CHARACTERIZATION OF BOVINE UTERINE AND
CONCEPTUS PROTEINS

By
FRANK FITZHUGH BARTOL

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
1983

■■*■■

ACKNOWLEDGEMENTS

For a period of now more than seven years the author has been a
graduate student in the Department of Dairy Science and a member of the
interdisciplinary reproductive biology group at the University of
Florida. It has indeed been a privilege, and a challenging and reward-
ing experience, to have had the opportunity to grow and learn in this
unique academic environment . The author is eternally grateful to all
members of this academic community whose combined interests, efforts,
and energies have served to create and continue to foster an enthusi-
astic spirit of cooperation and comradery in science that can only
inspire those fortunate enough to have experienced it.

The author expresses his sincerest appreciation and thanks to Dr.
William W, Thatcher, chairman of the advisory committee, mentor, and
friend throughout the author's graduate career. Dr. Thatcher's enthusi-
asm and intensity of interest in research are exceeded only by his
extreme patience, which becomes increasingly evident the longer it is
tried. For his constant faith and support the author is forever
grateful.

Special thanks are expressed to Dr. Fuller W. Bazer for his friend-
ship, insight, and guidance throughout the course of these studies.
Dr. Bazer is also acknowledged for technical support, having allowed
the author to take up residence in his laboratory for the past several
years. Conversations and experiences in this setting, both formal

IX

and informal, have been invaluable, and the author is grateful for having
had daily opportunities for such interaction.

Thanks are expressed to Dr. R. Michael Roberts for his technical
insight, guidance, and support throughout the course of these studies
and for helping the author feel at least a bit at home in the biochemistry
laboratory.

Dr. Daniel C. Sharp, III, is acknowledged for contributing to the
author's knowledge and appreciation of rhythm, and for shedding some
light on the complex interactions of reproductive physiology.

Thanks are due to Dr. Maarten Drost for surgical training and
assistance and for bringing his unique perspective to this research.

It is with deepest and most sincere appreciation that the author
acknowledges Dr. Donald H. Barron. The refreshing insight which Dr.
Barron brings to interpretation of physiological data has been a con-
stant source of inspiration and rejuvenation even in the darkest hours.
The author looks forward to the privilege of continued interaction with
Dr. Barron, whose unique philosophy and approach to research will serve
as a constant source of stimulation in the author's future endeavors.

Special thanks are due to fellow graduate students past and present
including Dr. Rod Geisert, Dr. Ron Kensinger, Dr. Randy Renegar, Jeff
Moffatt, and Jeff Knickerbocker; as well as post-doctoral fellows.
Dr. J.D. Godkin, Dr. G.S. Lewis, and Dr. A. Fazleabas. Thanks are also
extended to Louis Guilbault, Joan Curl, Linda Rico, Chuck Wallace, and
Dr. David Wolfenson. The friendship, support, and assistance provided
by these individuals are deeply appreciated.

iii

A special debt of gratitude is owed Warren Clark for his friend-
ship and technical expertise. Future generations of students will be
fortunate to "learn the ropes" from Mr. Clark. Thanks are also due to
Dr. M.S. A. Kumar, Candy Stoner, and Carol Underwood for their artistic
and technical assistance, and to June Wallace and Adele Koehler for
typing of this manuscript.

The author expresses his sincerest gratitude to his mother, Dorothy
F. Bartol, and Grace Z. Vogel for their support, faith, and patience.

Finally, the author acknowledges, with heart-felt gratitude, his
wife, Anne A. Wiley, for her invaluable assistance, patience, insight,
and love .

iv

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

LIST OF TABLES vll

LIST OF FIGURES vlii

ABSTRACT x

CHAPTER

I REVIEW OF LITERATURE ' 1

The Problem in Perspective 1

Endocrine and Physiological Aspects of the Estrous

Cycle and Early Pregnancy 8

The Uterus 26

The Conceptus 78

II UTERINE PROTEINS IN UNILATERALLY PREGNANT CATTLE 103

Introduction 103

Materials and Methods 104

Results 114

Discussion 135

III CHARACTERIZATION OF BOVINE ENDOMETRIAL PROTEINS 149

Introduction 149

Materials and Methods 150

Results 159

Discussion 180

IV CHARACTERIZATION OF PROTEINS PRODUCED IN VITRO BY PERI-
ATTACHMENT BOVINE CONCEPTUSES 195

Introduction 195

Materials and Methods 197

Results 203

Discussion 223

Page

V GENERAL DISCUSSION 238

LITERATURE CITED 256

BIOGRAPHICAL SKETCH 292

vi

LIST OF TABLES

Table

Page

2.1 Arithmetic means (± SEM) and ranges for uterine and
fetal-placental physical responses measured on day 270

of gestation for nine cows 116

2.2 Arithmetic means (± SEM) of steroid concentrations (Ej^
E2, E2S0^, P^) in uterine milk supernatant (UMS) and
peripheral plasma (PI) 117

2.3 Gross and partial within-cow correlations of steroid in
peripheral plasma and uterine milk supernatant 120

2.4 Concentration and content (X ± SEM and range) of selected
constituents of bovine uterine milk supernatant recovered

on day 270 of gestation 122

3.1 Distribution of cattle 153

3.2 Responses (X ± SEM) of endometrial explants from early
pregnant (P) and nonpregnant (NP) cattle 162

vii

LIST OF FIGURES

Figure Page

2.1 . Schematic diagram of surgical preparation used to

establish unilateral pregnancy in cattle 106

2.2 Representative Sephacryl S-200 elution profile of pro-
teins in unfractionated UMS (day 270) 124

2.3 Two-dimensional polyacrylamide gel electrophoresis
(12.5% acrylamide) of acidic (Panel A; lEF) and basic

(Panel B; NEPHGE) polypeptide components of day 270 UMS . . 127

2.4 Two-dimensional polyacrylamide gel electrophoresis

(10% acrylamide) of acidic (lEF) proteins characteristic

of UMS (A) and peripheral plasma (B) 129

2.5 Crossed-immunoelectrophoresis indicating serum-identical
proteins in UMS (top) and peripheral plasma (bottom) from

a day 270 unilaterally pregnant cow 132

2.6 DEAE-ion-exchange chromatograms of ammonium sulfate
precipitable proteins in UMS (A), bovine serum (B),

and bovine colostral whey (C) 134

2.7 Crossed-immunoelectrophoresis (C-IEP) revealing serum-
and immunoglobulin-identical components of day 270

bovine UMS 137

3.1 Release of nondialyzable H-labelled macromolecules by
bovine endometrial tissue during 48 h incubation in vitro . 161

3.2 Comparison of representative Sephacryl S-200 gel fil-
tration elution profiles of dialyzed MEM from day 19
pregnant (A) and day 270 unilaterally pregnant (ligated
uterine horn) endometrial explants (B) 167

3.3 Representative Sepharose CL-6B gel filtration elution
profile of bovine endometrial proteins from S-200 area I. . 169

3.4 Representative ion-exchange chromatograms of dialyzed

bovine endometrial explant medium 172

3.5 Two-dimensional polyacrylamide gel elctrophoresis (10%
acrylamide) and fluorography of polypeptides in bovine
endometrial explant MEM and tissues 175

Vlll

Figure Page

3.6 Fluorograph of a 2D-PAGE (lEF) gel (10% acrylamide)
depicting major bovine endometrial-specif ic polypeptides
present in MEM 179

3.7 Fluorographs prepared from 2D-PAGE (lEF) gels (10%
acrylamide) of polypeptides in bovine endometrial explant

MEM 182

4.1 Percent retention (least square mean ± SEM) of H-
leucine as nondialyzable product in 24 h cultures of

bovine conceptuses from days 16, 19, 22, and 24 205

4.2 Representative DEAE anion exchange chromatograms of
radiolabelled proteins in dialyzed MEM from day 16 (A) ,
19 (B) , 22 (C), and 24 (D) bovine conceptus cultures
following 24 h incubation in vitro 208

4.3 Fluorographs prepared from representative 2D-PAGE (lEF)
gels (10% acrylamide) of polypeptides in dialyzed bovine
conceptus culture MEM from days 16 (A), 19 (B) , 22 (C),

and 24 (D) 210

4.4 Fluorographs of 2D-PAGE (lEF) gels (10% acrylamide)
showing conceptus tissue polypeptides into which %-Leu

was incorporated in vitro 214

4.5 Fluorographs prepared from 2D-PAGE (lEF) gels (10%
acrylamide) of polypeptides in dialyzed bovine conceptus
MEM from days 19 (A) and 29 (B) , and isolated chorionic
culture MEM from day 69 (C) 216

4.6 Fractionation procedure for partial purification of
major low molecular weight, acidic polypeptides pro-
duced in vitro by bovine conceptuses 219

4.7 Characteristics of partially purified low molecular

weight, acidic bovine conceptus proteins 222

4.8 Isolation of the high molecular weight bovine conceptus
glycoprotein 225

ix

-:.-\::-^^r;^

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

CHARACTERIZATION OF BOVINE UTERINE AND CONCEPTUS PROTEINS

By
Frank Fitzhugh Bartol
April, 1983

Chairman: William W, Thatcher
Major Department: Animal Science

Studies were conducted to characterize proteins in bovine uterine
milk (UM) obtained from ligated (L) uterine horns (UH) of unilaterally
pregnant (UPx) cattle on days (D) 180, 210, 240 and 270; and produced
de novo from radiolabelled amino acid substrate (AAS) by endometrium
from nonpregnant (NPx; D4, 16, 19), pregnant (Px; D16, 19, 22, 24, 69),
and UPx (D270; LUH, PUH) cattle; and by bovine conceptuses (D16, 19,
22, 24, 29) and chorion (D69) in vitro.

