentrated and subjected to analysis for PAI-1 by ELISA. Hence, there was only one
observation per day. Variation in PAI-1 concentration in concentrated flushes due to
stage of early pregnancy was best described by a fifth order regression equation
(R2=0.55). As shown in Figure 4-2, PAI-1 in oviductal flushes may peak on Day 2 of
early pregnancy. Concentrations were low on Days 1, 5, 10, and 12 and were
undetectable on Day 0.
Hormonal Control of PAI-1 Synthesis and Secretion
To examine hormonal control of PAI-1 synthesis by the isthmus, tissue from
bilaterally OVX steroid-treated crossbred gilts was placed in culture and media analyzed
by 2D-SDS-PAGE, fluorography and densitometry. Densitometric analysis of
fluorographs revealed that estrogen stimulated synthesis of PAI-1 protein in the presence
of progesterone (p<0.10) but that an inhibition of PAI-1 synthesis occurred in the absence
of progesterone (Figure 4-3).
PAI-1 mRNA Steady-State Levels During the Estrous Cycle
Using dot-blot hybridization, steady state levels of PAI-1 mRNA were evaluated
in whole oviductal tissue throughout the estrous cycle (Days 0 to 18) in crossbred gilts.
Relative densitometric values are shown in Figure 4-4B. An effect of Day was found
(p<0.05) on PAI-1 mRNA expression throughout the estrous cycle, with significantly
greater levels of mRNA found on Days 1 and 2 than all other Days examined. No
difference could be detected in PAI-1 mRNA levels between Days 1 and 2.

100
To evaluate steady-state levels of PAI-1 mRNA In the isthmic segment (primary
site for PAI-1 synthesis), dot-blot hybridizations were evaluated during the estrous cycle
as above. An effect of day (p=0.08) was found for PAI-1 mRNA expression in the
Isthmus during the estrous cycle (Figure 4-4A). Unlike PAI-1 mRNA in the whole
oviduct, isthmic PAI-1 mRNA was significantly elevated on Days 2 and 12 of the estrous
cycle compared to all other days examined. For all dot-blot analysis, the positive control
(corpus luteum) had visible labeling, while yeast RNA did not hybridize with the PAI-1
cDNA probe.
PAI-1 mRNA Steady-State Levels in the Three Functional Segments
Levels of PAI-1 mRNA in crossbred gilts were also characterized in the three
oviductal segments on Days 0, 2 and 12 of the estrous cycle or early pregnancy. In cyclic
animals, an effect of day (p<0.01) and segment (p<0.01) were found, however, an
interaction was not detected (Figure 4-5). No differences were detected between
pregnant and cyclic animals. Orthogonal contrasts of least-squares means revealed that
in cyclic animals, Day 2 + Day 12 had a greater (p<0.01) expression of PAI-1 mRNA
than Day 0 in the infundibulum and ampulla. While mRNA expression on Days 2 + Day
12 were numerically greater than Day 0, no significant differences could be detected in
the isthmus on the three days examined. No significant differences could be detected
between Day 2 and Day 12 within the three oviduct segments of cyclic gilts.
In pregnant animals, an effect of day (p<0.01), segment (p<0.01) and an
interaction of day by segment (p<0.05) was detected for PAI-1 mRNA (Figure 4-5).
Similar contrasts as those for cyclic animals, revealed that Day 2 + Day 12 had a greater
(p<0.05) level of PAI-1 mRNA than Day 0 in the infundibulum and isthmus, but not the

101
ampulla. A difference between Day 2 and Day 12 was detected in the infundibulum
(p<0.05) and ampulla (p<0.01) but not in the isthmus, with a greater level of PAI-1
mRNA on Day 12 than Day 2.
Hormonal Regulation of PAI-1 mRNA
Oviducts from crossbred gilts, bilaterally OVX and treated with various steroid
regimens (com oil, estrogen, progesterone, or estrogen + progesterone), were examined
by dot-blot hybridization. Factorial analysis indicated an interaction (p<0.05) for
estrogen and progesterone. Progesterone alone significantly increased expression of PAI-
1 mRNA, however this expression is dependent upon the absence of estrogen (Figure 4-
6)-

102
0 2 5 0 2 5
Day of Early Pregnancy Day of Early Pregnancy
Figure 4-1. Densitometry of PAI-1 protein in fluorographs from 2D-SDS-PAGE
analyses of isthmic- and ampulla-conditioned medium (100,000 cpm) from Large White
and Meishan gilts during early pregnancy.
Densitometry of [3H]-leucine labeled PAI-1 from; A) Large White, ampulla, B) Large
White, isthmus, C) Meishan, ampulla and D) Meishan isthmus on Days 0, 2, and 5
(n=3/day/segment) of early pregnancy. Least-squares means + SEM of PAI-1 are
expressed as densitometric units/g tissue. Effects of segment (p<0.01), day (p<0.1),
breed by segment (p< 0.1), breed by day (p< 0.01), and breed by segment by day (p< 0.1)
were detected. Values without common superscripts are significantly different.

103
Days of Early Pregnancy
Figure 4-2. PAI-1 protein in oviduct flushes from crossbred gilts during early pregnancy.
Oviductal flushes from early pregnant crossbred gilts were pooled by day (0, 1, 2, 5, 8,
10, 12; n= 3, 6, 6, 3, 3, 6, 4 / day, respectively) and measured using an ELISA (see
Materials and Methods). Regression analysis is shown as a fifth order polynomial. PAI-1
levels are expressed as pg/pg oviduct protein. PAI-1 is shown to peak near Day 2 of
early pregnancy.

104
CO P E E + P
Steroid Treatment
Figure 4-3. Densitometry of PAI-1 protein in fluorographs from 2D-SDS-PAGE
analyses of isthmic-conditioned medium (100,000 cpm [3H]-leucine) from OVX
crossbred gilts.
Gilts (n=3/treatment) were treated with various steroid regimens (corn oil [CO], estrogen
[E], progesterone [P], or estrogen + progesterone [E+P]. Values were transformed and
data are expressed as means + SEM. Bars with an asterisk are significantly different (p<
0.05) from corn oil.

105
Day of the Estrous Cycle
Figure 4-4. Dot-blot hybridization analyses of oviductal total RNA from cyclic crossbred
gilts.
Total RNA from the A) isthmus, or B) whole oviduct, were collected from gilts
(n=3/day) on Days 0, 1, 2, 4, 8, 12, 15, and 18 of the estrous cycle (isthmus missing Days
15 and 18). Levels of PAI-1 mRNA are shown as least-squares means + SEM and were
analyzed with the 18S rRNA as a covariate. For whole oviduct, an effect of day was
found (p< 0.05) and the mean of Days 1 and 2 were significantly greater than all other
days examined (marked by asterisk). In the isthmus, an effect of day was found (p< 0.1),
and Days 2 and 12 were significantly greater than other days examined (marked by
asterisk). In order to detect PAI-1 mRNA in whole oviduct (comprised primarily of
ampulla tissue) cDNA probe concentrations were increased and hybridization stringency
modified compared to results shown for Isthmic PAI-1 mRNA. Therefore, PAI-1 mRNA
(relative units) should not be compared between panels A and B.

Figure 4-5. Dot-blot hybridization analyses of oviductal total RNA from the
infundibulum, ampulla, and isthmus of crossbred gilts.
Hybridization values were compared for gilts (n=3/day/segment) on Days 0, 2, and 12 of
the A) estrous cycle or B) early pregnancy for all 3 segments of the oviduct (as shown).
Levels of PAI-1 mRNA are shown as least-squares means + SEM and were analyzed
with the 18S rRNA as a covariate. No differences in mRNA expression were detected
between cyclic and pregnant gilts. Refer to results for effects of day, segment and all
interactions. The mean of Days 2 and 12 PAI-1 mRNA were found to be significantly
greater (p<0.05) than Day 0 in the infundibulum and ampulla of cyclic gilts and the
infundibulum and isthmus in early pregnant gilts. A representative dot-blot from early
pregnant cross-bred gilts is shown at the bottom of Figure 4-5 for both PAI-1 and the 18S
rRNA. Rows 1-3 (Day 0), rows 4-6 (Day 2), and rows 7-9 (Day 12); infundibulum (rows
1, 4, and 7), ampulla (rows 2, 5, and 8), and isthmus (rows 3, 6, and 9). A negative
control (yeast) and a positive control (corpus luteum) were also included.

PAI-1 mRNA, Relative Units
107
A. Estrous Cycle
B. Early Pregnancy
25000
20000
15000
10000
5000
0
Day
25000
20000
15000
10000
5000
0
0 2 12
Day
123456789
PAI-1
18S rRNA
i« • ♦ »
« # ♦
' - s
• ••••••••
*•••«•••• *4-
Yeast
Corpus Luteum
Yeast
Corpus Luteum
Dot-blot Analysis from Early Pregnant Gilts
PAI-1 mRNA, Relative Units

108
Figure 4-6. Dot-blot hybridization analysis of whole oviductal total RNA from OVX
steroid-treated crossbred gilts.
OVX gilts (n=3/treatment) were treated with various steroids (CO, E, P, E+P) as
described. Levels of PAI-1 mRNA are shown as least-squares means + SEM and were
analyzed with the 18S rRNA as a covariate. An interaction was detected for E+P
(p<0.05). The stimulation of P on PAI-1 mRNA is dependent upon the absence of
estrogen. Values with an asterisk are significantly different from corn oil.

109
Discussion
Several reports have suggested that the oviduct provides an important
microenvironment which supports development of early cleavage-stage embryos in vivo
[Gandolfi, 1995, Boatman, 1997, Buhi et al., 1997], These researchers suggest that
oviductal proteins such as growth factors, cytokines, and the oviduct-specific secretory
glycoprotein (OSP) secreted by the oviductal epithelium may sustain or facilitate the
processes of fertilization and early embryonic development. In the pig, fourteen de novo
synthesized and secreted proteins have been described in a limited fashion by relative
molecular weight and isoelectric point [Buhi et al., 1990], and steroid-modulated and
cycle-specific changes in oviductal secretory proteins have been reported [Buhi et al.,
1997], The beneficial effects of oviductal epithelial cell cocultures on embryonic
development to the blastocyst stage is well known [White et al., 1989, Nancarrow and
Hill, 1994, Suzuki and Foote, 1995] and the search for embryotrophic molecules secreted
by the oviduct is still actively under investigation [Liu et al., 1998], Recently, PAI-1 was
identified in the pig oviduct by western blotting and N-terminal amino acid
microsequence analysis [refer to Chapter 3], This protein was found to be a major de
novo synthesized and secreted product of the isthmus, was localized to the apical region
of oviductal epithelium, and associated with putative secretory granules and cilia of the
isthmus [refer to Chapter 3], With identification of PAI-1 in the pig oviduct, a closer
evaluation of its role in this tissue was examined by evaluating expression of its mRNA
and protein during the estrous cycle and early pregnancy.

110
Analysis of PAI-1 mRNA in the bovine oviduct showed a low steady-state
expression throughout the estrous cycle [Einspanier et al., 1997], In contrast, this study
clearly demonstrated that PAI-1 transcript and protein synthesis and secretion vary
throughout the estrous cycle and early pregnancy. Discrepancies between these studies
may be the result of differences between assays, more frequent time periods being
evaluated here, species differences, and examination of the three specific segments in our
study. Expression of PAI-1 mRNA was identified in all three segments of the oviduct,
supporting earlier work which localized PAI-1 protein to the apical region of the
infundibulum, ampulla, and isthmic epithelium [Chapter 3], However, while found in all
segments, levels of PAI-1 mRNA were found to be greater in the isthmus than the other
two segments indicating the isthmus as the primary site of expression of PAI-1. Day of
cycle-dependent expression of PAI-1 mRNA was found in both whole oviduct and
isthmic segments. A consistently high level of PAI-1 mRNA on Day 2 of the estrous
cycle and/or early pregnancy in both whole oviduct and the isthmus suggest the
importance of this protein during fertilization and early embryonic development.
Differences observed in PAI-1 mRNA on Day 12, between the isthmus and whole
oviduct, suggest that the hormonal regulation of PAI-1 in the isthmus may be different
from other segments or isthmic PAI-1 may be more sensitized to circulating ovarian
steroid concentrations during the estrous cycle. Progesterone, which peaks near Day 12
in the pig, was found to increase oviductal PAI-1 mRNA and may be the reason for
elevated levels on Day 12 in the isthmus. A proposed function for elevated expression of
PAI-1 mRNA on Day 12 in the isthmus is remodeling of oviductal ECM. No differences
could be detected in expression of PAI-1 mRNA between pregnant and cyclic gilts.

