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Bovine trophoblast protein-1

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

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Subjects / Keywords:
Cattle ( jstor )
Estrus ( jstor )
Estrus cycle ( jstor )
Ewes ( jstor )
Luteal cells ( jstor )
Pregnancy ( jstor )
Prostaglandins ( jstor )
Secretion ( jstor )
Sheep ( jstor )
Uterus ( jstor )
Animal Science thesis Ph.D
Corpus luteum ( lcsh )
Cows -- Reproduction ( lcsh )
Dissertations, Academic -- Animal Science -- UF
Estrus ( lcsh )
Trophoblast ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

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

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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20509982 ( OCLC )
AFQ1301 ( NOTIS )
AA00004807_00001 ( sobekcm )

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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




BOVINE TROPHOBLAST PROTEIN-1:
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 committe, 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 Dr*- R. Michel 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 Mr* 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
ii


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. Paris
for there continued love and support*
Special thanks and appreciation are expressed to the author's
wife, and best friend, Judith P. Helmer, for her unending love and
support
iii


TABLE OF CONTENTS
ACKNOWLEDGMENTS
ii
LIST OF TABLES... .............. *....*.v..vi
LIST OF FIGURES....... i... ^ t ^.. f .1i.. *.*.*'. vv..... i.. vii
ABSTRACT................ v.............. ...*.. ......... *mi* ...... lx
CHAPTERS
I LITERATURE REV IEW...*.... . v v.... 1
I nt roduct 1 on ^v. ... ...4 ....* . .... 1
The Bovine Estrous Cycle............................... v..* *2
Physiology and Endocrinology of the Ovary........... ** ...... 3
Uterine Regulation of CL Lifespan During
the Estrous Cycle........ *.... *... *. ¡9
Uterine Regulation of CL Lifespan During Pregnancy........39
Developement of the Bovine Conceptus. .. v . .. .. ..........43
Timing of the Conceptus Signal Relative to
"Maternal Recognition of Pregnancy"*-...*. w. ..... ... 4.44
Z IDENTIFICATION OF BOVINE TROPHOBLAST PROTEIN-I,
A SECRETORY PROTEIN IMMUNOLOGICALLY RELATED
TO OVINE TROPHOBLAST PROTEIN-1 ............4 ..........66
I nt r oduct i on 4 ................ v ..... v V........ v.............. 66
Materials and Methods. ...* .*...... **v.# ..... .. .... .. ...-v.* v *68
Results.-.*.. ...***'.... .. .. v 41*. .... v ... * . 7 4
Discussion . . ... ..... 8Z
3 DIFFERENTIAL GLYCOSYLATION OF THE COMPONENTS OF
THE BOVINE TROPHOBLAST PROTEIN-1 COMPLEX....-............. 87
Introduction.-. .v. w.. *. .*. t * ... ......... v ***.. v v...... 87
Materials and Methods. * vv. 4... *v..* . .. * vvvv. ... v*-.88
Results... ..... .. t............. .. v *.. v... v. v 4 .* v v 9 Z
Discussion.*. . ... . . 4 v v . v v.. .. v v **. v *... . 4 4 .* 98
iv


4 INTRAUTERINE INFUSION OF PURIFIED BOVINE
TROPHOBLAST PROTEIN-1 COMPLEX EXERTS AN
ANTILUTEOLYTIC EFFECT AND EXTENDS CORPUS
LUTEUM LIFESPAN IN CYCLIC CATTLE ,* a vv% vvvv 103
Introduction .. v. 103
Mat enals and Msthods v * ' v # v 4 w% ¥ vv v 104
Rs S U It S i t i t i1! V * ¥ ~ Discussion m 11 i iVi v v §; > v*. v-v v1 v>^ % v #*v w t v % 139
5 BOVINE TROPHOBLAST PROTEIN-1 COMPLEX ALTERS
ENDOMETRIAL PROTEIN AND PROSTAGLANDIN SECRETION
AND INDUCES AN INTRACELLULAR INHIBITOR OF
PROSTAGLANDIN SYNTHESIS IN VITRO.... v...v..w.142
Int roduct ion % v v * . % *v v v* -* % %-1 v- % %..14 2
Mat finals and mstHods v v > v v v v v ¥ v -* v¥ ¥¥ i* ¥ 144
ResuIt s .. * v v v # v¥ v ¥ i v v ¥ *. v ¥ v. ¥ v¥ . v ' ' 132
Discussi on .** v v **'f ¥ % v % v v % .. *% % i - i. ¥ ¡ 158
6 GENERAL CONCLUSIONS * .. . .. .. v 163
REFERENCES . §- % v % ¥ * v v .... v .. v .. * ^ v 175
BIOGAPHICAL SKETCH. % *. v v ^ v . v ¥ v v ^ *. ¥. % . 200
v


