Bovine trophoblast protein-1

MISSING IMAGE

Material Information

Title:
Bovine trophoblast protein-1 chemical characteristics and antiluteolytic effects on uterine and ovarian function
Physical Description:
x, 200 leaves : ill. ; 28 cm.
Language:
English
Creator:
Helmer, Stephen Dean, 1959-
Publication Date:

Subjects

Subjects / Keywords:
Trophoblast   ( lcsh )
Estrus   ( lcsh )
Cows -- Reproduction   ( lcsh )
Corpus luteum   ( lcsh )
Animal Science thesis Ph.D
Dissertations, Academic -- Animal Science -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 175-199)
Statement of Responsibility:
by Stephen Dean Helmer.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001151384
oclc - 20509982
notis - AFQ1301
sobekcm - AA00004807_00001
System ID:
AA00004807:00001

Full Text












BOVINE TROPHOBLAST PROTEIN-i:
CHEMICAL CHARACTERISTICS AND ANTILUTEOLYTIC
EFFECTS ON UTERINE AND OVARIAN FUNCTION















By


STEPHEN DEAN HELMER


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


1988
















ACKNOWLEDGEMENTS


The author wishes to express his sincere appreciation to the

chairman of his advisory committee, Dr. William W. Thatcher, for his

continuous guidance, support and most importantly, his friendship

throughout his doctoral program. Special thanks to Dr. Peter J.

Hansen for insight and friendship. He also expresses thanks to the

remaining members of his advisory committee Drs. Fuller W. Bazer and

William C. Buhi for their assistance during various phases of the

program of study.

Further appreciation goes to Dre R. Micheal Roberts for invaluable

guidance during the author's first two years of study, and to Dr.

Russel V. Anthony for advice, assistance and constant friendship.

Special thanks go to Austin Greene, Dale Hissem and Tom Bruce for

invaluable assistance in managing and making available cattle for the

study* For their willingness to extend to the author their clinical

expertise, thanks are extended to Drs. Martin Drost and Scott Norman.

The author also thanks Dr. Timothy Gross, Jesse Johnson, Mary

Ellen Hissem and Leslie Smith for their expert technical advice and

assistance. Thanks also go to Mri Larry Eubanks for use of the

slaughter facilities.

For their continual support and freindship the author extends

special thanks to the graduate students and postdoctoral fellows who









are too numerous to acknowledge individually, but made the author's

graduate program memorable In particular, thanks go to John

McDermott, Jeffery Knickerbocker, D. James Putney, Lokenga Badinga,

Deanne Morse, Matt Lucy, Jerry Malayer and Claire Plante, whom the

author feels privaledged to have known.

The author expresses sincere appreciation to his parents, Dean P.

Helmer and Marilyn T. Helmer, and to Mr; and Mrs. Frederick C. Pareis

for there continued love and supports

Special thanks and appreciation are expressed to the author's

wife, and best friend, Judith P. Helmer, for her unending love and

support
















TABLE OF CONTENTS


ACKNOWLEDGMENTS...... ........................ 4 .............1i

LIST OF TABLES........ .... .......... .,........., .......

LIST OF FIGURES. .. ..,,*.. w-.;.. ,**. i ..,.... .....r4.,, ..,vii

ABSTRACTs.,. ... ... ,...,........ ..,. ... ,.,,,. .... ix

CHAPTERS

I LITERATURE REVIEW....,... d i r.., ,..,.,..,v.; ...*...1

Introduction. ;. -., ..<....* .. ....,*.,... ......4 .....,, .. 1
The Bovine Estrous Cycle.... ... ,,. .....c... ...... .rz
Physiology and Endocrinology of the Ovary............, .....3
Uterine Regulation of CL Lifespan During
the Estrous Cycle.....*... ... ..,,...,...... ..v.29
Uterine Regulation of CL Lifespan During Pregnancy.........39
Development of the Bovine Conceptus ..v..-....e,...... ..43
Timing of the Conceptus Signal Relative to
"Maternal Recognition of Pregnancy".-,.....*s,. .,,....,.44

2 IDENTIFICATION OF BOVINE TROPHOBLAST PROTEIN-I,
A SECRETARY PROTEIN IMMUNOLOGICALLY RELATED
TO OVINE TROPHOBLAST PROTEIN-1 v ......,.., ...... *. ...66

Introductions. .......... ...,,.t, .w.o......l.... ....... 66
Materials and Methods. ... ..v..... i..., o. ,..., 68
Results... : .. ..'~ ... ,..... v ,ve.. ww .. .,, ,., i. ~ ., v .74
Discussion. ...... .. .,i... ., .. ,... .... .. e*,i ...V W 82

3 DIFFERENTIAL GLYCOSYLATION OF THE COMPONENTS OF
THE BOVINE TROPHOBLAST PROTEIN-1 COMPLEX.... ....v. 87

Introduction a w v e ,*.vr w d ,..... ..o, ww v '.t.wi *..wr.. .87
Materials and Methods-.c.... ,w, .v i ,re. .. Sv *. ,,l,,-88
Results.....* ... .., *... ...*., .. .--?* .. *..,,92
Discussion.;. 6 s .*vi.* ..*sw-.,'v .. ,,. 98










4 INTRAUTERINE INFUSION OF PURIFIED BOVINE
TROPHOBLAST PROTEIN-1 COMPLEX EXERTS AN
ANTILUTEOLYTIC EFFECT AND EXTENDS CORPUS
LUTEUM LIFESPAN IN CYCLIC CATTLE,.....,,,.+v.,....v103

Introduction......... ; .., .. i,, t. ., ... ., *..v ...... 103
Materials and Methods ... 6.vw.. ... .. r i. .. ,-..,104
Results i... ..* ..,.s. .. v, v.1. .ii, ..r r, 116
Discussion ........., t. .i ; Y i.*< *.v 139

5 BOVINE TROPHOBLAST PROTEIN-I COMPLEX ALTERS
ENDOMETRIAL PROTEIN AND PROSTAGLANDIN SECRETION
AND INDUCES AN INTRACELLULAR INHIBITOR OF
PROSTAGLANDIN SYNTHESIS IN VITROf.. .v. ..,r ....... .142

Introduction4... ,, *.. .... ,i. .. .. .. .., r .. .. 142
Materials and methods.. v ...v *~ ,;,....... .. ,. w 144
Results. ..... ,* v. o.*-'r.v. v..-. .,. -..,.v ..,. .. 152
Discussion.*... .. ... .... ;4 i i. i .. ..* i s.. : i. .. 158

6 GENERAL CONCLUSIONS *.. ....... ... .......,, .s ... 163

REFERENCES .. .,.. .e..... ... v.. ... .. ..- .. .., .. 175

BIOGAPHICAL SKETCH. .*u,.4 g ... ..*.. ri.. ..** ....,i W. ....200
















LIST OF TABLES


Table Page

4-1 Percent incorporation of [3H]leucine into nondialyzable
protein and protein content for the first, second,
third and fourth 24 h culture periods of conceptuses........117

4-2 Least square means + sem for characteristics of estrous
cycles for cows treated with BSA, bCSP or bTP-1 complex .....i31

4-3 Least squares means + sem of plasma PGF concentrations
and residual variances for BSA, bCSP and bTP-1 treated
cattle... ..............,.. ................................. 136

5-1 Analysis of variance for incorporation of [3H]-leucine
into secretary proteins by endometrial explants treated
with BSA, bCSP or bTP-1 complex.......... ..... ......... 150

5-2 Analysis of variance for PGF and PGE in medium for
endometrial explants incubated with BSA, bCSP or bTP-1......151

5-3 Least squres means of concentrations of PGF and PGE
secreted into medium of day 17 endometrial explants
incubated for 24 h with medium containing 4.8 pg
BSA/ml, 12.7 pg bCSP/ml or I 1g bTP-1/ml. ..... ... .......153

5-4 Least squares means for incorporation of [3H]-leucine
into TCA-precipitable macromolecules in medium (dpm/250
mg/15 ml) and tissue (dpm/250 mg/24 h) from endometrial
explants incubated for 24 h with medium containing no
BSA, 4.8 Pg BSA/ml, 12.7 pg bCSP/ml or 1 yg bTP-1/ml........ 157
















LIST OF FIGURES


Figure Page

2-1 Ouchterlony double-immunodiffusion analysis of concepts
secretary proteins from (a) sheep and (b) cattle. ... ,,....i75

2-2 Ouchterlony double-immunodiffusion analysis of bovine and
ovine concepts secretary proteins ..,vS.- CC.nss*-.76

2-3 Solid phase radiobinding assay of concepts secretary
proteins i;%.. WV'. 0 ;. .*W* #I. .**> ii64 svi* ;. ip e uwl I .* .'i Y q.i. P i*77

2-4 Analysis of immunoprecipitates from concepts secretary
proteins of cows and sheep by one-dimensional
polyacrylamide gel electrophoresis and fluorography. ,.. .. i .v79

2-5 Analysis of bovine CSP by two-dimensional polyacrylamide
gel electrophoresis and fluorography. .. ....v .e........,81

2-6 Electrophoretic analysis of cell-free translation products
of RNA isolated from cattle conceptuses;r.........v........*83

3-1 Fluorograph of SDS-PAGE of concepts supernatants from a
concepts cultured in medium containing 100 pCi [3H]
glucosamine ........... .. ....W .. .... ...... ..0. ..... .^. ..v93

3-2 Fluorograph of SDS-PAGE of concepts supernatants from
conceptuses cultured in the presence (lanes 2, 5, 6) or
absence (lanes 1, 3, 4) of tunicamycin for 24 hd-.iv...i..?s .* 95

3-3 Fluorograph of SDS-PAGE of concepts supernatants from
conceptuses cultured in the presence (lanes 2, 5, 6) or
absence (lanes 1, 3, 4) of DMM for 24 h.,,......,..96

3-4 Fluorograph of SDS-PAGE of concepts proteins treated
with Endo H. .r ..,,.... .. .1. .(. ...* .. ... 97

3-5 Conconavalin A-Sepharose 4B lectin chromatography of


4-1 HPLC gel filtration profiles of radiolabelled conceptus-
conditioned medium from the first, second, third and
fourth days of culture of a conceptus......*..w.s ,v,..,,.o.119


vii









4-2 Fluorograph of electrophoretogram of bCSPs subjected to
40% or 50% saturated ammonium sulfate (SAS) precipitation....122

4-3 Fluorograph of electrophoretogram of bCSP after
separation by HPLC gel filtration..., ; ....... ......,.*.l24

4-4 Silver-stained, two-dimensional electrophoretogram of
highly purified bTP-1 complex (16 jg, top left panel);
a gel which had been run with sample buffer, but no
protein (top right panel); total array of bCSPs (100 jg,
bottom left panel and bovine serum albumin (20 yg,
bottom right panel)..e..,. ...,*,..,..4... .. ..... 127

4-5 Autoradiograph of 2-D SDS-PAGE of purified bTP-1 complex
(8 ig/ml) transferred to nitrocellulose and immunoblotted
with a) rabbit anti-oTP-1 antiserum (1:100) or b) normal
rabbit serum.v.-... i... w..,t..6. cv......... ,...,. .... .,130

4-6 Progesterone profiles of cattle receiving intrauterine
infusion of bovine serum albumin (VSA, top panel); bCSP
(middle panel) or purified bTP-1 complex (bottom panel)
from days 15,5 to 21 of an estrous cycle...... b.,.........134

4-7 Representative profiles for plasma PGF of a BSA, bCSP,
and bTP-1 treated cow sampled every 15 min on day 19
after estrus ......., ...v.... W'OV.. W. .7..d ,V .,* I .V .... i 138

5-1 Least squares means (pooled sem=020) for PG synthesis
by the prostaglandin generating system in the presence of
cytosolic supernatants from day 17 endometrial explants
which had been treated with no BSA, 4;8 lg~BSA/ml,Pl2v7 g
bCSP/ml or 1 jg bTP-L/ml for 24 h.*..,............,. ..... ..155

6-1 Proposed model for differential glycosylation of the
components of the bTP-1 complex.... ,.... .......... 168

6-2 Proposed model for the bovine antiluteolytic pathway
during early pregnancy.-.. .... .... ......... ...... ..,173


viii
















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

BOVINE TROPHOBLAST PROTEIN-I:
CHEMICAL CHARACTERISTICS AND ANTILUTEOLYTIC
EFFECTS ON UTERINE AND OVARIAN FUNCTION

by

STEPHEN DEAN HELMER

AUGUST 1988

Chairman: William W. Thatcher
Major Department: Animal Science

The concepts must "signal" its presence to extend functional

lifespan of the corpus luteum (CL), a requirement of pregnancy

maintenance. Conceptus signals in both the ewe and cow are

proteinaceous in nature. In sheep this signal is ovine trophoblast

protein-1 (oTP-1). The antiluteolytic mechanisms for the ewe and cow

are similar because reciprocal transfer of extraembryonic membranes

into the uterus of sheep and cattle extend CL function in some cases.

However, the putative bovine antiluteolytic signal has not been

isolated. The research described in this dissertation was carried

out in an attempt to extend our understanding of concepts mediated

antiluteolytic mechanisms in the cow.

Components of bovine and ovine concepts secretary proteins (bCSP,

oCSP) cross-react with antiserum directed against oTP-1 The

immunologically cross-reactive components of bCSP (7 variants) are

defined as the bovine trophoblast protein-1 complex (bTP-1). This

ix


___________ _____









complex differs from oTP-1 in size (22 to 26 kDa vs.' 19 kDa) and

isoelectric points (pI 6.5-6.7 vs. 5.3-5.7). The size differences

result from the fact that bTP-1, unlike oTP-1, is glycosylated.

The 22 and 26 kDa species of bTP-1 were high-mannose and complex-type

glycoproteins, respectively. The deglycosylated form of bTP-1

migrated as an 18 kDa species during electrophoresis, a value close

to molecular weight estimates of oTP-1.

Antiluteolytic effects of bTP-1 were examined. Interestrous

intervals were longer for cyclic cows receiving intrauterine

infusions of bTP-1 compared to bCSP or BSA. A tendency toward

attenuated uterine PGF secretion was noted with a significant

decrease in residual variance in samples of bTP-1 treated cows

because luteolytic-type pulses of PGF were diminished.

In a separate experiment, bTP-1 and bCSP attenuated PGF release by

endometrial explant cultures and induced an intracellular inhibitor

of PG-synthesizing enzymes. Protein secretion also was decreased by

bTP-1 and bCSP treated explants.

Collectively, these data indicate that bTP-1 is the antiluteolytic

component of bCSP. This concepts signal regulates PGF secretion by

inducing an inhibitor of PG synthesizing enzymes in cattle to allow

for extension of CL function and associated progesterone secretion*
















CHAPTER 1
LITERATURE REVIEW



Introduction

The cow is afforded an opportunity to become pregnant every 21

days. This opportunity results from the release of an ovum from the

ovary at a time favorable for fertilization and subsequent

development in utero. The sequence of events leading up to and

including ovulation are regulated by complex mechanisms and have been

studied extensively in the past. Recently, there has been a greater

understanding of the mechanisms of intercommunication between the

concepts and maternal unit. That the concepts must "signal" the

maternal unit or uterine environment is evident because CL

regression and recurrent estrous cycles continue in the absence of

pregnancy. The nature of this concepts derived, antiluteolytic

"signal" appears to be proteinaceous in nature. However, its

identity, biological characteristics, and mechanism of action are not

fully understood. The research described in this dissertation was to

develop a greater understanding of the nature and mechanism of action

of these putative concepts "signals" in initiating the sequence of

events leading to CL maintenance and successful establishment of

pregnancy.









2

The Bovine Estrous Cycle

Cattle display a periodicity of sexual behavior which has formed

the basis for an exhaustive quest for knowledge. This period of

sexual receptivity, known as estrus, is a recurrent event and

delineates the boundaries of time referred to as the estrous cycle.

Observations of the cyclic nature of domestic cattle were made as

early as 1876 by Wallace (as cited by Marshall, 1922). The estrous

cycle, or interestrous interval, averages 21 days (Hansel et al.,

1973). Cattle display behavioral signs of estrus for an average

period of 16.9 + 4.9 hours (h) (Schams et al., 1977), but variability

between and within breeds can be high (for reviews, see Wishart,

1972). Estrous cycle lengths may be more repeatable on an individual

animal basis. Wishart (1972) reported that individual animal

variation was less than 2 days in 77.3 percent of 211 estrous cycles.

Chapman & Casida (1937) reported repeatability of estrous cycle

lengths to be 0.41. In a different study, the repeatability

estimate, described as the regularity of the occurence of estrus, was

0.18 (Pou et al., 1953).

The estrous cycle can be conveniently divided into four phases:

proestrus, estrus, metestrus and diestrus. The behavioral, endocrine

and biochemical changes which occur during the estrous cycle are

regulated by complex interactions of the hypothalamus, pituitary,

ovary, and uterus. This complex process results in release of a

fertilizable ovum, which may establish itself in utero and result in

the birth of young. An understanding of these processes might be

best obtained through a discussion of follicular development.












Physiology and Endocrinology of the Ovary

Dynamics of Follicular Growth

Two endocrine structures relevant to reproductive physiology are

found on the ovary, 1) the follicle and 2) corpus luteum (CL).

Follicles are present at all stages of the estrous cycle (Matton et

al., 1981) and can be classified according to their degree of

development. The following section on follicle development to the

mature Graafian follicle is based upon hisological observations of

Rajakoski (1960), Lobel & Levy (1968) and Marion et al. (1968). They

arise as primordial follicles which consist of the ovum and one layer

of epithelial cells. The primordial follicles comprise the pool of

all follicles present in the ovary at birth. These are depleted as

individual follicles are recruited to grow (Marion & Gier, 1971).

Once stimulated to grow, the cells surrounding the ovum, now referred

to as granulosa cells, become cuboidal in shape and are known as

primary follicles. Once mitosis of granulosa cells surrounding the

ovum results in formation of several cell layers, it is designated a

secondary follicle. During this time, the zona pellucida is formed

and vascularization of the stroma surrounding the granulosa and basal

lamina occurs. Further development to tertiary follicles is

characterized by formation of a fluid-filled antrum within the mass

of granulosa cells surrounding the ovum. As fluid accumulation

proceeds, internal and external thecal cell layers become more

organized and the ovum, surrounded by the corona radiata cells,

becomes suspended by the cumulus oophorus granulosa cells within the










4

developing antrum. Mature antral follicles, generally greater than

10 mm in diameter, are designated as Graafian follicles.