Ill
Although direct evidence for an association between the presence of an early cleavage-
stage embryo within the oviduct and changes in gene expression or protein synthesis by
this tissue has not been demonstrated in the pig, some evidence suggests that the oviduct
may respond to the presence of an embryo, as reviewed by Hunter (1988). Recently, the
presence of embryos in the oviduct has been shown to regulate endometrial receptivity
and increase the rate of implantation, suggesting communication between the early
oviductal embryo and the maternal system [Wakuda et al., 1999],
The present data show that de novo protein synthesis and secretion of PAI-1
varies between segments and in the isthmus during early pregnancy. Higher levels of
PAI-1 protein in the isthmus compared to the infundibulum or ampulla appears to mimic
protein synthesis and secretion of oviductal TIMP-1 [Buhi et al., 1996a], suggesting a
differential expression between the oviductal segments in expressing these protease
inhibitors. Interestingly, PAI-1 protein secretion in culture media and oviduct flushes
was found to be greater on Day 2 of early pregnancy coinciding with the time of
fertilization and early embryonic cleavage-stage development. Elevated levels of isthmic
PAI-1 and TIMP-1 on Day 2 suggest an important spatial and temporal role for these
protease inhibitors. In addition, pig oviduct flushes contain a greater amount of
plasminogen activator activity on Day 2 of early pregnancy than other days examined (as
detailed in Chapter 5). Thus, greater concentrations of PAI-1 in the oviduct on Day 2 of
early pregnancy, may be in response to the increased protease activity within the oviduct
or associated with the early cleavage-stage embryo. Mouse and rat pre-implantation
embryos have been shown to have uPA [Zhang et al, 1994, Harvey et al, 1995] and tPA
activity [Zhang et al, 1992, Carroll et al, 1993], hence, the presence of PAI-1 may be

112
required to regulate the discrete focal requirements of the PAs. The protease inhibitors,
PAI-1 and TIMP-1, may tightly regulate the remodeling and degradation of the ECM
within the oviduct or early cleavage-stage embryo. Potential functions might be to
prevent adhesion and implantation or protect the extracellular matrix of the zona
pellucida from digestion by proteolytic enzymes. Serine protease inhibitors have been
shown to stimulate DNA synthesis in endometrial glandular epithelial cells of pregnant
pigs [Badinga et al., 1999] while TIMP-1 was found to increase the in vitro development
of pig [Funahashi et al., 1997] and cow [Satoh et al., 1994] blastocysts. PAI-1 may be
having similar functions on DNA synthesis and/or development of the pre-implantation
embryo.
Breed-dependent variations (Large White vs. Meishan) in expression of PAI-1 in
the isthmus may be important relative to differences observed in time and synchrony of
estrus and ovulation between the two breeds [Martinat-Botte et al., 1989, Terqui et al.,
1990, Faillace et al., 1991, Wilmut et al., 1992, Hunter, 1993a], Hunter et al. (1993b)
suggests that the chronology of events within the estrus period may be critical to the
recognized prolificacy of Meishan pigs. However, the presence of greater PAI-1 protein
synthesis and secretion by the isthmus on Day 0 of pregnancy in Meishan gilts is difficult
to explain. As reviewed by Hunter et al. (1993b), Meishan pigs were shown to have
higher levels of circulating estradiol, greater estrogen concentrations in preovulatory
follicles, and greater aromatase activity than the Large White. Therefore, it is unclear
how the Chinese Meishan gilt secretes a greater level of PAI-1 under an elevated estrogen
environment, as estrogen was found to decrease PAI-1 protein synthesis and secretion in

113
crossbred gilts. This would suggest the possibility of other PAI-1 regulatory factors or
breed differences in hormonal regulation.
Oviductal PAI-1 appears to be regulated by changing levels of circulating ovarian
steroid hormones, estrogen and progesterone. Synthesis and secretion of PAI-1 in vitro
was inhibited in tissue cultured from OVX estrogen-treated gilts, however, this inhibition
could be relieved when progesterone was given in combination with estrogen. Similarly,
progesterone-mediated stimulation of PAI-1 mRNA levels could be inhibited when
estrogen was given in combination with progesterone. This data suggests that regulation
of transcription, synthesis, and secretion pathways for PAI-1 by estrogen or progesterone
is dependent upon the presence or absence of the inhibitory steroid. Additionally,
estrogen and progesterone may be temporally regulating these pathways through genomic
and non-genomic actions. Progesterone has been shown to increase PAI-1 mRNA in
endometrial stromal cells [Casslen et al., 1992, Schatz and Lockwood, 1993] and a
synergistic stimulation of PAI-1 mRNA has been described for estrogen and progesterone
in the endometrium [Schatz et al., 1994], Immunolocalization of PAI-1 in secretory
granules in the isthmus at Day 0 (high estrogen) and Day 9 (elevated progesterone)
[Chapter 3], suggested that while estrogen decreases PAI-1 protein synthesis and
secretion, it does not inhibit its accumulation and storage in secretory granules. Thus, this
supports our present data that estrogen does not inhibit transcription of PAI-1 mRNA.
While various in vitro studies have shown progesterone stimulates expression of PAI-1
mRNA and protein [Schatz and Lockwood, 1993, Casslen et al., 1992] our in vivo study
utilizing OVX animals was able to show a direct effect of this steroid on protein secretion
only in the presence of estrogen. Within the oviduct, we postulate that other regulatory

114
factors may be interacting in vivo to control PAI-1 synthesis and secretion. Regulators of
uterine PAI-1 mRNA and/or protein include, epidermal growth factor (EGF) [Miyauchi
et al., 1995] and transforming growth factor-beta (TGFp) [Bruner et al., 1995, Graham,
1997], These two growth factors have been immunolocalized in the pig oviduct [Buhi et
al., 1997] and measured in oviductal fluid [Swanchara et al., 1995, Buhi et al., 1997],
PAI-1 has also been shown to be regulated in extravascular tissues by gonadotropins,
cytokines, and dexamethasone, [Andreasen et al., 1990],
In summary, the presence of elevated levels of PAI-1 mRNA and protein in the
oviduct on Day 2 of early pregnancy coinciding with fertilization and subsequent early
cleavage-stage embryonic development, suggest that PAI-1 might play an important role
in facilitating or regulating one or both of these events. Additionally, the circulating
ovarian steroids, estrogen and progesterone, were shown to regulate the expression of
oviductal PAI-1 mRNA and protein in the oviduct.

CHAPTER 5
PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) AND uPA ACTIVITY IN THE
OVIDUCT AND ASSOCIATION OF PAI-1 WITH THE PREIMPLANTATION
EMBRYO
Introduction
The plasminogen/plasmin system has recently been shown to be an important
component of several proteolytic cascades which occur during the process of fertilization
[Huarte et al., 1993], Well-characterized proteolytic events during fertilization include
the acrosome reaction by spermatozoa and digestion through the zona pellucida matrix,
as well as the cortical granule reaction leading to the zona block to polyspermy
[Yanagimachi, 1988] However, besides acrosin, the proteolytic enzymes and their
inhibitors are relatively unknown. The zymogen, plasminogen, is relatively abundant in
uterine fluid [Finlay et al., 1983], ovarian follicular fluid [Beers, 1975] and seminal
plasma [Zaneveld et al., 1975, Kobayashi et al., 1992], Ovulated eggs have been shown
to secrete tissue plasminogen activator (tPA) [Huarte et al., 1985, Zhang et al., 1992] and
urokinase plasminogen activator (uPA) is associated with ejaculated spermatozoa in the
mouse [Huarte et al, 1987], human [Smokovitis et al., 1992] and pig [Smokovitis et al.,
1992], Although the exact nature of PA involvement during fertilization is unknown,
indirect evidence suggests that these enzymes, uPA and tPA, may have a role in
spermatozoal penetration through the zona pellucida. Male mice homozygous for a
targeted mutation in the acrosin gene remain fertile and spermatozoa penetrate the zona
115

116
pellucida in the complete absence of acrosin activity [Baba et al., 1994, Adham et al.,
1997]. Huarte et al. (1993) showed that addition of plasminogen during in vitro
fertilization in the mouse increased the percentage of eggs fertilized, while antibodies that
inhibit the catalytic activity of plasmin decreased fertilization. Plasminogen activators
have also been found to be associated with ovine [Bartlett and Menino, 1993] and bovine
embryos [Berg and Menino, 1992] and uPA mRNA is found in 2-cell rat embryos to the
blastocyst stage [Zhang et al., 1994],
The inhibition of the active uPA and tPA enzymes is an important element in
control of the plasminogen/plasmin system. Both uPA and tPA convert the proenzyme
plasminogen into the broad specificity enzyme plasmin. Plasminogen activator inhibitor-
1 is the primary physiological inhibitor of both uPA and tPA and is present in a wide
variety of tissues and cultured cells [Andreasen et al., 1992], PAI-1, a glycoprotein,
consists of 379 amino acids, corresponding to a 43 kDa polypeptide chain [Andreasen et
al., 1992], and is a member of the serine protease inhibitor superfamily (serpins). This
family, which includes the majority of plasma protease inhibitors, has been shown to be
involved in fibrinolysis, ovulation, and implantation [Andreasen et al., 1992], An
interesting feature of PAI-1 is that it exists in two forms - an active form which is
synthesized and secreted by cells into culture media or the extracellular space, and an
inactive or latent form [Hekman and Loskutoff, 1985] PAI-1 converts spontaneously to
the latent form shortly after secretion and can be subsequently reactivated by treatment
with denaturants and negatively charged phospholipids [Andreasen et al., 1992],
However, the active PAI-1 is stabilized by its association with extracellular matrix
[Mimuro and Loskutoff, 1989] or vitronectin in plasma [Declerck et al., 1988], The

117
major de novo synthesized and secreted protein of the pig isthmus was identified as PAI-
1 [Chapter 3], This protein was localized to the apical region of oviductal epithelium and
found to be associated with putative secretory granules of the isthmus. PAI-1 mRNA and
protein were found to be greatest on Day 2 of early pregnancy in gilts, coinciding with
the time of fertilization and early cleavage-stage embryonic development.
The temporal and spatial expression of PAI-1 in the oviduct coinciding with
fertilization and early embryonic development suggests that this protease inhibitor may
have an important function during these events. However, it is unknown if this protein
retains biological activity after secretion into the oviductal lumen or if it associates with
oviductal oocytes or embryos. The objectives of this study were 1) to characterize the
biological activity of oviductal PAI-1 and its interaction with uPA, 2) to evaluate PA
activity in the oviduct during early pregnancy, and 3) to examine PAI-1 association with
oviductal oocytes and embryos.
Materials and Methods
Materials
All culture materials used are described in Chapters 3 and 4. Unless otherwise
stated, all other chemical reagents and supplies were obtained from Sigma-Aldrich or
Fisher Scientific.
Animals and Collection of Oviductal Flush
Sexually mature crossbred gilts (Yorkshire X Duroc X Hampshire) were observed
daily for behavioral estrus for at least two estrous cycles in the presence of an intact boar.
The first day of standing estrus was designated Day 0. Gilts were bred at the onset of
estrus and again 24 h later to ensure fertile mating (except animals assigned to Day 0 of

118
pregnancy). Reproductive tracts were collected aseptically after sacrifice at the local
abbatoir or after surgery. For surgery, anesthesia was induced with a combination of
Telazol (Fort Dodge, IA, 2.2 mg/kg) and Xylazine (Fort Dodge, 2.2 mg/kg), administered
intramuscularly. Anesthesia was maintained via inhalation of Halothane (Halocarbon
Laboratories, River Edge, NJ)-oxygen mixture. Reproductive tracts were excised after
midline laparotomy, following which the incision was closed, and gilts allowed to
recover. Oviducts were flushed from the fimbria-infundibulum through the isthmus with
5 ml of (modified) Eagle's minimum essential medium (MEM) using a 10 ml disposable
syringe and 20-guage needle. Flushed material was collected into a sterile 15 ml conical
tube and stored at -20° C. Animal-use protocols were approved by the University of
Florida Institutional Animal Care and Use Committee.
Oviductal PAI-l/uPA Complex Formation
Semi-purified radiolabeled ([3FI]-leucine) porcine PAI-1 was obtained from
isthmic-conditioned oviduct explant culture medium after fractionation on a heparin-
agarose affinity column chromatography as previously described in Chapter 3. Heparin-
agarose fractions (0.2 M and 0.4 M NaCl elution) were resuspended in PBS (2 mg
protein/1.5 ml; pH 7.4). Latent porcine PAI-1 was activated by incubation with 4 M
guanidine-HCl for 2 h at room temperature, and then dialyzed against PBS (2 changes, 2
L each, 12 h each, 4° C). Non-activated PAI-1 was treated as above except for addition
of 4 M guanidine-HCl (volume was adjusted with PBS). After dialysis, samples were
counted for radioactivity by liquid scintillation spectrometry. To evaluate complex
formation, activated and non-activated PAI-1 (29,000 cpm each) were incubated with or
without 5 pg of human urokinase for 30 min at 37° C. Additionally, activated PAI-1

119
(29,000 cpm) was incubated with a polyclonal rabbit anti-human PAI-1 or normal rabbit
sera (15 pi each) followed by a 30 min exposure to urokinase as described. The purpose
of addition of the antibody to the activated PAI-1 was to test for inhibition of complex
formation. Samples were then run on a 10% non-reducing 1D-SDS-PAGE gel as
previously described [Buhi et al., 1989],
Inhibition of uPA Activity bv Oviductal PAI-1
Semi-purified PAI-1 was activated using guanidine-HCl as described above. To
evaluate the amount of exogenous uPA activity that could be inhibited by oviductal-
derived PAI-1 a direct peptidyl anilide assay was used according to Munch et al. (1993).
To compare inhibition of uPA activity between activated or latent PAI-1, various
amounts of activated or latent PAI-1 (0-40 pg) were incubated with uPA (0.5 pg/well) in
PBS (pH 7.4) for 1.5 h at room temperature in a 96-well Nunc-immuno plate (Nalgene
Nunc International, Rochester, NY) (final volume was 200 pi). After incubation, 10 pi of
the chromogenic substrate S-2444 (0.4 mM) (Sigma-Aldrich, St. Louis, MO) was added
to each well. Hydrolysis of the substrate to a tripeptide and the yellow p-nitroaniline
(pNA) was determined by following the absorbance at 405 nm on a ThermoMax
microplate reader (Molecular Devices, Menlo Park, CA). All samples were nm in
triplicate. Controls included uPA alone, substrate alone and PAI-1 alone (both activated
and non-activated). Additionally, activated PAI-1 (40 pg) was added to uPA + substrate
after a 30 minute co-incubation in order to determine if activated oviductal-PAI-1 could
inhibit the enzymatic reaction once initiated.