LIST OF TABLES
Table Page
4-1 Percent incorporation of [^HJleucine 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. .... 131
4-3 Least squares means +_ sem of plasma PGF concentrations
and residual variances for BSA, bCSP and bTP-1 treated
cattle. 136
3-1 Analysis of variance for incorporation of [%]-leucine
into secretory 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 means3 of concentrations of PGF and PGE
secreted into medium of day 17 endometrial explants
incubated for 24 h with medium containing 4.8 yg
BSA/ml, 12.7 yg bCSP/ml or 1 Ug bTP-1/ml.- ......153
5-4 Least squares means3 for incorporation of [^HJ-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 Og BSA/ml, 12.7 yg bCSP/ml or 1 yg bTP-l/ml......... 157
vi


LIST OF FIGURES
Figure Page
2-1 Ouchterlony double-immunodiffusion analysis of conceptus
secretory proteins from (a) sheep and (b) cattle..............75
2-2 Ouchterlony double-immunodiffusion analysis of bovine and
ovine conceptus secretory proteins.. v*v.... .. v .76
2-3 Solid phase radiobinding assay of conceptus secretory
pr oteins.. . .. .. ..*. .. .. .. -* -*, v . .. ... . v .. v.. 77
2-4 Analysis of immunoprecipitates from conceptus secretory
proteins of cows and sheep by one-dimensional
polyacrylamide gel electrophoresis and fluorography...........79
2-5 Analysis of bovine CSP by two-dimensional polyacrylamide
gel electrophoresis and fluorography...*.*......w.. ...........81
2-6 Electrophoretic analysis of cell-free translation products
of RNA isolated from cattle conceptuses... 83
3-i Fluorograph of SDS-PAGE of conceptus supernatants from a
conceptus cultured in medium containing 100 yCi [%]
glucosamine. .93
3-2 Fluorograph of SDS-PAGE of conceptus supernatants from
conceptuses cultured in the presence (lanes 2, 5, 6) or
absence (lanes I, 3, 4) of tunicamycin for 24 h. ...v-v...*...*95
3-3 Fluorograph of SDS-PAGE of conceptus supernatants from
conceptuses cultured in the presence (lanes 2, 5, 6) or
absence (lanes 1, 3, 4) of DMM for 24 h. ... * v.........v96
3-4 Fluorograph of SDS-PAGE of conceptus proteins treated
wi t h End o U...... ........... v........v .... v ..... w. 9 7
3-5 Conconavalin A-Sepharose 4B lectin chromatography of
bTP-1.... v..... .' v . v.... .. i.. .. ....... .*... .... .... .... .'.*99
4-1 HPLC gel filtration profiles of radiolabelled conceptus-
conditioned medium from the first, second, third and
fourth days of culture of a conceptus........................ 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 f iltration.... **.......... ........ **-124
4-4 Silver-stained, two-dimensional electrophoretogram of
highly purified bTP-1 complex (16 yg, top left panel);
a gel which had been run with sample buffer, but no
protein (top right panel); total array of bCSPs (100 yg,
bottom left panel and bovine serum albumin (20 yg,
bottom right panel)-.......... .............. v................. 127
4-5 Autoradiograph of 2-D SDS-PAGE of purified bTP-i complex
(8 yg/ml) transferred to nitrocellulose and immunoblotted
with a) rabbit anti-oTP-1 antiserum (1:100) or b) normal
rabbit serum, v.......... ........ m....................... 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..................... 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. .1.--.4- v *.138
5-1 Least squares means (pooled sem=0.20) 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 yg~BSA/ml,yl2v7 g
bCSP/ml or 1 yg bTP-l/ml for 24 h. 155
6-1 Proposed model for differential glycosylation of the
components of the bTP-1 complex..v 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-1:
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 conceptus 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 conceptus mediated
antiluteolytic machanisms in the cow.
Components of bovine and ovine conceptus secretory 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 6 kDa vs. 19 k.Da) and
isoelectric points (pi 6.5-6.7 vs. 5.3-5.7). The size differences
result from the fact that bTP-i, 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 conceptus 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*
x


CHAPTER 1
LITERATURE REVIEW
Introduction
The cow is afforded an opportunity to become pregnant every 2i
days. This opportunity results from the release of an ovum from the
ovary at a time favorable for fertilization and subsequent
development in tero. 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
conceptus and maternal unit. That the conceptus must "signal" the
maternal unit or uterine environement is evident because CL
regression and recurrent estrous cycles continue in the absence of
pregnancy. The nature of this conceptus 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 conceptus "signals" in initiating the sequence of
events leading to CL maintenance and successful establishment of
pregnancy.
L


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 tero and result in
the birth of young. An understanding of these processes might be
best obtained through a discussion of follicular development.