Accumulation of UM in LUH of UPx cattle (n=12) was detectable per
rectum by D166-180. Mean (± SEM) recovery of D270 UM from UPx cows
(n=9) was 205.3 ± 68.20 ml/LUH. Estrone and estradiol concentrations
in UM supernatant (UMS) and peripheral plasma were similar, while
estrone-sulfate and progesterone were higher in plasma. Day 270 UMS
was enriched in protein, prostaglandin F (PGF) , calcium and glucose.
Calcium concentration was positively correlated with protein concen-
tration (r=.68, P < .01) and PGF content (r=.92, P < .08). Bovine UMS

^'^^r.

was enriched in basic proteins of less than 40,000 molecular weight
(M ). Serum and UM-specific proteins were identified.

Endometrial explants (EE) from Px, NPx and UPx cattle produced
proteins de novo from AAS. In vitro responses of EE from P (D16, 19,
22, 24) and NP (D16, 19) cattle suggested a stabilizing conceptus

effect on endometrial protein synthesis. Medium (MEM) from all EE was

_3
enriched in polypeptides in four M (x 10 )/pH classes (I, -14/>7.2;

II, 19-24/5.4-6.3; III, 28-31/6.9-7.3; IV, >150/<5.1). Class II and

III polypeptides were identified in UMS.

Uptake of AAS and quality of polypeptides produced de^ novo by

conceptuses were related to stage of development. Day 16, 19, 22 and

24 conceptus-MEM was enriched in low M , acidic polypeptides (LM A;

22-26/6.5-5.6, 20-26/5.5-5,4, 16-20/5.0-4.5); not prominent products

— _3

of D29 and D69 tissues. A high M glycoprotein (X M x 10 ± SEM -

735 ± 22), produced by all conceptus tissues, and LM A were isolated
from MEM via anion exchange and gel filtration chromatography.

Results are discussed relative to conceptus-maternal interactions
necessary for establishment and maintenance of pregnancy.

xi

CHAPTER I
REVIEW OF LITERATURE

The Problem in Perspective
It was the purpose of research described in this dissertation to
examine components of the bovine uterine environment. Emphasis was
placed on proteins, particularly those contributed to this unique
milieu by the endometrium (uterine mucosal epithelium) and conceptus
(embryo and extraembryonic membranes) during the peri-attachment period
of early pregnancy. This period, defined as the time from initial
elongation of the trophoblast (extraembryonic membranes) until defini-
tive microvillar attachment of the outer trophoblastic membrane
(chorion) to maternal endometrium (approximately days 16 to 24; see
Wathes and Wooding, 1980; King et al., 1981), has been repeatedly
associated with a high incidence of embryonic mortality in cattle
(Greenstein and Foley, 1958a, b; Hawk et al., 1955; Ayalon, 1978; Hawk,
1979). Mossman (1937) observed that establishment of pregnancy in
eutherian mammals involved interactions between two interdependent
systems defined as the conceptus and uterus. Clearly, the consequence
of failure in either component is failure of pregnancy. Thus, the
importance of factors which may contribute to successful intercommuni-
cation of these two systems is evident. However, even after centuries
of investigation, the precise means by which the conceptus interacts
with its maternal environment to establish and maintain pregnancy is
Imprecisely understood.

-1-

-2-

Throughout history innumerable theories have been suggested to
explain the relationship between conceptus and maternal units. One
of the earliest recorded attempts to explain maternal support of the
developing conceptus was that of Diogenes of Apollonia (ca. 480 B.C.),
who suggested that the placenta (extraembryonic membranes) was the
organ of fetal nutrition (DeWitt, 1959). The source of nutrients,
however, remained a mystery. Students of Hippocrates believed the
fetus to be nourished per os by suckling on maternal cotyledons or
uterine paps (DeWitt, 1959). This theory of fetal nutrition suggested
that menstrual blood (in humans) served as a nutrient source to the
early conceptus with excess deposited as amniotic fluid. Later, as the
uterus grew, it was thought that pressure of the pregnant reproductive
tract against the breasts would cause them to pump milk into the
uterine arteries, to supply the uterine paps directly (DeWitt, 1959;
Needham, 1959).

Aristotle (384-322 B.C.) argued against Hippocratic theories. He
observed that the fetus was encased in several layers of membranes and
that such encasement would prevent fetal suckling in utero (DeWitt,
1959) . The teachings of Aristotle relative to embryology and maternal-
fetal relationships were set down in Peri Zoon Geneseos ("On the
Generation and Development of Animals") , and dominated the sciences of
natural history and development for nearly 2000 years (Haeckel, 1897;
Needham, 1959). The impact of these concepts, heavily influenced by
Hippocratic philosophies, is emphasized in the description and drawings
of female generative organs by both Leonardo da Vinci (1452-1518) and
Andreas Vesalius (1514-1564), which still showed arteries connecting
the breasts to the reproductive tract (Needham, 1959; DeWitt, 1959;
Saunders and O'Malley, 1982).

-3-

With the Renaissance came a legion of observations critical to
development of modern concepts of embryology and conceptus-maternal
relationships. Leonardo da Vinci (1452-1519) was familiar with the
ungulate placenta and observed that it was connected, but not united
with the uterus by little sponges or cotyledons (Steven, 1975a; DeWitt,
1959). It was also his contention that both fetal respiration and
nutrition took place via the umbilical cord (DeWitt, 1959). Jean
Fernel (1A85-1558) was perhaps the first to suggest that improper
uterine environment might adversely affect embryonic development.
Challenging both Hippocratic and Aristotelian doctrine, he theorized
that, in women, menstrual blood ^ utero might be deleterious to the
embryo (DeWitt, 1959). Thus was born the idea that the process of
conception followed a logical order and that condition of the uterine
environment was important to this order.

The frontispiece of De Generatione Animal ium (1650) by William
Harvey (1578-1657 A.D.) was inscribed with the words, "ex ovo omnia"
(p. 133; Needham, 1959). This major concept, that all living things
are born from eggs, predated Regnar De Graafs' investigations of the
mammalian ovary (De Mulierum Organis; 1672) by nearly 25 years, and
Karl Ernst Baers' description of the mammalian ovum (History of the
Evolution of Animals; 1828, 1837) by nearly 200 years (Haeckel, 1897;
Needham, 1959). In his treatise of 1650, Harvey observed "An egge is,
as it were, an exposed womb; wherein there is a substance concluded,
as the representative and substitute or vicar of the breasts" (p. 151;
Needham, 1959). Thus it was recognized that the conceptus was nourished
by substances within the womb (uterus) much as the neonate was nourished
by the products of the breasts.

-4-

Walter Needham (1631-1691) was described as the father of the
dynamic aspect of physico-chemical embryology (Needham, 1959) . In
his major treatise, De Forma tu Foetu (1667), he refuted the Hippocratic
theory of uterine paps and argued that the substance which could be
squeezed from uterine cotyledons was distinct from lymph and important
in fetal nutrition (Amoroso, 1952; DeWitt, 1959; Needham, 1959).
Needham is credited with naming this substance uterine milk (Amoroso,
1952; Needham, 1950, 1959). This view was supported by observations
of John Mayow (16A3-167 9) who, in De Respiratione Foetus in Utero et
Ovo (1674), also observed that the uterus was naturally adapted for
separation of gaseous oxygen from arterial blood, and that the placenta
was, therefore, the respiratory organ of the fetus _in utero (see
DeWitt, 1959; Needham, 1959).