120
Ovidiictal Plasminogen Activator Activity During Early Pregnancy
To examine PA activity in oviductal flushes obtained from early pregnant gilts
(Days 0, 1, 2, 5 and 12), a peptidyl anilide assay was employed using the chromogenic
substrate, 2-44x (0.4 mM) (American Diagnostica, Greenwich, CT). Oviductal flushes
were pooled by day in order to obtain enough antigen to accurately measure activity from
each day. Two different pools were established from separate animals according to day
and analysis are the means of two individual replicates. Pooled samples were centrifuged
(2,200 x g, 10 min, 4° C) and the supernatant dialyzed against dH20 (three changes, 4 L
each, 12 h each, 4° C). After dialysis, total protein was measured by the Bio-Rad protein
assay according to manufacturer's instructions. Aliquots (950 pg) from each day of early
pregnancy were lyophilized and stored at -20° C. Samples were resuspended in 325 pi
PBS (pH 8.8) and 100 pi of sample (300 pg protein) was added to a 96-well plate. After
a 10 min incubation with 10 pi of 2-44x, hydrolysis and formation of pNA was followed
over time (absorbance at 405 nm) on a ThermoMax microplate reader. All samples were
run in duplicate, and PA activity expressed as optical density/pg oviduct protein.
Controls included oviductal protein alone and substrate alone. This experiment was
replicated twice using two different oviductal flush pools. For number of animals within
each pool/day, see Figure 5-5 legend.
Amiloride, an inhibitor of uPA but not tPA [Vassalli and Belin, 1987] was
incorporated into the assay in order to determine which PA was being measured. Using a
fixed concentration of exogenous uPA (0.5 pg), various concentrations of amiloride (0,
0.25, 0.5, 1.25, 2.50 and 5.00 mM) were tested for inhibitory activity in order to
determine the appropriate level for the assay. Briefly, 10 pi of uPA was incubated with

121
100 pi of amiloride and PBS, pH 8.8 (final volume, 200 pi) for 30 min in a 96-well plate
and substrate formation read at 405 nm. A concentration of 2.5 mM was shown to almost
completely inhibit exogenously added uPA activity and was subsequently used in all
inhibition assays. Oviduct flushes from Days 0, 2, 8 and 15 were pooled together,
centrifuged, dialyzed, and lyophilized as described above. Aliquots (5 mg) were
resuspended in 1 ml PBS (pH 8.8) and pulse-centrifuged (10,000 x g) to remove insoluble
material. Pooled oviduct flushes were incubated with or without amiloride (2.5 mM) for
10 minutes prior to addition of 2-44x (0.4 mM). The assay was run in a 96-well plate and
absorbance (405 nm) recorded over time as described. The uPA activity in the pooled
oviduct flush was compared to a standard curve (0-5 IU human uPA), allowing for
estimation of the uPA concentration.
Association of Oviductal PAI-1 With the Oocvte or Embryo
Immunogold electron microscopy was utilized to localize PAI-1 in preovulatory
follicular oocytes (Day 0), oviductal oocytes (Day 2), oviductal embryos (Day 2), and
uterine embryos (Day 4). Two animals were used for each day and multiple (>3) oocytes
or embryos were evaluated from each animal. Preovulatory follicles were aspirated with
a sterile 10 ml disposable syringe and a 20-gauge needle. Oviductal oocytes and embryos
were flushed from the oviduct as described. Uterine embryos were collected by clamping
the bifurcation with a hemostat and the uterine horns flushed abovarian with 10 ml of
MEM. Oocytes and embryos were collected from sterile watch glasses using a dissecting
stereo-microscope, evaluated for cell number, and placed into fixative for 1 h in PBS, pH
7.4, containing 0.5% (v/v) glutaraldehyde, 4% (v/v) paraformaldehyde at 4° C. Samples
were submitted to the electron microscopy core (Interdisciplinary Center for

122
Biotechnology, University of Florida, Gainesville, FL) for processing within 1 h of
collection. After fixation, oocytes and embryos were rinsed in PBS, dehydrated through
a graded ethanol series, and embedded in Unicryl (British BioCell International, UK )
under UV light at -10° C for 2 days. Thin sections (0.5 mm) were cut and collected on
Formvar-coated 100 mesh nickel grids, and PAI-1 antigen detected by immunogold
labeling. The polyclonal rabbit anti-human PAI-1 (kindly donated by Dr. Schleef,
Scripps Institute, LaJolla, CA) and preimmune rabbit sera, diluted 1:1000 in a high salt
Tween buffer (0.02 M Tris-HCl, 0.5 M NaCl, 1% [v/v] Tween 20, pH 7.2) supplemented
with 1% (w/v) ovalbumin, were incubated overnight with grids in a humid chamber at 4°
C. Sections were then incubated with a secondary antibody (goat anti-rabbit IgG, 1:30
dilution in PBS) conjugated to 18 nm colloidal gold (Jackson ImmunoResearch
Laboratories, Inc, West Grove, PA) for 1 h at room temperature. Sections were post-
stained with 2% (w/v) uranyl acetate and Reynold's lead citrate. Grids were examined on
a Hitachi H-7000 transmission electron microscope (Hitachi Scientific Instruments,
Danbury, CT). Digital micrographs were taken on a Gatan BioScan/Digital Micrograph
2.5 (Gatan Inc, Pleasanton, CA). For antibody specificity, refer to Chapter 3.
Statistical Analysis
Data were analyzed by ANOVA using the General Linear Models procedure of
the Statistical Analysis System (SAS Institute Inc., Cary, NC, 1988). Data are expressed
as least-squares means + SEM and differences between treatment means evaluated using
the student Newman-Kuels T-test. A probability of p<0.05 was considered significant.

123
Results
PAI-l/uPA Complex Formation
To determine if oviductal-derived semi-purified radiolabeled PAI-1 could form a
stable complex with uPA, reactivated and latent PAI-1 were incubated with exogenous
uPA and complex formation evaluated using non-reducing 1D-SDS-PAGE and
fluorography. Activated PAI-1 was able to form a complex with uPA as shown by the
shift in the PAI-1 relative molecular weight and/or the formation of a previously
described substrate-like cleavage product (42,000 Mr) [Urano et al., 1992] located just
below PAI-1 (Figure 5-1). However, non-activated PAI-1 was unable to form a complex
with uPA and generate a cleaved substrate-like product (Figure 5-1). These results
indicate that complex formation between oviductal PAI-1 and uPA requires reactivation
of PAI-1 by denaturants such as guanidine-HCl.
PAI-1 Inhibition of uPA Activity
The ability of activated or latent PAI-1 to inhibit uPA activity was evaluated
using a peptidyl anilide assay as described in Materials and Methods. Semi-purified
activated PAI-1 was able to inhibit uPA activity over time (S h) at concentrations of 10-
40 pg, while 1 pg had no inhibitory effect (Figure 5-2A). However, latent PAI-1 at all
concentrations tested, was unable to inhibit uPA activity at any time during the
incubation (Figure 5-2B). The inhibition by activated PAI-1 was shown to be dose-
dependent with a greater inhibition of uPA activity at higher concentrations (15-40 pg
protein) (Figure 5-3). Addition of activated PAI-1 30 min after exposure of uPA to S-
2444 indicated that activated PAI-1 could specifically inhibit uPA activity after initiation
of the enzymatic reaction (Figure 5-4).

124
PA Activity in the Oviduct During Early Pregnancy
To determine PA activity in the oviduct during early pregnancy, oviduct flushes
were pooled by day and activity measured using the chromogenic substrate, 2-44X. An
effect of day (p<0.01) was detected for PA activity during early pregnancy, and was
found to be greater on Day 2 than on other days examined (Figure 5-5). Days 5 and 12
showed the least amount of PA activity during early pregnancy. In order to determine if
this activity was due to tPA or uPA, a specific inhibitor of uPA but not tPA was included
in the assay. At a concentration of 2.5 mM, amiloride was found to be sufficient to
inhibit an excess concentration of uPA (0.5 pg) (Figure 5-6). Treatment of pooled
oviduct flushes (Days 0, 2, 8, and 15) with amiloride inhibited 73% of the total PA
activity (9.5 h after initiation of reaction) (Figure 5-7). This indicates that the majority of
PA activity in the oviduct is uPA while the remaining activity may be due to tPA.
PAI-1 Association with Oocvtes and Embryos
Association of PAI-1 with oocytes or embryos was examined using immunogold
electron microscopy (EM) to determine site-specific localization. Figure 5-8 is an
electron micrograph of a preovulatory follicular oocyte surrounded by cumulus cells.
Oocytes retrieved from preovulatory follicles showed a sparsity of PAI-1 located
throughout the zona pellucida and perivitelline space. The oocyte as well, showed an
infrequent scattering of PAI-1 throughout the cytoplasm (Figure 5-9). The compact mass
of cumulus cells that surrounded the follicular oocytes had an intense nuclear localization
of PAI-1, while the cytoplasm was only moderately stained (Figure 5-9). Oviductal
oocytes, flushed from the oviduct on Day 2 of the estrous cycle, had a moderate level of
PAI-1 localized throughout the zona pellucida, perivitelline space, and cytoplasm of the

125
oocyte (Figure 5-10). Oocytes retrieved from Day 2 gilts, had a high density of gold
particles were associated with the outer edge of the zona pellucida (Figure 5-10).
Oviductal embryos (2-4 cell) showed very similar localization of PAI-1 to that of
oviductal oocytes. PAI-1 was densely associated with the outer rim of the zona pellucida
and moderately localized throughout the interior of the zona matrix, perivitelline space,
and blastomere cytoplasm (Figure 5-11). PAI-1 was also densely associated with the
head region of boar spermatozoa bound to or embedded within the zona pellucida in these
embryos (Figure 5-11). However, the sperm midpiece or tail showed no localization of
PAI-1. Embryos (8-16 cell) having traversed through the isthmus, the primary site of
PAI-1 synthesis and secretion, flushed from the tip of the uterine horn on Day 4 of early
pregnancy, showed localization patterns similar to those of oviductal embryos. PAI-1
was densely associated with the outer surface portion of the zona pellucida and
moderately associated throughout the zona pellucida matrix, perivitelline space, and
blastomere cytoplasm (Figure 5-12). Spermatozoa, still attached to the zona pellucida
were shown to have PAI-1 localized along the head region, but not detected on the
midpiece or tail (Figure 5-12). Controls for localization of PAI-1 in association with
preovulatory follicular oocytes, oviductal oocytes and embryos, or uterine embryos are
shown in Figure 5-13.

126
96
66
45
29
Activated PAI-1 Latent PAI-1
- uPA + uPA - uPA +uPA
Figure 5-1. Representative fluorograph of denaturant-activated or latent PAI-1 incubated
in the presence of uPA after non-reducing 1D-SDS-PAGE separation.
Activated PAI-1 (Lanes 1 and 2) or latent PAI-1 (Lanes 3 and 4) were incubated with (+)
or without (-) 5 pg of uPA as described in Materials and Methods. Activated PAI-1
incubated with uPA resulted in a complex formation and the generation of a substrate¬
like cleavage product (arrow) just below the 45,000 molecular weight marker (x 103).
Activated PAI-1 not exposed to uPA did not show a substrate-like cleavage product, nor
did latent PAI-1 incubated with uPA.

Figure 5-2. Oviductal PAI-1 inhibition of uPA activity over time.
Various concentrations (0-40 pg protein) of semi-purified latent (A) or activated (B) PAI-
1 were incubated over time in the presence of uPA and S-2444 (chromogenic substrate).
Generation of the chromophore, p-nitroaniline, was recorded at 405 nm absorbance and is
expressed as relative uPA activity. Latent PAI-1 at all concentrations tested was unable
to inhibit uPA activity while activated PAI-1 inhibited uPA at concentrations greater than
1 pg. All samples were run in triplicate.

uPA activity (Optical Density / 200 pi)
128
Time (minutes)
■■ • uPA alone
uPA + PAI-1 (1 pg)
—r— uPA + PAI-1 (10 pg)
uPA +PAI-1 (15 pg)
uPA + PAI-1 (20 pg)
uPA + PAI-1 (40 pg)

129
Figure 5-3. Dose-dependent inactivation of uPA activity by activated PAI-1.
Increasing concentration of semi-purified activated PAI-1 (1-40 pg) was shown to
decrease relative uPA activity. Latent PAI-1 was unable to inhibit uPA activity. All
samples were run in triplicate and absorbance (405 nm) was determined at 45 min after
addition of S-2444.

130
Figure 5-4. Inhibition of uPA activity with activated PAI-1 (40 pg) 30 minutes after
initiation of the enzymatic reaction (cleavage of the chromogenic substrate, 2-44x, by
uPA).
Absorbance (405 nm) was determined over time and all samples were read in duplicate.
Arrow indicates point of addition of activated PAI-1. PAI-1 was able to completely
inhibit the enzymatic cleavage of 2-44x by uPA.

131
Day of Early Pregnancy
Figure 5-5. Plasminogen activator activity in oviduct flushes from early pregnant
crossbred gilts.
Oviduct flushes were pooled (1 flush/animal/oviduct) randomly by day (0, 1, 2, 5, and 12;
n = 2, 5, 5, 3, and 4 animals/day respectively) and measured for PA activity using a
peptidyl anilide assay as described in Materials and Methods. Values shown are least-
squares means + SEM and this experiment was replicated twice using different pools for
each day. Samples from each replicate were run in duplicate. An effect of day (p<0.05)
was determined for PA during early pregnancy. Bars with different superscripts are
significantly different.

132
Amiloride Concentration (mM)
Figure 5-6. Dose-dependent inhibition of uPA activity by amiloride.

133
0123456789 10
Time (hours)
Figure 5-7. Inhibition of oviductal flush PA activity by amiloride.
Oviduct flushes from Days 0, 2, 8, and 15, were pooled together and PA activity was
measured using a peptidyl anilide assay as described in Materials and Methods. Oviduct
flushes were incubated with or without amiloride (2.5 mM) for 10 min prior to addition
of 2-44x. All samples were run in duplicate and absorbance (405 nm) recorded over
time.

Figure 5-8. Electron micrograph of a pig preovulatory follicular oocyte with surrounding
cumulus cells.
Cumulus cells surrounding the oocyte are shown by an arrow. OC (oocyte), ZP (zona
pellucida).