3
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 (i960) 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: i) 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., L988). 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 conceptus 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 interna 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, L966).
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 L 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
the cow. Thecal cells specifically utilize the pathway for
conversion of pregnenolone to androstenedione via 17 Ob hydroxy-


9
pregnenolone and dehydro-epiandrosterone as opposed to the A ^
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 a hydroxylase. These findings are consistent with those
of Falck (1959), and support the theory that thecal cells synthesize
androgens via the & 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 [^^1] 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


u
preovulatory follicles during estrus, when estrogen synthesis is
highest (Chenault et al., 1975).
Of recent interest are studies relating follicular fluid
concentrations of 3-carotene, vitamin E, cholesterol, and vitamin A
to follicular function (Schweigert et al., L987; Schweigert & Zucker,
1988). Concentrations of 3-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 3-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 3carotene. 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 conceptus development. While the follicle served
as a source of nourishment for the ovum, the uterus and oviduct
assume this role during early conceptus 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.


u
The formation of the CL has been described (Donaldson & Hansel,
1965a; Priedkalns et al., L968). 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., L968; 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 interna and large luteal cells from
granulosa cells of the ovulatory follicle. Small luteal cells are
15-18 jjn and large cells 18-45 jam in diameter for cattle (Chegini et
al., 1984). Similar estimates for small and large luteal cells of
sheep were obtained (12-22 Pm and 23-25 pm, 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 interna and granulosa
cells within the CL could be assessed. Percentage of large luteal
cells binding granulosa specific antibody at days 4-6, LO-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 al., 1988) and sheep (Fitz et al., 1982; 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
activites 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 [^-*1] 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-2& (PGF-201) 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 al. (1979) also
reported that the relative affinity of PGF-2CX 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 aearly 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 othas
been examined in cattle (Alila et al., 1988). Alila et al. (1988)
observed that PGF-2 astimulated 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-201'
(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., L982). The
relative affinity of PGF-20. 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-2<*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.


17
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


18
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-D has been shown to
have a luteotrophic effect on small luteal cells in vitro (Hixon &


19
Hansel, 1979; Benhaim et al., 1987; and Alila et al., 1988). The
stimulation of P4 production by PGF-20t 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-2 ot 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, 1983; Reynolds et al.,
1983; Chenault et al., 1984). In one study, (Chenault, 1983), 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-176 and PGE-2, which represent conceptus
secretory 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 PGH2 (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 Ln vitro (Milvae & Hansel, 1980). Bovine luteal cells
collected on days 5, 10, 15, and 18 after estrus produced 128 +_ 12,
87 +_ 18, 38 +_ 9, and 54 +_ 7 ng/10^ 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, 1986).
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 secretory 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 preceeding 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 conceptus 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., 1983b; Harrison et al.,
1987). Measurement of oxytocin concentrations in the vena cava of
cows revealed pulsatile secretory responses which parallel
progesterone secretion (Walters & Schallenberger, 1984; Walters et
al., 1984). The release of oxytocin also paralleled release of PGF-
2 aduring the period of luteolysis. Thus, oxytocin is not only
involved in the luteolytic mechanism of PGF-2 c; 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-20t synthetic


IL
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


24
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 withdrawl 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, L980) 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 occured 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,