It is believed that mature Graafian follicles arise as the result

of waves of follicular development. Dynamics of the process of

follicular recruitment, atresia and ovulation have been extensively

reviewed (Rajakoski, 1960; Choudary et al., 1968; Dufour et al.,

1972; Matton et al., 1981, Ireland & Roche, 1983a,b; Ireland, 1987).

Early observations as to the timing of follicular waves yielded

conflicting results. Rajakoski (1960) suggested that there were two

"waves" of follicular growth during the bovine estrous cycle. The

first of these was initiated on day 3 of the cycle and ended at mid-

cycle with the development of an ovulatory sized follicle which

eventually underwent atresia. A second wave of development begins

around mid-cycle and ends in the formation of the preovulatory

Graafian follicle. Contrary to these findings, Choudary et al.

(1968) and Marion & Gier (1971) reported that follicular growth was

continuous and independent of the phases of the cycle. They found

that normal follicles greater than 5 mm in diameter were only present

during the follicular phase. Large atretic follicles were present at

all times in the follicular and luteal phases.

The biphasic theory of follicular development of Rajakoski (1960)

has been supported by results of several other laboratories (Dufour

et al., 1972; Matton et al., 1981; and Pierson & Ginther, 1984).

Utilizing ultrasound to characterize follicular development, Pierson

& Ginther (1984) reported that mean number of follicles among days

differed for the 4-6 mm and greater than 10 mm categories. These










5

differences appeared to be due to: 1) an increase in 4-6 mm follicles

at early diestrus which grow to ovulatory size and regressed at mid-

diestrus and 2) an accelerated growth of the follicle destined to

ovulate four days prior to ovulation. More recently, evidence for

existence of three waves of follicular growth and selection have been

reported (Fortune et al., 1988). Sirois & Fortune (1988), using

ultrasonography, found that 7 of 10 heifers studied had three waves

of follicular development with the third resulting in ovulation. The

first two began on about days 1.9 and 9.4 with the ovulatory wave

beginning on day 16.1.

Growth rates of follicles have also been studied (Lussier et al.,

1987). They found that growth rate varied with follicular size.

Follicle growth from 0.13-0.67 mm, 0.68-3.67 mm and 3.68-6.50 mm

required 27, 6.8, and 7.8 d, respectively. The entire process (that

period of development from recruitment to attainment of ovulatory

size) requires a period of time equivalent to two estrous cycles.

The relatively slow growth of small follicles suggests a selective

control over their growth. This leads to a discussion of follicle

dominance as related to the hormonal control of follicular

development. The rapid rate of development for preovulatory sized

follicles has lead to a further understanding of follicular dynamics.

Rapid replacement of large follicles has been noted by several

laboratories (Dufour et al., 1972; Matton et al., 1981; Lussier et

al., 1987). Dufour et al. (1972) and Matton et al. (1981) marked the

largest and second largest follicles with india ink and determined if

these remained the largest and second largest later in the cycle.










6

The results of these studies indicated that the ovulatory follicle

could not be predicted prior to 4 days before ovulation.

Ovulation then results in a separation of ovum and associated

cumulus oophorus and corona radiata cells from the follicle. The

fate of the ovum and subsequent concepts development will be

discussed later. The thecal and granulosa cells of the follicle

become the corpus luteum (CL) through cellular reorganization and

proliferation under hormonal control of the pituitary.

Dynamics of Corpus Luteum Development

The corpus luteum develops from the cells of the follicle

following ovulation. Donaldson & Hansel (1965a) reported that luteal

cells are derived from both the theca internal and granulosa cells of

the ovulatory follicle. Similar histological studies by Priedkalns

et al. (1968) support this hypothesis. Luteinization is directed by

the effects of luteinizing hormone (LH) from the anterior pituitary

gland (Hansel, 1966).

Luteinization begins approximately 6 h after onset of estrus

(Donaldson & Hansel, 1965a). Mitotic activity increases in both the

thecal and granulosa layers, but is more frequent in the granulosa

cells. Nuclei of granulosa cells enlarged during the first 4 days of

the estrous cycle (estrus= day 0). The greater mitotic activity of

the granulosa cells was associated with an increased ability to bind

LH during this period (Niswender et al., 1981).

The remainder of this section is based upon the histological

observations by Donaldson & Hansel (1965a) and Priedkalns et al.

(1968) on development and regression of the CL.










7

Immediately following ovulation, which occurs 24 to 30 h after the

preovulatory LH surge (day 1; Chenault et al., 1975; Schams et al.,

1977; Hansel & Convey, 1983), the follicle walls collapse and become

deeply folded with a loss of distinction between granulosa and thecal

cells by 24 to 48 h after ovulation (Donaldson & Hansel, 1965a).

Mitotic activity was intense in all tissue elements: luteal,

stromal, and vascular endothelium and connective tissue trabeculae in

the center of the folds were distinct. By day 3 after estrus, folds

of the walls met, and by day 4 the cavity was obliterated.

Connective tissue trabeculae of thecal externa origin were still

apparent.

By day 5-8 after estrus, the corpus luteum becomes more

homogeneous. While mitosis of all elements was still high early in

this period, they decreased for granulosa luteal cells while there

was an increase in the rate of hypertrophy of these cells. Later,

large luteal cells were found to be associated with several small

luteal cells of thecal origin, a blood vessel and lymphatic duct.

Mitosis was confined to the small luteal cells. Toward the end of

this period, mitosis was confined to stromal elements and hypertrophy

of luteal cells (particularly large luteal cells of granulosa origin)

and nuclei was noted.

Corpus luteum growth continued until about day 12, but the CL was

fully developed and vasularized by day 9 after ovulation. As the

cycle progressed (day 12-18), connective tissue continued to

infiltrate the CL and hypertrophy of the blood vessels occurred,









8

increasing the thickness of vessel walls and in some cases

obliterating their lumen.

The period from day 18 through 21 was characterized by degradation

of the CL. Connective tissue invasion and hypertrophy of blood

vessels continued. The first indications of degradation are a

decrease in cytoplasmic stippling and rounding of the cell outlines.

This was followed by cell shrinkage, cytoplasmic darkening and

nuclear pyknosis with frequent presence of mast cells and phagocytes.

The process of degradation or regression was very rapid and was

completed by 2 days after estrus of the new estrous cycle.

Hormonal Control of Follicular Growth

Follicular development, endocrine secretion and ultimate

transformation to luteal tissue is tied intimately to release of

luteinizing hormone (LH) and follicle stimulating hormone (FSH) from

the anterior pituitary. A complete understanding of how LH and FSH

stimulate follicular steroidogenesis necessitates some explanation of

the two cell theory of estrogen biosynthesis.

The two cell theory was first proposed by Falck (1959), who

reported that neither the granulosa or thecal cells of the follicle

are independently capable of estrogen biosynthesis. The combined

activities of these cell types are required such that thecal cells

metabolize C-21 steroids to androstenedione which are subsequently

utilized by granulosa cells for production of estrogens. These

observations were supported by studies of Lacroix et al. (1974) in

5-
the cow. Thecal cells specifically utilize the A5- pathway for

conversion of pregnenolone to androstenedione via 17c- hydroxy-










9

pregnenolone and dehydro-epiandrosterone as opposed to the A4

pathway. Consistent with the two-cell theory, thecal cells have a

very low aromatase enzyme capacity for converting androstenedione to

estrogen. However, granulosa cells are highly efficient in

aromatizing androstenedione to estradiol, and have a limited ability

to metabolize pregnenolone to androgens due to deficiency of C-21

steroid, 17 ahydroxylase. These findings are consistent with those

of Falck (1959), and support the theory that thecal cells synthesize

androgens via the 5 pathway which are then aromatized by granulosa

cells for estrogen production (Hansel & Convey, 1983).

Estrogen production by the follicle is linked directly to

gonadotrophin stimulation. To complement the two-cell estrogenic

model, Armstrong & Dorrington (1977) proposed that both

gonadotrophins, LH and FSH, are required for estrogen production.

They reported that LH binds specifically to thecal cells and

stimulates androgen production by these cells. Granulosa cells then

undergo stimulation by FSH to metabolize the thecal androgens to

estrogen. A specificity of binding of gonadotrophins to the specific

cell types also was observed by Ireland & Roche (1983b). In this

study, they observed that at day 17, binding of FSH to granulosa

cells and human chorionic gonadotrophin (hCG),an LH-like hormone, to

thecal cells was high. Binding of hCG to granulosa cells was very

low in comparison. As the period of CL regression approached,

specific binding of hCG increased in granulosa cells, indicating a

transformation of granulosa cells from follicular to luteal status.

Those follicles which had increased binding of hCG in granulosa cells










10

produced more estrogen in follicular fluid and had larger diameters

(Ireland & Roche, 1982a,1983a,b). These data have led to a model for

steroid control of receptor dynamics in the follicle. Richards et

al. (1987) theorized that FSH induces LH receptors on granulosa

cells, but FSH will not cause receptor synthesis without estrogen to

synergize the FSH effect. Therefore, large estrogen synthesizing

follicles acquire LH receptors as a result of increased estradiol

secretion which is supported by research from Ireland & Roche

(1982a,1983a,b) who found positive correlations between follicular

diameter, estradiol (E2) and progesterone (P4) concentrations in

follicular fluid and binding of [1251] iodo-hCG to granulosa cells.

In contrast, estrogen inactive follicles bound little hCG. So it

appears that FSH, in conjuction with estrogen, stimulates synthesis

of LH receptors. Increased binding of LH then leads to increased

capacity to secrete estrogens (Ireland & Roche, 1982a, 1983a,b).

These data suggest a positive correlation between estrogen

production and follicular viability. Henderson et al. (1987)

reported that cells from atretic follicles of all sizes produced low

amounts of estrogen and high amounts of P4 in comparison to normal

non-atretic follicles. These results are supported by other reports

(Staigmiller et al., 1982; Ireland & Roche, 1982a, 1983a,b; Tsonis et

al., 1984). Aromatase activity was measured in granulosa cells, and

found to be higher in non-atretic than atretic follicles (Tsonis et

al., 1984). Staigmiller et al. (1982) reported a positive

correlation between binding of hCG to thecal cells and estrogen

secretion (r=0.68), reflecting receptor populations present on










11

preovulatory follicles during estrus, when estrogen synthesis is

highest (Chenault et al., 1975).

Of recent interest are studies relating follicular fluid

concentrations of 8-carotene, vitamin E, cholesterol, and vitamin A

to follicular function (Schweigert et al., 1987; Schweigert & Zucker,

1988). Concentrations of s-carotene, .vitamin E, and cholesterol

concentrations in follicular fluid of viable and atretic follicles

did not differ, but vitamin A was elevated in follicular fluid of

viable follicles (Schweigert & Zucker, 1988). It was hypothesized

that $-carotene, vitamin E and cholesterol were transported into

follicular fluid from the blood by passive transfer while bound to

high density lipoproteins. However, elevated concentrations of

vitamin A in follicular fluid, probably represents a metabolic

conversion from s-carotene. Vitamin A may then influence follicular

development and thereby act as a factor regulating recruitment,

selection, and growth of dominant follicles.

Hormonal Control of the CL

Origin of the cell types. The end result of follicular growth and

development is ovulation. This affords an opportunity for

fertilization and concepts development. While the follicle served

as a source of nourishment for the ovum, the uterus and oviduct

assume this role during early concepts development. Establishment

of a uterine environment conducive to embryonic development is

dependent upon P4 secretion from the CL. In effect, the follicle,

which becomes the CL, is still serving the role of supporting not

only development of the ovum, but the products of conception as well.









12

The formation of the CL has been described (Donaldson & Hansel,

1965a; Priedkalns et al., 1968). The relevance of transformation of

the follicle to a CL, as it relates to CL function and development,

will now be addressed. In cattle (Donaldson & Hansel, L965a;

Preidkalns et al., 1968; Koos & Hansel, 1981; Alila & Hansel, 1984;

Fields et al., 1985; Chegini et al., 1984; Alila et al., 1988), sheep

(O'Shea et al., 1980; Fitz et al., 1982; Rodgers and O'Shea, 1982;

Rodgers et al., 1983a), and other species the CL has been described

as containing two distinct, steroidogenic cell populations (large and

small luteal cells). It has been hypothesized that small luteal

cells are derived from thecal internal and large luteal cells from

granulosa cells of the ovulatory follicle. Small luteal cells are

15-18 pn and large cells 18-45p m in diameter for cattle (Chegini et

al., 1984). Similar estimates for small and large luteal cells of

sheep were obtained (12-221-m and 23-251 m, respectively; Fitz et

al., 1982). These cell types differ also in that small luteal cells,

during mid-cycle: 1) possess eccentrically located, indented, cup-

shaped nuclei with heterochromatin lining the nuclear envelop; 2)

lack granules in the cytoplasm; 3) have relatively few microvilli on

a relatively smooth surface; 4) contain both smooth and rough

endoplasmic reticulum; and 5) possess a large golgi complex and

pleomorphic mitochondria with tubular cristae situated in an arc

opposite the nucleus. In contrast, the large luteal cells possess:

1) centrally located, round nuclei, with dispersed chromatin and

prominent nucleoli; 2) numerous electron dense granules in the

cytoplasm; 3) highly convoluted cell surfaces; 4) extensive smooth










13

endoplasmic reticulum and 5) two types of mitochondria (Koos &

Hansel, 1981).

Alila & Hansel (1984) developed specific monoclonal antibodies to

granulosa and thecal cells of the bovine follicle. They used these

to develop an assay whereby the fate of theca internal and granulosa

cells within the CL could be assessed. Percentage of large luteal

cells binding granulosa specific antibody at days 4-6, 10-12, and 16-

18 were 77 + 6, 47.5 + 3, and 30.2 + 2, respectively. Percentage of

small cells bound by granulosa cell specific antibody was 14% on days

4-6 and none were labelled thereafter. When antibody specific to

thecal cells was introduced to large luteal cells, binding increased

10 + 1.3% between days 4-6 and 46 + 3% between days 10-12. Thecal

specific antibody bound a majority of small luteal cells on days 4-6,

10-12, and 16-18 (70 + 4, 69 + 3, and 58 + 6%, respectively).

Binding of granulosa specific antibody to large luteal cells

decreased during pregnancy, but binding to thecal specific antibody

increased (Alila & Hansel, 1984). These results indicate that small

luteal cells and large luteal cells are derived from thecal and

granulosa cells, respectively, and that small luteal cells develop

into large luteal cells as the CL matures. Some controversy exists

as to the validity of the latter part of this hypothesis due to

inability of any other laboratory to repeat these results. It

represents an eloquent model, but needs to be tested further.

Binding to all cells for either antibody decreased with CL maturity,

and it can not be ruled out that the antigenic domains of the luteal

tissues recognized by the antibodies might be altered to some degree









14

or lost during the transformation from follicular to luteal cells or

during maturation of these cells.

Characteristics of the cell types. Receptor populations and

endocrine secretion by luteal tissue and specific cell types have

been characterized in cattle (Spicer et al., 1981; Milvae & Hansel,

1983; Alila et al., 1988) and sheep (Fitz et al., 1982; Rodgers &

O'Shea, 1982; Rodgers et al., 1983a; Harrison et al., 1987). In both

species, P4 is produced by the small and large luteal cells. Basal

secretion rates of P4 are much higher for large luteal cells than

small luteal cells (Harrison et al., 1987; Alila et al., 1988).

Luteinizing hormone stimulates P4 secretion by small luteal cells but

not large luteal cells of both cattle and sheep. These results can

be explained based on receptor populations on the luteal cell types.

In sheep (Fitz et al., 1982), LH receptor binding sites per cell were

greater for small luteal cells than for large luteal cells (33,260

vs. 3,074, during the breeding season). In contrast to these

results, Harrison et al. (1987) reported that the number of LH

receptors per cell were not different for large or small luteal

cells. They also were not different on days 10 or 15 of the estrous

cycle. They hypothesized that this discrepancy may be due to the

source of luteal cells. That is, Fitz et al. (1982) obtained luteal

cells from superovulated CL, from cyclic ewes, whereas Harrison et

al. (1987) obtained them following spontaneous ovulations. The

effects of PMSG have been reported (Cran, 1983) and described as

causing luteinized granulosa cells leading to CL composed of

hypertrophied thecal-lutein cells. One consistent result for both









15

cattle (Alila et alb, 1988) and sheep (Fitz et al., L982; Henderson

et al., 1987) is that large luteal cells have higher basal secretion

rates of P4 than small luteal cells, and that LH-stimulated P4

secretion is high in small luteal cells while large luteal cells are

unresponsive to LH. This probably reflects differences in metabolic

activities of the cell types* However, the presence of LH receptors

on non-responsive large luteal cells is not consistent with their

responsiveness to LH and requires further research

In support of the theory proposed by Alila & Hansel (1984) that

small luteal cells develop into large luteal cells is a study by

Chegini et al (1984). They found that basal, hCG-stimulated, or

cyclic AMP-stimulated P4 production; apparent dissociation constants

for [1251] hCG binding and total number of available binding sites

for hCG on small and large luteal cells during pregnancy were

similar. Also, morphological characteristics were more similar than

dissimilar for the two cell types during pregnancy. Increasing

similarities between the cell types during pregnancy lends additional

support to the theory proposed by Alila & Hansel (1984).

Fitz et al. (1982) also quantified prostaglandin F-2a (PGF-2a) and

prostaglandin E-2 (PGE-2) receptor numbers on ovine luteal cells.

They reported that specific binding sites for both PGF-2a and PGE-2

were approximately 32-fold and 12-fold higher for large than for

small luteal cells. Rao et al* (1979) and Bartol et al, (1981)

reported that PGF-2a binding to membranes of CL tissue increased from

day 3 to day 20 and decreased on days 21-24. Rao et alv (1979) also

reported that the relative affinity of PGF-2a binding was low on days









16

3 and 13 compared to day 20. Relative affinity was actually 203-fold

higher on day 20 compared to day 13. Collectively, these data

suggest that the CL is less responsive to PGF-2 early compared to

late cycle and that large luteal cells respond to PGF-2 more than

small luteal cells. This will be of significance in a later

discussion of luteolysis.

The responsiveness of large and small luteal cells to PGF-2 ahas

been examined in cattle (Alila et al., 1988). Alila et al. (1988)

observed that PGF-2 stimulated P4 production by small luteal cells.