Though many erroneous theories relative to the nature and means
by which the mammalian conceptus was supported in utero persisted
throughout the 17th and 18th centuries (see Needham, 1959), the ob-
servations of William Harvey, Walter Needham and John Mayow set the
stage for development of more modern concepts of the relationship
between the conceptus and its uterine environment. With publication
of A Theory of the Structure of the Placenta, by Charles Sedgwick Minot
(1852-1914) in 1891, many of the diverse opinions relative to nature
of the interface between the conceptus and its environment were recon-
ciled (DeWitt, 1959). According to DeWitt (1959), this paper
correctly described the nature of early associations between the chorion
and underlying endometrium during the peri-attachment period in both
ungulates and man, and correctly identified the nature of chorionic
vascular izat ion .

'^^

-5-

During this same period potential sources of nutrient materials
provided to the conceptus Jji utero were more clearly defined as re-
viewed by Amoroso (1952). Bonnet (1882) coined the term "embryotrophe"
and defined it as any substance which could be regarded as a product
of degeneration of maternal tissues or as glandular secretions of the
endometrium (Amoroso, 1952). Later, Bonnet (1899; see Amoroso, 1952)
was the first to demonstrate active phagocytosis of embryotrophe by
the ruminant trophoblast. Meyer (1925) used the term embryotrophe to
describe all available material supplied to the conceptus ^^i utero
(Amoroso, 1952). Grosser (1927) observed that such material could be
supplied either directly from maternal blood (hemotrophe) , or as a
result of endometrial synthetic activity (histotrophe; Amoroso, 1952).
Whatever the source, the extent to which substances provided to the
conceptus specifically by the maternal reproductive tract are Important
for embryonic growth, appears to be related to the nature of the
maternal-fetal interface (placental type) and responsively demonstrated
by stage of maturity of the neonate.

Grosser (1909; In: Steven, 1975b) classified placentae of mammals
based upon number of maternal and fetal tissue layers separating the
two bloodstreams. The epitheliochorial placenta, now known to be
characteristic of cattle, sheep, pigs and horses (Wathes and Wooding,
1980; Steven^ 1975b) , was shown to consist of six tissue layers
Including (1) endothelium of fetal capillaries; (2) fetal connective
tissue (mesenchyme) ; (3) fetal chorionic epithelium; (4) maternal
uterine epithelium; (5) maternal connective tissue; and (6) maternal
endothelium. Other general classifications of placentae included the
endotheliochorial type (most carnivores) , and the hemochorial type

-6-

(Man, primates, and rodents). The former were characterized by absence
of both maternal epithelium and connective tissue, and the latter by
absence of all maternal tissue layers except blood (Steven, 1975b).

An early but persistent view in comparative placentation suggested
that the six layered epitheliochorial placenta of ungulates presented
the most formidable barrier to diffusion and transfer of nutrients
between mother and fetus (Steven, 1975b). Barcroft (1946) disagreed
with this view observing that, in general, the greater the number of
placental layers the more fully developed the animal at birth. Further-
more, animals with epitheliochorial placentae do not support ectopic
(extra-uterine) pregnancy and undergo a characteristically prolonged,
free-living period in utero prior to attachment (Cook and Hunter, 1978;
Steven, 1975b). By comparison, animals with hemochorial placentae (in
which fetal chorion is apposed directly to maternal blood) do not
experience a prolonged free-living period ±n utero, do support pregnancy
in ectopic sites, have characteristically shorter gestation lengths
(except Man) , and are born in an immature, helpless, often embryonic
state (Barcroft, 1946; Mossman, 1937; Needham, 1950). Consequently,
these species require intense, prolonged, postnatal care to reach
maturity. For these reasons it is apparent that, in mammals with
epitheliochorial placentae (including the cow), the relationship be-
tween the conceptus and its uterine environment is unique. Furthermore,
substances provided the conceptus by the maternal reproductive tract
are at least permissive, if not trophic, with respect to embryonic
growth.

More recently, embryo transfer (Seidel, 1981; Betteridge et al. ,
1980) and in vitro culture studies (Wright and Bondioli, 1981) further

-7-

emphasized importance of the uterine environment for maintenance of
conceptus and establishment of pregnancy in domestic farm species.
Yet, the suggestion of Harvey over 300 years ago that the mammalian
uterus, by providing substances to the conceptus, acts as a "vicar of
the breasts" (p. 151; Needham, 1959) to the developing embryo, remains
as nearly precise a statement as is currently possible relative to the
nature and function of components of the uterine environment.

From an evolutionary standpoint, tendency toward increasingly
longer periods of uterine gestation in eutherian mammals suggested a
requirement for interdependence of conceptus and maternal units (Moss-
man, 1934) . Conceptus adaptation to prolonged uterine gestation, was
suggested to involve development of ability to produce biologically
active agents necessary to effect maintenance of pregnancy (Amoroso and
Perry, 1975). Interdependence between conceptus and maternal units
might be considered most highly developed in species, such as cattle,
which possess epitheliochorial placentae since (1) the uterus is the
preferred site for establishment of pregnancy; (2) conceptuses exper-
ience a prolonged free-living period in utero early in gestation;
(3) attachment is superficial; and (4) gestation is prolonged (Mossman,
1934; Needham, 1950; Cook and Hunter, 1978). The critical nature of
the peri-attachment period in bovine gestation (Ayalon, 1978; Hawk,
1979) and the necessity for synchrony in bovine embryo transfer
(Seidel, 1981; Betteridge et al., 1980; Sreenan, 1978) emphasize im-
portance of both the uterine environment and conceptus "signals" in
pregnancy recognition (Short, 1976). Like the uterine environment,
little is known of the nature or function of products of the peri-
attachment bovine conceptus.

-8-

Historically, study of structure has preceded that of function.
It was in this spirit that research described herein was conducted.

Endocrine and Physiological Aspects of the Estrous
Cycle and Early Pregnancy

As suggested by Short (1976) , the sexual cycle displayed by
mammalian females may have developed as a natural mechanism for maxi-
mizing reproductive efficiency by providing multiple opportunities for
establishment of pregnancy. In large domestic farm species, including
seasonally polyestrus sheep and horses and polyestrus pigs and cattle,
it is the recurring estrus period (period of sexual receptivity) that
provides this opportunity. The condition of recurring estrus in non-
pregnant farm species is referred to as the estrous cycle, and occurs
as a consequence of the integrative action of anterior pituitary
polypeptide and ovarian steroid hormones which must either (1) stimu-
late conditions required to provide a non-hostile intrauterine
environment for sustenance of the conceptus; or (2) in the absence of
pregnancy, induce changes necessary to insure resumption of cyclicity
so as to provide another opportunity for conception. In this light,
it is the objective of the following section to review general endo-
crinological and physiological aspects of the estrous cycle and early
pregnancy with particular emphasis on the cow. Endocrine profiles
associated with recurrent estrus in major domestic farm species have
been extensively characterized. The reader is referred to reviews by
Schams et al. (1977), Britt et al. (1981), Hansel et al. (1973), Bazer
et al. (1981b) and Ginther (1979), for extended bibliographies and
comparison of endocrine profiles characteristic of cattle, sheep, pigs
and horses. Endocrine profiles characteristic of later stages of

-9-

pregnancy and the periparturient period in cattle were presented by
Smith et al. (1973), Robertson and King (1979) and Eley et al.
(1981a, b).

Cyclicity

The sexually mature bovine female (Bos taurus) has an estrous
cycle length of 20 to 21 days with a normal range of 17 to 25 days
(Salisbury et al., 1978; Robinson, 1977). Bovine estrous cycle length
is not affected by season of year, although reproductive phenomena
such as age at first estrus and interval to first postpartum estrus
may be affected by interactions of season with plane of nutrition
(Harrison et al., 1982; Grass et al. , 1982; Rzepkowski et al. , 1982;
Peters et al. , 1980; Tucker and Ringer, 1982). Heifers (nulliparous
females) tend to exhibit estrous cycles approximately 1 day shorter
than cows (parous females; Robinson, 1977). Four general phases of
the bovine estrous cycle are associated with characteristic alterations
in peripheral plasma endocrine profiles which are responsible for
changes in animal behavior (Arave and Albright, 1981) and morphology,
histology and biochemistry of the reproductive organs necessary for
establishment of pregnancy or reestablishment of cyclicity (Britt
et al., 1981).