30
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, L964). 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.
C1985) 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-iPt was infused
into the uterine lumen of ewes, elevated levels of PGF-itot 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 ais the uterine luteolysin of
cattle and sheep. This concept is based upon several lines of
evidence. Exogenous administration of PGF-2^ is luteolytic when
administered to cattle (Hansel et al., 1973; Lauderdale, 1974;
Thatcher & Chenault, 1976; King et al., 1982). Elevated
concentrations of PGF-2a in uterine venous drainage (Nancarrow et
al., 1973; Sheraesh & 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-2a 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 (0A) 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-2a
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-ifct 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 aand increase the concentration of peripheral PGFM when
administered late in the estrous cycle (Barcikowski et al., 1974;
Thatcher et al., 1984b; Bartol et al., L981b; Thatcher et al., 1986b;
Hixon & Flint, 1987; Lafranee & 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 1 to 5 days after estrus resulted in
short estrous cycles (Ginther, 1968; Lawson & Cahill, 1983).
Capacity of the uterus to release PGF-2W 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 EZ
receptors on the uterus thereby blocking the luteolytic action of EZ
(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-2a release (Milvae & Hansel, 1980), and
elevating peripheral PGFM concentrations (Lafranee & Goff, 1985,
1988). Additionally, it was reported that E2 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 ltea was greatest at days 3-6 of the estrous


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 occured, 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-2X release. The luteolytic
action of PGF-2ais to decrease P4 secretion and PGF-231 is also
associated with further oxytocin release by the CL. The second
release of oxytocin may then reinforce the secretion of PGF-<& from


35
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-lfct release includes increased turnover of
phosphoinositides, a possible, but limited, source of arachidonic
acid for PGF-2 a synthesis (Flint et al., L986; Hixon & Flint, 1987).
Flint et al. (1986) incubated slices of caruncular endometrium from
steroid-treated, ovariectomized ewes with [^HJinositol to radiolabel
tissue phosphatidylinositol. Treatment with oxytocin was shown to
increase incorporation of [^HJinositol 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 [^HJphosphatidylinositol 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
O Q
[JH]arachidonyldiacylglycerol was converted to [ H]arachidonic acid
by caruncular endometrium. In a similar experiment, caruncular
endometrium was incubated with [^H]inositol in the presence of E2 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-2a 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-201 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-201
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-201, and was followed 2 h later by
lowered peripheral P4 concentrations (Niswinder et al., L976).
McCracken et al. (1979) and Einer-Jensen & McCracken (1981) reported
that P4 secretion decreased 50% by 1 to 2 h after PGF-2a
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-2a 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-20t elicited production of a
chemoattractant for eosinophiles by luteal tissues. Eosinophiles
have been reported to release substances which mediate tissue injury
(Gleich & Loegering, L984). 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-
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-ZX is that it
may antagonize local gonadotrophic support of the CL resulting in
luteolysis. It was thought that PGF-22 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-£i 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-2a induced luteolysis (Henderson & McNatty, 1975). It
has also been shown by X-ray diffraction that membrane structure of
luteal cells is changed by PGF-2X (Buhr et al., 1979; Carlson et al.,
1981). The action of PGF-2&might be to either limit accessability
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-2X induced suppression of P4 contradicts data of
others who reported increased P4 secretion by small luteal cells in
response to PGF-2 a (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 0, First, receptors for PGF-2X 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 conceptus may release or induce a luteotrophic
factor which counteracts the luteolytic effect of PGF-Zx. (Henderson &
McNatty, 1975). Although PGE-2 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-2 a during early pregnancy
(Inskeep et al., 1975; Mapletoff et al., 1976; Pratt et al., 1977;
Silvia & Niswender, 1984) and this refractoriness to PGF-H also was
reported after PGE-2 administration (Henderson et al., 1977; Pratt et


40
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 conceptus 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-201, transfer of PGF-201 from the ovarian
vein to the ovarian artery is reduced during pregnancy resulting in
lower PGF-201 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-201 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-201 secretion is suppressed
during pregnancy.
Uptake of PGF-201 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-201 from cyclic and


41
pregnant ewes was reported and it therefore seems possible that in
sheep, PGF-2CC secretion might be altered in a manner different than
that for cattle. This was supported by others who found mean PGF-2CX
in ovarian venous blood to be higher (Wilson et al., 197Z; 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 rayometrial 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-iP is elevated, but luteolytic pulses are
absent during pregancy 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-22 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


4 2
PGF-ZOt 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.t 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 E2 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-2CX 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-2CX by the countercurrent exchange system of uterus and ovary may
decrease. Collectively, these results indicate that, in some
fashion, the conceptus 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 conceptus mediates its
antiluteolytic effects for CL survival. Understanding of this
mechanism begins with knowledge of critical stages in conceptus
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., 1981). 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 conceptus 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 conceptus development* Further
examination to determine if this wastage was associated with
conceptus 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 conceptus must make its presence known if
successful establishment of pregnancy is to occur. If the conceptus
does not signal its presence, luteolytic pulses of PGF-20t are
released by the uterus resulting in demise of the CL. Successful
establishment of pregnancy involves secretion of nutrients from the
uterus for conceptus 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 ocsynthesis, 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 conceptus "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 12% 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 conceptus
must be present in tero 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)