This is contrary to the currently accepted theory of the luteolytic

mechanism, but the authors hypothesized that the preponderance of

small luteal cells early in the estrous cycle might act in a

paracrine manner for regulation of P4 production. It has been

clearly demonstrated that the CL has the capacity to secrete PGF-2a

(Shemesh & Hansel, 1975; Milvae & Hansel, 1983). It has also been

shown that the majority of receptors for PGF-2a for luteal tissues of

sheep reside on the large luteal cells (Fitz et al., 1982). The

relative affinity of PGF-2a for its receptor on the CL is low early

in the estrous cycle and increases to the time of luteal regression

(Rao et al., 1979). It seems possible that during the early stage of

the estrous cycle, when receptor affinities for PGF-20 on large

luteal cells are low, PGF-2a could stimulate P4 production by small

cells as demonstrated in vitro by Alila et al. (1988).

Luteotrophic substances. Several substances have been described

as being luteotrophic. Luteinizing hormone is the classic example of

a luteotrophin; a substance which stimulates P4 production by the CL.












Numerous studies have demonstrated that LH and hCG are stimulatory to

P4 production in cattle (Schomberg et al., 1967; Milvae et al., 1983)

and sheep (Suter et al., 1980). Early studies evaluated the effect

of LH or hCG administration on estrous cycle length of cattle

(Wiltbank et al., 1961; Donaldson & Hansel, 1965b). They found that

LH and hCG extended CL lifespan and increased embryonic survival

rates. Estrous cycle extension following treatment with LH or hCG

initially may have been ascribed to a direct extension of CL

lifespan, but in view of the recent findings of McDermott et al.

(1986) a different conclusion may be drawn. McDermott et al. (1986)

administered hCG (3,300 IU) to cattle on day 15 after estrus and

reported that this treatment regime caused follicular luteinization

and extended cycles, due to a reduction in follicular estrogen

secretion to initiate uterine PGF-2a secretion. This effect of hCG

is probably the same which brought about cycle extension in the

earlier studies.

Though LH is widely accepted as having a luteotrophic role, many

other substances have also been described as having luteotrophic

activities. Among these the catecholamines epinephrine (E; Black &

Duby, 1965) and norepinephrine (NE; Auletta et al., 1972) prevent the

normal effect of oxytocin induced luteolysis in the cow. Similarily,

E, NE and isoproternol (IPNE) all stimulated P4 production by luteal

tissues cultured in vitro from day 8 to 16 of the estrous cycle

(Condon & Black, 1976). In this study, they also determined that the

catecholamine induced stimulation in P4 production was mediated by

beta-adrenergic receptors. Preincubation of luteal tissue with











propranolol (a beta-adrenergic receptor blocker) inhibited

catecholamine (E, NE, IPNE) and also gonadotrophic (LH) stimulated P4

production. Condon & Black (1976) also evaluated the effect of

phenoxybenzamine (an alpha-adrenergic receptor blocker) on P4

production and found no inhibitory effect of phenoxybenzamine

preincubation on E, NE, IPNE, or LH stimulated P4 production. In a

separate study, Jordon et al. (1978) evaluated effects of propranolol

preincubation on LH stimulated P4 production. They utilized a much

lower dose of propranolol than Condon & Black (1976) and found no

effect on P4 production by luteal tissue. The exact role of the

beta-andrenergic receptor in P4 production by luteal tissues is yet

to be fully understood.

In a more recent study, Milvae et al. (1983) again demonstrated

catecholamine (E, IPNE) and LH stimulated increases in P4 production

by luteal cells. To understand this mechanism further, they tested

the effect of a methylation inhibitor (S-adenosylhomocysteine, SAH)

on catecholamine and LH stimulated P4 production and found that

presence of SAH inhibited E and IPNE stimulated P4 production.

Similarly, incubation of tissues with S-adenosylmethionine (SAM, an

endogenous stimulator of methylation in membranes) and LH caused an

elevation of the stimulatory effect of LH on luteal cell P4

production. Therefore, methylation appears to be involved in the

mechanisms whereby LH stimulates luteal cells to produce P4.

Some prostaglandins were ascribed with having a role as

luteotrophic agents. As mentioned earlier, PGF-2c has been shown to

have a luteotrophic effect on small luteal cells in vitro (Hixon &











Hansel, 1979; Benhaim et al., 1987; and Alila et al., 1988). The

stimulation of P4 production by PGF-2a was similar to that obtained

by addition of phorbol ester or phospholipase C. Davis et al. (1987)

reported that PGF-2a acts by stimulating phosphatidylinositol 4,5-

trisphosphate hydrolysis in the small luteal cells which results in

release of diacylglycerol, an activator of protein kinase C. Thus,

PGF-2a stimulated P4 production appears to be mediated through

protein kinase C activation in small luteal cells.

Several studies have evaluated the role of prostaglandin E-2 in CL

function (Chenault, 1983; Gimenez & Henricks, L983; Reynolds et al.,

1983; Chenault et al., 1984). In one study, (Chenault, L983), it was

reported that intrauterine infusion from day 14 to 24 or 28 after

estrus of PGE-2 delayed luteolysis, though not for a period of time

exceeding the period of infusions. In contrast to these findings,

Reynolds et al. (1983) found no extension of cycle length due to

intrauterine infusion of PGE-2 alone, but did report extension of CL

lifespan for cows receiving PGE-2 and estradiol in combination. They

theorized that estradiol-17B and PGE-2, which represent concepts

secretary products, may act synergistically through a luteotrophic

mechanism during early pregnancy to maintain luteal function.

Interest in the role of luteal prostacyclin (PGI-2) has increased

since Sun et al. (1977) reported that the predominant form of

prostaglandin produced by luteal membrane preparations when incubated

with PGH-2 (the endoperoxide precursor of prostaglandins for the F,

E, D, and I series) was PGI-2. Prostacyclins have been shown to

display luteotrophic activities (Milvae, 1986). Injection of PGI-2









20

directly into the CL on day 10 of the estrous cycle caused elevated

P4 concentrations in jugular vein plasma within 5 min (Milvae &

Hansel, 1980). Similarly, PGI-2 increased P4 production by dispersed

luteal cells in vitro (Milvae & Hansel, 1980). Bovine luteal cells

collected on days 5, LO, 15, and 18 after estrus produced 128 + 12,

87 + 18, 38 + 9, and 54 + 7 ng/106 cells of PGI-2, respectively

(Milvae & Hansel, 1983), and concentrations of PGI-2 and P4 secretion

followed similar trends in this experiment. Of additional interest

is that luteal production of PGI-2 was elevated on day 25 of

pregnancy compared to day 25 of the cycle (Milvae, L986).

Collectively, these data show relations between PGI-2 and P4

secretion and implicate PGI-2 as having an important luteotrophic

role.

Products of the CL. The CL has been identified as the source of

several hormones, but primarily P4. Progesterone is secreted by

large and small luteal cells which are variably responsive to the

luteotrophic action of LH (Hansel et al., 1973). A brief description

of the P4 secretary pattern of the cow follows. This topic has been

extensively reviewed (Henricks et al., 1970; Lemon et al., 1975;

Chenault et al., 1975). Briefly, peripheral P4 concentrations are

low (less than 0.5 ng/ml) from approximately 2 days proceeding to 3

days following ovulation. Following ovulation, luteinization of

thecal and granulosa elements ensues. Progesterone concentrations

rise from day 4 to about day 12 (Schams et al., 1977). Henricks et

al. (1970) reported that P4 concentrations rose 0.73 ng/ml/day and

0.69 ng/ml/day from days 0 to 8 after estrus for pregnant and non-









21

pregnant cattle and then rose 0.61 and 0.15 ng/ml/day thereafter.

This gives some evidence that the concepts might be mediating

luteotrophic mechanisms as its presence was associated with elevated

P4 secretion. Peak levels of P4 were reached by day 16 to 18 after

estrus and thereafter steadily declined to less than 0.5 ng/ml/day at

the next estrus (Henricks et al., 1970).

Recently, oxytocin has been identified as being a product of the

CL of cattle (Fields et al., 1983; Wathes et al., 1983; Hansel &

Dowd, 1986) and sheep (Rodgers et al., 1983b; Harrison et al., 1987;

Schams et al., 1987). Oxytocin production was localized to large

luteal cells of sheep, and its secretion was maximal during the early

phase of the estrous cycle (Rodgers et al., L983b; Harrison et al.,

1987). Measurement of oxytocin concentrations in the vena cava of

cows revealed pulsatile secretary responses which parallel

progesterone secretion (Walters & Schallenberger, 1984; Walters et

al., 1984). The release of oxytocin also paralleled release of PGF-

2 during the period of luteolysis. Thus, oxytocin is not only

involved in the luteolytic mechanism of PGF-2 q but may also be

involved in regulation of P4 secretion early in the estrous cycle

(Schams, 1987). Schams (1987) proposed that by regulating P4

secretion early in the estrous cycle, extension of the cycle may

occur which is supported by results of others who have shown that P4

supplementation early in the estrous cycle (day 1-5) caused short

cycles (Woody et al., 1967; Ginther, 1968,1969; Lawson & Cahill,

1983). Progesterone supplementation was believed to shorten the

estrous cycle by prematurely activating the PGF-2a synthetic









22

mechanism of the uterus resulting in luteolysis (Baird et al., 1976;

Ottobre et al., 1980). Therefore, oxytocin secretion early in the

estrous cycle may act to reduce early P4 secretion and thereby extend

the functional lifespan of the CL.

Other products of the CL recently identified are GnRH-like

proteins (Aten et al., 1987; Ireland et al., 1988). These proteins

exhibit potent antigonadotrophic activities thereby suppressing the

stimulatory effects of LH. The concentrations of P4 in blood and the

capacity of luteal tissues to respond to LH by increasing P4

production have been shown to increase with advancement of the cycle

(Milvae & Hansel, 1983). The concentrations of GnRH-like peptides

decrease coincidentally in a similar manner (Ireland et al., 1988).

It might be hypothesized then that the GnRH-like peptides regulate

steroidogenic capacity of the CL early in the cycle possibly allowing

longer interestrous intervals than would occur in the absence of

GnRH-like peptides. GnRH-like proteins have also been found in

granulosa cells of the cow (Ireland et al., 1988) and are actually

present in higher concentrations in granulosa than luteal cells. Due

to their potent antigonadotrophic activities they may act similarly

to regulate growth and atresia of follicles.

Intraovarian Regulation

Increasing evidence suggests that intraovarian regulation of

follicles occurs. Research directed at elucidating mechanisms

whereby a follicle is selected to ovulate has led to studies of

follicular dominance. As previously described, follicles undergo

recruitment from the dormant pool of primordial follicles in response









23

to a stimulus, probably FSH. Recruited follicles then develop to the

antral stage and selection of one of these to develop to ovulatory

size ensues. The follicle selected then suppresses growth of the

other follicles until it ovulates or undergoes atresia (Ireland &

Roche, 1987). That the largest follicle inhibits growth of lesser

follicles was reported by Matton et al. (1981) who, after cauterizing

all follicles on the ovaries of heifers, noted rapid proliferation of

new follicles to replace them. Follicular factors have been

identified which may play a role in establishing dominance of a

single follicle on the ovary at any time. The interesting aspect of

this theory is that the dominant follicle itself must continue to

develop while inhibiting development of other follicles.

One possible regulatory factor which has been identified is

inhibin. Henderson & Franchimont (1983) reported that granulosa

cells from cattle produce inhibin in vitro. Furthermore, follicular

fluid, which contains inhibin-like activity, caused decreased FSH

secretion in ovariectomized heifers (Ireland et al., 1983).

Therefore, inhibin may act to depress FSH secretion from the anterior

pituitary thereby suppressing growth of, or recruitment and selection

of, additional dominant follicles (Padmanabhan et al., 1984).

Another intraovarian regulator, follicular regulatory protein (FRP),

has been identified in ovarian venous blood (diZerega et al., 1982),

follicular fluid (diZerega et al., 1983b) and medium from cultured

granulosa cells (diZerega, 1983a) of humans. This compound appeared

to inhibit the aromatase activity of granulosa cells in vitro

(diZerega & Wilks, 1984). Also, concentrations of FRP of estrogen











inactive (atretic) follicles was twice that of estrogen active

follicles (Ireland & Roche, 1987). A model, proposed by Ireland &

Roche (1987) to explain the phenomenon of dominance, theorized that

the dominant follicle, at any particular moment, secrets inhibin

which suppresses pituitary release of FSH which blocks recruitment

and selection of new dominant follicles. Then, FRP is secreted by

the dominant follicle which impairs the aromatase system of other

non-dominant follicles resulting in their atresia. Estradiol

secretion by the dominant follicle, in turn, enhances its sensitivity

to gonadotrophic stimulation thus ensuring its survival. The GnRH-

like peptides described by Aten et al. (1987) and Ireland et al.

(1988) may also play a role in this process because these molecules

have been shown to possess antigonadotrophic activities, specifically

to attenuate LH-induced cyclic adenosine monophosphate (cAMP)

accumulation in rat luteal cells. Massicotte et al. (1980) also have

shown GnRH or its agonists suppress FSH-induced accumulation of cAMP

in porcine granulosa cells. Based on knowledge that GnRH-like

peptides are found in granulosa cells and that agonists to GnRH

affect FSH stimulation of granulosa cells, it does not seem unlikely

that GnRH-like peptides might be involved in control of follicular

steroidogenesis and thereby control dominance. Although it is not

known at this time, GnRH-like peptides might be secreted by the

dominant follicle to suppress other non-dominant follicles.

If a follicle has established itself and gained dominance, it will

either ovulate or undergo atresia. If the follicle does not ovulate,

FRP continues to accumulate in the follicular fluid of the dominant









25

follicle, impairing its estrogen producing ability and it eventually

undergoes atresia. This relieves the negative inhibition on FSH

secretion by the pituitary and results in recruitment of a new set or

wave of follicles in response to FSH release.

Gonadotrophin Secretion during the Bovine Estrous Cycle

The release pattern of LH from the anterior pituitary has been

studied extensively. Generally, LH secretion is low during most of

the estrous cycle and becomes elevated for a short period of time

immediately following onset of estrus (Henricks et al., 1970; Spicer

et al., 1981). Serum P4 and specific binding of hCG to the CL

increase from 1.9 to 4.5 days after the preovulatory LH surge,

remained unchanged between 8.3 and 12.4 days post-LH surge, and

declined thereafter (Spicer et al., 1981) indicating that the CL

becomes more responsive to LH concomittant with elevated P4

secretion. Secretory patterns of LH have revealed pulsatile patterns

that are dependent upon the steroids being secreted. The early

luteal period, day 3, was characterized by low amplitude (LH, 0.3-

1.8 ng) and high frequency pulses (16-30 pulses every 24 hours (Rahe

et al., 1980; Walters et al., 1984). During the mid-luteal phase of

the estrous cycle, days 10 to 11, LH pulses were classified as high

amplitude ( LH, 1.2-7.0 ng) and low frequency (6-8 pulses every 24

hours). Ireland & Roche (1982b) found that insertion of

progesterone-releasing intravaginal devices (PRID) caused decreased

pulse frequencies, and the number of pulses increased after PRID

removal, which supports the hypothesis that P4 inhibits pulses of









26

LH, but does not itself affect pulse amplitude (Ireland & Roche,

1982b).

Luteolysis was induced in a group of cattle which resulted in a

preovulatory surge of LH 59 h after administration of the

prostaglandin analogue, cloprostenol (Walters & Schallenberger,

1984). This pulse of LH was the result of simultaneous increases in

the pulse frequency (pulse interval = 38-40 min versus 200 min for

mid-luteal period) and amplitude (7-32 ng/ml) of LH just prior to the

LH surge. Rahe et al. (1980) also reported increased LH pulse

frequencies and amplitude at the time of LH surge and theorized that

LH secretion is probably modulated by ovarian steroids.

Estrogen release was correlated positively to LH pulses (Walters &

Schallenberger, 1984; Walters et al., 1984) which was not surprising

when viewed in light of the previous discussion of follicular

steroidogenesis. One product of the follicle is E2, and its

secretion is tied directly to gonadotrophic stimulation of the

follicle. It is also likely that E2 modulates LH release. Karsh et

al. (1983) evaluated the effect of E2 and P4 in ovariectomized ewes

and observed that P4 withdrawal increased pulse frequencies and E2

administration caused a further increase in pulse frequencies and

decrease in pulse amplitudes. Collectively, these data support the

theory that intimate control of LH pulsatile release is a function of

steroid regulation of the anterior pituitary.

The high amplitude, low frequency pattern of LH secretion during

the luteal phase is thought to be the result of P4 negative feedback

on hypothalamic release of gonadotrophin releasing hormone (GnRH;









27

Knobil, 1980) and sensitivity of the pituitary to GnRH (Padmanabhan

et al., 1982). Prior to the preovulatory surge, E2 results in

decreased sensitivity of the pituitary to GnRH and reduced LH pulse

amplitudes (Kesner & Convey, 1982). Then when E2 secretion is

maximal, sensitivity of the pituitary gland to GnRH reaches its

maximum resulting in increased frequency, but not amplitude of LH

release (Walters & Schallenberger, 1984; Kesner & Convey, 1982;

Padmanabhan et al., 1982). Walters & Schallenberger (1984) suggested

that negative feedback of E2 on the hypothalamus might explain the

endocrine patterns observed during the periovulatory period.

Regulation of.FSH secretion is less understood. Generally, FSH

secretion is higher than LH during the estrous cycle (Walters et al.,

1984; Walters & Schallenberger, 1984). Pulse frequencies for FSH

were similar to those of LH during the early luteal phase (8.5 vs.

8.0 pulses every 12 hours; Walters et al., 1984). In contrast to LH,

FSH pulse frequencies changed little during the mid-luteal phase (LH

3.6 vs. FSH = 6.3 pulses every 12 hours). Walters et al. (1984)

also found that 90-100% of all LH/FSH and separate FSH pulses were

associated with pulses of P4. Pulse amplitude for FSH increased as

ovulation approached (Walters & Schallenberger, 1984), and a second

surge was reported to occur 4 to 12 h after the LH-surge. This was

thought to be due to an increase in amplitude and not frequency of

pulses (Walters & Schallenberger, 1984).