Estrus, generally designated day 0, is the period of sexual
receptivity. This phase is usually 16h to 21h in duration (Schams
et al., 1977), but may be as short as 9h to lOh during periods of high
environmental temperature in summer months of the northern hemisphere,
or in tropical climates (Chenault et al., 1975). Endocrinologically,
estrus is characterized by high peripheral plasma estrogen (E) and low
progesterone (P^) (Robinson, 1977; Schams et al. , 1977). Peripheral

■10-

plasma estradiol (E ) begins to increase about 3 days prior to onset
of estrus and reaches maximal levels (- 10 pg/ml) during estrus
(Wetteman et al. , 1972; Hansel et al. , 1973; Chenault et al. , 1975;
Schams et al., 1977). This period of rising E is associated with in-
creasing uterine blood flow (UBF) to peak levels at estrus in cattle
(Ford et al., 1979), sheep (Greiss and Anderson, 1969) and pigs (Ford
and Christenson, 1979) . Onset of estrus precedes ovulation, which is
spontaneous, by 20h to 30h. Estrus is associated with a surge of
peripheral plasma luteinizing hormone (LH) of 10 to 25 ng/ml above
basal concentrations of approximately 1 ng/ml (Schams et al., 1977;
Rahe et al. , 1980; Britt et al., 1981). Both estrus and the LH surge
are precipitated by declining peripheral plasma P, and increasing E
(Britt et al. , 1981; Schams et al., 1977).

Metestrus (approximately days 1 to 3) is associated with waning
peripheral plasma E concentrations, cessation of estrous behavior,
ovulation, and initiation of luteinlzation of ovarian follicular granu-
losa cells (Hansel et al., 1973; Britt et al., 1981; Robinson, 1977).
Chenault et al. (1975) detected a 50% decline in plasma E by 5h after
the LH peak and basal E concentrations (1 to 3 pg/ml) obtained by 14h
(Chenault et al. , 1975). Luteinlzation results in formation of the
ovarian corpus luteum (CL) and initiation of P, production. Histolo-
gical and cytological descriptions of luteinlzation were presented by
Priedkalns and Weber (1968), and Channing (1970). Enzymatic changes
associated with this phenomenon were described for the cow by Lobel and
Levy (1968).

Diestrus, the longest phase of the estrous cycle (approximately
days 4 to 18) , is characterized endocrinologically by increasing pe-
ripheral plasma P, concentrations from around 1 to 2 ng/ml in late

-11-

metestrus to 6 to 10 ng/ml by days 12 to 15 during mid-diestrus. The
CL establishes full functionality during this period as reflected by
peaks in luteal tissue weight (Bartol et al. , 1981a; Hansel et al.,
1973), ovarian blood flow, and P, production (Wise et al. , 1982; Ford
and Chenault, 1981; Hansel et al. , 1973).

Regular waves of ovarian follicular development during the estrous
cycle are preceded by increases in peripheral plasma follicle stimu-
lating hormone (FSH) and responsively demonstrated, especially during
late diestrus, by rises in peripheral plasma E concentrations (Glen-
cron et al., 1973; Hansel et al. , 1973; Ireland et al. , 1979; Schams
et al. , 1973; Wetteman et al. , 1972) and increases in UBF (Ford et al. ,
1979). Consequently, the final phase of the estrous cycle, proestrus
(approximately days 19 and 20) , is a period of waning plasma P, and
rising E concentrations, associated with demise of the CL (luteal re-
gression) and development of the ovulatory Graafian follicle (Ireland
et al., 1979; Dufour et al. , 1972; Schams et al., 1977; Hansel et al. ,
1973).

Central to recurring estrus in cattle is the process of luteal
regression. This process is thought to occur as a consequence of the
action of products of the ovarian follicle(s) (presumably E2) on
uterine endometrium leading to uterine production of a luteolysin,
prostaglandin-F (PGF„ ) , which effects luteolysis via a local utero-
ovarian pathway.

Considerable data exist to support the concept that ovarian
follicular growth is continuous throughout the estrous cycle (Marion
et al., 1968; Choudry et al. , 1968; Ireland et al. , 1979). Rajakoski
(1960) maintained that there were two waves of bovine follicular
growth: one between days 9 and 12; and a second between days

-12-

12 and 16 which gave rise to the ovulatory follicle. The dynamic
nature of ovarian follicle growth in cyclic cattle was supported by
the work of Dufour et al. (1972). When largest ovarian follicles
present were marked in situ on different days throughout the estrous
cycle, the ovulatory follicle could be predicted only if marked after
day 18. Hence, follicular growth and atresia was occurring throughout
the estrous cycle.

Follicular growth is stimulated by FSH, while a combination of
FSH and LH appeared to induce maximal E„ biosynthesis (Thibault, 1977;
Merz et al. , 1981). Data of Lacroix et al. (1974) support a two-cell
mechanism for E synthesis by bovine ovarian follicles involving the
A -pathway (conversion of pregnenalone to androstenedione through
A -3 3-hydroxy steroids; Ryan and Smith, 1965). It was suggested that
ovarian thecal cells were responsible for testosterone production which
would then be aromatized to estrogen. in granulosa cells (Lacroix et al. ,
1974). Recent data of Merz et al. (1981) indicated that 85% of bovine
ovarian follicles (> 8 mm diameter) from cyclic cattle on days 6, 12
or 18 bound FSH in granulosa and human chorionic gonadotropin (HCG;
LH-like activity) in theca cells. Additionally, 38% of follicles
bound HCG in granulosa cells, and this binding was correlated with
increased E concentration in follicular fluid. In vitro production
of E by the two largest follicles obtained from cyclic cattle on days
4, 8, 12, 14, 16 and 19, was unaffected by cycle stage (Bartol et al. ,
1981a) . Data suggested that follicles were present on ovaries of
cattle throughout the estrous cycle which were capable of producing
significant amounts of E (Bartol et al., 1981a). Evidence for the
necessity of follicles in luteal regression was presented by Villa-
Godoy et al. (1981). Data indicated that electrocautery of visible

-13-

follicles combined with X- irradiation of ovaries in cyclic cattle on
days 9, 12 or 15 postestrus produced heavier CL at day 24 compared to
those from non-treated controls (5.5g vs. l.lg). Data also indicated
a luteolytic effect of ovarian follicles after day 15 (Villa-Godoy
et al., 1981),

Since the landmark observations of Loeb (1923) , presence of the
uterus or uterine tissue was found to be essential for luteal regression
in many species including cattle (Wiltbank and Casida, 1956; Malven
and Hansel, 1964), sheep (Anderson et al., 1969), pigs (Spies et al.,
1958; Du Mesnil Du Buisson, 1966) and horses (Ginther and First, 1971).
Initial investigations demonstrated that surgical removal of the uterus
prior to day 16 resulted in prolonged luteal lifespan in cattle
(Wiltbank and Casida, 1956), as well as sheep, pigs and mares (Anderson
et al., 1969; Ginther, 1981). Subsequent reports showed that cattle
(Bland, 1970) and ewes (McCracken and Caldwell, 1969) with congenitally
absent uterine horns adjacent to CL bearing ovaries had prolonged
luteal lifespans. These studies confirmed earlier observations from
surgical preparations, and established the concept of local utero-
ovarian control of CL lifespan in these species. This concept suggests
that the uterine luteolysin is transferred from the uterine venous
drainage to the ovarian arterial supply via a countercurrent mechanism.
Current evidence for such a mechanism in cattle is strong but still
circumstantial. In this regard, the reader is referred to reviews by
Ginther (1967, 1981) and the studies of Hixon and Hansel (1974) and
Shemesh and Hansel (1975b),

Distension or irritation of uterine mucosa with intrauterine
devices (lUD) , chemical irritants, or bacterial infections shortened

-14-

cycle length and luteal lifespan in cattle and sheep (Hawk, 1968;
Ginther, 1967; Moore and Nalbandov, 1953; Hansel and Wagner, 1960;
Anderson et al., 1969). Administration of E or oxytocin to cattle
during early diestrus resulted in premature luteolysis only if the
uterus was present (Brunner et al., 1969; Wiltbank et al. , 1961;
Armstrong and Hansel, 1959). Ablation of endometrium with caustic
chemicals or as a consequence of chronic bacterial infection resulted
in lengthened estrous cycles in cattle, pigs, sheep and mares (Ander-
son et al., 1969; Hawk, 1968; Hughes et al., 1977). Substances such
as estrogen and oxytocin, or devices such as lUD's and chemical
irritants appeared to precipitate premature luteal regression through
a common path. Whether physiological or mechanical, stimulation of
endometrium during diestrus in the uterine horn ipsilateral to the
CL was sufficient to cause production and release of the luteolysin
as reflected by shortened estrous cycles in treated cattle. Hence, the
endometrium was established as a major functional component of the
bovine luteolytic system.