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, conceptus 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 conceptus removal on days 13 or 15 after estrus. These
results indicate that the bovine conceptus, like the ovine conceptus,
"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 conceptus 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 conceptus signals is in order.
The bovine conceptus 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 conceptus, could
increase delivery of nutrients for conceptus growth and development.
Therefore, an increase in blood flow to the uterus would in effect
dilute PGF-2 oi 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 [^H]-P4 as precursor, produced small amounts of
estradiol, estrone, and estriol, in order of prevalence* Eley et al.
(1983) was unable to detect conceptus 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 conceptus may be rendered inactive by
conjugation or act locally to alter endometrial function to favor
conceptus survival. Secretion of estrogen by the bovine conceptus 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., L982), 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 ^-reduced
androstanes were also produced, but not to a great extent. The high
activity of the 5 8-reductase system in conceptus tissue was distinct
from that of the uterine endometrium which utilizes the 5 0rreductase
system (Eley et al., 1983). It was hypothesized that 5 ^-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 8-
reductase system in conceptuses might be to reduce availability of
precursors for E2 production by the conceptus which would be
luteolytic if released in sufficient amounts (Thatcher et al.,
1984a).
Prostaglandin production by the bovine conceptus 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-


49
2, the production of which increases with age of the conceptus
(Shemesh et al., L979). 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-2a, 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 [%] PGF-2a to
[^H] PGFM, respectively.
To further characterize PG production by the maternal-conceptus
O
unit, Lewis & Waterman (1983) evaluated conversion of [ H]arachidonic
acid to PGs in co-cultures of conceptus and endometrial tissues. In
this experiment, both conceptus 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 conceptus. When blastocysts were cultured in endometrial
O
conditioned culture medium, incorporation of [ H] arachidonic acid
was not altered. In contrast, co-culture of blastocysts with
o
endometrium resulted in increased metabolism of [ H] arachidonic acid
to PGFM and PGE-2 with decreased metabolism to PGF-2 a. This
represented alterations in metabolism of arachidonic acid to PG
products with no alteration in the proportion of arachidonic acid
metabolized by the conceptus. Results indicated that endometrium is
capable of metabolizing [^H] PGF-2a to PGFM and of shifting
metabolism of [%] arachidonic acid away from [^H] PGF-2 ot and towards


50
[^H] PGE-2. This shift from PGF-2a production to PGE-2 production
may represent metabolism of conceptus derived PGF-20. to PGE-2,
because metabolism of arachidonic acid by the conceptus to PGF-2 ,
PGE-2 and PGFM was not altered by incubation with endometrial culture
supernatants. Only when endometrium and conceptus 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 conceptus is probably
minimal compared to the secretory 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., L979; 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 Oi, The antiluteolytic effect of the
conceptus in ewes was to increase basal release of PGF-2a from the
uterus and eliminate release of pulses of PGF-2 a that would be
luteolytic (Zarco, 1984).
Uterine flushings from pregnant cattle (Bartol et al., 1981b) were
reported to contain considerable quantities of PGF-2 o* This
accumulation of PGF-2 ot could be the result of conceptus-derived PGF-
2 a(Lewis & Waterman, 1983) or result from decreased transfer of


51
endometrial PGF-2^ 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-201 (Henderson et al., 1977; Pratt et al., 1977; Reynolds et
al., 1981). In cattle, PGE-2 attenuates the luteolytic effects of
PGF-201 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-3. secretion in cattle would cause
an increase in the PGE-2:PGF-2a 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-2 administration (Gimenez & Henricks, 1983; Reynolds et
al., 1983).
A luteotrophic substance produced by day 13 to 18 bovine
conceptuses has been identified (Hickey & Hansel, 1987). This
conceptus product, characterized as a small (<10 kDa), heat-labile,


and lipid soluble molecule, was shown to stimulate P4 synthesis by
dispersed bovine luteal cells In vitro and suggests that the
conceptus 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, conceptus 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., L981b,
1985; Geisert et al., 1988; Godkin et al., 1988; and sheep: Godkin
et al., 1982). Godkin et al. (1982) characterized secretory 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 conceptus produced one major protein. This major


53
secretory 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 conceptus secretory
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 secretory 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 conceptus 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 conceptus
secretory 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 conceptus size increased from
40 to 100 mm and greater than 100 mm in length. Complexity of the
secretory pattern of proteins increased greatly in conceptuses
greater than 100 mm in length.