Figure 5-9. Immunogold localization of PAI-1 in association with pig preovulatory
follicular oocytes and cumulus cells.
Gold particles (marked by arrows) can be seen primarily in the nucleus (nu) of cumulus
cells (Panel A) and associated with the perivitelline space (PS) and oocyte (OC) (Panel
B). Controls (NRS) are shown in Panel C (cumulus cells) and Panel D (PS + OC).

Li I

Figure 5-10. Immunogold localization of PAI-1 in association with oviductal oocytes.
Gold particles (marked by arrow) can be seen densely associated with the outer surface
portion of the zona pellucida (ZP) (Panels A and B). Gold particles were also seen
associated with the perivitelline space (PS) and oocyte (E should be OC; typing error)
(Panel C) and within the oocytes cytoplasm (Panel D).

6£l

Figure 5-11. Immunogold localization of PAI-1 in association with oviductal embryos
(2-4 cell) and spermatozoa attached and embedded in the zona pellucida.
Gold particles can be seen densely associated with the outer surface portion (arrow) of
the zona pellucida (ZP) (Panel A) and also within the ZP matrix (Panel B). Additionally,
gold particles (arrow) were associated with the perivitelline space (PS) and embryo (E)
(Panel C). PAI-1 was also localized to the head region (arrow)of boar spermatozoa
(Panel D).

in

Figure 5-12. Immunogold localization of PAI-1 in association with early uterine
embryos (8-16 cell).
Gold particles are seen densely associated with the outer surface portion (arrow) of the
zona pellucida (ZP) (Panel A), moderately associated with the ZP matrix (Panel B), and
embryo (Panel C). PAI-1 is also associated with the head region (arrow) of boar
spermatozoa (Panel D) similar to oviductal embryos.

m

Figure 5-13. Representative controls (NRS) for immunogold localization of PAI-1 in
association with follicular oocytes, oviductal oocytes, and embryos, and uterine embryos.
No gold particles were seen associated with the zona pellucida (ZP) (Panel A),
perivitelline space (PS) (Panel B), embryo (Panel C), or spermatozoa (Panel D).

SPI

146
Discussion
This study demonstrates clearly that porcine oviductal-derived PAI-1, semi-
purified from conditioned explant culture media, retains its biological activity and can
interact with uPA. PAI-1, after activation with guanidine-HCl, bound to uPA and
resulted in a shift in the molecular weight of PAI-1. Additionally, the interaction of
activated PAI-1 with uPA resulted in formation of a cleaveable, substrate-like form
(42,000 Mr) of PAI-1. PAI-1 preparations are heterogeneous and are a mixture of at least
three different forms: active PAI-1 that can form complexes with PAs; latent PAI-1
which remains intact after incubation with PAs; and a population which is cleaved as a
substrate by PAs [Urano et al., 1992], Denaturant-activated PAI-1 can therefore act not
only as an inhibitor but also as a substrate, and once exposed to catalytic amounts of PA
is cleaved proteolytically in the reactive center [Munch et al., 1993], This study
demonstrates that radiolabeled oviductal PAI-1 can be purified from explant culture
media and used for functional studies in the oviduct. Oviductal PAI-1 was able to inhibit
the catalytic activity of uPA which was dependent upon the active or latent conformation.
Non-activated (latent) PAI-1 fractions showed a much greater uPA activity than the
exogenous uPA included in the assay. This is most likely due to the presence of
endogenous PA in the semi-purified preparation. However, endogenous and exogenous
PA activity are inhibited once PAI-1 is denaturant-activated. The activity of PAI-1 in
vivo is likely stabilized by binding to the oviductal ECM or vitronectin [Declerck et al.,
1988, Mimuro and Loskutoff, 1989], However, the presence of vitronectin in the oviduct
or its lumen has not been examined.

147
Plasminogen activator activity was identified in oviduct flushes throughout early
pregnancy. Total PA activity was greatest in gilts on Day 2 of pregnancy that coincides
with ovulation, fertilization, and early cleavage-stage embryonic development. The
source of PA is unknown, although it could be from one of the three oviductal segments
and/or selective serum transudate. Inhibition studies using a specific inhibitor of uPA,
amiloride, revealed that uPA and likely some tPA are present in oviductal luminal fluid,
however, the majority of this activity apparently belonged to uPA. Due to protein
requirements to examine PA activity, the ratio of uPA/tPA on individual days of early
pregnancy was not measured. The presence of PA in the oviduct and the possibility of
plasminogen/plasmin involvement in fertilization [Huarte et al., 1993] and early
embryonic development indicates the potential need of regulatory elements such as PAI-
1. Interestingly, oviductal flush and culture media PAI-1 were greater on Day 2 of early
pregnancy as described in Chapter 4. Therefore, increased expression and synthesis of
PAI-1 mRNA, protein, and PA activity on Day 2 of early pregnancy compared to other
days examined, suggest an important role in the regulation of the plasminogen/plasmin
proteolytic cascade in the oviduct.
To the author's knowledge this study is the first demonstration of PAI-1 in
association with oviductal oocytes and embryos. There were no observed differences in
PAI-1 localization between oviductal oocytes and embryos, as both showed PAI-1 to be
moderately localized throughout the interior of the zona pellucida, perivitelline space,
and cytoplasm of the ooycte or blastomeres. The outer portion of the zona pellucida
matrix in both oviductal oocytes and embryos had a high density of gold particles,
suggesting that PAI-1, originating from the oviduct, coated the exterior of the zona

148
pellucida. Interestingly, PAI-1 was found to be densely associated with the head region
of boar spermatozoa. PAI-1 has been shown to be in association with human
spermatozoal membranes and boar spermatozoal membranes were shown to contain uPA
and tPA [Smokovitis et al., 1992], The localization of PAI-1 on spermatozoa here may
be due to; 1) the presence of oviductal PAI-1 bound to spermatozoa, 2) endogenous PAI-
1 associated with spermatozoal membranes, or 3) oviductal or testicular PAI-1
complexed to either tPA or uPA. Additionally, vitronectin, which associates with human
spermatocytes, spermatids [Nuovo et al., 1995] and spermatozoa [Fusi et al., 1994,
Bronson and Preissner, 1997], binds PAI-1 and stabilizes its activity both in vitro and in
vivo [Declerck et al., 1988], Evidence suggests that oolemal integrins and their ligands
on spermatozoa play an integral role in gamete recognition, binding and subsequent
fertilization [Sueoka et al., 1997], As reviewed by Sueoka et al. (1997), numerous
integrins have been identified on the oolema including psav and p60t„, both of which bind
vitronectin. Vitronectin is located in the acrosomal region of ejaculated spermatozoa and
is liberated into culture medium following induction of the acrosome reaction by
ionomycin [Fusi et al., 1994], These findings, in addition to our data, suggest that
oviductal PAI-1 may be bound to vitronectin on the spermatozoa. Therefore, PAI-1 is
positioned to play a strategic role in gamete interactions leading to fertilization such as
sperm binding to the zona pellucida, penetration through the zona pellucida matrix,
and/or binding and fusion with the oolema.
PAI-1, shown to be densely associated with the outer portion of the zona
pellucida, may function to protect the zona pellucida matrix from proteolytic degradation.
Oviductal oocytes and embryos had a greater resistance to proteolytic digestion by

149
pronase than were either follicular oocytes or uterine embryos [Broermann et al., 1989],
This suggests that protease inhibitors secreted by the oviduct bind to the zona pellucida
and stabilize the matrix. Fertilization and early embryonic development occur in a
protease-rich environment when PA activity is greatest. In addition, numerous
proteolytic enzymes are released from spermatozoa following the acrosome reaction.
Thus, PAI-1 in the outer rim of the zona may help regulate proteolytic events to specific
focal areas such as spermatozoa penetration of the zona pellucida matrix.
Supplementation of culture medium with proteases has been shown to improve
the hatching rate of mouse embryos in vitro [Lee et al., 1997], and proteases are
implicated in hatching and zona lysis [Perona and Wassarman, 1986, Menino and
Williams, 1987, Gonzales and Bavister, 1995], PAI-1 may act to stabilize the zona
matrix and prevent premature hatching prior to blastocyst development. Plasminogen
activators are secreted by oocytes following fertilization or by the early cleavage-stage
embryo [Zhang et al., 1992, 1994] and are present in the oviductal microenvironment.
These data suggest a regulatory molecule in the oviduct, such as PAI-1, may be required
to prevent digestion of the zona pellucida matrix and premature hatching.
While follicular oocytes had sparse PAI-1, they did not have a dense coat of PAI-
1 encircling the zona pellucida as seen in oviductal oocytes or embryos. This suggests
that the heavy concentration of PAI-1 on oviductal oocytes and embryos was obtained
from oviductal secretions. Cumulus cells were found to contain a high density of PAI-1
within their nucleus, suggesting PAI-1 associated with the follicular oocyte may be of
cumulus cell origin. PAI-1 and PAI-2 mRNA has been detected in human cumulus and
granulosa-luteal cells [Piquette et al., 1993] while PAI-1 mRNA has been detected in the

150
cow cumulus-oocyte complex [Bieser et al., 1998], Uterine embryos (8-16 cell) collected
on Day 4 of early pregnancy had a localization of PAI-1 similar to oviductal embryos.
This suggests that the oviductal embryo may retain oviductal-derived PAI-1 in the uterine
environment and did not dissociate from the embryo. However, it cannot be ruled out
that this PAI-1 associated with the embryo is not of uterine origin.
In summary, functionally active PAI-1 is secreted by the oviductal epithelium at
the time of ovulation, fertilization, and early cleavage-stage embryonic development.
Active PAI-1 can then bind to and regulate the catalytic activity of plasminogen
activators, such as uPA, which are present in the oviductal microenvironment.
Additionally PAI-1 can associate with oviductal oocytes and embryos and may regulate
the processes of sperm binding and/or penetration through the zona pellucida matrix.
This process is possibly facilitated through the PAI-1 binding protein, vitronectin, that
has been found associated with spermatozoa. Additional functions of PAI-1 may involve
stabilization of the zona pellucida matrix to prevent proteolytic degradation or premature
hatching.

CHAPTER 6
EFFECTS OF A PORCINE OVIDUCT-SPECIFIC GLYCOPROTEIN ON
FERTILIZATION, POLYSPERMY AND EARLY EMBRYONIC DEVELOPMENT IN
VITRO
Introduction
The porcine oviduct, responds to ovarian steroid hormones by synthesizing and
releasing several specific proteins into the lumen [Buhi et al., 1990], The cumulative
synthesis and transport of these proteins into the oviductal lumen during proestrus, estrus
and metestrus creates a microenvironment capable of supporting important reproductive
events which include fertilization and early cleavage-stage embryonic development.
Several de novo synthesized proteins of the porcine oviduct have been identified and
characterized [Buhi et al., 1997], including the pig OSP. This protein is highly conserved
across species including the human [Arias et al., 1994], sheep [DeSouza and Murray,
1995], mouse [Sendai et al., 1995], cow [Sendai et al., 1994], hamster [Suzuki et al.,
1995], baboon [Donnelly et al., 1991] and rhesus monkey [Verhage et al., 1997],
Ovariectomy and steroid hormone replacement studies have shown that pOSP mRNA
and protein synthesis are estrogen-dependent and that expression is greatest during the
preovulatory period and at ovulation [Buhi et al.,1996]. The function of this protein
remains unknown. Immunolocalization studies in the pig, however, have revealed pOSP
to associate with the zona pellucida, perivitelline space and vitelline and blastomere
151

152
membranes of ovulated oocytes and oviductal embryos, respectively [Buhi et al., 1993],
suggesting a role for this protein during fertilization and early embryonic development.
Embryo in vitro culture systems have been shown to result in successful in vitro
maturation/fertilization (IVM/IVF) and subsequent development of the early cleavage-
stage pig embryo to the blastocyst stage [Abeydeera and Day, 1997, Aberydeera et al.,
1998a, Abeydeera et al., 1998b, Abeydeera et al., 1999], However, a high incidence of
polyspermy (often >50%) remains a major impediment in porcine IVF [Wang et al.,
1991, 1994, 1997, Abeydeera and Day, 1997], Differences in polyspermy rates have
been shown to be much greater with in vitro matured oocytes (65%) than for ovulated
oocytes flushed from the porcine oviduct and fertilized in vitro (28%) [Wang et al.,
1998], Co-culture of oocytes with oviductal epithelial cells [Kano et al., 1994] or
preincubation of ooyctes with oviductal fluid [Kim et al., 1996] significantly reduced the
incidence of polyspermy in pigs. A functional block to polyspermy in vivo has been
suggested to be due to a factor of oviductal origin. Several investigators have speculated
that this activity may be due to a specific function of the OSP [Hunter, 1991, Dubuc and
Sirard, 1995, Kim et al., 1996, Wang et al., 1998], The association of pOSP with
ovulated oocytes and early cleavage-stage embryos [Buhi et al., 1993] suggests this
glycoprotein may be a likely candidate for such an activity. Funahashi and Day (1997)
speculated that oviductal proteins in preincubation and/or fertilization media compete
with sperm receptors for binding of zona pellucida ligands, stimulate the rate of sperm
acrosome reaction and, thus, reduce the number of capacitated spermatozoa attaching to
the surface of pig oocytes.