Administration of LH or hCG has been shown to profoundly affect

luteal function (Wiltbank et al., 1961; Donaldson & Hansel, 1965b).

In these studies, LH or hCG administration resulted in prolongation









28

of CL lifespan and increased embryonic survival. Some of this effect

may have been due to the luteotrophic action of LH but other

mechanisms have been suggested (Schomberg et al., 1967; McDermott et

al., 1986). Schomberg et al. (1967) found that administration of LH

or hCG resulted in prolonged CL lifespan but also caused new

ovulations resulting in accessory CL. McDermott et al. (1986) also

reported formation of accessory CL in response to administration of

hCG on day 15 after estrus.

Exogenous administration of hCG early in the estrous cycle has

been shown to affect the developing CL and probably not affect

follicles as much. Moody & Hansel (1971) reported that

administration of 11,000-15,000 IU hCG during days 1 to 7 after

estrus increased CL size. Studies in vitro also have indicated that

hCG increases P4 content, but not concentration in CL tissue (Moody &

Hansel, 1971; Veenhuizen et al., 1972). Therefore, it appears that

hCG, given during CL development, causes increased CL size and P4

production, but does not affect the steroidogenic capability of the

CL tissue on a per unit mass basis. Helmer & Britt (1987) reported

that hCG given on days 2 to 4 did, in fact, increase P4 secretion in

heifers given 1000 IU on each day. In contrast, hCG administered

during the luteal phase causes cycle extension (Wiltbank et al.,

1961; Donaldson & Hansel, 1965b), but this effect was due to new

ovulations resulting in formation of accessory CL (Schomberg et al.,

1967; McDermott et al., 1986). Thus, hCG seems to have variable

effects. Early in the estrous cycle it has luteotrophic effects on

the developing CL while in mid-luteal periods it affects










29

luteinization of follicles and or ovulations resulting in assessory

CL formation and cycle extension.

Uterine Regulation of CL Lifespan during the Estrous Cycle

It is generally accepted that the uterus plays an active role in

the luteolytic mechanisms of several species. One of the first

reports of this association was by Loeb (1923) who noted that removal

of the uterus from guinea pigs extended CL function. Surgical

removal of the uteri of both sheep (Wiltbank & Casida, 1956; Anderson

et al., 1969) and cattle (Wiltbank & Casida, 1956; Anderson et al.,

1965) also resulted in extended CL function. The process of uterine-

mediated luteolysis also has been ascribed to being a local rather

than a systemic phenomenon. Ginther et al. (1967) unilaterally

hysterectomized heifers and found that oxytocin- induced CL

regression occurred only when the remaining uterine horn was

ipsilateral to the CL bearing ovary. Regression did not occur when

the remaining uterine horn was contralateral to the CL bearing ovary.

That uterine-mediated luteolysis is a local versus systemic effect

has been unequivocally demonstrated for the cow and ewe (Ginther,

1981).

The vasculature of the uterus and ovaries have been studied to

elucidate the local mechanism of luteolysis (Wallmerhaus, 1964;

McCracken et al., 1971; Ginther et al., 1973; Ginther, 1974, 1981).

Passage of the luteolytic substance from uterus to ovary was

theorized to occur via transfer from uterine venous drainage into

ovarian arterial supply by a countercurrent exchange mechanism just

below the ovarian vascular pedicle. This countercurrent,











venoarterial transfer is possible because the ovarian artery follows

a convoluted and tortuous path on the surface of the uteroovarian

venous drainage, the venous drainage of uterus and ovary, at this

point, sharing common vessels. Futhermore, walls of the major

vessels are thinner in the region of venoarterial apposition and

connective tissue bundles of the two vessels form a single stratum

such that demarcation of the vessels is no longer apparent

(Wallmerhaus, 1964). Results of these studies strongly support the

idea that the uterus is the source of the luteolytic agent causing

demise of the CL and that this occurrs by a local venoarterial

transfer in sheep and cattle.

Some evidence exists which would modify the model for luteolysis

as described thus far. Abdel Rahim et al. (1984) and Heap et al.

(1985) suggested that lymphatic drainage of the uterus plays an

integral role in the luteolytic mechanism of sheep. Abdel Rahim et

al. (1984) reported that extension of CL lifespan occurred in ewes

which had all connections between uterus and ovary severed except the

uteroovarian vascular system. Connections between the uterine horn

and ipsilateral ovary, i.e. oviduct and accompanying vessels, broad

ligament, nerves, lymphatics, and arteries, were all severed. If the

uteroovarian countercurrent mechanism was all that was required for

luteolysis, this model system should have resulted in normal

luteolysis. Since it did not, some additional systems may be

required for luteolysis to occur. Heap et al. (1985) supplied

evidence that the lymphatic drainage of the uterus plays an integral

role in uterine mediated luteolysis by infusion of radiolabelled PGF-










31

2a into a uterine lymphatic vessel or uterine vein or injection of it

into the uterine lumen of anesthetized ewes. When PGF-A2 was infused

into the uterine lumen of ewes, elevated levels of PGF-2x were

observed within 20 min in both uterine lymphatics and uterine venous

plasma, but persisted longer in lymphatic secretions. Infusion of

PGF-23 into uterine afferent lymphatics resulted in transfer to

ovarian artery within 10 min and transfer rates were 0.4% from the

lymphatic vessel. This was actually higher than the transfer rate

from the uterine vein in this study (0.3%).

It is widely accepted that PGF-2 atis the uterine luteolysin of

cattle and sheep. This concept is based upon several lines of

evidence. Exogenous administration of PGF-2A is luteolytic when

administered to cattle (Hansel et al., 1973; Lauderdale, 1974;

Thatcher & Chenault, 1976; King et al., 1982). Elevated

concentrations of PGF-Mt in uterine venous drainage (Nancarrow et

al., 1973; Shemesh & Hansel, 1975), uterine tissue (Shemesh & Hansel,

1975), and uterine flushings (Lamothe et al., 1977; Bartol et al.,

1981b) are associated with the period of luteal regression.

Prostaglandin F-M2 was metabolized from arachidonic acid and

prostaglandin endoperoxides (prostaglandin H-2) in the uterus and the

conversion of PGH-2 to PGF-2a was very efficient (Wlodawer et al.,

1976). Concentrations of PGF were also shown to be elevated in the

ovarian artery (OA) compared to concentrations in the peripheral

circulation (Wolfenson et al., 1985). The difference in

concentrations of PGF between ovarian artery and peripheral vein (PV)

were highest (OA-PV 160 pg/ml) during luteal regression (days 19-









32

20). Wolfenson et al. (1985) reported that about 1% of the PGF in

the uterine venous drainage was transferred to the ovarian vein

during luteolysis. Collectively, these results indicate that PGF-Za

is the uterine luteolysin in cattle and sheep, since it is produced

by and secreted from the uterus during periods corresponding to

luteolysis and is shown to have luteolytic properties in vivo.

Although PGF-4x is generally recognized as being the uterine

luteolysin, this does not yield insight into how this molecule

affects luteolysis. A great deal of effort has gone into the study

of the luteolytic mechanism. A working hypothesis incorporates the

actions of several ovarian and uterine hormones and their actions on

one another. Estradiol has been shown to cause uterine release of

PGF-2 cand increase the concentration of peripheral PGFM when

administered late in the estrous cycle (Barcikowski et al., 1974;

Thatcher et al., 1984b; Bartol et al., 1981b; Thatcher et al., 1986b;

Hixon & Flint, 1987; Lafrance & Goff, 1988) and result in luteal

regression (Wiltbank, 1966; Eley et al., 1979; Thatcher et al.,

1986b). However, E2 administration early in the estrous cycle does

not cause premature luteolysis (Loy et al., 1960). Furthermore, P4

administration during the first I to 5 days after estrus resulted in

short estrous cycles (Ginther, 1968; Lawson & Cahill, 1983).

Capacity of the uterus to release PGF-2t is believed to be an effect

of P4 priming of the uterus such that after a period of approximately

10 days of P4 exposure, the luteolytic mechanism becomes functional.

Control of susceptability to E2-induced luteolysis seems to be

stage specific and probably dependent upon prior ovarian steroid









33

modulation. It is known that E2 stimulates synthesis of its own

receptors and of P4 receptors (Clark et al., 1977). Furthermore, P4

suppresses estrogen receptor synthesis (Clark et al., 1977) as well

as synthesis of its own receptors (Schrader & O'Malley, 1978).

Elevated P4 during the luteal phase inhibited formation of E2

receptors on the uterus thereby blocking the luteolytic action of E2

(Henricks & Harris, 1978). Receptors for P4 were also suppressed

during the late luteal phase of the estrous cycle presumably by the

action of P4 (Zelinski et al., 1982) which would allow E2 from

developing follicles to induce E2 receptors, thus making the

luteolytic mechanisms operative.

A role for oxytocin in the luteolytic mechanism has also been

identified. Administration of exogenous oxytocin has been shown to

be luteolytic in cattle (Armstrong & Hansel, 1959; Auletta et al.,

1972) by inducing uterine PGF-2; release (Milvae & Hansel, 1980), and

elevating peripheral PGFM concentrations (Lafrance & Goff, 1985,

1988). Additionally, it was reported that EZ caused formation of

oxytocin receptors following a period of P4 priming in sheep (Roberts

et al., 1976; McCraken et al., 1984; Hixon & Flint, 1987). Oxytocin

release into the vena cava paralleled P4 secretion during the luteal

phase and pulse frequency, but not amplitude, increased from 2.0 to

4.7 pulses every 12 h from days 4 to 11 after estrus (Walters et al.,

1984). However, oxytocin concentrations during the periovulatory

period were very low or undetectable (Walters & Schallenberger,

1984). Transcription of oxytocin messenger ribonucleic acid (mRNA)

in bovine corpora lutea was greatest at days 3-6 of the estrous


1









34

cycle, while oxytocin content (ng/g tissue) was greatest on days 11-

18 of the estrous cycle (Schams et al., 1987). Oxytocin content of

corpora albicantia, early developing CL, and CL of pregnant animals

was low (Schams et al., 1987). Of interest is that oxytocin has also

been demonstrated in follicular fluid (Schams et al., 1985) and it

has been localized to granulosa cells by immunocytochemistry (Kruip

et al., 1985). Large luteal cells, which are responsible for luteal

oxytocin secretion in sheep (Rodgers et al., 1983b) are derived from

granulosa cells (Alila & Hansel, 1984). Collectively, these data

indicate that the synthetic capacity of the CL to produce oxytocin

originates in the follicle. Oxytocin accumulates in the CL

following mRNA transcription early in the estrous cycle, reaches

maximal concentrations at the mid-luteal phase (Schams et al., 1987)

and then is depleted from luteal tissues around the time of

luteolysis and is low after CL regression and during pregnancy.

Collectively, these data led McCracken et al. (1981, 1984) to

propose a working hypothesis or model for the luteolytic mechanism of

cyclic ewes. After P4 priming has occurred, inhibition of E2 receptor

formation is relieved. Estrogens stimulate synthesis of their own

receptor which when bound by estrogen results in oxytocin receptor

synthesis. Oxytocin from the CL and or pituitary interact with the

endometrial receptor which stimulates PGF-&t release. The luteolytic

action of PGF-Z ais to decrease P4 secretion and PGF-;P is also

associated with further oxytocin release by the CL. The second

release of oxytocin may then reinforce the secretion of PGF-Ax from












the uterus. This cycle would continue until the CL is no longer

capable of oxytocin synthesis and secretion.

It has been proposed that one mechanism whereby oxytocin and

estrogen stimulates PGF-2a release includes increased turnover of

phosphoinositides, a possible, but limited, source of arachidonic

acid for PGF-2 atsynthesis (Flint et al., L986; Hixon & Flint, 1987).

Flint et al. (1986) incubated slices of caruncular endometrium from

steroid-treated, ovariectomized ewes with [3H]inositol to radiolabel

tissue phosphatidylinositol. Treatment with oxytocin was shown to

increase incorporation of [3H]inositol into phosphatidylinositol.

Phosphoinositides are normally hydrolysed to inositol phosphates and

diacylglycerol, the latter of which can be metabolized to arachidonic

acid. Tissue slices preincubated with [3H]phosphatidylinositol had

increased incorporation of radiolabel into inositol mono-, bis- and

tris-phosphates, the latter being the prevalent form after addition

of oxytocin to incubations. Furthermore, 72% of

[3H]arachidonyldiacylglycerol was converted to [3H]arachidonic acid

by caruncular endometrium. In a similar experiment, caruncular

endometrium was incubated with [3H]inositol in the presence of EZ and

or oxytocin (Hixon & Flint, 1987). In this study, E2 enhanced the

oxytocin-induced increase in phosphoinositide turnover which

coincided with PGF-2a release and functional luteolysis in sheep

(Hixon & Flint, 1987). These data indicate that PGF-2c synthesis by

the uterine endometrium may result from increased phosphoinositide

turnover, resulting in liberation of arachidonic acid, the precursor

of prostaglandins.









36

Pharris et al. (1970) hypothesized that PGF-Za mediates its

luteolytic effect by constriction of ovarian venous drainage. This

would result in decreased nutrient supply to the CL and lead to its

demise. McCracken et al. (1971) found that injection of PGF-2c

directly into the CL resulted in decreased P4 secretion without any

corresponding reduction in ovarian blood flow. However, this did not

rule out alteration in intraovarian blood flow. Subsequently,

Niswender et al. (1976) measured blood flow to the ovaries of sheep

and found that blood flow to the CL-bearing ovary increased as the CL

developed, was sustained while P4 was elevated, declined within 4 h

of intrauterine infusion of PGF-2a, and was followed 2 h later by

lowered peripheral P4 concentrations (Niswinder et al., 1976).

McCracken et al. (1979) and Einer-Jensen & McCracken (1981) reported

that P4 secretion decreased 50% by I to 2 h after PGF-Za

administration and well before any change in capillary blood flow was

detected. It appears unclear as to what role decreased blood flow

has on inducing luteolysis. It is most likely associated with

structural events associated with luteolysis rather than initiation

of the event. Along with functional luteolysis (decline in P4

secretion), PGF-20 also induces structural luteolysis (degradation of

the luteal tissue). Murdock (1987) demonstrated that the

immune/inflammatory system of sheep is active in the process of

luteolysis. They reported that PGF-Za elicited production of a

chemoattractant for eosinophiles by luteal tissues. Eosinophiles

have been reported to release substances which mediate tissue injury

(Gleich & Loegering, 1984). Eosinophile accumulation in ovine luteal









37

tissue is supported by others (Nett et al., 1976a; McClellan et al.,

1977). Lysosome which appear to play a role in luteolysis of ovine

CL (McClellan et al., 1977) might be released in response to PGF-i2

and unmask autoimmune sites (possibly covered by sialic acid

residues) on luteal cells which could be recognized by preexisting

antibodies. This process could elicit an inflammatory reaction

resulting in the observed accumulation of eosinophiles. Macrophages

have also been implicated in cellular luteolysis. Paavola (1979)

reported that autophagy (cellular self-digestion) and heterophagy

(removal of cells by macrophages) were active components on

luteolysis in the guinea pig. Lysosomal release resulting in

autophagy was detected in this study and concurred with results of

McClellan (1977) in sheep. In the study by Paavola (1979),

macrophages played an integral role in the luteolytic mechanism by

removing luteal cell fragments as well as whole luteal cells.

Murdock (1987) also reported the appearance of macrophages during

luteolysis, but only after the peak of eosinophila.

Another mechanism for the luteolytic action of PGF-ar is that it

may antagonize local gonadotrophic support of the CL resulting in

luteolysis. It was thought that PGF-2x might bind to the luteal cell

membrane and prevent the regulatory unit of adenylate cyclase from

activating its catalytic unit thereby suppressing P4 production

(Henderson & McNatty, 1975). This theory was supported by evidence

that PGF-2X does inhibit LH-induced accumulation of intracellular

cAMP in luteal cells (Lahav et al., 1976; Thomas et al., 1978;

Hamberger et al., 1979). This antigonadotrophic effect is likely










38

receptor-mediated as gonadotropins have also been demonstrated to

inhibit PGF-Z induced luteolysis (Henderson & McNatty, 1975). It

has also been shown by X-ray diffraction that membrane structure of

luteal cells is changed by PGF-iX (Buhr et al., 1979; Carlson et al.,

1981). The action of PGF-2 might be to either limit accessibility

of LH to its receptor (through altered membrane structure) or by

uncoupling of the regulatory unit of adenylate cyclase of the LH

receptor as suggested by Henderson & McNatty (1975). In either case,

the effect would be to withdraw LH support and cause luteal

regression.

Prostaglandin F-;a induced suppression of P4 contradicts data of

others who reported increased P4 secretion by small luteal cells in

response to PGF-2 c(Alila & Hansel, 1984). It is important to note

that in the experiment by Alila & Hansel (1984) enriched small and

large luteal cell populations were utilized. Pate & Condon (1984)

reported a decrease in P4 synthesis for unseparated bovine luteal

cells in response to PGF-2 These results raise several

possibilities for control of CL function by a luteal source of PGF-

2 4 First, receptors for PGF-ZX are present in highest numbers on

large luteal cells (Fitz et al., 1982), but receptor affinities are

low until the period of luteal regression approaches (Rao et al.,

1979). This suggests that the effect noted for small luteal cells

could regulate P4 production early in the estrous cycle without

initiating luteolysis. As receptors on large cells acquire higher

affinites for PGF-2x later in the estrous cycle (Rao et al., 1979),

large cells could become responsive to luteolytic action of PGF-2X.










39

Results of Pate & Condon (1984) also imply that intercommunication

occurs between large and small luteal cells of the CL to regulate

responsiveness of each other.



Uterine Regulation of CL Lifespan During Pregnancy

The presence of a functional CL with continued P4 secretion is an

absolute requirement for pregnancy maintenance in the cow and ewe.

It has been reported that P4 concentrations in peripheral circulation

are elevated as early as day 10 of pregnancy compared to non-pregnant

animals (Henricks et al., 1970, 1971; Lukaszewska & Hansel, 1980).