54
Protein production by cultured bovine conceptuses ( jg/mg wet
weight) increased significantly from day 16.5 (4.9 +_ 2.4) to day 17.0
(15.8 +5.2; Knickerbocker et al., L986b). 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 conceptus 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 [^H]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 [^H] or [^S] 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-i. 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
similiar on days 8 through 16 for pregnant and cyclic cattle.
However, on day 19 four protein species (relative molecular weights x
1CT3 = 15.2, 306.8, 322.2, and 342.8) specific to uterine flushings
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 conceptus. Knickerbocker et al.
(1986b) found that conceptus wet weight and protein synthetic
capacity increase significantly on day 17 of pregnancy. Thus, it
would appear that presence of the conceptus 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 (ty- = 14-16,000; pi = 7.2-6.8) and a
polypeptide with a Mr = 35,000 (pi = 8.4-7*3) by endometrium from the
uterine horn ipsilateral to the CL during pregnancy was observed.
More recently, Gross et al- (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 [^H]
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 conceptus in tero or intrauterine infusion of
secretory components of the conceptus 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 conceptus
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 22*4 days for ewes receiving
homogenates compared to 166 days for controls (Rowson & Moor, 1967).
Daily infusions of day 25 ovine conceptus 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, conceptus 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
conceptus homogenates and termed the antiluteolytic substance of the
conceptus "Trophoblastin". They found the signal to be stage-
specific, as day 21-23 conceptus 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 conceptus must be present in tero by day 15 to 17 after
estrus for establishment of pregnancy to occur. To determine the
nature of the conceptus signal, they carried out intrauterine
infusions of one homogenized day 17 to 18 bovine conceptus 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. conceptus homogenate; 21.1 +_0.74
verus 24.0 +_ 0.38 days). Although, infusion of conceptus 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 conceptus homogenates utilized in the experiments
described would contain fewer of the typical, conceptus secretory
proteins, yet cycles were extended by 3 to 4 days. In a similar


58
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 secretory
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 conceptus signal is stage specific.
The ovine and bovine conceptus "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


59
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 conceptus signals for these species may be similar.
Data from the preceeding studies indicated that the antiluteolytic
conceptus 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 conceptus secretory 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-i (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 occured 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 8-reduced steroid from bovine conceptuses, 5 8-
pregnane-3 Qt-ol-20-one, had no effect on estrous cycle lengths*
Collectively, these studies indicate that the major, conceptus
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-i 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., 1986a).
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 conceptus
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 conceptus 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
conceptus. However, the theory that the PG synthesis regulator is a
humoral-factor should be veiwed with caution as it is possible that
conceptus 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


pregnant than cyclic cattle. The inhibitor was in the high speed
cytosolic supernatant fraction of endometrial tissues and was
proteinaceous.
To determine if conceptus 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 conceptus signal and there seems to be
similarities between antiluteolytic mechanisms of ovine and bovine
conceptuses, it seems possible that analogous conceptus protein
systems may exist.
A great deal is known about the ovine conceptus antiluteolytic
signal. Rowson & Moor (1967) first proposed that the conceptus
antiluteolytic signal might be proteinaceous in nature since heat-
treatment of conceptus 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 conceptus secretory product (19 kDa; pi = 5.3-
5.7) which was referred to as oTP-1 (Godkin et al., 1982). This


63
conceptus signal was demonstrated to attenuate luteolytic PGF release
by the uterus (Godkin et al., L984b; Vallet et al., 1.988b) and PGF
release in response to E2 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-i 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-i 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 conceptus 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-i 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-a-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-a-I) and class
II (IFN-Oi-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 activites 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-C* had no effect on release of PGF (Dore-
Duffy et al., 1983). 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-ot-l 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


65
L after estrus (Plante et al., 1987). However, In vitro effects of
IFN-ot-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-ct-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-
ot-I, which is not as homologous to the inferred primary sequence of
oTP-1 as that of IFN-cc-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 conceptus 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
conceptus became the objective of research contained in this
dissertation.