153
Specific objectives of this in vitro study were to; 1) evaluate the effects of pOSP
on fertilization responses including penetration rate and the incidence of polyspermy, 2)
examine the effect of pOSP on zona solubility and sperm binding to the zona, 3)
determine if a polyclonal antibody generated against pOSP could negate specific pOSP
effects, and 4) determine if pOSP has the capacity to enhance blastocyst development.
Materials and Methods
Materials
Acrylamide, N,N' diallyltartardiamide, urea, Nonidet P-40, and sodium dodecyl
sulfate were acquired from Gallard-Schlesinger (Carle Place, NY); X-Omat AR film and
photography reagents were a product of Eastman Kodak Co. (Rochester, NY); amino
acids and protein standards were purchased from Sigma-Aldrich (St. Louis, MO);
ampholines were from Pharmacia-Biotech (Piscataway, NJ); all other supplies and
reagents for gel electrophoresis were procured from either Bio-Rad Laboratories
(Richmond, CA) or Fisher Scientific (Orlando, FL); and all medium and culture supplies
were obtained from Life Technologies (Grand Island, NY). L-[4,5-3Fl]leucine (specific
activity, 120 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Unless
otherwise stated, all other chemicals and reagents were acquired from Sigma or Fisher.
Culture Media
Basic in vitro oocyte maturation (IVM) medium was protein-free Tissue Culture
Medium 199 [Abeydeera et al., 1999] supplemented with 10 ng/ml epidermal growth
factor, 0.57 mM cysteine, 0.1% (w/v) polyvinyl alcohol (PVA), 0.5 ng/ml leutenizing
hormone (LH), 0.5 |tg/ml follicle stimulating hormone (FSH), 75 ng/ml potassium
penicillin G, and 50 ng/ml streptomycin sulfate, 3.05 mM D-glucose, and 0.91 mM

154
sodium pyruvate. IVF medium was essentially that of Abeydeera and Day [1997],
designated modified Tris-buffered medium (mTBM), pH 7.2-7.4 at 39°C, 5% CO2 (v/v)
in air. The mTBM consists of 113.1 mM NaCl, 3 mM KC1, 7.5 mM CaCl2 2H2O, 20 mM
Tris (crystallized free base; Fisher Scientific, Fair Lawn, NJ), 11 mM glucose, 5 mM
sodium pyruvate, and no antiobiotics. Embryos were cultured in North Carolina State
University (NCSU) 23 medium [Petters and Wells, 1993], supplemented with 4 mg/ml
BSA (designated IVC medium). Medium (IVM, IVF and IVC) were covered with
paraffin oil and equilibrated at 39°C, 5% CO2 (v/v) in air at least 12 h prior to use.
In Vitro Maturation and In Vitro Fertilization
Ovaries from prepubertal gilts were collected from an abattoir and immediately
transported to the laboratory at 25-30°C in 0.9% saline containing 75 pg/ml potassium
penicillin G and 50 pg/ml streptomycin sulfate. Oocytes were aspirated from follicles (3-
6 mm diameter) using a 20-gauge needle connected to a 10-ml disposable syringe,
transferred to a 50 ml conical tube and allowed to sediment at room temperature (25° C).
Supernatant was discarded and follicular contents were washed with Tyrode's Lactate
(TL)-Hepes medium supplemented with 0.01% (w/v) PVA (TL-Hepes-PVA). Oocytes
with an evenly granulated cytoplasm and surrounded by compact cumulus cells were
washed twice with TL-Hepes-PVA, and three times in IVM medium. Oocytes (50-70)
were suspended in 500 pi of IVM medium supplemented with LH and FSH in a 4-well
multidish (Nunc, Roskilde, Denmark) and cultured for 42-44 h.
Upon completion of IVM, cumulus cells were removed by treatment with 0.1%
(w/v) hyaluronidase in basic IVM medium and vortexed for 1 min. Denuded oocytes
were washed three times in 500 pi of IVM medium followed by three washes in IVF

155
medium containing 1 mM caffeine and 1 mg/ml BSA. Oocytes (n=35) were placed into
50 pi drops of the same medium, that had been covered with warm paraffin oil in a 35 x
10-mm2 polystyrene culture dish (Becton Dickinson & Co., Lincoln Park, NJ) and pre¬
equilibrated (39°C, 5% [v/v] CO2 in air) overnight. A frozen semen pellet was thawed
and washed three times by centrifugation at 1,900 x g for 4 min in Dulbecco’s PBS
(DPBS; Life Technologies, Grand Island, NY) supplemented with 1 mg/ml BSA, 75
pg/ml potassium penicillin G, and 50 pg/ml streptomycin sulfate (pH 7.2). After
washing, the sperm pellet was resuspended in IVF medium supplemented with caffeine
(1 mM) and BSA (0.1%, w/v) and 50 pi of the sperm suspension was added to 50 pi
drops of IVF medium containing the oocytes so that the final sperm concentration was
2.5-3.5 x 105/ml. Spermatozoa and oocytes were coincubated for 6 h at 39°C in 5% CO2
(v/v) in air.
Tissue Collection and Protein Purification
Sexually mature crossbred gilts (Yorkshire x Duroc x Hampshire) were observed
daily for behavioral estrus for at least two estrous cycles in the presence of an intact boar.
The first day of standing estrus was designated as Day 0 and animals were taken to the
abattoir for slaughter on Day 0 or 1 of the estrous cycle. After exsanguination,
reproductive tracts were collected aseptically, opened longitudinally, and oviductal tissue
washed in several volumes of (modified) Eagles minimum essential medium (MEM).
Tissue was cut into 1- to 3-mm3 sections and explants (500 mg) were cultured in 15 ml of
leucine-deficient MEM containing 100 pCi of [3H]-leucine in petri dishes on a rocking
platform at 39°C under a defined atmosphere of 50% N2 (v/v): 47.5% O2 (v/v): 2.5% CO2
(v/v) [Buhi et al., 1990], Non-labeled cultures were generated as above in complete

156
MEM, without addition of [3H]-leucine. After 24 h of culture, media was aspirated and
frozen at -20° C until purification.
Recent molecular cloning and analysis of porcine pOSP cDNA revealed a
putative heparin-binding concensus sequence in the protein [Buhi et al., 1996], Utilizing
a heparin-agarose affinity column, highly purified pOSP was obtained (Alvarez and Buhi,
unpublished). Briefly, culture media from Days 0 and 1 whole oviduct cultures were
thawed, centrifuged (2,200 x g, 10 min, 4° C), pooled, diluted (1:3) with 20 mM Tris-HCl
(pH 7.6, 4° C) containing 0.02% (w/v) NaN3 and slowly loaded onto the heparin-agarose
column (2.5 x 8.2 cm) at 4° C. Porcine OSP was eluted utilizing stepwise increments of
NaCl (0.1-3.0 M) and purification was monitored by 2D-SDS-PAGE and fluorography as
previously described [Buhi et al., 1990], Purified preparations from the 0.4 M NaCl
elution were utilized for all experiments detailed herein. Highly-purified pOSP was
dialyzed against (ÍH2O (3 changes, 4 L each, 24 h each, 4° C), pooled, measured for total
protein content by the Bio-Rad microassay (according to manufacturer’s instructions) and
radioactivity by liquid scintillation spectrophotometry. Pooled samples were lyophilized
and stored at -20° C.
Experiments 1, 2, 3 and 4 utilized radiolabeled pOSP obtained from whole
oviduct cultures as detailed above. Experiment 4, designed to evaluate pOSP effects on
embryonic development to the blastocyst stage, used pOSP purified from the ampulla
segment of the oviduct and was not labeled with [3H]-leucine. Purification of ampulla-
derived pOSP was identical to that of the whole oviduct. Protein (5 mg) from either
pOSP preparation was resuspended in 3 ml of mTBM (39° C) for IVF or NCSU 23
(39°C) for IVC and total protein content was measured by the Bio-Rad protein assay

157
according to the manufacturer’s instructions. Aliquots were stored at -20° C. Oviductal
pOSP resuspended in IVF medium was evaluated by 2D-SDS-PAGE and fluorography
[Buhi et al., 1990],
IeG Purification
Polyclonal anti-pOSP antiserum was prepared as described by Buhi et al. (1993).
The IgG fraction was separated by ammonium sulfate precipitation and purified by
diethylamino ethyl sepharose (DEAE-Sepharose,Pharmacia-Biotech, Piscataway, NJ)
ion-exchange chromatography [Harlow and Lane, 1988], The isolated IgG fraction was
dialyzed against dH20 (2 changes, 2 L each, 24 h each, 4° C), total protein content was
measured as described and purity determined by 1D-SDS-PAGE. Aliquots containing
100 pg of IgG were lyophilized and stored at -20° C. A dose-response of radiolabeled
pOSP (10 pg/ml) binding was evaulated with various concentrations of purified anti-
pOSP IgG (10-200 pg/ml). Incubation was allowed to extend overnight at 39° C in order
to mimic conditions established for IVF. Antibody-pOSP complex was precipitated
using Protein A-Sepharose (1 h incubation, gentle rotation, room temperature) and
centrifuged (2,200 x g, 5 min). Protein A-Sepharose complexes and supernatant were
analyzed by 1D-SDS-PAGE and fluorography [Buhi et al., 1989], The pOSP antiserum
was shown previously to cross-react with only a couple of other oviductal proteins
released into culture [Buhi et al., 1993] and these proteins were not purified during
heparin-agarose affinity purification (data not shown). The pOSP antiserum does not
cross-react with any of the radiolabeled proteins shown in Figure 6-1 [Buhi et al., 1993],

158
Experiment 1: dOSP Effects on In Vitro fertilization
Pig oocytes, matured in vitro, were exposed to various concentrations of pOSP
before and during IVF. In vitro matured oocytes, after 42-44 h of culture, were removed
of cumulus cells as described, washed three times in IVF medium, and randomly
assigned to each of six treatments. Oocytes (n=3 5/treatment) were placed into 50 pi
drops of IVF medium containing 0, 0.1, 1, 10, 50 or 100 pg/ml pOSP, and preincubated
for 4 h at 39° C, 5% (v/v) CO2 in air. Oocytes were then inseminated with spermatozoa
(50 pi, 2.5-3.5 x lOVml) essentially halving the concentration of pOSP used during
preincubation. Six hours after insemination, oocytes were washed three times in IVC
medium (100 pi) and incubated in IVC medium (100 pi) for an additional 4 h. After
incubation in IVC medium, sperm cells attached to the zona were removed by washing
three times in TL-Hepes-PVA (39° C) with a small bore pipette, mounted, and placed into
fixative (25% [v:v] acetic acid in ethanol, room temperature) for 72 h. Oocytes were then
stained with 1% (w:v) orcein in 45% (v:v) acetic acid and examined under a phase-
contrast microscope at x200 and x400 magnification. The meiotic stage of the oocytes
were assessed according to Hunter and Polge [1966], Oocytes were considered
penetrated when one or more sperm heads and/or male pronuclei and corresponding
sperm tails were present. The rate of polyspermy, male pronuclei formation, and mean
number of sperm/oocyte, are determined from those ooyctes which are penetrated.
Experiment 1 was replicated five times with multiple observations for each replicate.
Experiment 2; Anti-pOSP IaG Inhibition of the Decrease in Polyspermy
Preliminary experiments testing various concentrations of pOSP IgG (10, 50, 100,
200 and 400 pg/ml) indicated that 50 pg/ml of IgG could inhibit the effects seen on

159
polyspermy without affecting penetration rates. For this experiment, a concentration of
10 pg/ml pOSP was used. Treatments consisted of control (no addition), pOSP, pOSP +
IgG (50 pg/ml), or antibody alone and were evaluated as a 2 x 2 factorial design.
Cumulus-free oocytes were assigned randomly to each of the four treatments
(n=3 5/treatment), preincubated for 4 hours, inseminated, and fertilization parameters
evaluated as above. Experiment 2 was replicated 3 times with multiple observations
within each replicate.
Experiment 3; pOSP Effects on Zona Pellucida Solubility
To test zona pellucida solubility, cumulus-free in vitro matured oocytes were
washed three times in 500 pi drops of IVF medium and transferred to 50 pi drops of IVF
medium containing various concentrations of pOSP (0, 0.1, 1, 10, 50 or 100 pg/ml) for 4
h at 39° C, 5% (v/v) CO2 in air. Oocytes were then washed three times in TL-Hepes-
PVA (39° C) and oocytes (n=l 5/treatment) placed into 100 pi of a 0.1% (w:v) pronase
solution in phosphate-buffered saline (PBS). Zona digestion was observed continually at
room temperature (25°C) with an inverted microscope. When the zona pellucida was no
longer visible at 200x magnification, the zona pellucida dissolution time was recorded
(total number of oocytes missing zona pellucidas at designated time points - see Table 6-
2 for time points). Experiment 3 was replicated 3 times.
Experiment 4: Effects of pOSP on Sperm Binding to the Zona Pellucida
To examine for pOSP effects on sperm binding, cumulus-free in vitro matured
oocytes (n=3 5/treatment) were washed and preincubated with various concentrations of
pOSP (0, 0.1, 1, 10, 50 or 100 pg/ml) for 4 h at 39° C and coincubated with spermatozoa
as described above. After fertilization, putative zygotes were washed twice in 500 pi of

160
IVC medium and pipetted in and out (10 times) of a wide bore pipette to remove loosely
bound sperm. Putative zygotes were then placed into 50 pi drops of TL-Hepes-PVA
containing Hoescht 33342 (bisBenzamide; 1.3 mg/ml) and incubated for 30 min at 39° C
in 5% (v/v) C02 in air. Putative zygotes were then washed twice in 300 pi of TL-Hepes-
PVA, mounted, and the number of tightly bound sperm/zygote counted under a phase-
contrast microscope (Leitz Laborlux D) equipped with ultraviolet illumination (excitation
at 330-380 nm, emission at 420 nm). Experiment 4 was replicated 3 times with 15
spermatozoa counted from each replicate.
Experiment 5: nOSP Effects on Cleavage Rate and Embryonic Development
In vitro matured oocytes were removed of cumulus cells, washed and randomly
assigned to each of two treatments; control (0 pg/ml of pOSP during preincubation/TVF,
n=35 oocytes/50 pi drop, n=2 drops) or pOSP (10 pg/ml during preincubation/TVF, n=35
oocytes/50 pi drop, n=10 drops). A concentration of 10 pg/ml of pOSP was chosen
because, compared to the control, this concentration significantly decreased the incidence
of polyspermy yet had no effect on the penetration rate. Preincubation and fertilization
were carried out as described. Six hours after fertilization, control putative zygotes were
washed three times in 100 pi drops of IVC medium, transferred to a fresh 100 pi drop
(n=35 oocytes/drop) and incubated at 39° C in 5% (v/v) C02 in air. Putative zygotes
exposed to pOSP during preincubation/IVF were pooled, washed three times in 500 pi of
IVC medium and randomly assigned to 100 pi IVC drops containing 0, 1, 10, 50 or 100
pg/ml of pOSP (n=70 zygotes/treatment, 35 embryos/drop). At 48 and 144 h after
insemination, cleavage rate and blastocyst formation, respectively, were evaluated under

161
a stereomicroscope. Blastocyst formation was calculated from the number of oocytes.
Experiment 5 was replicated 9 times with multiple observations within each replicate.
Statistical Analyses
Data were analyzed by ANOVA using the General Linear Models procedure of
the Statistical Analysis System (SAS Institute Inc., Cary, NC, 19S8). All percentage data
were subjected to arcsine transformation before statistical analysis. Data are expressed as
means ± SEM or least squares means + SEM. Differences between means were
evaluated by the Student Newman-Kuels T-test. The model for analysis included the
main effects of treatment (0-100 pg/ml pOSP), replicate, and replicate x treatement.
Replicate and replicate x treatment was not significant in any of the analysis. Therefore,
replicate was not included in the final model (main effect of treatment alone) as the
analyses were no longer significant when included in the model. A probability of p<0.05
was considered significant. A 2 x 2 factorial design was used to evaluate the effects of
pOSP and anti-pOSP IgG on polyspermy. A set of pre-planned orthogonal contrasts were
used to evaluate pOSP concentrations on embryonic development. Contrast one
evaluated blastocyst development between oocytes exposed to 10 pg/ml pOSP during
preincubation/IVF and the control (0 pg/ml pOSP). Contrast two evaluated oocytes
exposed to pOSP during preincubation/IVF alone vs. those oocytes exposed to pOSP
during preincubation/IVF and embryonic development. Contrast three evaluated the two
lowest concentrations of pOSP added during embryo development (1 and 10 pg/ml
pOSP) vs. the higher concentrations of pOSP (50 and 100 pg/ml).