Current data support the notion that the endogenous bovine uterine

luteolysin is PGF- . Since the observations of Babcock (1966), and
2a

Phariss and Wyngarden (1969), exogenous PGF„ was shown to be luteolytic
in cattle (Hafs et al., 1974; Lauderdale, 1974; Thatcher and Chenault,
1976), as well as pigs (Diehl and Day, 1974; Moeljono et al. , 1976a, b),
ewes (Coding, 1974) and mares (Douglas and Ginther, 1972; Allen and
Rowson, 1973; Noden et al. , 1974). Arachidonic acid, the principle
substrate in PG synthesis (Ramwell et al., 1977; Granstrom, 1981; Nelson
et al., 1982), was suggested by Hansel et al. (1975) to be the bovine
luteolysin. In other studies, however, the abrupt increase in bovine

-15-

endometrial content of arachldonic acid, noted to occur between days
10 and 14 (Hansel et al. , 1975), was found to precede striking in-
creases in endometrial tissue (Shemesh and Hansel, 1975a,b) and uterine
luminal flushing PGF content (Lamonthe et al. , 1977; Bartol, 1981a), as
well as uterine venous plasma concentrations of PGF„ (Shemesh and
Hansel, 1975b; Nancarrow, 1972) that occur after day 15. Furthermore,
evaluation of peripheral plasma concentrations of 13, 14-dihydro-15-
keto-PGF (PPGFM) , the inactive metabolite of PGF , revealed temporal
associations between elevated PPGFM and declining P, accompanying
luteolysis in cyclic cattle (Kindahl et al. , 1976; Thatcher et al. ,
1979) and sheep (Peterson et al. , 1976). Bartol et al. (1981a)
demonstrated that, in cyclic cattle, uterine flushing PGF content
increased between days 16 and 19 at a time when a stable population
of high affinity PGF -specific binding sites were present in luteal
tissues. Hixon and Hansel (1974) observed that uterine infusion with
PGF„ per cervix on days 12 or 13 postestrus caused increases in PGF^^
concentrations in ovarian and carotid arterial and jugular venous
plasma. Collectively, these studies provided strong evidence favoring
PGF„ as the endogenous bovine uterine luteolysin.

Thatcher et al. (1979) reported that E^-HB (3 mg I.V.) given to
cyclic cattle on day 13 postestrus caused an increase in PPGFM which
peaked at 6h post-injection and returned to pre- injection levels by 9h
post- inject ion. Bartol et al. (1981b) demonstrated that the predictable
E induced 6h increase in PPGFM was accompanied by a concommitant in-
crease in uterine luminal content of the parent compound, PGF2^, as
well as total protein. Recently, Knickerbocker et al. (1982), using
an identical protocol, measured UBF and uterine venous concentration

-16-

of both PGF and PGFM, as well as PPGFM. The 6h increase in PPGFM was
reflected directly, at the level of the uterus, by increased uterine
production of both PGF„ and PGFM. These studies demonstrated that
bovine uterine PG synthesis and metabolism in response to E could be
accurately assessed by monitoring PPGFM.

The E induced increase in bovine UBF seen by Knickerbocker et al.
(1982) agreed with previous studies (Roman-Ponce et al., 1978), and
resembled similar UBF responses shown to occur in normal cyclic cattle
during late diestrus as follicular E increased (Ford et al., 1979).
Bearing in mind that E induced increases in uterine production and
metabolism of PG (Knickerbocker et al., 1982) required prior induction

of uterine enzymes including cyclooxygenase (Huslig et al., 1979),

13
15-hydroxyprostenoate dehydrogenase, and A -reductase (Anggard, 1971),

normal increases in UBF during late diestrus in cyclic cattle (Ford
et al., 1979) might be considered to reflect increased uterine meta-
bolic activity associated with enhanced production of the luteolysin,

PGF„ . Data reviewed above are consistent with the concept that E ,
2a 2

of ovarian follicular origin, acts on diestrus endometrium to induce
increases in metabolic activity. Increased endometrial metabolic
activity may be met by increased UBF and reflected by enhanced produc-
tion of PGF„ from tissue stores of arachidonic acid. Luteolysis may
then result from binding of endometrially produced PGF„^ at specific
receptors in luteal tissues. It should be mentioned, however, that
recent data, summarized by McCracken et al. (1981), suggest an important
role for pituitary oxytocin in this luteolytic system.

Early Pregnancy

If conception occurs following mating, luteal maintenance and not
luteal regression becomes the goal if pregnancy is to be maintained.

-17-

Levels of peripheral plasma steroids (P, and E) and LH in early pregnant
cattle approximated those seen in cyclic (nonpregnant) cattle during
the same period post-estrus (Wetteman and Hafs, 1973; Wetteman et al. ,
1972; Folman et al., 1973; Hasler et al., 1980), but P did not decline
after days 18 to 19. In this respect the period of bovine pregnancy
of primary concern in this review, between conception and early concep-
tus attachment around day 22 to 24 (Wathes and Wooding, 1980),
corresponds in a temporal sense to periods of metestrus and diestrus
in nonpregnant (cyclic) cattle. Early pregnancy might, therefore, be
considered the beginning of a prolonged pregnancy-associated diestrus
period during which P, appears as the predominant maternal peripheral
plasma steroid.

Maintenance of peripheral plasma P, concentrations is, to date,
the earliest measurable manifestation of pregnancy in large domestic
farm species (Sauer, 1979). The CL is required for maintenance of
pregnancy in all large domestic species (Aitken, 1979; Hansel et al. ,
1973). Luteal tissue is required throughout gestation in the cow and
sow, but not in the ewe and -mare in which ovariectomy during the second
half of gestation does not result in abortion (Hafez and Jainudeen,
1974). Necessity of the CL for establishment and maintenance of
pregnancy in mammals was recognized even before the discovery of P, .
In a recapitulation of the Born-Frankel theory, Hartman (1924) stated
that product (s) of the CL effected those changes in the uterus which
made implantation possible. Hartman (1924) observed that ovaries (CL)
were necessary for continuance of pregnancy since ovariectomy during
early pregnancy "invariably" caused early embryonic death. Furthermore,
Hartman (1924) indicated that the direct cause of death in such cases
was "malnutrition of the embryos" (p. 449) as a consequence of collapse

-18-

of the uterine mucosa. These observations clearly indicate that, in
order for the conceptus to survive and mature, at least two important
processes must occur (1) prevention of luteolysis and (2) induction
of a supportive, non-hostile uterine environment (Cook and Hunter,
1978).

The obvious difference between a cyclic and pregnant animal is
presence of a conceptus in utero. Consequently, luteal maintenance,
characteristic of pregnancy, reflects response of the CL to physiolo-
gical conditions initiated by the conceptus and/or its products within
the uterine lumen. Short (1969) referred to the process by which the
peri-attachment conceptus signaled its presence to the maternal unit,
as reflected by luteal maintenance, as maternal recognition of preg-
nancy. In cattle, the precise mechanism(s) whereby "recognition" is
accomplished is not well understood. Clearly, this event must occur
very early in gestation, and may require both luteotrophic and anti-
luteolytic factors.

In cattle, sheep and pigs, presence of conceptus tissues in utero
prior to days 17 (Northey and French, 1980; Betteridge et al., 1980),
12 (Rowson and Moor, 1967; Martal et al. , 1979) and 13, respectively
(Dhinsda and Dziuk, 1968; Bazer et al. , 1982), resulted in prolongation
of the estrous cycle as a consequence of luteal maintenance. Inter-
estrus interval was extended in cattle from which conceptuses were
removed on day 17 (25 ±1.2 days) as compared to either non-mated
controls (21 days) or cattle from which conceptuses were removed on day
15 (20.2 ± 0.8 days; Northey and French, 1980). Additionally, intra-
uterine infusion of conceptus homogenates on days 15 through 17 extended
inter-estrus Interval (Northey and French, 1980) . Surgical transfer of

'T

-19-

84 single and 51 twin bovine embryos to synchronous (± 1 day) recipients
resulted in pregnancies only when performed before day 17 (Betteridge
et al., 1980). Thus, the bovine conceptus must be present in utero
at least from days 15 through 17 if luteal maintenance is to be ini-
tiated.

Location of the conceptus as well as timing appeared important
for successful recognition. Sreenan (1978) reported that a signifi-
cantly greater percentage of single bovine embryos were either still
present ±n utero at slaughter on day 40 or 60, or represented as live
calves at term, when transferred to the uterine horn ipsilateral (60%)
rather than contralateral (24%) to the CL bearing ovary in synchronous
recipients. In surgically prepared, unilaterally pregnant cattle,
luteal maintenance resulted when the gravid uterine horn was ipsilateral
but not contralateral to the CL (Ginther, 1981). Similar observations
were made for sheep (Rowson and Moor, 1962; Ginther, 1981), but not
pigs in which infusion of porcine embryonic extracts was effective in
maintaining bilateral luteal function in unilaterally pregnant gilts
(Ball and Day, 1982a). Studies suggest that, in early pregnant cattle
and sheep, a local utero-ovarian venoarterial pathway is involved with
luteal maintenance.