CHAPTER 2
IDENTIFICATION OF BOVINE TROPHOPLAST PROTEIN-1, A SECRETORY 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., 1984; 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 secretory protein of the
sheep conceptus, 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


67
extension of luteal lifespan when introduced in purified form into
uteri of nonpregnant recipient ewes (Godkin et al., 1984b; Vallet et
al., L988b). 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 substance(s) which
is recognized by the respective species. The object of the


68
experiments described in this chapter was to determine whether oTP-i
crossreacts immunologically with component(s) 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 37C 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 artifically
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


69
where they were trimmed of excess tissue, including the ovaries and
oviducts. The cervix was closed by applying a large, curved,
Rochester-Ochsner forcep 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 1 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 Ug/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 yg/ml),
choline chloride (100 yg/ml), folic acid (100 Ug/ml), i-inositol
(200 Ug/ml), nicotinamide (100 Ug/ml), pyridoxal-HCl (100 ug/ml),
riboflavin (10 ug/ml) and thiamine (100 ug/ml). Content of leucine
was limited to 0.1 times normal (5.15 mg/1) to enhance uptake of L-
3
[ Hjleucine when added to cultures. Medium was filter sterilized
(0.22 Um) and stored at 4C.
In-Vitro Culture of Conceputses
After collection, conceptuses from cows and ewes were transferred
to plastic culture dishes containing 15 ml fresh modified (leucine
deficient), Eagle's MEM and 100 UCi L- [^H]leucine. Conceptuses


70
were cultured for 24 h in a controlled atmosphere chamber (Model
number 7741-10010, Blico Biological Glassware, Vineland, NJ,
U.S.A.), flushed for 10 min with 50% : 47.5% 0^ :2.5% CO., (v/v)
and maintained at 37C in the dark on a rocking platform.
Incubations were terminated by removing conceptuses from culture
medium. The medium was frozen at -20C until used in subsequent
studies.
Purification of oTP-1
Ovine TP-i was purified by the method of Godkin et al. (1982).
Purity was confirmed by polyacrylamide gel electrophoresis (Godkin et
al., 1982).
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 (1^. cutoff 1,000)
extensively (4 liters, changed 4 times) against 10 mM Tris-HCl buffer
(pH 8.2) at 4C 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.


71
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; 0v2 g
KC1/1;2.16 g KH^po^.7 H^O/l, 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 Radiobinding Assay
Bovine conceptus secretory proteins, purified oTP-1 and bovine
serum albumin were each adjusted to a concentration of 50 yg/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 NaCl 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 y 1 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 1 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 4C overnight. These solutions were removed
and plates were washed with PBS containing bovine serum albumin.
After washing, ^I-la belled, sheep anti-rabbit IgG antibody (50,000
c.p.m.; sp.act. 10^ c.p.m./yg) which had been affinity purified was
added to each well and left for 1 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 ^ I measured.
Immunoprecipitation
Duplicate aliquots of ovine and bovine conceptus culture medium
(80 yg and 120 yg protein, respectively) were lyophilized and
redissolved in immunoprecipitation buffer [0.35 ml 0.3 M NaCl, 0.05 M
Tris-acetate (pH 7.5), L mM phenylmethylsulphonyl fluoride, 1 mM
disodium EDTA, 0.L mg bovine serum albumin/ml, 0.02% (w/v) NaN^ and
2% (v/v) Nonidet P-40]. Anti-oTP-i 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 4C. 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
1 min) and washed five times with 1 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 NaCl and 0.02% NaN^]. 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 raM K^CO^, 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 conceptus 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 -
20C with 25 )1 1 M acetic acid and 750 yl 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 (20C) 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, 1 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 conceptus 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 yl 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, 1000 M GTP,
5.5 mM creatine phosphate, 200 yg phosphokinase/ml, 80 yM spermidine
phosphate, 1 mM BME, 50 yM each of 19 amino acids (minus methionine),
35
600 yCi L-[ S]methionine/ml and 13.5y g placental RNase inhibitor/ml
35
in a total volume of 15 yl. After translation, the [ SJmethionine-
labelled products were analysed by i-D SDS-PAGE as total translation
products or following immunoprecipitation with anti-oTP-i antiserum
(10 yl) 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-i antiserum then was added at increasing dilutions
and bound immunoglobulin detected by means of ^^I-labelled sheep
antirabbit IgG (Fig. 2-3). The results confirmed that the anti-oTP-1


75
a
b
Fig. 2-1. Ouchterlony double-immunodiffusion analysis of conceptus
secretory proteins from (a) sheep and (b) cattle. Total
conceptus secretory proteins (a, sheep; b, cattle) (15 lil;
1.5yg) 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 1 at increasing
dilutions in phosphate-buffered saline (1:3, 1:7, 1:15,
1:31, 1:63) respectively.