162
Results
Purification of dOSP
A representative 2D-SDS-PAGE fluorograph of conditioned oviductal culture
media fractionated using a heparin-agarose affinity column resulted in a highly purified
preparation of pOSP (Figure 6-1). The 0.4 M NaCl elution showed an 80-85%
purification of pOSP protein from oviductal culture media (Alvarez, Buhi, unpublished).
Porcine OSP is the primary protein product that was resuspended in IVF medium.
Several minor proteins from the oviduct that contain heparin-binding regions or are
complexed with pOSP were found to be co-purified along with pOSP (not shown). The
majority of these minor proteins appear not to be de novo synthesized products of the
oviduct as they are not labeled with [3H]-leucine, suggesting that they are from serum
transudate. Four fractions of pOSP were identified in the purified preparation: pOSP 1-3
previously identified [Buhi et al., 1990] and a fourth protein that concentrated during
purification that can react with pOSP antibody (Buhi, Alvarez, unpublished). The
radiolabeled proteins which are not pOSP (possibly PAI-1, see Chapter 3 fluorograph),
shown in Figure 6-1, do not react with the pOSP antibody [Buhi et al., 1993],
Experiment 1: Effects of pOSP on In Vitro Fertilization
To examine pOSP effects on in vitro fertilization, IVM pig oocytes were
preincubated with various concentrations of pOSP (0-100 pg/ml) and then fertilized in
the presence of pOSP. Effects of pOSP on fertilization responses are shown in Table 6-1.
A pOSP treatment effect (p<0.05) was found for sperm penetration. Concentrations of
pOSP from 0-50 pg/ml had no effect on penetration, while 100 pg/ml of pOSP
significantly decreased sperm penetration of pig oocytes (41%) compared to control

163
(74%) (Table 6-1). A treatment effect (p<0.05) for pOSP also was determined on the
incidence of polyspermy. Concentrations of pOSP from 10-100 pg/ml significantly
decreased polyspermy (24-29%) compared to control (61%) (Table 6-1). No effect of
pOSP was determined on the formation of the male pronucleus. A treatment effect
(p<0.05) with pOSP was also determined for the mean number of sperm (MNS)/oocyte.
A concentration of 10 pg/ml pOSP was selected for all subsequent experiments since this
concentration significantly decreased polyspermy yet maintained penetration rates similar
to control. A photograph of a polyspermic porcine oocyte is shown in Figure 6-2.
Experiment 2: Anti-pOSP IgG Inhibtion of the Decrease in Polvsnermv
To determine whether the decrease in polyspermy was due to a specific effect of
pOSP and not an effector which copurified with pOSP, a polyclonal anti-pOSP IgG was
included during preincubation/IVF. For cross-reactivity see IgG purification in Methods
and Materials. Experiments testing various concentrations of IgG (10, 50, 100, 200 and
400 pg/ml) indicated that concentrations of 200 pg/ml or greater reduced penetration of
oocytes, adversely affecting polyspermy rates (data not shown). Flowever, addition of an
IgG concentration of 50 pg/ml maintained a penetration and polyspermy rate similar to
that of the control. Similar to the previous experiment (Table 6-1), an effect of pOSP
(p<0.05) (Figure 6-3) was found on polyspermy and an interaction was detected for pOSP
and anti-pOSP IgG (p<0.05), indicating that the effect of pOSP on polyspermy is
dependent on the presence or absence of antibody. This result demonstrates that the
pOSP-induced decrease in polyspermy rate was due to a specific effect of pOSP and not
an effector molecule, if any, that co-purified with pOSP. Here, 10 pg/ml pOSP decreased

164
the incidence of polyspermy (23%), while addition of specific pOSP antibody showed a
polyspermy rate similar to that of the control (46%) (Figure 6-3).
Experiment 3: Effects of pOSP on Zona Pellucida Solubility
Effects of pOSP on zona pellucida solubility were examined to determine if
enzymatic digestion of the zona pellucida was altered by pOSP association. When
incubated for 4 hours in the presence of various concentrations of pOSP (0-100 pg/ml),
pOSP concentrations tested failed to alter zona pellucida disestion time (Table 6-2).
Experiment 4: Effects of pOSP on Sperm Binding to the Zona Pellucida
To determine if the decrease in polyspermy in vitro is due to a change in the
number of spermatozoa that are attached to the zona pellucida, pOSP effects on sperm
binding were examined. The number of sperm bound to each zygote/treatment was
evaluated and determined by fluorescent microscopy (Figure 6-5) as detailed in Materials
and Methods. A treatment effect (p<0.05) of pOSP on sperm binding was demonstrated.
The number of sperm bound/zygote was significantly reduced with a decrease in sperm
binding observed with increasing concentrations of pOSP (Figure 6-4). The decrease in
the number of spermatozoa bound to the zona pellucida with increasing concentrations of
pOSP appears to have reached a plateau by 1 pg/ml of pOSP.
Experiment 5; Effects of pOSP on Embryonic Development
To test whether pOSP had an effect on embryonic development in vitro, pOSP
was included during preincubation/IVF alone or during preincubation/IVF + IVC and
both the cleavage rate and development to blastocyst examined. There were no treatment
effects observed on cleavage rates of oocytes fertilized and cultured in the presence of
pOSP (Figure 6-6). There was, however, a treatment effect of pOSP (p<0.05) on

165
development of embryos to blastocysts. A significant increase (p<0.05) in blastocyst
number was found when pOSP was included during preincubation/IVF compared to
control (Figure 6-7). No additional effect of pOSP (1, 10, 50 or 100 pg/ml) added during
IVC could be detected. Higher concentrations of pOSP (50 and 100 pg/ml) during IVC
tended (p=0.08) to decrease the effect observed on development to blastocyst when pOSP
was added during preincubation/IVF, however, the blastocyst development was still
above those of the control.

166
Figure 6-1. Representative fluorograph of [3H]-luecine labeled proteins (500 pg) from
whole oviduct explant-conditioned culture media (Day 0/1) subjected to heparin-agarose
affinity column chromatography and separated by 2D-SDS-PAGE.
Lyophilized proteins were resuspended in IVF media prior to electrophoresis. Porcine
OSP (1-4) are marked by arrows. Molecular weight markers (xlO3) are indicated and the
pH gradient runs from left (pH 8) to right (pH 4)

167
Table 6-1. Effect of pOSP on Fertilization Parameters of Pig Oocytes Matured and
Fertilized In Vitro.
pOSP
(pg/ml)
No.
oocyte
Penetrated
(%)
Polyspermic
(%)
(%) Male
Pronucleus
MNS/
Oocyte
0.0
160
74.4 + 5.2*
61.2 + 4.0*
92.0 ±3.5*
1.9 ±0.11 *
0.1
168
59.4 + 4.8
49.6 + 7.0*
88.0 + 4.3 *
1.8 +0.20 *’b
1.0
176
67.4 + 5.3 *-b
46.6 + 6.8*
91.8 + 1.1 “
1.7 ± 0.16 *-b
10.0
162
63.0 + 5.5 a'b
29.2 + 4.3 b
90.0 + 3.4“
1.4 +0.05 *’b
50.0
166
52.0 + 7.4 *-b
24.0 +3.8 b
95.4 + 2.0*
1.4 ± 0.06 *-b
100.0
178
41.8 ± 8.0 b
26.2 ± 4.8 b
92.6 ±3.5*
1.3 ± 0.07 b
MNS, mean number sperm.
Within a column, values with different superscripts are significantly different (p<0.05).
Values are expressed as means ± SEM of 5 replicates.
The percentage of polyspermlc oocytes, male pronuclei and MNS/Oocyte are calculated
from the number of penetrated oocytes.

168
\
A
b
Figure 6-2. Photograph of polyspermic porcine oocyte matured and fertilized without
exposure to pOSP.
Panel A shows a porcine oocyte with two male pronuclei (arrowhead) and one female
pronuclei (arrow). Panel B is the same oocyte at a different microscopic field showing
the presence of two polar bodies. MPN, male pronuclei, FPN, female pronuclei, PB,
polar body. X 40.

169
Treatment
Figure 6-3. Effect of in vitro incubation of pOSP and anti-pOSP IgG on the polyspermy
rate of porcine oocytes matured and fertilized in vitro.
The concentration of pOSP was 10 pg/ml and 50 pg/ml for the anti-pOSP IgG treatment.
A treatment effect (p< 0.05) and an interaction (p<0.05) was detected for pOSP and anti-
pOSP IgG. Bars indicate the least-squares means ± SEM of 3 replicates. A total of 210
ooyctes are represented for each treatment. Different superscripts indicate significant
differences (p<0.05). The rate of polyspermy was determined from the number of
penetrated oocytes.

170
Table 6-2. Effect of pOSP on Zona Pellucida Solubility of Oocytes Matured In Vitro.
pOSP
(pg/ml)
No.
oocyte
% Oocytes that Underwent Zona Digestion (minutes)
1-3
3-6
6-10
10-20
0.0
35
75.7 + 08
15.3 + 09
6.7 ±6.7
2.3 ±2.3
0.1
34
73.3 + 10
26.6 + 10
0.0 ±0.0
0.0 ± 0.0
10.0
35
80.0+ 10
13.3 ±04
6.7 ±6.6
0.0 ± 0.0
50.0
34
62.0 ±31
31.3 ± 24
6.7 ±6.6
0.0 ±0.0
100.0
35
73.0 ± 16
24.6 ± 17
2.3 ±2.3
0.0 ± 0.0
Values are expressed as means ± SEM of 3 replicates.

171
pOSP concentration (pg/ml)
Figure 6-4. Effect of in vitro incubation of pOSP on sperm binding to porcine putative
zygotes.
Oocytes were in vitro matured and then pre-incubated (4 h) and fertilized (6 h) in the
presence of increasing concentrations of pOSP (0-100 pg/ml). A treatment effect
(p>0.05) was detected for pOSP on sperm binding. Bars indicate the least-squares means
+ SEM of 3 replicates. A total of 45 oocytes are represented for each concentration of
pOSP. Different superscripts indicate significant differences (p<0.05).

Figure 6-5. Photograph of sperm binding to putative zygotes after pre-incubation and
fertilization in the presence of 0 pg/ml pOSP (A) or 100 pg/ml pOSP (B).
Panel B is a representative photograph of the lower end of the range observed for this
treatment (about 14 sperm/zygote). Panel A is a representative photograph of the average
sperm bound/zygote in the control (about 46). X 200.

173

174
80
70
g 60
í 50
CD
Ctl „
a) 40
CD
> 30
CO
CD
8 20
10
1)pOSP
2) pOSP
10
10
1
10
10
10
50
10
100
Figure 6-6. Effect of in vitro incubation of pOSP on cleavage rate using porcine oocytes
matured, fertilized and cultured in vitro.
1) In vitro matured oocytes were pre-incubated and fertilized in the absence (-) or
presence of 10 pg/ml pOSP as described in Materials and Methods. 2) In vitro fertilized
oocytes were then cultured in the absence or presence of pOSP (1-100 pg/ml) during
early cleavage-stage (2- to 4-cell) development. Bars indicate the least-squares means +
SEM of 9 replicates for each treatment. Shown in each bar are the number of oocytes
within each treatment. No treatment effect was detected for pOSP on cleavage rates.

175
0
TO
cc
to
>,
o
o
w
_ro
CQ
D
2)
40
35
30
25
20
15
10
5
0
pOSP - 10 10 10 10 10
pOSP 1 10 50 100
Figure 6-7. Effect of in vitro incubation of pOSP on blastocyst development using
porcine oocytes matured, fertilized and cultured in vitro.
1) In vitro matured oocytes were pre-incubated and fertilized in the absence (-) or
presence of 10 pg/ml pOSP as described in Methods. 2) In vitro fertilized oocytes were
then cultured in the absence or presence of pOSP (1-100 pg/ml) during embryonic
development to the blastocyst. Bars indicate the least-squares means ± SEM of 9
replicates for each treatment. Shown in each bar is the number of oocytes within each
treatment. Differences between treatments were evaluated by orthogonol contrasts and p
values corresponding to these analyses are shown. Blastocyst formation was calculated
as a % of ooyctes inseminated (not cleaved embryos).