Several studies indicated that peripheral plasma concentrations of
P, were greater in pregnant than in nonpregnant or cyclic (non-bred)
cattle after day 10 post-mating or estrus (Boyd et al. , 1969; Henricks
et al., 1970; Holness et al. , 1977; Ford et al., 1979; Lukazewska and
Hansel, 1980), although such differences were not detected by others
(Batson et al. , 1972; Folman et al. , 1973; Hasler et al. , 1980). Work
reviewed recently by Ginther (1981) suggested that venous blood from

bS,!^;

:.>«jwr

-20-

the gravid uterine horn of cattle and sheep contained a "luteotrophic
factor." In these studies, luteal maintenance was consistently achieved
when venous effluent from the gravid uterine horn was provided access
to contralateral uterine venous or ovarian arterial supply. Beal
et al. (1981) reported that a heat-labile component of homogenates and
extracts of day 18 bovine conceptuses stimulated production of P by
dispersed bovine luteal cells in vitro. Similarly, Godkin et al.
(1978) detected a factor in day 13 to 15 ovine conceptuses which
stimulated ±a vitro production of P, by ovine luteal slices. Protein
extracts from pig conceptuses were shown to bind LH receptors in por-
cine luteal tissues (Saunders et al. , 1980). However, Ball and Day
(1982a, b) presented evidence for a non-thermolabile luteotropin in
aqueous extracts of porcine conceptuses.

In contrast to these studies, several reports indicated that
luteostatic and/or antiluteolytic mechanisms might be associated with
conceptus induced CL maintenance. In cyclic cattle, uterine infusion
of conceptus homogenates from days 15 through 17 delayed decline in
peripheral plasma P., but did not stimulate it as compared to uninfused
controls (Northey and French, 1980). Smith et al. (1982) failed to
detect any simple relationship between length of day 16 bovine concep-
tuses and ability of dispersed luteal cells to respond to LH in vitro.
Poffenbarger et al. (1982), using a mouse Leydig cell bioassay, did
not detect LH activity in homogenates of day 16 to 20 bovine conceptuses
or in culture medium in which day 16 and 17 conceptuses were incubated
for 12h. Similarly, no evidence of LH or prolactin activity was de-
tected in ovine conceptus homogenates by radioreceptor assay, nor did
homogenates stimulate P, sjmthesis or CAMP in isolated ovine luteal
cells (Ellinwood et al. , 1979a).

-21-

In both cattle (Kindahl et al., 1976) and ewes (Peterson et al.,
1976) PPGFM levels were depressed during early pregnancy as compared
to the same period post-estrus during which plasma P, levels were de-
clining in cyclic controls. Kindahl et al. (1981) suggested that, in
cattle, P, might regulate duration of uterine release of PGF^^ as
reflected by suppressed PPGFM. It was further suggested that chronic
P., characteristic of early pregnancy, might suppress gonadotropic
support to accessory ovarian follicles which, otherwise, could provide
estrogenic stimulation necessary for a luteolytic surge of uterine
PGF (Kindahl et al. , 1981). Alternatively, maternal P^, products of
the conceptus, or both were suggested to stimulate production or
activity of an endogenous inhibitor of uterine PG synthesis (Kindahl
et al., 1981). Wlodawer et al. (1976) provided evidence for presence
of such an inhibitor in bovine endometrium.

Rico et al. (1981) presented evidence supporting the idea that
the bovine conceptus could modify uterine response to an endocrine
stimulus. Plasma PGFM response of day 18 pregnant cattle given £^-173
(3 mg, I. v.), as described above (see Thatcher et al., 1979; Bartol
et al., 1981b; Knickerbocker et al. , 1982), was suppressed as compared
to that of E -173-treated, day 18 cyclic controls (Rico et al. , 1981).
Data from the University of Florida (D. Wolfenson, personal communica-
tion) indicated that increases in ovarian arterial concentrations of
PGF„ , observed during the normal period of luteolysis in cyclic cattle
(days 19-20), were absent in pregnant cattle at the same stage. A
conceptus effect on uterine production and/or transport of the luteo-
lysin (PGF ) was suggested. Evidence that PG may be subject to active,
carrier -media ted transport in reproductive tissues of various species

-22-

was presented by Bito and coworkers (Bito, 1972; Bito and Spellane,
1974; Bito et al., 1976).

Present data clearly indicate that maternal recognition of
pregnancy in the cow, as defined by maintenance of a functional CL,
involves complex interactions between the peri-attachment conceptus
and underlying uterine tissues. This process undoubtedly involves
both luteotrophic and antiluteolytic phenomena. Products of the
conceptus may act directly on the CL, either to stimulate luteal
function or antagonize the luteolysin. Additionally, interaction of
conceptus and maternal units as early as day 16 or 17 may initiate
modifications in uterine tissue and/or utero-ovarian vascular dynamics
which alter production, metabolism and transport of the luteolysin.
Elucidation of the mechanism(s) responsible for pregnancy recognition
in cattle will require further investigation of the nature and function
of substances produced by both maternal and conceptus units during the
peri-attachment period. For a comparison of mechanisms thought to be
involved with maternal recognition of pregnancy in cattle, sheep, pigs
and mares, the reader is referred to Bazer et al. (1981c).

Importance of the Uterine Environment

Early pregnancy (pre-attachment stage) in domestic farm animals
corresponds in a temporal sense to the period of diestrus in nonpreg-
nant (cyclic) animals. Hence, successful reproduction depends initially
upon uterine response to the integrative action of circulating ovarian
steroids, and other important endocrine agents present during this
period, appropriate to establish an environment capable of sustaining
the pre-attachment conceptus. The unique embryotrophic quality of the
uterine environment was demonstrated by failure of embryos from sheep

-23-

(Winterberger-Torres, 1956) and pigs (Murray et al, , 1971; Pope and
Day, 1972), as well as mice, rats and rabbits (Heap et al., 1979), to
develop to, or past, the blastocyst stage when confined surgically to
the oviduct. Similarly, an exhaustive review of culture media and
conditions used for support of bovine, ovine and porcine conceptuses
in vitro indicated that no system supported development beyond the
hatched blastocyst stage (Wright and Bondioli, 1981). Such observa-
tions lead Heap et al. (1979) to suggest that mammalian ova could
develop independently of their uterine environment prior to blastu-
lation.

As reviewed above, the bovine conceptus must be present in utero
prior to day 17 post-estrus if pregnancy is to be established (Better-
idge et al. , 1980). Furthermore, removal of conceptuses from pregnant
cattle on day 17, but not on day 15, resulted in prolongation of the
estrous cycle (Northey and French, 1980) . Therefore, the bovine
conceptus does not appear to interact detectably with its uterine
environment prior to day 15 post-estrus. However, a strict requirement
for sjmchrony between donors and recipients in bovine embryo transfer
(± 2 days; Sreenan, 1978; Seidel, 1981), which must be accomplished
prior to day 17 (Betteridge et al., 1980), emphasizes the importance
of the uterine environment during this period. That such an environ-
ment can be established independently of the presence of a conceptus
is demonstrated by the fact that embryos can be transferred and preg-
nancies established in cyclic recipient cattle which are reproductively
synchronous (±2 days) with pregnant donors (Sreenan, 1978; Seidel,
1981). In these cattle, as in early pregnant cattle during the same
period post-estrus uterine response to circulating endocrine agents

-24-

of maternal origin results in establishment of a supportive, embryo-
trophic, uterine environment. Thus, "the stage is set before the
play begins" (p. 175; Barcroft, 1934).

The maternal endocrine agent essential to establishment of an
embryotrophic uterine environment is P, . While Sauer (1979) indicated
that preovulatory ovarian E was important in potentiating uterine
response to this hormone (see below) , P, replacement therapy alone
maintained pregnancy in ovariectomized cattle (Hawk et al. , 1963),
sheep (Foote et al,, 1957; Gumming et al. , 1974) and pigs (Gentry
et al. , 1973) . In cattle, several workers reported more successful
conception following estrous cycles in which plasma P, was higher than
usual (Gorah et al. , 1974; Holness et al., 1977). Data support the
notion that the P, -dominated, diestrus uterus is required to insure
support to the developing conceptus.