76
Fig. 2-2. Ouchterlony double-immunodiffusion analysis of bovine and
ovine conceptus secretory proteins. Anti-oTP-1 antiserum
was placed in the center well (Godkin et al., L982).
Total bovine conceptus secretory proteins were placed in
well 1 (30 H, 6Ug) and wells 3 and 5 (15 yl, 1.5 yg).
Total ovine conceptus secretory proteins were placed in
wells 2, 4, and 6 (15 yl, 1.5 yg; 10 yl, 1.0 yg and 7.5
yl, 0.75 yg) respectively.


I BOUND / WELL (CPM)
77
Fig. 2-3. Solid phase radiobinding assay of conceptus secretory
proteins. Anti-oTP-i 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 simi|^ at greater dilutions.
Binding was measured using 3I-labelled sheep
anti-rabbit IgG.


78
antiserum bound to some component in bovine CSP and crossreacted with
purified oTP-i. 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-i, the crossreactivity displayed here is
biologically significant as BSA is a major component of conceptus
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 ^^I-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-
3
[ HJluecine 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 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


MOLECULAR WEIGHT x 10
79
CO
1 2
1 16h
97-
77
45-
3 4
Fig. 2-4. Analysis of immunoprecipitates from conceptus secretory
proteins of cows and sheep by one-dimensional
polyacrylamide gel electrophoresis and fluorography. Lanes
1 and 3 contained material from bCSPs and oCSPs
respectively, which had been immunoprecipitated by
anti-oTP-i 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.


Fig. 2-5. 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.


MfX10" Mrx 10"
81
ro
97:
77"
45-
d-tPCOOWNO^OWrO
csj o ^
NN(((u5(D((D((iO
I I 1 I I J
TOTAL BOVINE-CSP


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.
Immunopreclpitation of Polypeptides from In-Vitro Translation
Products
Total poly (A)+ conceptus mRNA was translated in vitro in a wheat-
germ translation system in which L-[Jmethionine 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
conceptus mRNA a dominant translation product of M about 18,000 was
noted which crossreacted with anti-oTP-i 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


83
1 23456
116m
9 7.
77m
45-
Fig. 2-6. 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 conceptus 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 12.5% polyacrylamide gels and detected by florography.


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 conceptus
(Thatcher et al., 1986b).
In both the ewe and cow, low molecular weight acidic proteins are
major components of the conceptus 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 conceptus products. Until the present study
these low molecular weight acidic polypeptides produced by the ewe
and cow conceptus had not been compared.
Ouchterlony double immunodiffusion clearly showed that antiserum
to oTP-1 crossreacted immunologically with some component(s) of bCSP.
When ovine and bovine conceptus secretory 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


85
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-i 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-1 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-i (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 conceptus
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-l(Bartol et al., 1985), we suggest that the protein complex
secreted by the bovine conceptus which crossreacts with antiserum to
oTP-1 should be called bovine trophoblast protein-1 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 conceptus, therefore,
must signal its presence to attenuate uterine PGF-& 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 conceptus 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 alv, 1984b). Cattle
secrete immunologically cross-reactive molecules similar to oTP-1
called the bTP-i 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-1 is not glycosylated (Anthony et al., 1988), bTP-1
appears to be glycosylated since conceptuses cultured with
87


88
[^HJglucosamine 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-i has undergone evolutionary divergence from the nonglycosylated
ovine signal and other related interferons which are nonglycosylated,
O-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-[^S]methionine was from Amersham (Arlington Heights, IL) or New
England Nuclear (Boston, MA); endo-8 -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 oi-N-acetylgalactosaminidase (0glycanase) 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.lx) medium supplemented with 50-100 OCi
[^SJmethionine 0r complete MEM with 100 UCi [^H]
glucosamine/culture/24 h. Conceptus-conditioned medium was
centrifuged for 15 min at 2,600 x g to remove particulate matter and
dialyzed (Mj- 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 (1-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
Ug/ml in MEM. Medium was prepared by adding 10 mg tunicamycin to 1
ml dimethylsulfoxide (DMSO) to create a 10 yg/ jl stock, of which 100
l1! was added to 50 ml methionine deficient MEM and filter sterilized.


Full Text
UNIVERSITY OF FLORIDA
3 1262 08554 2891



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