Although this observation has not been fully supported by

observations of others (Folman et al., 1973; Sreenan & Diskin, 1983),

it.suggests that the concepts may release or induce a luteotrophic

factor which counteracts the luteolytic effect of PGF-Zt (Henderson &

McNatty, 1975). Although PGE-Z has been shown to extend CL function

in cattle under some circumstances (Chenault, 1983; Reynolds et al.,

1983), its effects are not consistent. Also, no effect of pregnancy

on secretion of PGE-2 from cultured (Curl et al., 1983) or perifused

(Gross et al., 1988c) bovine endometrium has been demonstrated. In

contrast, intrauterine infusion of PGE-2 to ewes has been shown to

extend luteal lifespan (Pratt et al., 1979; Huie et al., 1981;

Magness et al., 1981). The CL of sheep also becomes more refractory

to luteolytic effects of injected PGF-Z during early pregnancy

(Inskeep et al., 1975; Mapletoff et al., 1976; Pratt et al., 1977;

Silvia & Niswender, 1984) and this refractoriness to PGF-21 also was

reported after PGE-Z administration (Henderson et al., 1977; Pratt et












al., 1977; Reynolds et al., 1981). These data, along with results of

Ellinwood et al. (1979) and Silvia et al. (1984) indicating that PGE-

2 is elevated in uteroovarian venous plasma of pregnant ewes, suggest

that the concepts may exert its antiluteolytic effect, in part, by

secretion of PGE-2 which may act as a luteoprotective substance.

Prostaglandin E-2 is not elevated during pregnancy in cattle (Curl et

al., 1983), but with suppression of uterine PGF secretion the net

effect would be to increase PGE-2:PGF ratios.

In addition to the attenuated response of the CL of pregnancy to

luteolytic effects of PGF-2a, transfer of PGF-2a from the ovarian

vein to the ovarian artery is reduced during pregnancy resulting in

lower PGF-20 concentrations reaching the ovary (Wolfenson et al.,

1985). Concentrations of PGFM in peripheral circulation of pregnant

cattle (Kindahl et al., 1976; Betteridge et al., 1984; Thatcher et

al., 1984b) also were lower. Similarly, PGFM and PGF-Za secretions

by endometrial tissue cultured (Thatcher et al., 1984b) or perifused

(Gross et al., 1988c) from pregnant cattle were reduced compared to

cyclic cows from day 17 after estrus. Collectively, these data

indicate that bovine endometrial PGF-2a secretion is suppressed

during pregnancy.

Uptake of PGF-2 by uterine veins and ovarian arteries of pregnant

cattle also was found to be suppressed (Thatcher et al., 1984b).

Although differences were not significant, Lewis et al. (1978)

reported a trend for selective retention of PGF-2a in ovarian vein

during the comparable period for luteolysis in cyclic ewes. No

difference in endometrial production of PGF-2a from cyclic and










41

pregnant ewes was reported and it therefore seems possible that in

sheep, PGF-2a secretion might be altered in a manner different than

that for cattle. This was supported by others who found mean PGF-2a

in ovarian venous blood to be higher (Wilson et al., 1972; Ellinwood

et al., 1979) or not different (Nett et al., 1976b; Silva et al.,

1984) between pregnant and cyclic ewes at the time of normal

luteolysis. Uterine endometrium from pregnant ewes was found to

contain more PGF (Lewis et al., 1977; Ellinwood et al., 1979; Findlay

et al., 1983) than comparable cyclic endometrium. Endometrium from

pregnant ewes secreted more PGF in vitro than cyclic endometrium

(Ellinwood et al., 1979; Findlay et al., 1981). Furthermore, rate of

secretion of PGF was higher and more was released toward the luminal

versus myometrial side of ovine endometrium utilizing a perifusion

device (Lacroix & Kann, 1983). Frequent blood sampling led to a

theory that, in sheep, PGF-;Z secretion by the uterus is not altered

by pregnancy, but its pattern of release from the uterus is altered

(Zarco et al., 1984). Results of Zarco et al. (1984) indicate that

basal secretion of PGF-&t is elevated, but luteolytic pulses are

absent during pregnancy so the that ovary does not receive a

luteolytic signal leading to demise of the CL. Lacroix & Kann (1986)

also found that PGFM pulses were absent in early pregnant ewes. This

attenuation of the luteolytic mechanisms in ewes is, therefore,

different from that of cows in which synthesis and secretion of

uterine PGF-2O is inhibited. In cattle (Thatcher et al., 1984b;

Wolfenson et al., 1985) and sheep (Lacroix & Kann, 1983,1986; Zarco

et al., 1984) responsiveness of the uterus to agents which stimulate











PGF-2a release in a lytic pulsatile pattern is attenuated by

pregnancy.

The model for luteolysis includes estrogen induction of oxytocin

receptor and oxytocin activation of the prostaglandin synthesizing

machinery of the endometrium. One of the strongest lines of evidence

for involvement of oxytocin in luteolysis is extension of luteal

function following passive or active immunization against oxytocin in

sheep (Sheldrick et al., 1980; Schams et al., 1983). Additionally,

stimulation of PGFM release following oxytocin administration is

suppressed during pregnancy in both cattle (Lafrance & Goff, 1985)

and sheep (Fairclough et al., 1984). Similarly, PGFM release was

attenuated in pregnant cattle compared to cyclic cattle in response

to E2 injections (Rico et al., 1981; Thatcher et al., 1984b,1986b).

Kittock & Britt (1977) reported that the luteolytic effect of EZ was

decreased in pregnant verus cyclic ewes. More recently, Lacroix &

Kann (1986) found that pregnancy completely abolished PGFM pulses in

response to estradiol. Collectively, these data indicate that

mechanisms for initiating luteolysis are attenuated in both sheep and

cattle. However, the exact mechanisms for blocking luteolytic pulses

of PGF-20 are slightly different. In cattle, synthesis and secretion

of PGF by endometrial tissue is suppressed during pregnancy (Thatcher

et al., 1984b) as is transfer of PGF from uterine vein to ovarian

artery (Wolfenson et al., 1985). In sheep, synthesis of PGF does not

seem to be altered. However, there seems to be enhanced secretion of

PGF toward the uterine lumen (Lacroix & Kann, 1983) in association

with increased basal secretion of PGF (Zarco et al., 1984) and










43

attenuation of pulsatile release of PGF by the endometrium (Lacroix &

Kann, 1980b). In both species, responses to luteolytic doses of

oxytocin and E2 are decreased. It also appears that transport of

PGF-2a by the countercurrent exchange system of uterus and ovary may

decrease. Collectively, these results indicate that, in some

fashion, the concepts is activating an antiluteolytic mechanism to

attenuate PGF-2a secretion and or its potential accessibility to the

CL.



Development of the Bovine Conceptus

With the above discussion in mind, it seems appropriate now to

address the mechanism whereby the concepts mediates its

antiluteolytic effects for CL survival. Understanding of this

mechanism begins with knowledge of critical stages in concepts

development.

Fertilization rate does not represent a significant factor

accounting for embryonic death or "wastage." Henricks et al. (1971)

reported that fertilization rate was 89%, but the proportion of

embryos surviving to day 42 after insemination was only 60%.

Similarly, first service conception rates were 50 to 55% for heifers

(Roche et al., 1977; Wishart et al., 1977) and 52 to 57% for dairy

cows (Mawhinney & Roche, 1978). The exact period of development in

which the majority of pregnancy wastage occurs has been evaluated

(Diskin & Sreenan, 1980; Roche et al., [981). Diskin & Sreenan

(1980) reported that survival rates of conceptuses were 93%, 56%,

66%, and 58% on days 8, 12, 16, and 42, respectively. They concluded









44

that the majority of early embryonic death occurs between days 8 and

16. Similarly, Roche et al. (1981) evaluated time of concepts death

by recovering conceptuses following slaughter. Percentage of animals

pregnant on days 3, 8, 14, 18, and 28 after insemination were 81, 84,

75, 60, and 62, respectively In concurrence with Diskin & Sreenan

(1980), they concluded that 10 to 20% of all ovulated ova are not

fertilized, These data demonstrated that the majority of remaining

embryonic wastage occurs between day 8 and 18, and indicate that this

represents a critical period in concepts development Further

examination to determine if this wastage was associated with

concepts communication with the uterus to signal pregnancy is

warranted.



Timing of the Conceptus Signal Relative to "Maternal Recognition of
Pregnancy"

It is apparent that the concepts must make its presence known if

successful establishment of pregnancy is to occur. If the concepts

does not signal its presence, luteolytic pulses of PGF-20 are

released by the uterus resulting in demise of the CL. Successful

establishment of pregnancy involves secretion of nutrients from the

uterus for concepts growth as well as an alteration of the

luteolytic mechanism for continued CL function and P4 secretion.

This process of events has been referred to as "Maternal Recognition

of Pregnancy" (Short, 1969). The majority of research in this area

has been directed at understanding mechanisms for extension of CL

function, which represents only one aspect of maternal recognition of

pregnancy. Several mechanisms for extension of CL function have been










45

reported. The mechanisms most understood are antiluteolytic-antiPGF,

antiluteolytic-luteoprotective and luteotrophic ones (see Thatcher et

al., 1986a). Luteotrophic mechanisms are those which increase P4

secretion. Antiluteolytic-luteoprotective mechanisms are those which

protect the CL or make it less responsive to luteolytic actions of

PGF-2 a Antiluteolytic-antiPGF mechanisms are those mechanisms which

attenuate PGF-2 synthesis, secretion and or type of release by the

uterus. Evidence exists for each of these mechanisms and discussion

of these comprise the remainder of this review.

Studies in both cattle and sheep have been carried out to

determine if there is a critical time when the concepts "signals"

its presence to the uterus. Moor & Rowson (1966a) found that 65% of

all embryo transfers in sheep made on day 12 of the estrous cycle

resulted in pregnancies whereas only i2% of the ewes receiving

embryos on day 13 became pregnant. The effect of embryo removal from

5th to 15th day of the estrous cycle on interestrus interval was

examined (Moor & Rowson, 1966b). The mean interestrous interval for

all ewes from which embryos were removed between days 5 and 12 of

pregnancy was 18.0 + 0.3 days. In contrast, 93% of ewes from which

embryos were removed on days 13, 14, or 15 had extended cycles (24.5

+ 0.8 days). Collectively, these results indicated that a concepts

must be present in utero on day 12 after estrus if proper signalling

is to occur in the ewe.

Similar evidence for critical timing of conceptus-uterine

communication in establishment of pregnancy exists for cattle.

Betteridge et al. (1984) reported that synchronous transfer (+ 1 day)


L










46

of bovine conceptuses before day 17 after estrus resulted in

successful pregnancies, whereas transfer on day 17 resulted in no

pregnancies at day 42. Humblot and Dalla Porta (1984) observed that

embryo removal on days 9 or 14 had no effect on interval to return to

estrus compared to noninseminated, uterine flushed, cyclic controls.

In contrast, concepts removal on day 16 extended interestrous

intervals 4 to 7 days. Similarly, Northey and French (1980) reported

a cycle extension in cattle from which conceptuses were removed on

days 17, 18, or 19 after estrus, but observed no cycle extension

following concepts removal on days 13 or 15 after estrus. These

results indicate that the bovine concepts, like the ovine concepts,

"signals" its presence in a precise and defined time period. This

period, which is critical for maintenance of the CL appears to occur

between days 15 and 17 after estrus.

Evidence for conceptus-induced maintenance of the CL has

predictably lead to a search for the "signals" responsible for

mediating associated events; these being establishment of a uterine

environment conducive to concepts development, alteration of the

uterine luteolytic mechanism for CL regression, and continuation of

P4 secretion. "Maternal Recognition of Pregnancy" encompasses all of

these processes. However, this discussion and the majority of

research in this area has been dedicated to specifically

understanding the process of CL maintenance. With this in mind a

review of putative concepts signals is in order.

The bovine concepts has been shown to secrete several steroid

hormones between days 13 and 16 after estrus. These include P4,










47

testosterone and small amounts of E2 (Shemesh et al., 1979). Blood

flow to the gravid uterine horn is elevated between days 14 and 18

after estrus (Ford et al., 1979) and the rise may be temporarily

associated with changes in P4 to E2 ratios in cattle. Indeed, E2

stimulates uterine blood flow of cyclic cattle (Roman-Ponce et al.,

1978, Knickerbocker et al., 1986c). A localized increase in uterine

blood flow, induced by estrogen secreted by the concepts, could

increase delivery of nutrients for concepts growth and development.

Therefore, an increase in blood flow to the uterus would in effect

dilute PGF-Za in the blood draining the uterus, thereby reducing the

likelihood of a luteolytic concentration reaching the ovary.

Knickerbocker et al. (1985) reported that bovine conceptuses in

culture, using [3H]-P4 as precursor, produced small amounts of

estradiol, estrone, and estriol, in order of prevalence' Eley et al.

(1983) was unable to detect concepts production of estrogens, but

this may have due to the low sensitivity of their test system. Due

to relatively low secretion rates of estrogens by bovine conceptuses,

a definitive role for them is unclear at this time. That estrogens

are luteolytic when administered late in the estrous cycle (Wiltbank,

1966; Eley et al., 1979; Thatcher et al., 1986b) is evidence for the

importance of having low levels of estrogen present during pregnancy.

Estrogens secreted by the concepts may be rendered inactive by

conjugation or act locally to alter endometrial function to favor

concepts survival. Secretion of estrogen by the bovine concepts is

distinct from that of pig conceptuses which secrete large quantities

of estrogens (Gadsby et al., 1980),that accumulate in uterine










48

flushings (Ford et al., 1982; Geisert et al., 1982), and appear to be

responsible for initiating maintenance of CL function in this species

(Thatcher et al., 1986a).

Other reports have provided evidence for conceptus-mediated

conversion of radiolabelled androstenedione (Chenault, 1980; Gadsby

et al., 1980; Eley et al., 1983), testosterone (Chenault et al.,

1980) and P4 (Knickerbocker et al., 1980; Eley et al., 1983) to an

array of 5 reduced metabolites. The significant point of these

reports was that conversion of P4 to 5 -reduced pregnanes was

favored. Smaller amounts of androstenedione and 5 s-reduced

androstanes were also produced, but not to a great extent. The high

activity of the 5 -reductase system in concepts tissue was distinct

from that of the uterine endometrium which utilizes the 5 0-reductase

system (Eley et al., 1983). It was hypothesized that 5 a-reduced

metabolites might act by stimulating erythropoietic activity

(Gorshein & Gardner, 1970), hemoglobin synthesis (Necheles & Rai,

1969) and reduce activity of the uterine myometrium (Kubli-Garfias et

al., 1979). Another possible role for high activity of the 5 -

reductase system in conceptuses might be to reduce availability of

precursors for E2 production by the concepts which would be

luteolytic if released in sufficient amounts (Thatcher et al.,

1984a).

Prostaglandin production by the bovine concepts has also been

described (Lewis et al., 1982; Lewis & Waterman, 1983; Shemesh et

al., 1979). Bovine conceptuses recovered on day 13, 15, or 16 of

pregnancy and cultured for 48 h are capable of producing PGF and PGE-











2, the production of which increases with age of the concepts

(Shemesh et al., 1979). Lewis et al. (1982) also reported PGF-2a,

PGE-2, and PGFM production by bovine conceptuses collected on days 16

and 19 after mating and cultured for 24 h. Production of these PGs

were greater by day 19 verus day 16 conceptuses. Endometrial slices

from day 16 and 19 of pregnancy produced PGF-2c, PGFM and PGE-2, but

production was similar for these two days. Endometrial tissues and

blastocysts metabolized 34.3 + 1.5% and 7.5 + 1.6% of [3H] PGF-2C to

[3H] PGFM, respectively.

To further characterize PG production by the maternal-conceptus

unit, Lewis & Waterman (1983) evaluated conversion of [3H]arachidonic

acid to PGs in co-cultures of concepts and endometrial tissues. In

this experiment, both concepts and endometrium cultured alone

produced PGFM, PGF-2a, and PGE-2, however, production per mg of

tissue weight was very low for endometrium compared blastocysts.

Total PG secretion by uterine endometrium would, however, exceed that

of the concepts. When blastocysts were cultured in endometrial

conditioned culture medium, incorporation of [3H] arachidonic acid

was not altered. In contrast, co-culture of blastocysts with

endometrium resulted in increased metabolism of [3H] arachidonic acid

to PGFM and PGE-2 with decreased metabolism to PGF-2o. This

represented alterations in metabolism of arachidonic acid to PG

products with no alteration in the proportion of arachidonic acid

metabolized by the concepts. Results indicated that endometrium is

capable of metabolizing [3H] PGF-2a to PGFM and of shifting

metabolism of [3H] arachidonic acid away from [3H] PGF-2a and towards










50

[3H] PGE-2. This shift from PGF-2a production to PGE-2 production

may represent metabolism of concepts derived PGF-Za to PGE-2,

because metabolism of arachidonic acid by the concepts to PGF-2 ,

PGE-2 and PGFM was not altered by incubation with endometrial culture

supernatants. Only when endometrium and concepts tissue were

incubated together was the array of PGs produced altered. This

suggests that the endometrium can regulate amounts and ratios of PGs

in the uterine lumen during pregnancy.

In cattle and sheep, PG production by the concepts is probably

minimal compared to the secretary capacity of the entire uterine

endometrium. With this in mind, the function of conceptus-derived

PGs remains obscure. The effects of pregnancy on endometrial PG

production as discussed in this review are more dramatic.

In sheep, endometrial PGF-2a production does not appear to be

attenuated. In fact, endometrial production is greater in pregnant

verus cyclic ewes (Ellinwood et al., 1979; Findlay et al., 1981).

Secretion was preferentially directed toward the uterine lumen rather

than the blood stream (Lacroix & Kann, 1983), which would result in

uterine accumulation of PGF-2 0 The antiluteolytic effect of the

concepts in ewes was to increase basal release of PGF-2a from the

uterus and eliminate release of pulses of PGF-2 that would be

luteolytic (Zarco, 1984).

Uterine flushings from pregnant cattle (Bartol et al., 1981b) were

reported to contain considerable quantities of PGF-2 This

accumulation of PGF-2t could be the result of conceptus-derived PGF-

2 a(Lewis & Waterman, 1983) or result from decreased transfer of









51

endometrial PGF-2a out of the uterus as proposed by Thatcher et al.

(1984a). Wolfenson et al. (1985) demonstrated that ovarian arterial

concentrations of PGF were lower for pregnant than non-pregnant

cattle supporting the theory that uterine release of PGF-2 was

decreased during pregnancy. Similarly, peripheral concentrations of

PGFM in pregnant cattle were attenuated (Kindahl et al., 1976;

Betteridge et al., 1984; Thatcher et al., 1984a). These results

indicate that PGF production by the endometrium, as reported for

endometrial tissue in culture (Thatcher et al., 1984b) or perifused

(Gross et al., 1988c), is attenuated.