Perhaps the earliest physical response of the maternal unit to
presence of a conceptus in the uterine lumen is an increase in uterine
blood flow and/or vascular permeability indicating initiation of inter-
action between the conceptus and its uterine environment (Sauer, 1979).
Transient 2- to 3-fold increases in blood flow to the gravid uterine
horn(s) were observed in cattle, between days 15 and 17 (Ford et al.,
1979); sheep, between days 12 and 16 (Greiss and Anderson, 1970); and
pigs, between days 12 and 13 (Ford and Ghristenson, 1979). Addition-
ally, Boshier (1970) described a positive Pontamine Blue reaction in
uteri of pregnant ewes between days 15 and 16. Increased blood flow
and uterine vascular permeability, induced locally by the conceptus,
may enhance potential for exchange of substances between uterine and
vascular compartments important for initiation of luteostatis and/or
maintenance of a supportive intrauterine environment.

-25-

Removal of the uterus before, or the conceptus after that period
post-estrus associated with conceptus- induced transient increases in
bovine UBF (days 15-17) resulted in prolongation of the estrous cycle
(Wiltbank and Casida, 1956; Northey and French, 1980), Consequently,
uterine responsiveness during this period, whether to maternal or
conceptus signals, is crucial for successful reproduction. Psychoyos
and Casimiri (1980) noted that, in species which display a decidual
response, the period associated with increased uterine vascular
permeability was also the only period during which trauma-induced
deciduogenesis would occur. Endometrial sensitivity to both mechanical
and physiological stimuli were suggested to be maximal during this
time (Psychoyos and Casimiri, 1980), Present data suggest a similar
period of uterine sensitivity in cattle between days 15 and 17.
Uterine responses to endogenous stimuli during this period dictate
whether a luteolytic or embryotrophic (luteostatic) path will be
followed. Either is consistent with the presumed goal of the sexual
(estrous) cycle, that of maximizing reproductive efficiency. If,
during this period, signals integrated by uterine tissues are of
maternal origin alone, the luteolytic path is taken and another oppor-
tunity for conception (estrus) provided. In contrast, if appropriately
timed signals integrated by uterine tissues are of both maternal and
conceptus origin, the embryotrophic path is taken and pregnancy estab-
lished. A wider or less well defined period of uterine sensitivity
might decrease reproductive efficiency by providing fewer opportunities
for conception as a consequence of irregularities in estrous cycle
length.

As established by Hartman (1924), conceptus-induced prevention of
luteolysis is a necessary prerequisite to maintenance of pregnancy.

-26-

It is difficult, if not impossible, to separate those conceptus-induced
maternal responses associated with luteostasis from those required for
induction of an embryotrophic uterine environment. Indeed these two
phenomena are far from independent, and mechanisms associated with one
may well insure the other. In cattle, these mechanisms are especially
unclear. However, the bovine uterus clearly serves as a major integra-
tor of goal -directed activity as defined by phenomena necessary for
maximal reproductive success.

The Uterus
Bovine Uterine Anatomy

According to Mossman (1980), "the sole purpose of the uterus of
a placental mammal is to furnish a conduit through which spermatozoa
reach the eggs, and to contain and sustain the developing conceptus
from the morula or early blastocyst period until parturition" (p. 3) .

The uterus of the cow is bipartite, consisting of two distinct
uterine horns (cornua) , approximately 25 cm in length, joined poster-
iorly in a short, common uterine body and suspended from the pelvis by
the broad ligament (corpus uteri; Skjerven, 1956a; Priedkalns, 1976).
The uterine wall consists of three basic, histologically distinguish-
able layers including (1) the serosa, or perimetrium; (2) the
muscularis, or myometrium; and (3) the mucosa, or endometrium. The
perimetrium consists primarily of semidense collagenous tissue and is
covered by peritoneal mesothelium except in posterior portions adjacent
to the cervico-vaginal area. Underlying the perimetrium, the myometrium
consists of both an outer longitudinal and inner circular layer of
smooth muscle fibers (Priedkalns, 1976). The inner-most mucosal layer,
or endometrium, is of particular importance to this discussion since it

-27-

is this layer which relates mother to conceptus both structurally and
functionally (Mossman, 1980) .

In ruminants two morphologically distinct areas are apparent on
the luminal surface of the endometrium. These are the caruncular and
intercaruncular areas (Priedkalns, 1976). Histologically, both areas
are comprised of three tissue layers. Surface epithelium (propria
mucosa) may be cuboidal, columnar and pseudostratif ied columnar
(Priedkalns, 1976; King et al., 1981; Wathes and Wooding, 1980; Mossman,
1980). This layer is continuous with the submucosa (stratum compactum) .
There is no uterine mucosal muscular is (Priedkalns, 1976). Below the
stratum compactum, the stratum spongiosum is seen as a layer of loosely
arranged collagenous and few reticular connective tissue fibers. This
layer extends from the stratum compactum to the myometrium and is some-
times referred to as the deep submucosa (Priedkalns, 1976) .

In cattle, caruncular areas appear as isolated thickenings of the
propria submucosa (Priedkalns, 1976) . Atkinson et al. (1982) described
these structures as pedunculated nodules, present on the bovine fetal
endometrium as early as day 200 of gestation. From 70 to 140 of these
morphologically distinct, aglandular areas may be present in a single
bovine uterus (Amoroso, 1952). These discrete structures, rich in
fibroblasts and extensively vascularized, serve as maternal components
of placentomes, the principle sites of attachment and maternal-fetal
gas exchange during gestation (see Kingman, 1948; Bjorkman, 1973;
King et al., 1980; Wooding and Wathes, 1980; King et al. , 1981).

Intercaruncular areas of the bovine endometrium represent the
glandular component of the uterus. These areas are penetrated by many
simple, coiled, tubular glands lined with ciliated and nonciliated
epithelium. Such glands are present throughout the endometrium.

-28-

extending into the stratum spongiosum and even the myometrium depending
upon reproductive status. Mouths of endometrial glands open only on
the intercaruncular surface (Priedkalns, 197 6).

Uterine vascular anatomy of the cow, as well as other major
domestic farm and laboratory species, was reviewed by Ginther (1976).
Further descriptions of bovine uterine vasculature were presented by
Ginther and Del Campo (1974), Yamauchi and Fumihiko (1968) and Yamauchi
and Sasaki (1969a, b). The following description is based largely upon
these publications.

Each side of the bovine uterus receives blood from three major
vessels. These include (1) the ovarian artery (OA) ; (2) the uterine
artery (UA) ; and (3) the vaginal artery (VA) . Branches of each of
these arteries course through the broad ligament to provide vascular
support to different regions of the reproductive tract. The OA orig-
inates from the dorsal aorta and divides in the broad ligament to
form the uterine, tubal and ovarian branches of the OA (UBOA, TBOA and
OBOA) . The UBOA provides blood to central and anterior ends of each
horn. A branch of the internal iliac artery, the UA also divides into
primary branches (UBUA) within the broad ligament. These vessels
supply major arterial support to the uterine body and horns. Toward
the uterus UBUA divide forming arcuate arteries (AA) which penetrate
the myometrium, run between longitudinal and circular muscle layers
and encircle each uterine horn. Longitudinal myometrial smooth muscle
is supplied by AA while basal arteries (BA) , branches of the AA, supply
the inner circular smooth muscle layer. Further branching and coiling
of the BA give rise to the tortuous radial arteries (RA) which supply
inner-most endometrial tissue layers. Since the RA are most intimately

-29-

associated with endometrial tissues it was suggested that these vessels
might be responsible for the phenomenon of metestrus bleeding (Hansel
and Asdell, 1951) . These vessels were also suggested to serve as im-
portant components of the maternal vascular supply to the placenta
(Yamauchi and Fumihiko, 1968; Carter et al. , 1971; Ginther, 1976).
Anastomoses of branches of the VA (UBVA) with UBUA may also supply
blood to the uterine body. Venous drainage of the uterus is essentially
parallel and opposite that of the arterial supply. However, primary
drainage of the uterus occurs through the ovarian vein (OV) . It is
the unique anatomical relationship between the UBOV, OV, and OA that
is thought to permit countercurrent exchange of the uterine luteolysin
(see Ginther, 1976) .

Uterine lymphatics may be found running essentially parallel to
blood vasculature (Reynolds, 1949). Although these vessels are un-
doubtedly important physiologically, few if any studies of bovine
uterine lymphatic supply have been conducted. However, the reader is
directed to recent work of Staples et al. (1982) in which uterine
lymphatic drainage of both the sheep and goat was described.

While it is established that reproductive organs are well supplied
with lumbar and sacral nerves (Getty, 1975), the integrative role of
neural input to the bovine uterus remains unclear. Primary innervation
of the uterus arises from the hypogastric plexus which provides sympa-
thetic adrenergic input. Structural similarities between certain
catechol-estrogens (Fishman, 1977) and the sympathetic neurotransmitters
(catecholamines; e.g., norepinephrine), however, suggest that uterine
response to nervous input may be more important than previously realized
(see Ball et al., 1975; Davies et al. , 1975; Kelly and Abel, 1980).