176
Discussion
In the present study, it was demonstrated that pOSP administered in vitro
decreased the incidence of polyspermy in pig oocytes matured and fertilized in vitro.
This anti-polyspermic effect of pOSP was inhibited by anti-pOSP IgG, indicating that the
reduction was specific to pOSP and could not be ascribed to an effector molecule that
may have co-purified with pOSP. Polyspermy has remained a persistant problem of pig
oocytes matured and fertilized in vitro, often reaching levels greater than 50% [Wang et
al., 1991, 1994, 1997, Abeydeera and Day, 1997], However, polyspermic fertilization in
vivo is uncommon in pigs mated at the onset of estrus prior to ovulation [Hunter, 1967],
A recent study reported that pig oocytes flushed from the oviduct on Day 2 of the estrous
cycle and subsequently fertilized in vitro, had a much lower incidence of polyspermy
(28%) than oocytes matured and fertilized in vitro (62%) [Wang et al., 1998], Likewise,
oocytes incubated in the presence of cultured oviductal epithelial cells had a decreased
rate of polyspermy [Kano et al., 1994], In addition, when spermatozoa are co-incubated
with oviductal epithelial cells [Nagai and Moore,1991, Dubuc and Sirard, 1995] or
oviductal fluid [Kim et al., 1996] the incidence of polyspermy is reduced significantly.
These reports strongly suggest that an unknown factor of oviductal origin can associate
with either oocytes and/or spermatozoa, and effectively decrease the incidence of
polyspermy. In the pig, the in vivo block to polyspermy is thought to be due to a
restriction in the number of spermatozoa that reach an egg, and the zona pellucida block
to multiple spermatozoa penetration [Hunter, 1990], However, our findings suggest that
a third factor, pOSP synthesized and secreted de novo by the oviductal epithelium, may
be responsible for the in vivo prevention of polyspermic fertilization. Porcine oviduct-

177
conditioned culture media collected from cultured oviducts of periovulatory gilts was
shown to improve in vitro maturation, reduce polyspermy, and increase normal
fertilization rates, while oviduct-conditioned media from oviducts of luteal gilts did not
improve polyspermy or fertilization rates [Vatzias and Hagen, 1999],
Eggs ovulated after luteal phase gonadotrophin treatment show a high incidence
of polyspermy (60.6%) and animals treated with progesterone systemically or by local
microinjection into the oviduct wall during estrus, show elevated levels of polyspermic
fertilization (40% and 32.3%, respectively) [Hunter, 1991], It has been suggested that
under these conditions, the isthmus is more patent allowing more spermatozoa to reach
the site of fertilization. During the luteal phase, pOSP mRNA and protein expression are
at basal levels, and progesterone has been shown to rapidly down regulate pOSP mRNA
and protein synthesis and secretion in ovariectomized gilts [Buhi et al., 1992, 1996],
Thus, the in vivo observations noted, may be due to decreased or an absence of pOSP
synthesis by the progestational oviduct during fertilization. For most mammals studied to
date, in vitro fertilization requires large numbers of spermatozoa to efficiently fertilize a
high percentage of oocytes. In the case of the pig, these large numbers of spermatozoa
lead to a greater incidence of polyspermy [Niwa, 1993], The observation that in vitro
administration of pOSP reduces the rate of polyspermy is an important finding for pig
IVM, IVF and culture of embryos. However, in vivo, where the sperm/egg ratio is very
low [Buhi unpublished, Hunter, 1993], the exact nature of pOSP involvement in the block
to polyspermy becomes less clear.
In the pig, in vitro fertilized oocytes when compared to in vivo fertilized oocytes,
show an incomplete corticol granule exocytosis [Kim et al., 1996], It was proposed that a

178
factor, such as pOSP, might facilitate a more synchronous exocytosis of corticol granule
contents or augment the response of the zona matrix to corticol granule exudate. This
may be one plausible explanation for pOSPs prevention of in vivo polyspermy. However,
recent studies utilizing a different maturation system showed no difference in corticol
granule exocytosis after fertilization in monospermic or polyspermic eggs [Wang et al.,
1998], Thus, it is unknown why the zona pellucida does not respond in vitro as it does in
vivo to corticol granule exudate. Hamster OSP has been shown to associate with
flocculent material in the perivitelline space of oviductal oocytes only after fertilization
[Kan and Roux, 1995], This observation suggested to these investigators that OSP may
interact with the contents of corticol granules and facilitate the block to polyspermy.
Therefore, corticol granule contents may be interacting with pOSP in the zona pellucida
and perivitelline space to modify the ZP receptors and prevent polyspermy.
Oviductal-derived oocytes and embryos have been shown to be more resistant to
in vitro proteolytic degradation than either follicular oocytes or embryos recovered from
the uterine lumen [Broermann et al., 1989], Similarly, the zona pellucida of in vitro
matured oocytes are more susceptible to protease digestion by pronase than non-fertilized
oocytes flushed from the oviduct on Day 2 of the estrous cycle [Wang et al., 1998],
Therefore, an unknown factor of oviductal origin may be involved in providing zona
stability which may also contribute to the functional block to polyspermy. Our results
however, suggest that pOSP may not be this factor, as pOSP had no effect on retarding
protease digestion of the zona. However, an effect of pOSP on the zona pellucida matrix
cannot be ruled out as a titration analysis of various concentrations of pronase was not
examined. The concentration of pronase might be too high to detect subtle differences in

179
modifications of the zona pellucida by pOSP. In addition, this experiment did not include
an in vivo control (oviductal oocytes) for comparison. Future experiments will include
pronase titration analysis and an evaluation of zona hardening after fertilization rather
than before fertilization. Modification in the zona pellucida of oviductal oocytes and
embryos that provide protease resistance is likely due to known and unknown protease
inhibitors of the oviduct, such as PAI-1 and TIMP-1 [Buhi et al., 1996b, Chapters 3-5],
Data in this study indicate that added pOSP decreased the number of
spermatozoa bound to the zona pellucida after in vitro fertilization. This may be another
explanation for the observed decrease in polyspermy. While addition of 0-50 pg/ml of
pOSP did not effect penetration of oocytes, it did significantly reduce the number of
sperm that attach to the zona pellucida. This function may be the result of a physical
block to sperm attachment as pOSP is known to associate with the zona pellucida of
ovulated oocytes [Buhi et al., 1993], Another possibility is that pOSP assists in the
modification of zona pellucida sperm receptors immediately after fertilization in a
process known as "zona hardening" [Hinsch, 1997], Corticol granule exudate might
therefore interact with pOSP in the conversion of ZP2 and ZP3 to ZP2f and ZP3f. These
modified zona receptors appear shortly after fertilization and are cleavage products of
ZP2 and ZP3, resulting in inhibition of sperm binding [Hinsch, 1997], Association of
pOSP with the zona pellucida [Buhi et al., 1993] may have a role in the initial recognition
and binding of spermatozoa by the zona pellucida sperm receptor (ZP1). Porcine OSP,
together with ZP1, may assist in a specific selection mechanism of sperm that are highly
motile (hyperactivated) and capacitated. Results from these studies appear to be
contradictory to findings in the hamster [Schmidt et al., 1997a, 1997b] and human

180
[O'Day-Bowman et al., 1996], These studies found an increase in number of spermatozoa
bound to the oocyte when exposed to OSP. The differences observed between our data
and previous findings may be due to a species-specific difference, purity of OSP, and the
use of in vitro matured and fertilized oocytes in our study.
The in vitro penetration rate of pig oocytes by sperm was not affected by oocyte
exposure to pOSP except at a high concentration, where decreased penetration was
observed. This high concentration of pOSP and association of large amounts of pOSP
with the zona may lead to a physical block of sperm binding. Previously reported data in
other species on the penetration rate vary. In the hamster, partially-purified OSP was
shown to both increase [Boatman and Magnoni, 1995] and decrease [Kimura et al., 1994]
the penetration rate, while in the cow, the penetration rate was found to increase with
OSP [Martus et al., 1998], Differences may be due to means utilized to score penetration
rates. In one study, the penetration rate included sperm found within the perivitelline
space [Boatman and Magnoni, 1995], while another included eggs having completed
meiosis with fused sperm attached [Kimura et al., 1994], When the penetration rate in
the porcine IVF system was low (45-50%), addition of pOSP was observed to increase
the penetration rate (65-75%) (data not shown). Bovine OSP (100 pg/ml) was found to
increase penetration rates in a cow IVF system (about 51% vs 37% in control) when low
concentrations of spermatozoa (0.125 x 106) were used [Martus et al., 1998], By creating
a condition in which the penetration rate was low in the control, an observable increase
on penetration rate in the presence of bovine OSP was found. Thus, OSP may be able to
maintain high penetration rates after decreasing the number of bound sperm to the zona

181
pellucida. The increased penetration rate observed in vitro by Martus et al. (1998) was
due to a specific effect on the oocyte and not the spermatozoon.
This study was not designed to evaluate whether a decrease in polyspermy was
due to an effect on one or both gametes. In most domestic species studied thus far, OSP
has been shown to associate with the zona pellucida [Boice et al., 1990, Weger and
Killian, 1991, Boice et al., 1992, Gandolfi et al., 1991, Murray and Messinger, 1994,
Buhi et al., 1993, Leveille et al., 1987], The association of OSP with spermatozoa is less
clear. Species-specific OSPs were found to bind to bovine [King and Killian, 1994] and
hamster [Boatman and Magnoni, 1995] spermatozoa, but the human OSP did not
associate with human spermatozoa [Reuter, 1994], There is no evidence to date
indicating that pOSP associates with boar spermatozoa. However, incubation of boar
spermatozoa with porcine oviductal epithelial cells decreased the incidence of
polyspermy in this species [Dubuc and Sirard, 1995], Further research will be needed to
clarify species-specific effects of OSP on individual gametes in relation to fertilization
and polyspermy.
Results in this study further demonstrate that pOSP provided a significant
increase in post-cleavage development of embryo to blastocyst. This observed increase
in blastocyst development may be due to decreased polyspermy. The pathological
condition of polyspermy, the penetration of the vitellus by more than one spermatozoa, is
known to be a very early cause of death for the zygote [Beatty 1957, Bomsel-Helmreich
1965, Hunter 1991], However, recent studies indicate that poly-pronuclear pig eggs can
develop normally to the blastocyst stage [Han et al., 1999a] and establish pregnancy [Han
et al., 1999b], The addition of pOSP during IVC had no effect on development to

182
blastocyst, and high concentrations of pOSP tended to decrease the observed effect on
development. Physiologically, in vivo embryos are no longer exposed to oviductal pOSP
after the 4-cell stage that coincides with entry into the uterus in the pig. Therefore,
beneficial effects of pOSP on development to blastocyst may have accrued during
fertilization and early cleavage-stage cell division (1- to 4-cell). In other domestic
species, bovine OSP was found to increase the number of morula and blastocysts on Day
6 but not Day 7 of development [Martus et al., 1998] or had no effect on blastocyst
development [Vansteenbrugge et al., 1997], Similarly, ovine OSP was shown to have no
effect on blastocyst development of bovine embryos [Hill et al., 1997], Thus, a species-
specific effect of OSP is observed. The interpretation of many of these studies becomes
complicated when preparations of OSP are varied. In this study, and those of Schmidt et
al. (1997a, 1997b) and Martus et al. (1998), antibodies specific to that species OSP were
used to eliminate the possibility of observed effects being due to co-purified or
contaminant proteins. No such experiments have been reported in other studies.
In summary, this study indicates that pOSP significantly reduces the incidence of
polyspermy in pig eggs in vitro matured and fertilized. This decrease may be due to a
reduction in sperm binding to the zona pellucida and not a protective proteolytic-
modification of the zona pellucida matrix prior to fertilization. A post-cleavage increase
in development to blastocyst was observed when pOSP was included during
preincubation/IVF, however addition of pOSP during IVC had no synergistic stimulation
on development. These data indicate that pOSP may play an important role in vivo in the
fertilization process including a block to polyspermy.