In sheep, PGE-2 secretion by the uterus is elevated (Ellinwood et

al., 1979; Silvia et al., 1984) and attenuates the luteolytic effect

of PGF-21 (Henderson et al., 1977; Pratt et al., 1977; Reynolds et

al., 1981). In cattle, PGE-2 attenuates the luteolytic effects of

PGF-20 only for a short period of time (Chenault, 1983; Reynolds et

al., 1983) and is not produced in greater amounts by cultured (Curl

et al., 1983) or perifused (Gross et al., 1988c) pregnant

endometrium. The reduction of PGF-Za secretion in cattle would cause

an increase in the PGE-2:PGF-Za ratio in the circulation. Therefore,

PGE-2 may play some role in luteal maintenance and hence embryonic

survival in cattle, which is supported by limited cycle extensions

following PGE-Z administration (Gimenez & Henricks, 1983; Reynolds et

al., L983).

A luteotrophic substance produced by day 13 to 18 bovine

conceptuses has been identified (Hickey & Hansel, 1987). This

concepts product, characterized as a small (<10 kDa), heat-labile,










52

and lipid soluble molecule, was shown to stimulate P4 synthesis by

dispersed bovine luteal cells in vitro and suggests that the

concepts secretes luteotrophic signals, to enhance luteal P4

production, prior to secreting the antiluteolytic signal. The nature

of this molecule has not been determined, but it was speculated to be

either a steroid, luteotrophic prostaglandin, concepts derived

platelet activating factor (PAF) or some combination of these.

Plante et al. (1987) demonstrated that conceptuses produce

luteotrophic substances. Trophoblastic tissue from bovine

conceptuses were cultured and supernatants tested for luteotrophic

activity by culturing with rat granulosa cells. Supernatants

increased P4 secretion indicating that luteotrophic substances were

secreted by bovine conceptuses.

Although the luteotrophic factor described by Hickey & Hansel

(1987) has not been identified, it is not proteinaceous and

increasing evidence implicates PAF as a possible candidate for this

role.

Conceptuses also have been shown to secrete a variety of proteins,

the array of which is age dependent (cattle: Bartol et al., 1981b,

1985; Geisert et al., 1988; Godkin et al., 1988; and sheep: Godkin

et al., 1982). Godkin et al. (1982) characterized secretary proteins

produced by ovine conceptuses collected on of days 13, 14, 21, and 23

of gestation and cultured for 24 h. They noted on day 13, a time

just following the critical period for maternal recognition of

pregnancy, that the concepts produced one major protein. This major









53

secretary component had a low molecular weight, migrated as an

acidic protein (pI = 5.5) and was initially called protein X.

Bartol et al. (1985) characterized bovine concepts secretary

proteins (bCSP) released into medium by days 16, 19, 22, and 24

bovine conceptuses. On day 16, one group of proteins, low molecular

weight (22 to 26 kDa) with an isoelectric point of 6.5 to 5.6,

represented the major secretary component of bCSP. These proteins

may be critical to maintenance of the CL and will be discussed in

depth later in this review. Other less prominent polypeptides were

present at this stage and as age of the concepts increased, the

distribution of proteins became more complex. The lower molecular

weight, acidic proteins increased in abundance by day 19 and declined

to days 24 and 29.

More recently, Godkin et al. (1988) characterized bovine concepts

secretary proteins from day 17 to day 38 of pregnancy. Conceptuses

were shown to secrete a major low molecular weight acidic protein

that represented the major product from days 17 to 22 of pregnancy.

In this study, these low molecular weight, acidic proteins were still

detectable in trace quantities on day 38. Geisert et al. (1988)

collected bovine conceptuses on days 15, 16, and 17 and classified

them by length. The low molecular weight acidic proteins were

detectable in small quantities from conceptuses averaging 14 mm in

length and then secretion increased as concepts size increased from

40 to 100 mm and greater than 100 mm in length. Complexity of the

secretary pattern of proteins increased greatly in conceptuses

greater than 100 mm in length.










54

Protein production by cultured bovine conceptuses ( g/mg wet

weight) increased significantly from day 16.5 (4.9 + 2.4) to day 17.0

(15.8 + 5.2; Knickerbocker et al., 1986b). These alterations in

protein synthetic capacity from day 16.5 to 17.0 correspond to the

time of maternal recognition of pregnancy in cows. Results of these

studies led to a further examination of effects of pregnancy-specific

proteins of concepts origin on endometrial protein production and

function.

The major, low molecular weight acidic protein of day 14-16 ovine

conceptuses (hereafter referred to as oTP-1) was purified by Godkin

et al. (1982) and its effect on endometrial protein production

evaluated (Godkin et al., 1982; Vallet et al., 1987; Salamonsen et

al., 1988). Incubation of endometrial tissue from day 12 nonpregnant

ewes, with oTP-1 increased incorporation of [3H]leucine into secreted

macromolecules (28 to 48%) and decreased incorporation of radiolabel

into tissue proteins (4 to 17%) compared to endometrium incubated

with BSA (Godkin et al., 1984a). Specifically, oTP-1 caused

enhancement or induction of 6 proteins from culture of endometrium.

More recently, Vallet et al., (1987) performed a similar experiment

in which day 12 cyclic endometrium was incubated with oTP-1 or BSA.

Although percent incorporation of either [3H] or [35S] methionine

into secreted macromolecules was unaffected by treatment, incubation

of endometrium with oTP-1 selectively increased secretion of 11

proteins and decreased secretion of 6 proteins compared to BSA-

treated tissues. The most striking amplification was of a 70 kDa

protein (pI = 4) whose secretion was increased 370% by incubation










55

with oTP-1. Salamonson et al. (1988) also demonstrated that oTP-1

selectively stimulated secretion of 5 proteins by dispersed

endometrial cells from sheep.

Bartol et al. (1981b) characterized proteins present in uterine

flushings of cyclic and pregnant cattle on days 8 to 19. Total

recoverable protein tended to increase toward the end of the estrous

cycle (days 14, 16, 19) in cyclic cattle, but there was no

significant effect of day on recoverable protein during pregnancy.

Generally, recoverable protein was lower in pregnant versus cyclic

cattle from days 8 through 16 but amounts were comparable on day 19.

The array of proteins recovered on day 19 of the cycle and pregnancy

also was analysed by one-dimensional sodium dodecyl sulfate

polyacrylamide gel electrophoresis. Generally, protein patterns were

similar on days 8 through 16 for pregnant and cyclic cattle.

However, on day 19 four protein species (relative molecular weights x

10-3 = 15.2, 306.8, 322.2, and 342.8) specific to uterine flushing

from pregnant cattle were identified. Results from this study are

difficult to interpret because uterine proteins in uterine flushings

may be from endometrium or the concepts. Knickerbocker et al.

(1986b) found that concepts wet weight and protein synthetic

capacity increase significantly on day 17 of pregnancy. Thus, it

would appear that presence of the concepts prior to day 19 decreased

protein production by the uterus since total recoverable protein was

the same or lower.

Geisert et al. (1988) characterized the qualitative array of

proteins secreted into medium after culture of endometrial tissue of









56

day 17 non-pregnant and pregnant cattle, Induction of a group of low

molecular weight peptides (M- = 14-16,000; pi = 7.2-6.8) and a

polypeptide with a Mr= 35,000 (pI = 8.4-703) by endometrium from the

uterine horn ipsilateral to the CL during pregnancy was observed.

More recently, Gross et alw (1988a) incubated endometrium from day 17

cyclic cattle with bCSP to assess effects upon protein and PG

synthesis. They reported that bCSP decreased incorporation of [3H]

leucine into both secreted and tissue proteins of endometrial

explants compared to BSA treated controls. While secretion of

protein into medium was decreased by bCSP, secretion of two proteins

(10 and 13 kDa) was amplified.

Presence of a concepts in utero or intrauterine infusion of

secretary components of the concepts in cattle and sheep have a

profound effect on endometrial protein synthesis and secretion. The

role of these proteins, whether for alteration of PG metabolism or

for establishing an optimal environment for the developing concepts

requires further study.

Embryonic homogenates (1 to 2 embryo equivalents/infusion) of day

14-15 ovine conceptuses when infused daily into uterine lumen of

nonpregnant sheep beginning on day 12, extend CL function; estrous

cycle lengths were greater than 224 days for ewes receiving

homogenates compared to 16.6 days for controls (Rowson & Moor, 1967).

Daily infusions of day 25 ovine concepts homogenates, day 14 pig

homogenates or day 14-15 heat treated ovine conceptuses did not

extend the interestrous interval. These results reaffirm that the

antiluteolytic, concepts signal is stage and species specific and










57

was possibly proteinaceous in nature. Martal et al. (1979) reported

extension of CL function by intrauterine infusion of day 14-16 ovine

concepts homogenates and termed the antiluteolytic substance of the

concepts "Trophoblastin". They found the signal to be stage-

specific, as day 21-23 concepts homogenates would not extend cycles.

The signal molecule was heat labile and inactivated by pronase.

As previously described, Northey and French (1980) determined that

the bovine concepts must be present in utero by day 15 to 17 after

estrus for establishment of pregnancy to occur. To determine the

nature of the concepts signal, they carried out intrauterine

infusions of one homogenized day 17 to 18 bovine concepts twice

daily between days 14 and 18 after estrus in cycling heifers. This

treatment regime resulted in an extension of CL function and

interestrous intervals (control vs. concepts homogenate; 21.1 + 0.74

verus 24.0 + 0.38 days). Although, infusion of concepts homogenates

extended CL lifespan, there was no evidence of luteotrophic

stimulation since concentrations of P4 in serum did not differ

between treatment groups. The cycle extension observed in this and

other similar experiments is somewhat surprising. Bartol et al.

(1985) demonstrated by 2-dimensional polyacrylamide gel

electrophoresis that typical bCSP were was not easily detected in

tissue homogenates of day 22 conceptuses while bCSP were actively

secreted into culture medium at this same time. Therefore, it seems

likely that concepts homogenates utilized in the experiments

described would contain fewer of the typical, concepts secretary

proteins, yet cycles were extended by 3 to 4 days. In a similar


M












experiment, Humblot and Dalla Porta (1984) introduced into the uterus

whole day 12 conceptuses (2 per infusion) or day 16 conceptuses (1

per infusion), which had been frozen and thawed, transcervically to

evaluate their effect on CL lifespan. Day 16 but not day 12

conceptuses extended CL lifespan based on examination of P4 secretary

profiles. The interestrous interval for heifers receiving no

treatment, or intrauterine infusion of saline, day 12 or day 16

conceptuses twice daily from days 15 to 19 were 22.3, 19.0, 20.0, and

27.0 + 1.0 days, respectively. Administration of day 16 conceptuses

extended interestrous intervals 4 to 7 days. That day 12 conceptuses

did not extend interestrous intervals lends support to the theory

that the antiluteolytic concepts signal is stage specific.

The ovine and bovine concepts "signals" were later shown to be

secreted by the trophoblast rather than the inner cell mass or embryo

proper (Heyman et al., 1984). Trophoblastic vesicles, which had been

derived from sections of day 14 cow and days 11-13 sheep conceptuses

cultured for 24 h in vitro, were transferred to recipient cattle and

sheep, respectively, on day 12 after estrus. In cattle,

trophoblastic vesicles caused extension of cycle length in 8 of 12

recipients which had 25 to 37 day estrous cycles. In sheep,

trophoblastic vesicles caused extension of estrous cycles in 7 of 12

recipients, which had 20 to 54 day estrous cycles. Interestrous

intervals were extended for 2 of 11 ewes receiving 2 trophoblastic

vesicles on day 12 after estrus from day 13 bovine conceptuses

(Martal et al., 1984). Similarly, interestrous intervals were

extended in 2 of 10 heifers receiving 2 trophoblastic vesicles on day












12 of the estrous cycle from day 11 to 13 ovine conceptuses. This

interspecies transfer resulted in extended estrous cycles in about

20% of the cases studied (Martal et al., 1984) compared to 60%

extended cycles for intraspecies transfers (Heyman et al., L984).

That interspecies transfers of trophoblastic vesicles is partly

effective in extending CL lifespan which implies that the

antiluteolytic concepts signals for these species may be similar.

Data from the proceeding studies indicated that the antiluteolytic

concepts signal for the ewe is proteinaceous in nature. The theory

that the antiluteolytic signal is proteinaceous was further tested by

Godkin et al. (1984b) who infused ovine concepts secretary proteins

(oCSP) and purified oTP-1, which represents the major, low molecular

weight (17 kDa) acidic (pI 5.5) protein produced by day 16 ovine

conceptuses and described by Godkin et al., (1982), into the uterine

lumen of cyclic day 12 ewes. Intrauterine infusion of oCSP from days

12 to 18 resulted in extension of CL function to days 24, 34, and

beyond day 52 in treated ewes. Treatment with oTP-1 extended CL

function 4 days longer than controls. These results were confirmed

and extended by Vallet et al. (1988b) who infused oCSP from day 16

conceptuses, oTP-1, oCSP minus oTP-1, or serum protiens into cyclic

ewes from days 12 to 14. Intrauterine infusion of oCSP or oTP-1

extended the interestrous interval to 25 and 27 days, respectively,

compared to control ewes (19 days) and ewes receiving oCSP-oTP-1 (19

days). Ewes in this experiment received injection of E2 on day 14

and oxytocin of day 15 to test uterine response following infusions

which may have reduced cycle extension by oTP-1 compared to the cycle









60

extension which may have occurred without oxytocin and E2. These

studies indicate that oTP-1 is the major antiluteolytic agent in oCSP

as cycle length was unaffected by infusion of oCSP from which oTP-1

had been removed.

Proteins secreted by days 16 to 18 bovine conceptuses also

extended interestrous intervals of cattle receiving intrauterine

infusions from days 15 to 21 after estrus (Knickerbocker et al.,

1986b). The total complex of bCSP secreted into medium from cultured

conceptuses extended interestrous intervals 33,4 + 2.5 versus 23.5 +

0.5 days for controls receiving serum proteins. Intrauterine infusion

of the primary 5 B-reduced steroid from bovine conceptuses, 5 8-

pregnane-3 c-ol-20-one, had no effect on estrous cycle lengths.

Collectively, these studies indicate that the major, concepts

derived, antiluteolytic signal responsible for extending CL function

is proteinaceous in nature.

Conceptus proteins have also been shown to alter uterine PG

secretion in vivo. Release of PGF from the uterus (Vallet et al.,

1988b) and peripheral PGFM concentrations (Fincher et al., 1986;

Vallet et al., 1988b) in response to E2 or oxytocin was evaluated in

cyclic ewes receiving oCSP or oTP-1 on days 12 to 14 of the cycle.

In both studies the release of PGF and PGFM was decreased in response

to estradiol and oxytocin challenges In cattle, Knickerbocker et

al. (1986b) reported that PGF release by cattle receiving

intrauterine infusion of bCSP from days 15 to 18 was attenuated on

days 18, 19 and 20 compared to controls receiving serum proteins.

Release of PGFM also was decreased in cattle receiving bCSP infusions










61

from day 15.5 and injected with E2 on day 18 after estrus

(Knickerbocker et al., L986a).

A component of bCSP and oCSP (oTP-1 in sheep) appears to be the

antiluteolytic signal of bovine and ovine conceptuses. One remaining

question is, how are these proteins acting to decrease uterine PG

synthesis and or secretion? One possibility is that concepts

antiluteolytic signals stimulate induction of an endometrial

intracellular inhibitor of enzymes involved in PG synthesis. An

inhibitor of PG synthesis was identified in uterine preparations of

cyclic cows by Wlodawer et al. (1976). This inhibiting factor was

found to suppress the fatty acid cyclooxygenase system. Shemesh et

al. (1984) also found an endogenous, heat-labile, inhibitor of PG

synthesis in maternal caruncular tissue of placemtones that modulated

placental PG synthesis during pregnancy. Basu and Kindahl (1987)

demonstrated the existence of an endogenous endometrial inhibitor of

PG synthesis during the bovine estrous cycle and reported that its

activity increased during early pregnancy. They also reported that

presence of a concepts increased activity of the inhibitor in the

non-gravid uterine horn, suggesting the existence of a humoral factor

which regulates PG synthesis rather than the physical presence of the

concepts. However, the theory that the PG synthesis regulator is a

humoral-factor should be veiwed with caution as it is possible that

concepts products might be carried to the non-gravid uterine horn

through the uterine body. Recently, Gross et al. (1988b)

demonstrated the presence of an intracellular endometrial inhibitor

of PG synthesis which was, again, more active in tissues from










62

pregnant than cyclic cattle. The inhibitor was in the high speed

cytosolic supernatant fraction of endometrial tissues and was

proteinaceous.

To determine if concepts proteins were responsible for induction

of the intracellular PG synthesis inhibitor, Gross et al. (1988a)

evaluated effects of incubating day 17 cyclic endometrial explants

with bCSP, compared to BSA as a control, on induction of the

inhibitor. Cytosol from endometrium incubated with bCSP reduced PG

synthesis by the cotyledonary microsomal PG generating system (Gross

et al., 1988a).

These data indicate that the antiluteolytic agent of bovine

conceptuses is proteinaceous, a component of bCSP, and likely acts by

inducing an endometrial inhibitor of PG synthesis. As oTP-1 has been

identified as the ovine concepts signal and there seems to be

similarities between antiluteolytic mechanisms of ovine and bovine

conceptuses, it seems possible that analogous concepts protein

systems may exist.

A great deal is known about the ovine concepts antiluteolytic

signal. Rowson & Moor (1967) first proposed that the concepts

antiluteolytic signal might be proteinaceous in nature since heat-

treatment of concepts homogenates prior to intrauterine infusions

abolished their antiluteolytic effect. This was confirmed later by

Martal et al. (1979) who termed the putative substance

"Trophoblastin". Electrophoretic analysis of oCSP confirmed the

presence of a major concepts secretary product (19 kDa; pi = 5.3-

5.7) which was referred to as oTP-1 (Godkin et al., 1982). This










63

concepts signal was demonstrated to attenuate luteolytic PGF release

by the uterus (Godkin et al., L984b; Vallet et al., 1988b) and PGF

release in response to EZ and oxytocin administration (Fincher et

al., 1986; Vallet et al., 1988b) in cyclic ewes. The source of oTP-1

was identified as the trophectoderm as trophoblastic vesicles will

extend CL function in cyclic ewes (Heyman et al., 1984). Godkin et

al. (1984a) demonstrated that oTP-1 is taken up by endometrial

surface epithelium and superficial glandular epithelium presumably by

binding to specific receptors. The antiluteolytic mechanism is not

the result of cAMP, cGMP or inositol phospholipid turnover in

endometrial tissue (Vallet et al., 1987). Further characterization

of oTP-1 demonstrated that three isomeric forms exist and that none

are glycosylated (Anthony et al., 1988). In addition to its

antiluteolytic effects, oTP-1 has been shown to induce or enhance

secretion of several endometrial proteins (Godkin et al., 1982;

Vallet et al., 1987; Salamonsen et al., 1988). Hence, oTP-1 appears

to be the antiluteolytic signal from the ovine concepts responsible

for initiating events associated with maintenance of CL function. Of

particluar importance to this review are similarities between the

antiluteolytic mechanism for the ewe and cow.

Evidence now exists suggesting that oTP-1 is an interferon-like

molecule (Imakowa et al., 1987; Stewart et al., 1987; Charpigny et

al., 1988). In these studies, the nucleotide sequence of oTP-1 was

determined and the inferred primary amino acid sequence was found to

have 45-55% homology with a variety of interferons (IFN) of the alpha










64

family. The greatest degree of sequence homology was with bovine

IFN-O-II (70.3%).

Interferons are molecules which are classically ascribed as having

antiviral activity. Three major classes of interferons have been

described. These are 1) leukocyte or alpha IFN, 2) fibroblast or

beta IFN, and 3) immune or gamma IFN (Pestka & Baron, 1981). As the

inferred primary amino acid sequence of oTP-1 indicates homology to

the alpha IFN family, the remaining discussion will be limited to

this class of IFN. The genes coding for bovine alpha IFN (IFN-a) can

be divided into two classes of molecules, class I (IFN-t-I) and class

II (IFN-a-II; Capon et al., 1985)v Activites for IFN other than

their antiviral activities have also been identified, some of which

include, antigrowth activity, stimulation of cytotoxic activities of

lymphocytes and macrophages and of natural killer cell activity as

well as increased expression of some tumor-associated antigens (see

Pestka et al,, 1987).

Of particular interest to this discussion are IFN effects on PG

synthesis. Alpha IFN's have been shown to suppress PGE-2 secretion

by mouse monocyte-macrophage (Boraschi et al., 1985) and human

mononuclear leukocytes (Dore-Duffy et al., 1983). However, treatment

of human leukocytes with IFN-a had no effect on release of PGF (Dore-

Duffy et al., 1983)b In contrast, Salamonsen et al. (1988) reported

suppression of both PGE and PGF-2a secretion of ovine endometrial

cells treated with oTP-1 or IFN-o-II. Treatment with IFN-c-I also

resulted in extension of interestrous intervals from 22.8 + 0.8 to

26.8 + 1.4 days for cyclic cows receiving infusions from days 15.5 to


I











65

21 after estrus (Plante et al., 1987). However, in vitro effects of

IFN-a-I are in contrast to effects of bCSP. Treatment with bCSP

decreased PGF secretion by endometrial explants, induced an

intracellular inhibitor(s) of PG synthesis but had no effect on PGE-2

secretion (Gross et al., 1988a). IFN-a-I had no effect on PGF

secretion, did not induce the intracellular inhibitor of PG synthesis

and enhanced PGE-2 secretion (Plante et al., 1988). The differences

may be ascribed to the IFN used in these experiments, that being IFN-

a-I, which is not as homologous to the inferred primary sequence of

oTP-1 as that of IFN-a-II (Stewart et al., 1987; Imakawa et al.,

1987; Charpigny et al., 1988) which was used by Salamonsen et al.

(1988).

It seems clear that oTP-1 from the ovine concepts and some

component of bovine conceptuses mediate antiluteolytic regulatory

mechanisms in the uterine endometrium. It also is apparent that the

regulatory system of the ewe and cow are similar in many respects.

In view of these findings, identification, characterization and

function of the putative bovine antiluteolytic signal of the

concepts became the objective of research contained in this

dissertation.

















CHAPTER 2
IDENTIFICATION OF BOVINE TROPHOPLAST PROTEIN-1, A SECRETARY PROTEIN
IMMUNOLOGICALLY RELATED TO OVINE TROPHOBLAST PROTEIN-1



Introduction

Embryo transfer experiments have established that maternal

recognition of pregnancy in the ewe occurs 12-13 days after onset of

estrus (Moor & Rowson, 1966a,b), The corresponding period for the

cow is day 15-16 (Betteridge et al., L984; Northey & French, 1980).

In both species the estrous cycle can be extended significantly if

extracts of conceptuses (Rowson & Moor, 1967; Martal et al., 1979;

Northey & French, 1980; Humblot & Dalla Porta, 1984) or trophoblast

tissue (Martal et al., 1984; Heyman et al., 1984) are introduced into

the uteri of nonpregnant recipients just before this critical period

during which maternal recognition of pregnancy occurs. Evidence has

accumulated that in sheep the active substance is proteinaceous and

produced for a limited period, not extending beyond days 21-23 of

pregnancy (Rowson & Moor, 1967; Martal et al.,1979; Godkin et al.,

1982).

Attention has focused on one particular secretary protein of the

sheep concepts, oTP-1 (Godkin et al., 1984a,b; Hansen et al., 1985).

This polypeptide is released as a major product by cultured sheep

conceptuses between days 13 and 21 and is produced maximally around

days 15-17 (Godkin et al., 1982; Hansen et al.,1985). It causes

66


I









67

extension of luteal lifespan when introduced in purified form into

uteri of nonpregnant recipient ewes (Godkin et al., 1984b; Vallet et

al., 1988b). The protein consists of a group of 3-4 isoelectric

variants (pI 5.4-5.7) of relative molecular weight 19,000 (Godkin et

al., 1982). Evidence has been presented to indicate that oTP-1 is a

hormone-like substance which acts, in a paracrine manner, on the

maternal endometrium (Godkin et al., 1984a).

The conceptuses of cows cultured in vitro also release a group of

acidic, low molecular weight polypeptides. As with oTP-1, their

synthesis is limited to a short, 7-10 day period (days 16-26) which

coincides with the time at which maternal recognition of pregnancy

occurs (Bartol et al., 1985). Introduction of total unfractionated

proteins released by cultured bovine conceptuses into nonpregnant

recipient cows causes a significant extension of the interestrous

interval (Knickerbocker et al., 1986b). Since the low molecular

weight acidic proteins are the major component of bCSP, Bartol et al.

(1985) suggested that they may be homologous to oTP-1. In addition,

Heyman et al. (1984) have shown that transfer of trophoblastic

vesicles, comprised of trophectoderm and extraembryonic endoderm

derived from day 11-13 sheep conceptuses, to day 12 recipient cows

resulted in extension of luteal lifespan in a significant number of

animals. They further demonstrated that reciprocal interspecies

transfer of bovine trophoblastic tissue to recipient ewes had a

similar effect. Together these results suggest that the trophoblast

of ewes and cows produces a functionally similar substances) which

is recognized by the respective species. The object of the










68

experiments described in this chapter was to determine whether oTP-1

crossreacts immunologically with components) of the bCSP.



Materials and Methods

Materials

All materials used were supplied by vendors as noted by Godkin et

al. (1984a), Bartol et al. (1985) and Hansen et al.(1985).

Animals

Adult crossbred ewes, primarily of Rambouillet breeding, were

checked twice daily for estrus with vasectomized rams. Ewes were

mated at behavioral estrus (day 0) and every 8 to 12 h thereafter to

two intact rams'at each breeding period. Pregnant ewes were

anaesthetized and reproductive tracts were exposed by midventral

laparotomy. Intact conceptuses were flushed from uteri on day 16 of

pregnancy with a modified minimum essential medium (MEM, see p. 69)

at 370C and collected in sterile serum bottles (Godkin et al., 1982).

Cows and heifers of Holstein, Jersey and crossbred beef breeding

from the University of Florida research herds were utilized for

collection of bovine conceptuses. The cattle were maintained on

pasture and checked for estrus by using visual observation and bulls

with penile shunts. All animals were mated at first observation of

estrus (day 0) to an intact Brown Swiss bull and artificially

inseminated about 12 h later.

Cows and heifers were slaughtered on day 17 or 18 of pregnancy.

Reproductive tracts recovered after exsanguination were placed in

plastic bags and transported on ice to a sterile, laminar flow hood











where they were trimmed of excess tissue, including the ovaries and

oviducts. The cervix was closed by applying a large, curved,

Rochester-Ochsner force to the anterior cervix. A plastic, 50-ml

syringe fitted with a 16-guage needle was then used to inject 40 ml

sterile MEM into the uterine lumen through the tip of the uterine

horn contralateral to the ovary bearing the corpus luteum. The

anterior tip (about I cm) of the horn ipsilateral to the corpus

luteum had been removed to provide an enlarged opening. Conceptuses

were flushed through this opening into sterile, plastic, culture

dishes.

Medium Preparation

Eagle's minimum essential medium (MEM, Gibco custom formula #86-

5007) was modified by supplementation with penicillin (100 units/ml),

amphotericin B (250 ng/ml), streptomycin (100 Pg/ml), insulin (0.2

units/ml), non-essential amino acids (1%, v/v) and glucose (5 mg/ml).

Medium also was supplemented with D-Ca pantothenate (100 pg/ml),

choline chloride (100 pg/ml), folic acid (100 Pg/ml), i-inositol

(200 Pg/ml), nicotinamide (100 Pg/ml), pyridoxal-HCl (100 pg/ml),

riboflavin (10 pg/ml) and thiamine (100 pg/ml). Content of leucine

was limited to 0.1 times normal (5.15 mg/l) to enhance uptake of L-

[ H]leucine when added to cultures. Medium was filter sterilized

(0.22 Pm) and stored at 40C.

In-Vitro Culture of Conceputses

After collection, conceptuses from cows and ewes were transferred

to plastic culture dishes containing 15 ml fresh modified leucinee

deficient), Eagle's MEM and 100 PCi L- [3H]leucine. Conceptuses









70

were cultured for 24 h in a controlled atmosphere chamber (Model

number 7741-10010, Bellco Biological Glassware, Vineland, NJ,

U.S.A.), flushed for 10 min with 50% N : 47.5% 0 :2.5% CO2 (v/v)

and maintained at 370C in the dark on a rocking platform.

Incubations were terminated by removing conceptuses from culture

medium. The medium was frozen at -200C until used in subsequent

studies.

Purification of oTP-1

Ovine TP-1 was purified by the method of Godkin et al. (1982).

Purity was confirmed by polyacrylamide gel electrophoresis (Godkin et

al., L982).

Preparation of Conceptus Culture Medium for Analysis

Medium from cultured conceptuses was centrifuged at 12,000 g for

10 min to sediment cellular material. Supernatant fractions pooled

from several cultures then were dialysed (M cutoff 1,000)

extensively (4 liters, changed 4 times) against 10 mM Tris-HCl buffer

(pH 8.2) at 40C to remove low molecular weight compounds, e.g. salts

and unincorporated radiolabelled precursors. Whenever the protein

concentration of a sample was too low for immediate analysis, samples

were dialysed (M cutoff 1,000) against double distilled water,

lyophilized and resolubilized in an appropriate buffer.

Protein Determination

Protein concentrations were determined by the method of Lowry et

al. (1951) using bovine serum albumin as the standard.












Immunodiffusion Techniques

Immunodiffusion plates containing 5 ml 1% (w/v) agarose in 0.07 M

sodium phosphate buffer (pH 7.4) were prepared using 6-well tissue

culture plates (diameter 35 mm) according to the method of

Ouchterlony & Nilsson (1974). Antibody specificity was determined by

placing antiserum raised in rabbits against oTP-1 (Godkin et al.,

1984a) in the center well of each plate with various dilutions of

ovine CSP and bovine CSP introduced into the outer wells. The plates

were placed in a container to maintain a moist atmosphere. Resultant

precipitation bands were observed at 24-48 h at room temperature.

Plates were extensively washed in Dulbecco's saline (PBS; Ov2 g

KCl/l;2.16 g KH2PO4.7 H20/1, 8 g NaCl/1; Dulbecco & Vogt, 1954) to

elute nonprecipitated material, and the bands stained with Coomassie

blue. After destaining, gels were photographed.

Solid-Phase Radidbinding Assay

Bovine concepts secretary proteins, purified oTP-1 and bovine

serum albumin were each adjusted to a concentration of 50 pg/ml in

PBS. Anti-oTP-1 antiserum was enriched for immunoglobins by

precipitation with ammonium sulphate and subsequent fractionation on

a DEAE-cellulose column (Good et al., 1980). Proteins were eluted

with a 150 ml linear salt gradient (0-0.25 M NaC1 in 10 mM Tris-HCl

buffer, pH 8.2) and 2.0 ml fractions collected. The early eluting

IgG-rich peak of anti-oTP-1 (fractions 3-19) was pooled and utilized

in subsequent steps. Bovine CSP, purified oTP-1 or bovine serum

albumin (50 11 of each per well) were allowed to adsorb passively to

the wells of 96-well flexvinyl plates ( Falcon brand; Fisher









72

Scientific, Orlando, FL) for I h at room temperature. These protein

solutions were withdrawn, and plates washed once with 0.1% bovine

serum albumin (w/v) in PBS (pH 7.4). Anti-oTP-1 antiserum or normal

rabbit serum (NRS) then was added at increasing dilutions and allowed

to stand in the wells at 40C overnight. These solutions were removed

and plates were washed with PBS containing bovine serum albumin.

After washing, 25I-labelled, sheep anti-rabbit IgG antibody (50,000

c.p.m.; sp.act. 06 c.p.m./pg) which had been affinity purified was

added to each well and left for I h at room temperature. The unbound

labelled second antibody was then removed and the plates washed three

times with PBS-bovine serum albumin. Wells were cut from the plates

and the amount of bound 5I measured.

Immunoprecipitation

Duplicate aliquots of ovine and bovine concepts culture medium

(80 Pg and 120 pg protein, respectively) were lyophilized and

redissolved in immunoprecipitation buffer [0.35 ml 0.3 M NaC1, 0.05 M

Tris-acetate (pH 7.5), 1 mM phenylmethylsulphonyl fluoride, I mM

disodium EDTA, 0.1 mg bovine serum albumin/ml, 0.02% (w/v) NaN3 and

2% (v/v) Nonidet P-40]. Anti-oTP-1 antiserum (0.05 ml) or normal

rabbit serum (0.05 ml) was added to ovine and bovine samples and the

tubes were placed on a tube turner overnight at 4 C. Subsequently,

0.1 ml of a 10% (v/v) suspension of Protein A-Sepharose was added to

each tube and allowed to incubate for 6 h at room temperature on a

tube turner. The Sepharose suspension was centrifuged (15,000 g for

I min) and washed five times with I ml detergent buffer [0.05 M Tris-

acetate (pH 7.5), 0.5% (v/v) Nonidet P-40, 0.1% sodium dodecyl









73

sulphate, 0.3 M NaCI and 0.02% NaN3]. Protein absorbed to the gel

beads was solubilized in 0.05 ml 5 mM Tris-HCl (pH 6.8), 15% (w/v)

glycerol, 5 % (w/v) sodium dodecyl sulphate and 5% (w/v) 2-

mercaptoethanol before one-dimensional electrophoresis, or in 0.05 ml

5 mM K2C03, 5 mg dithiothreitol/ml, 2% (v/v) Nonidet P-40 and 9.16 M

urea before two-dimensional electrophoresis. One-dimensional and

two-dimensional polyacrylamide gel electrophoresis (1-D SDS-PAGE and

2-D SDS-PAGE), using 12.5% (w/v) acrylamide gels, were performed

according to procedures described in detail by Roberts et al. (1984).

Isolation and Translation of Conceptus mRNA

Isolation and translation of concepts mRNA was accomplished by

the methods described by Hansen et al. (1985). Conceptus tissue was

homogenized with 1 ml 4 M guanadinium thiocyanate, 0.5% (w/v) sodium

N-lauroyl sarcosine, 25 mM sodium citrate buffer (pH 7.0), 0.1 M 2-

mercaptoethanol and 0.1% (v/v) antifoam A, and then precipitated at -

200C with 25 1I M acetic acid and 750 pl absolute ethanol (Chirgwin

et al. 1979). The ethanol precipitate was collected by

centrifugation (12,000 g), redissolved in the homogenization buffer

(see above), layered over a 5.7 M cesium chloride cushion and

centrifuged for 20 h at 100,000 g (200C) to harvest total cellular

RNA (Chirgwin et al., 1979). Polyadenylated RNA was isolated from

the total cellular RNA by two cycles of binding to (50 mM potassium

citrate, pH 7.5, 0.5 M potassium chloride, L mM disodium EDTA) and

elution from 10 mM potassium citrate, pH 7.5, 1 mM disodium EDTA)

oligodeoxythymidylate cellulose (Aviv & Leder, 1972).









74

Translation of concepts poly A RNA was accomplished in a cell-

free system using wheat-germ lysate (Roberts & Patterson, 1973). The

translation mixture contained 0.25-1.0 pg poly A RNA, 6 ip wheat

germ lysate, 48 mM potassium chloride, 27 mM Hepes (pH 7.5), 67 mM

potassium acetate, 2.7 mM magnesium acetate, 1.2 mM ATP, 100P M GTP,

5.5 mM creatine phosphate, 200 pg phosphokinase/ml, 80 VM spermidine

phosphate, 1 mM BME, 50 1M each of 19 amino acids (minus methionine),

600 XCi L-[ 35S]methionine/ml and 13.5p g placental RNase inhibitor/ml

in a total volume of 15 pl. After translation, the [ 35S]methionine-

labelled products were analysed by 1-D SDS-PAGE as total translation

products or following immunoprecipitation with anti-oTP-1 antiserum

(10 p1) as described in the preceding section.



Results

Ouchterlony Double-Immunodiffusion Analysis

Rabbit antiserum prepared against highly purified oTP-1 gave a

single immunoprecipitation band against both oCSP and bCSP (Fig. 2-

la,b). When the ovine and bovine CSP were placed in adjacent outer

wells of the immunodiffusion plate, with the antiserum in the central

well, the immunoprecipitation band was discontinuous and had clearly

defined spurs (Fig. 2-2).