CHAPTER 7
SUMMARY AND CONCLUSIONS
The mammalian oviduct has long been recognized for its importance in
establishing an optimal environment (pH, temperature, osmotic pressure, nutrients and
oxygen tension) for fertilization and early cleavage-stage embryos. Yet, emerging
evidence suggests that the oviduct may have a more active role in union of gametes,
fertilization, and embryo development. Previous studies have shown that the porcine
oviduct invests energy in the de novo synthesis and secretion of macromolecules into the
oviductal lumen or culture media [Buhi et al., 1997], The majority of these
macromolecules have, until recently, remained unidentified and were characterized in a
limited fashion only by molecular weight and isoelectric point. One unidentified protein,
of 45,000 molecular weight was shown to be the major secretory product of the isthmus
[Buhi et al., 1990],
In Chapter 3, the identification of this unknown isthmic protein was described. A
96% sequence identity was found between this isthmic protein and porcine PAI-1, as
determined by N-terminal amino acid sequence analysis. Immunoprecipitation and
western blotting further confirmed the identity of this protein. PAI-1, a member of the
serpin family of serine protease inhibitors, is the primary inhibitor of the proteases, uPA
and tPA. Hence, this protein most likely has an integral role in the regulation of ECM
remodeling/degradation and fibrinolysis. Based on previous studies of its activity, the
183

184
primary role of PAI-1 in the oviduct should be to regulate PA activation of plasminogen.
Both tPA and uPA initiate proteolysis by converting plasminogen to the broad-spectrum
enzyme plasmin. Plasmin is responsible for the degradation of fibrinectin and laminin
within the ECM and is also capable of activating promatrix metalloproteinases (MMPs).
When evaluating PAI-1 function within the oviduct, consideration must be given to the
fact that this protein is one inhibitor of a proteolytic cascade leading to the activation of
numerous enzymes.
With the identification of PAI-1 in the oviduct, our next objective was to
characterize this protein for potential insights into its function. PAI-1 has been shown to
be produced by a number of tissues and cells in culture [Andreasen et al., 1990], Chapter
3 describes the localization of PAI-1 within the pig oviduct. Although this protein is
primarily a secretory protein of the isthmus, it can be localized to epithelium of all three
segments of the oviduct. Immunocytochemistry revealed that PAI-1 was heavily
concentrated in the apical region of epithelium, which suggested possible release into the
lumen. No differences could be detected in the localization of PAI-1 between pregnant
and non-pregnant animals indicating that its presence in the oviduct was not dependent
on the presence of an embryo. PAI-1 was found to be associated with putative secretory
granules and cilia of the isthmic epithelium supporting the proposal that this protein is
packaged into vesicles for exocytosis and released into the lumen. Localization of PAI-1
in the lumen, near the apical membrane and associated with cilia, may act to: 1) prevent
embryonic invasion of oviductal ECM due to oviductal or embryonic PA activity, 2)
maintain gamete transport by preventing adhesion of embryos to oviductal fibrin deposits

185
and 3) regulate the cyclic remodeling of porcine oviductal epithelium in response to the
changing steroid environment.
To understand PAI-l's role in relation to fertilization and early cleavage-stage
embryo development, expression of PAI-1 mRNA and protein were evaluated during
early pregnancy. In Chapter 4, it was shown that synthesis and secretion of PAI-1 protein
into culture media varies during early pregnancy (Days 0, 2 and 5). PAI-1 in the Large
White was shown to be greatest on Day 2, coinciding with the time of fertilization. The
Meishan, numerically showed a greater secretion on Day 0 of early pregnancy, although
this value was not significantly different from Day 2. It is unknown why the Meishan
would have a greater secretion of PAI-1 on Day 0; possibilities are highlighted in the
discussion of Chapter 4. Interestingly, PAI-1 mRNA collected during various days of the
estrous cycle in crossbred gilts, were greatest on Day 2 in whole oviduct and isthmic
segments. Therefore, expression of PAI-1 mRNA and protein are greatest at the time of
fertilization and early embryonic development. Huarte et al. (1993) suggests that there is
an important, yet undefined, role for the plasminogen/plasmin system during fertilization.
It may be that the elevated expression of PAI-1 at this time contributes to the regulation
of this proteolytic cascade. If the proteolytic activity of certain oviductal enzymes, uPA
and/or tPA, are not controlled, the zona pellucida matrix could possibly destabilize
leading to the possible death of the oocyte or embryo. PAI-1 has a similar mRNA and
protein expression to a previously identified metalloproteinase inhibitor of the oviduct,
TIMP-1 [Buhi et al., 1996], The spatial (isthmus) and temporal (Day 2 of early
pregnancy) expression of these two inhibitors suggests the importance for some

186
regulatory measure of proteolysis during fertilization and early cleavage-stage embryonic
development.
In the ovary and uterus, PAI-1 has been shown to be regulated by steroids,
estrogen and progesterone, and by gonadotropins [Ny et al., 1993, Schatz and Lockwood,
1993], Chapter 4 describes the regulation of PAI-1 by ovarian steroids in the oviduct.
PAI-1 mRNA expression was shown to be stimulated by progesterone while estrogen
inhibited this progesterone-mediated effect. PAI-1 protein expression was shown to be
inhibited by estrogen, however, the combination of estrogen and progesterone abrogated
the effect of estrogen on PAI-1 secretion. In the cycling gilt, estrogen peaks between
Days 18-19 and reaches basal levels by Day 1. Therefore, the relief of estrogen
inhibition on Day 2 appears to initiate expression of PAI-1 mRNA and protein.
An important question addressed in Chapter 5, "Does oviductal PAI-1 retain
biological activity"? It was observed that the majority of PAI-1 was released into culture
media in the inactive "latent" form which could be reactivated by denaturants.
Denaturant-activated PAI-1 was active functionally and it could: 1) form a complex with
uPA 2) be cleaved by uPA in its reactive center and 3) inhibit uPA activity. Additionally,
inhibition of uPA activity by PAI-1 was found to be dose-dependent. This evidence
suggested that PAI-1 was indeed functionally active once released from the oviductal
epithelium. It may be that PAI-1 is stabilized by binding to the proteins in the oviductal
or zona ECM.
To evaluate the physiological amount of PAI-1 and PA activity that may be
present during early pregnancy, oviduct flushes were obtained on various days (0, 1, 2, 5,
8, 10 and 12). Chapter 5 showed that PA activity was greatest on Day 2 of early

187
pregnancy. Further examination of this activity using a specific inhibitor of uPA,
amiloride, revealed that the majority of this activity is uPA. However, tPA activity may
also be present. Use of anti-tPA would be required to confirm this suggestion. Oviductal
PAI-1 was also found to be greatest on Day 2 of early pregnancy. In review of these
data, it appears that PA activity, PAI-1 mRNA and protein (from explant culture media
and oviduct flushes) are elevated during the time of fertilization in the pig.
In light of these results, we suggest that PAI-1 may interact with the oocyte or
sperm prior to and during fertilization. To examine this hypothesis, ovulated oviductal
oocytes or embryos were collected and evaluated by immunogold electron microscopy.
Results of this study are detailed in Chapter 5. PAI-1 was associated with oviductal
oocytes and embryos in a similar fashion. The outer rim of the zona pellucida showed
intense labeling with PAI-1 while the inner zona, perivitelline space and oocyte or
embryo showed moderate levels of PAI-1 localization.
Follicular oocytes did not show the intense labeling of PAI-1 on the outer portion
of the zona, indicating that in oviductal oocytes or embryos, PAI-1 was probably
obtained from oviductal secretion. Cumulus cells surrounding follicular oocytes were
intensely labeled with gold particles, especially in the nuclear region. PAI-1 and PAI-2
mRNA have both been described in bovine cumulus cells [Piquette et al., 1993], It may
be that PAI-1 and/or PAI-2 secretion by cumulus cells protects the preovulatory oocyte
from proteolytic degradation due to increased levels of PA at ovulation that are believed
to be significant contributors to rupture of the follicle wall through proteolysis of the
collagen framework.

188
Embryos collected from the uterus on Day 4 of early pregnancy (8- to 16-cell
stage) show a similar localization of PAI-1 as did oviductal oocytes or embryos (2- to 4-
cell). Because the isthmus is the primary source of PAI-1 synthesis and secretion, we
hypothesized that the cleavage-stage embryo would sequester PAI-1 from its immediate
environment during its transit into the uterus. It cannot be excluded, however, that PAI-1
associated with the uterine embryos is not in part derived from the uterus. One
preliminary experiment, not reported here, indicates that PAI-1 may be a primary de novo
synthesized and secreted product of the Day 4 pregnant porcine endometrium.
Another interesting observation of this study was that PAI-1 associated with the
head region of boar spermatozoa when attached to the zona pellucida. Vitronectin, which
binds PAI-1, has been shown to be associated with human sperm membranes and is
released upon initiation of the acrosome reaction [Fusi et al., 1994], We propose that
oviductal PAI-1 may be binding to vitronectin once released from sperm cells thus
stabilizing PAI-1 activity PAI-1 bound to vitronectin is strategically placed to have an
important role in the fertilization process.
The association of PAI-1 with oviductal oocytes and embryos may therefore,
prevent: 1) proteolytic degradation of the zona pellucida, 2) premature hatching due to
inherent PA activity of the embryo, or 3) adhesion to oviductal epithelium. Additionally,
PAI-1 may function in sperm recognition/association, binding and penetration of the zona
pellucida.
The second aspect of this proposal was to examine the functionality of a well-
characterized porcine oviduct protein, pOSP, during in vitro fertilization and culture. In
porcine IVF, polyspermy and low blastocyst development (about 30%) remain a major

189
impediment to the successful culture of pig embryos [Funahashi and Day, 1997], Several
investigators have suggested that oviductal constituents, such as pOSP which are absent
in the artificial culture system, may reduce polyspermy and enhance blastocyst
development [Hunter, 1991, Dubuc and Sirard, 1995, Kim et al., 1996, Wang et al.,
1998], Chapter 6 describes the results of these experiments. Addition of pOSP to IVM
oocytes prior to and during fertilization showed a significant decrease (62% vs 24-29%)
in the incidence of polyspermy. Several concentrations of pOSP were found to decrease
the rate of polyspermy, and yet did not affect the penetration rate. Higher concentrations
of pOSP, however, decreased the penetration rate. This decreased incidence of
polyspermy when oocytes are fertilized in the presence of pOSP could be blocked when
co-incubated with anti-pOSP IgG. These results indicated that the observed effect on
polyspermy was specific to pOSP and not a co-purified product.
The decreased incidence of polyspermy in oocytes incubated with pOSP was
possibly due to an effect on sperm binding. Oocytes exposed to pOSP show decreased
numbers of sperm bound to the zona pellucida when compared to controls. Porcine OSP
has been shown to associate with the zona pellucida; hence this protein may act as a
physical barrier to the binding of multiple spermatozoa. However, there are other
possible explanations for this result. One is that pOSP interacts with corticol granule
contents after fertilization occurs, leading to a more efficient block of multiple sperm
penetrations. This may occur through modifications of the ZP3 and ZP2 receptors
causing the release of spermatozoa from their binding sites and inhibition of subsequent
sperm attachment. In the hamster, OSP becomes associated with flocculent material in
the perivitelline space only after fertilization and is suggested to have a role in the block

190
to polyspermy [Kan and Roux, 1995], Another interesting observation of these
experiments was that addition of pOSP to IVM oocytes prior to and during fertilization,
increased development to blastocyst stage. This was a post-cleavage (2- to 4-cell)
phenomenon and may be the result of a decreased incidence of polyspermy. Polyspermy
is known to cause the demise of early preimplantation embryos [Beatty, 1957, Bomsel-
Helmreich, 1965, Hunter, 1991], Addition of pOSP, at several concentrations, during
IVC had no synergistic stimulation on embryonic development. This suggests that the
beneficial effects of pOSP are accrued during fertilization and not cleavage-stage
development.
Chapters 3-6 have detailed an active role for the pig oviduct in the synthesis and
secretion of proteins that may, through their association with the oocyte or embryo,
facilitate the process of fertilization and protect the embryo in a proteolytic rich
environment. A developing model of proteins of the pig oviduct is shown in Figure 7-1.
Several de novo synthesized and secreted proteins of the oviduct have been identified
including; PAI-1, TIMP-1, pOSP, clusterin, IgA, procollagen, and Complement C3.
Circulating ovarian steroids, such as estrogen, may act to control the expression and
release of these proteins in cycle-dependent and stage-specific patterns. Once released
into the lumen, these proteins can interact with the oocyte, spermatozoa, or
preimplantation embryo and affect their biology. Potential targets include fertilization
and cleavage-stage development of oviductal embryos. Further examinations of these
proteins will help to elucidate specific roles of the oviduct that may contribute to the
previously mentioned processes.

Figure 7-1. Model for the de novo synthesis and secretion of proteins by the porcine
oviduct.
Several de novo synthesized proteins of the porcine oviduct have been identified by N-
terminal amino acid sequence analysis or western blotting including; 1) procollagen, 2)
clusterin, 3) IgA, 4) complement C3, 5) porcine oviduct-specific secretory glycoprotein
(pOSP), 6) tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and 7) plasminogen
activator inhibitor-1 (PAI-1). Also several growth factors and cytokines have been
immunolocalized to the epithelium or measured in oviduct flushes by radioimmunoassay.
Some of these de novo synthesized proteins (PAI-1, TIMP-1 and pOSP) have been shown
to be regulated by ovarian steroids, such as estrogen, and have stage-specific, regional-
specific, and cycle-dependent expression of their mRNAs. These proteins depicted may
interact with the oviductal oocyte, spermatozoa, or early embryo and facilitate the
processes of fertilization and early-cleavage stage embryonic development.


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BIOGRAPHICAL SKETCH
Andrew J. Kouba was bom in Omaha, Nebraska, on December 13, 1968. He
earned a Bachelor of Science degree at Northwest Missouri State University (NWMSU)
with two majors, wildlife ecology/conservation and zoology. After completing his
undergraduate degree at NWMSU, Andrew earned a Master’s degree in animal
physiology at Clemson University, Clemson, South Carolina, in 1995 with a thesis
entitled "Insulin-like Growth Factor-1 (IGF-1) in Bovine Seminal Plasma and its
Receptor on Spermatozoa,” under the guidance of Dr. Don Henricks. He then joined Dr.
Bill Buhi's laboratory in August 1995 as a Ph D student in the Department of Animal
Sciences and the Interdisciplinary Concentration in Animal Molecular and Cell Biology
at the University of Florida and completed the requirements for the degree of Doctor of
Philosophy in December 1999. He will pursue postdoctoral research with Dr. Terri Roth
at the Center for Reproduction of Endangered Wildlife (CREW) at the Cincinnati Zoo,
Cincinnati, Ohio, beginning October 1999.
220

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adecúate, ¡í scope~and-qyalit;
as a dissertation for the degree of Doctor of Philosophy.
William C. Buhi, Chair
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, is scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Frank A. Simmen
Professor of Animal Science
I certify that 1 have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, is scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Professor of Animal Science
Peter J. Hansen
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, is scope and quality,
as a dissertation for the degree of Doctor of Philosophy. /
William W. Thatcher
Graduate Research Professor
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 fully adequate, is scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
/ Kenneth C. Drury *
Assistant Professor
Obstetrics/Gynecology and
Endocrinology

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
December 1999
Dean, College of Agriculture
Dean, Graduate School

LD
1780
1991
K5?g
UNIVERSITY OF FLORIDA
3 1262 08556 6072



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