Solid-Phase Radiobinding Assay

Total bCSP was adsorbed passively to the wells of flexvinyl

plates. Anti-oTP-1 antiserum then was added at increasing dilutions

and bound immunoglobulin detected by means of I5-labelled sheep

antirabbit IgG (Fig. 2-3). The results confirmed that the anti-oTP-1


















a


Ouchterlony double-immunodiffusion analysis of concepts
secretary proteins from (a) sheep and (b) cattle4 Total
concepts secretary proteins (a, sheep; b, cattle) (15 1;
1.5 Pg) were placed in the center well. Phosphate-
buffered saline was placed in well 2 and antiserum to oTP-
1 was placed in wells 3,4,5,6 and I at increasing
dilutions in phosphate-buffered saline (1:3, 1:7, 1:15,
1:31, 1:63) respectively.


Fig. 2-1.











































Ouchterlony double-immunodiffusion analysis of bovine and
ovine concepts secretary proteins. Anti-oTP-1 antiserum
was placed in the center well (Godkin et al., L982).
Total bovine concepts secretary proteins were placed in
well 1 (30 U, 6 1g) and wells 3 and 5 (15 1l, 1.5 pg).
Total ovine concepts secretary proteins were placed in
wells 2, 4, and 6 (15 1, 1.5 pg; 10 pi, I.0Olg and 7.5
l1, 0.75 1g) respectively.


Fig. 2-2.




















































1:9 1:19 1:39 1:79 1:159 1:319 1:639 1:1,279 1:2,559 1:5.119


SERUM DILUTION


Solid phase radiobinding assay of concepts secretary
proteins. Anti-oTP-1 antiserum was serially diluted and
tested for binding to purified oTP-1 ( -), bCSPs
( --- ), or bovine serum albumin ( --- ). Normal rabbit
serum, used in a control, was serially diluted and tested
for binding to purified oTP-1 (not shown), bCSPs (not
shown) or bovine serum albumin ( -- ). Results of
curves not shown were approximately 400 c.p.m. greater
than the bovine serum albumin-non-immune rabbit serum at a
1:10 dilution and were similar at greater dilutions.
Binding was measured using I-labelled sheep
anti-rabbit IgG.



0.
0

-I
-J
w




0
CL


z
M


0

CM
LO


10,000


9,000


8,000


7,000


6,000


5,000


4.000


3,000


2,000


1,000


0


Fig. 2-3.











antiserum bound to some component in bovine CSP and crossreacted with

purified oTP-1. At dilutions of antiserum below 1:20 ,there was

detectable binding of antiserum to bovine serum albumin. Although

the relative affinity of the antiserum for BSA was low compared to

that of BCSP or oTP-1, the crossreactivity displayed here is

biologically significant as BSA is a major component of concepts

cultures. Preimmune serum tested over a similar range of dilution

failed to bind either oTP-1 or total bCSP. Half-maximal binding to

bCSP and oTP-1 was detected at antiserum dilutions of about 1:80 and

1:160 respectively. The binding curves for these two protein

fractions appeared parallel. At the initial 1:10 dilution of.

antiserum the wells containing adsorbed oTP-1 bound about twice as

much 5I-labelled second antibody as did the wells containing bCSP.

Immunoprecipitation of Polypeptides from Ovine and Bovine CSP

Sheep and cattle conceptuses were incubated in the presence of L-

[3H]luecine and polypeptides in the dialyzed culture medium

immunoprecipitated by successive addition of anti-oTP-1 antiserum and

protein A-Sepharose. For oCSP, the antiserum specifically

crossreacted with polypeptide(s) with a 4 about 19,000 (Fig. 2-4).

With bCSP, two bands of polypeptides were detected by 1-D SDS-PAGE

analysis. These bands had apparent molecular weights of 22,000 and

24,000 (Fig. 2-4). Gels were also stained with coomassie blue and

after destaining specific precipitation of protein with relative

molecular weight identical to BSA was visualized.

When the polypeptides from day 17-18 conceptuses (Fig. 2-5, upper

panel) were immunoprecipitated and analysed by 2-D SDS-PAGE, 7

















9
77-


45-


29-



20-


12-


2


3 4


Analysis of immunoprecipitates from concepts secretary
proteins of cows and sheep by one-dimensional
polyacrylamide gel electrophoresis and fluorography. Lanes
I and 3 contained material from bCSPs and oCSPs
respectively, which had been immunoprecipitated by
anti-oTP-1 antiserum followed by Protein A-Sepharose.
Lanes 2 and 4 were control lanes in which bCSPs and oCSPs,
respectively, had been treated with normal rabbit serum
followed by Protein A-Sepharose,


I
0


I-
I
0




LJ

r
-j

0


Fig. 2-4.


I



























Analysis of bovine CSP by two-dimensional polyacrylamide
gel electrophoresis and fluorography. Horozontal scale
represents pi values. Top panel represents total array
of proteins secreted by day 17-18 conceptuses. Lower panel
represents immunoprecipitable material from bCSP. Bovine
CSP was treated with anti-oTP-1 antiserum, immune
complexes were collected on Protein A-Sepharose and the
radioactive proteins analysed.


Fig. 2-5.


_______~_








pH (D O kc 4 q
a ao!

II TOTAL BOVINE-CSP f


45 .



20
T







7
0












polypeptides could be visualized on fluorographs (Fig. 2-5, lower

panel). The majority of these were localized in two parallel rows of

apparent M 22,000 and 24,000. Their approximate isoelectric points

ranged from 6.7 to 6.5, These polypeptides were the major components

present on gels of total bCSP at day 17-18 of pregnancy.

Immunoprecipitation of Polypeptides from In-Vitro Translation
Products

Total poly (A)+ concepts mRNA was translated in vitro in a wheat-

germ translation system in which L-[35S]methionine was provided as a

source of labelled amino acid. The products of translation were

analysed by one-dimensional PAGE and fluorography. Translation of

bovine mRNA gave rise to a range of translation products with

molecular weights ranging from 130,000 to 12,500 (Fig, 2-6).

During translation of ovine mRNA, the dominant translation product

had a M of about 21,000 (Hansen et al., 1985) and this component was

specifically immunoprecipitated with anti-oTP-1 antiserum. For cow

concepts mRNA a dominant translation product of M about 18,000 was

noted which crossreacted with anti-oTP-1 antiserum.



Discussion

A considerable body of evidence has accumulated to suggest that

the mechanisms involved in maternal recognition of pregnancy and

luteal maintenance in the ewe and cow are similar (Roberts et al.,

1985; Bazer et al., 1986; Thatcher et al., 1986a). Secretory

components, proteinaceous in nature, are thought to be involved as

antiluteolytic substances in both species. These substances probably

act locally on the uterine endometrium and, by mechanisms still not


i











1 23456


116m
97-
77-


Electrophoretic analysis of cell-free translation products
of RNA isolated from cattle conceptuses. Lane 1
represents total translation products when no bovine mRNA
was present. Lanes 2 and 3 show translation products when
total poly (A)+ bovine concepts mRNA was used as a source
of exogenous mRNA. Lane 4 shows the material that was
immunoprecipitated from such products by using normal
rabbit serum. Lanes 5 and 6 show material that was
immunopprecipitated from the total translation mixture by
anti-oTP-1 antiserum. The proteins were analyzed
in 12.5Z polyacrylamide gels and detected by fluorography.


45-


29m




20m


f)
I

O

L_


12.5m


1 uccoTP-1


Fig. 2-6.


'-' -L '"


f


f~










84

understood, reduce the pulsatile release of the presumed uterine

luteolysin prostaglandin F-2 from the gravid uterine horn (Thatcher

et al., 1986a; Fincher et al., 1985; Knickerbocker et al., 1986b).

Other antiluteolytic, luteoprotective and luteotrophic mechanisms may

be regulated by these unique proteins secreted by the concepts

(Thatcher et al., 1986b).

In both the ewe and cow, low molecular weight acidic proteins are

major components of the concepts secretions during the critical time

that the corpus luteum is rescued. The question has arisen as to

whether these proteins are antiluteolytic, either alone or in

combination with other concepts products. Until the present study

these low molecular weight acidic polypeptides produced by the ewe

and cow concepts had not been compared.

Ouchterlony double immunodiffusion clearly showed that antiserum

to oTP-1 crossreacted immunologically with some components) of bCSP.

When ovine and bovine concepts secretary proteins were placed in

adjacent outer wells, with the antiserum to oTP-1 located in the

center well, fusion of immunoprecipitation lines was incomplete and

spurs were evident. This indicated that a protein is present in bCSP

which is not identical to oTP-1, but is related serologically to it.

This result was confirmed by solid-phase radiobinding assay which

showed that half-maximal binding of antiserum to adsorbed oTP-1

occurred at about twice the antiserum dilution as observed with bCSP.

The protein serologically related to oTP-1 in bCSP was clearly

present as a high proportion of the total protein. In addition,

binding curves obtained with increasing antiserum dilutions were



.1 /












parallel, a result which suggests that relative affinity of antibody

towards oTP-1 and crossreacting protein in bCSP is similar under

these conditions. Recent evidence exists suggesting that the

relative affinity of oTP-1 and the crossreactive component of bCSP

are not similar. Vallet et al. (1988a) developed a radioimmunoassay

for oTP-1 and was not able to detect displacement in the assay by

undiluted bCSP while oTP-1 was detectable in oCSP diluted 1:1,000.

Similarly, binding of bCSP to an immunoaffinity column constructed of

IgG enriched, anit-oTP-1 antiserum, bound to cyanogen bromide

activated sepharose, was very low and binding was inhibited by low

salt concentrations (0.15 M) normally used to reduce non-specific

binding. The fact that antiserum did bind to bovine serum albumin in

the solid-phase radiobinding assay (Fig. 2-3) indicated that part of

the reaction between bCSP and antiserum observed by ouchterlony

immunodiffusion and solid phase binding assay may have been directed

nonspecifically towards some plasma component. Crossreactivity

against polypeptides released by day 12-14 pig conceptuses (Vallet et

al., 1988a) has not been demonstrated. The antiserum does not,

therefore, appear to have broad crossreactivity with trophoblast

proteins from all species, but results should be viewed with caution

due to its crossreactivity to BSA. Although anti-oTP-1 antiserum

does not appear to be a probable aid in purification of crossreactive

components of bCSP, it may still represent a useful tool for

identification of crossreactive components of bCSP during

characterization of these problems.









86

Anti-oTP-I antiserum specifically immunoprecipitated a group of 6-

8 polypeptides from bCSP which, when analysed by 2-D SDS-PAGE, fell

into two major molecular weight classes (22 and 24 kDa). A trace of

a 26 kDa component was also present. These bovine polypeptides were

slightly more basic and of higher molecular weight than oTP-1 (Godkin

et al., 1982). The fact that only a single molecular weight band

(18,000 M) of protein is immunoprecipitated from cell-free

translation of bovine poly (A)+ mRNA by anti-oTP-1 antiserum suggests

that heterogeneity in molecular size of the bovine protein may be the

result of some post-translational processing of the initial

translation product. This cell-free translation product has a

relative molecular weight 4,000-6,000 less than the products

immunoprecipitated from bCSP. This is in contrast to the

immunoprecipitable translation product for ovine poly(A+) mRNA, in

which the translation product has a molecular weight 4,000 larger

than the products immunoprecipitated from oCSP (Hansen et al., 1985).

These differences suggest that post-translation processing of the

ovine and bovine proteins is dissimilar. One explanation is that

bovine proteins are glycosylated whereas ovine protein simply

undergoes proteolytic cleavage, eg. removal of signal sequence. In

conclusion, results of this study demonstrate that the cow concepts

secretes a complex protein mileau part of which is immunologically

related to oTP-1. Because of immunological similarity and because

it is synthesized at a developmentally equivalent stage of pregnancy

as oTP-1(Bartol et al., 1985), we suggest that the protein complex

secreted by the bovine concepts which crossreacts with antiserum to

oTP-1 should be called bovine trophoblast protein-i or (bTP-1).
















CHAPTER 3
DIFFERENTIAL GLYCOSYLATION OF THE COMPONENTS OF THE
BOVINE TROPHOBLAST PROTEIN-1 COMPLEX



Introduction

The presence of a functional corpus luteum is essential for

pregnancy maintenance in the cow and ewe. The concepts, therefore,

must signal its presence to attenuate uterine PGF-A secretion that

would otherwise lead to demise of the corpus luteum, termination of

pregnancy, and resumption of ovarian cyclicity. For both the cow and

ewe, signaling by the concepts occurs via secretion of proteins

(Godkin et al., 1984b; Knickerbocker et al., 1986b). The "signal"

proteins are similar for both species since interspecies transfer of

trophoblastic vesicles between the ewe and cow will cause cycle

extension (Martal et al., 1984). In sheep, the anti-luteolytic

molecule has been identified as oTP-1 (Godkin et al., 1984b). Cattle

secrete immunologically cross-reactive molecules similar to oTP-1

called the bTP-1 complex. This complex consists of seven isomers

secreted in two size classes (Chapter 2), appear to be members of the

a -interferon family (Imakawa et al., 1987; Stewart et al., 1987;

Charpigny et al., 1988) and likely represent a novel role for

interferon molecules.

While oTP-I is not glycosylated (Anthony et al., 1988), bTP-1

appears to be glycosylated since conceptuses cultured with

87











[3H]glucosamine incorporated the radioisotope into bTP-1 (Anthony et

al., 1988). In addition, the primary translation product of bovine

mRNA, which migrated as a single 17-18 kDa protein species during gel

electrophoresis, was smaller than its secreted form (22 and 24 kDa;

chapter 2; Anthony et al., 1988). Therefore, it seems likely that

bTP-1 has undergone evolutionary divergence from the nonglycosylated

ovine signal and other related interferons which are nonglycosylated,

0-glycosylated and rarely N-glycosylated (Bielefeldt Ohmann et al.,

1987; Langer and Pestka, 1985). Alternatively, bTP-1, oTP-1 and

interferons may all represent molecules which arose from a common

molecule and diverged simultaneously. The results of experiments,

described in this chapter indicate that bTP-1 is glycosylated in an

N-linked manner and that differences in relative molecular weight

between classes of bTP-1 are due to differential post-translational

processing to result in bTP-1 with high-mannose and complex-type

carbohydrate moieties.



Materials and Methods

Materials

L-[35S]methionine was from Amersham (Arlington Heights, IL) or New

England Nuclear (Boston, MA); endo- -N-acetylglucosaminidase H (Endo

H) from Streptomyces plicatus was purchased from Miles Laboratories

(Naperville, IL); desoxymannojirimycin-HCl (DMM) from Bacillus

species was obtained from Boehringer Mannheim (Indianapolis, IN);

Conconavalin A-Sepharose 4B (Con A) and tunicamycin were from Sigma

(St. Louis, MO), neuraminidase from Vibrio cholerae was from Life









89

Technologies and endo a-N-acetylgalactosaminidase (0-glycanase) from

Diplococcus pneumoniae was purchased from Genzyme (Boston, MA). All

other materials were supplied as noted previously (chapter 2) or were

reagent grade or better.

In vitro culture of conceptuses

Conceptuses collected from Angus and Brangus cows at day 17-18 of

pregnancy were cultured as previously described (chapter 2) for 72 h

with fresh Eagle's modified or complete minimum essential medium

(MEM) being replaced every 24 h. Cultures were carried out with

methionine-deficient (O.x) medium supplemented with 50-100 VCi

[35S]methionine or complete MEM with 100 1Ci [3H]

glucosamine/culture/24 h, Conceptus-conditioned medium was

centrifuged for 15 min at 2,600 x g to remove particulate matter and

dialyzed (Mr cutoff 3,500) extensively to remove low molecular

weight compounds. Immunoprecipitation (IMP) of proteins in medium

was carried out as previously described (chapter 2). One-dimensional

and two-dimensional sodium dodecyl sulfate polyacrylamide gel

electrophoresis (I-D and 2-D SDS-PAGE), using 12,5% (w/v)

polyacrylamide gels, were performed as described elsewhere (Roberts

et al., 1984). Radioactive polypeptides were detected by

fluorography using Kodak XAR film.

Culture of conceptuses with tunicamycin

Conceptuses were cultured in presence or absence of tunicamycin 20

Jg/ml in MEM. Medium was prepared by adding 10 mg tunicamycin to I

ml dimethylsulfoxide (DMSO) to create a 10 1g/ 1 stock, of which 100

1 was added to 50 ml methionine deficient MEM and filter sterilized.









90

After 24 h of culture, tissue and medium were separated by

centrifugation and aliquants of medium dialyzed and lyophilized for

analysis of total bCSP (50,000 cpm, 0.736 ml) and immunoprecipitable

components of bCSP (200,000 cpm, 2.946 ml). Lyophilized total bCSP

and immunoprecepitates were solubilized in gel loading buffer

(chapter 2) in preparation for electrophoresis.

Culture of conceptuses with DMM

A stock solution of DMM (2.5 mg/ml) was prepared by dissolving 10

mg DMM in 4 ml methionine deficient MEM. Conceptuses were initially

cultured in complete MEM for 24 h without treatment, and subsequently

cultured for 4 h in the presence or absence of I mM (200p g/ml) DMM

(1.2 ml of the 2.5 mg/ml stock solution added to 13.8 ml methionine

deficient MEM). After 4 h of preincubation with DMM, 100 iCi L-

[35S]methionine was added to culture medium. After 24 h of culture

in presence or absence of DMM, tissue and medium were separated and

medium dialyzed. Aliquants of dialyzed medium from control (total

bCSP = 100,000 cpm, 0.561 ml; bCSP for immunoprecipitation 350,000

cpm, 1.963 ml) and DMM-treated (total bCSP = 100,000 cpm, 0.242 ml;

and bCSP for immunoprecipitation 350,000 cpm, 0.847 ml) conceptuses

were lyophilized in preparation for electrophoresis.

Glycosidase treatment of concepts culture medium

Conceptus-conditioned medium from the second 24 h of culture in

which conceptuses had been cultured in methionine deficient MEM

supplemented with 100 Ci L-[35S]methionine were subjected to Endo H

treatment. Endo H was prepared as a stock solution containing 6

mU/30 pi. Aliquants of total bCSP (50,000 cpm, 158 1) and bCSP for