-30-

Steroids and Endometrial Response

Predictable cyclic fluctuations in endometrial histology, cytology
and metabolic activity occur largely as a result of the integrative
action of steroid hormones. Consequently, prior to a review of uterine
histology and components of -the uterine environment, a brief description
of mechanism of action of steroid hormones is warranted. Information
presented below was obtained largely from reviews by o'Malley and Means
(1974), O'Malley and Schrader (1976), Clark et al. (1977), Schrader and
O'Malley (1978), Stormshak (1979) and Walters and Clark (1980).

Entry of steroids from extracellular space into uterine cells is
believed to occur by passive diffusion (Clark et al. , 1977; Schrader
and O'Malley, 1978; Stormshak, 1979). Evidence was presented, however,
to suggest that E entry might be protein mediated (Milgrom et al. ,
1973) and that E-specific binding sites might be present on the endo-
metrial cell surface (Pietras and Szego, 1977). Upon entry, steroids
may bind specifically to high-affinity, low capacity cytosol receptors,
or nonspecif ically to low-affinity, high capacity components of the
intracellular milieu (Stormshak, 1979'; Clark et al., 1977). The
latter components may be important in maintaining availability of
steroids within target tissues.

High affinity cytosol receptors for both P, and E are thought to

be dimeric proteins consisting of an A and B subunit, each of which

can bind one molecule of steroid (Schrader and O'Malley, 1978). Binding

of hormone (H) to the cytosol receptor (R ) forms an H- R complex which

is then translocated from the cytosol to the nucleus (H' R ) . Once

within the nuclear envelope the B subunit of the H*R binds specifically

n

to an acceptor site on nonhistone protein of nuclear chromatin (Schrader
and O'Malley, 1978; Stormshak, 1979). Binding of the B subunit at an

-31-

acceptor site is thought to effect exposure of a specific sequence of
nucleotide base pairs on DNA. This event is accompanied by dissocia-
tion of the A subunit which subsequently binds to exposed DNA in a
nucleotide sequence-specific manner and initiates transcription of
specific RNA molecules necessary for synthesis of steroid-induced
proteins (Clark et al, , 1977; Schrader and O'Malley, 1978; Stormshak,
1979). Fate of the H-R complex following these events remains unclear,
but may involve recycling of receptors from nucleus to cytosol (Walters
and Clark, 1980; Schrader and O'Malley, 1978).

Nuclear translocation of E-R requires 2 to 3 min. (Clark et al. ,
1977; Katzenellenbogen et al. , 1980). Following translocation, uterine
responses to E include both early and late uterotrophic events (Clark
et al. , 1977; Katzenellenbogen et al. , 1980). Early uterotrophic
events generally occur within 4-6h of tissue exposure to E and include
hyperemia, lysosome labilization, and increases in water imbibition,
precursor uptake and synthesis of phospholipids, RNA and E-specific
proteins (Clark et al. , 1977; Kirkland et al., 1978, 1981; Katzenellen-
bogen et al. , 1980). These events are thought to be prerequisite to
tissue growth. Late uterotrophic events including increases in net
cellular content of RNA, DNA and protein, as well as cell division,
reflect E- induced growth phenomena (Katzenellenbogen et al. , 1980;
Kirkland et al. , 1978, 1981). These events may be detected within
5h to lOh of E exposure and continue for 24h to 30h (cell division;
Katzenellenbogen et al. , 1980).

Increases in nuclear content of RNA polymerase II, within 15 min.
of E exposure, characteristically precede early E-induced uterotrophic
responses (Katzenellenbogen et al . , 1980; Stormshak, 1979). This

-32-

enzjraie catalyzes transcription of mRNA prerequisite to synthesis of
E-specific proteins (Clark et al. , 1977). Increases in nuclear content
of RNA polymerase I, responsible for transcription of rRNA, are detec-
table within 2h to 6h post E exposure and precede late uterotrophic
events (Clark et al. , 1977; Katzenellenbogen et al., 1980). Ovine
endometrial RNA polymerase I activity increased within 6h postadminis-
tration of E -173 (500 ug, l.M.) to diestrus ewes (Luebke et al. , 1980).
Less immediately dramatic changes in protein and RNA synthesis

are seen following P exposure and nuclear translocation of P • R

't 4 c

stimulation of specific mRNA occurs within 4h to 6h of P, exposure,
with maximal stimulation not seen before 18h to 20h (Schrader and
O'Malley, 1978; Clark et al., 1977). Endometrial response to P, re-
quires E priming (Walters and Clark, 1980). Since P, may also regulate
tissue E receptor levels (Walters and Clark, 1980), temporal patterns
of appearance and relative amounts of each hormone (E and P.) may
dictate extent and nature of endometrial response.

Estrogen stimulates endometrial synthesis of both its own and
P,-specific cytosol receptors (Janne et al. , 1978; Clark et al., 1977).
Consequently E potentiates uterine responsiveness both to itself and
P, . Uterine sensitivity to ovarian steroids (E and P ) is modified
through the integrative action of E and P, . Estrogen-induced increases
in endometrial P, cytosol receptors augment uterine response to P,
(Clark et al., 1979). Progesterone, in turn, modifies uterine respon-
siveness to E both by inhibiting synthesis of E receptors and decreasing
nuclear retention time of E-R (Clark et al., 1977; Katzenellenbogen
et al., 1980). Consequently, P, decreases ability of the uterus to
respond in a totally E directed fashion (Clark et al. , 1977).

-33-

Uterine sensitivity to E may also be modified by other hormones.
Walters and Clark (1980) indicated that certain androgens, such as
dihydroxytestosterone, if present in very high levels, may compete
successfully for cytosolic E receptors. Alternatively, local conversion
of androgens to estrogens could also provide additional estrogenic
support (Walters and Clark, 1980). To date, however, no such conversion
has been reported for bovine, ovine or porcine endometria (see below).
Recent studies (Gardner et al., 1978; Kirkland et al. , 1981) indicated
that, in ovariectomized rats, hypothyroidism diminished late utero-
trophic responses to E -173. Diminished responsiveness was restored
by administration of thyroid hormone (T, ) . Thyroid hormone alone did
not elicit a uterine response. However, T restored E -17 3- induced
uterine responsiveness in a dose dependent manner. Thus, uterine
responsiveness to E may be modified by metabolic honnones.

In addition to its effect on uterine E receptor synthesis, P,
suppresses endometrial synthesis of its own cytosol receptor (Walters
and Clark, 1980; Schrader and O'Malley, 1978). A short term effect of

P, on E-primed endometrial tissue included a rapid increase in trans-

4

location of P,-R to P,-R (Walters and Clark, 1980). Levels of P , • R„
4 c 4 n t n

were maximal at Ih and returned to basal levels by 9h post-P^. Rapid
increase in P, • R was accompanied by a depression in P.- R , followed by
replenishment within 12h post-P, . This rebound phenomenon was suggested
to indicate recycling of nuclear receptor to the cytosol (Walters and
Clark, 1980). However, following the 12h post-P replenishment of P^
cytosol receptors, levels decreased to those characteristic of unprimed
(E) tissue (Walters and Clark, 1980). Progestational reduction in P^
receptors was not irreversible and was overcome by administration of
E (Walters and Clark, 1980; Schrader and O'Malley, 1978). Tissue P^

-34-

receptor levels may also be affected by products of the adrenals
induced during conditions of environmental stress (Walters and Clark,
1980).

Clearly, uterine responsiveness to E and P depends upon temporal
pattern of appearance and amount of each hormone present (E/P, ratio).
Following E-induction of high levels of endometrial P, receptors, P,
may eliminate or modify E-induced uterotrophic responses. However, as
P. suppresses endometrial production of its own receptors, E-induced
events become less suppressed. Hence, P, receptor levels dictate P
response as reflected by E-induced uterotrophic events (Walters and
Clark, 1980). In this respect, the P, dominated uterus of pregnancy
presents an intriguing problem in steroid receptor regulation which is
not well understood. Walters and Clark (1980) suggested that non-
estrogenic uterotrophic stimuli may act alone or synergistically with
low levels of endogenous E to maintain an adequate P receptor popula-
tion throughout gestation. In cattle and other species, such stimuli
may be provided by the conceptus. Recently, Findlay et al. (1982)
presented evidence that the ovine conceptus could affect caruncular
and intercaruncular tissue levels of E receptors as early as day 9 of
gestation.

Studies of endometrial content of cytosol receptors for E and P.
in normally cyclic domestic farm species revealed predictable patterns
with respect to the review of receptor djmamics presented above. Endo-
metrial content of cytoplasmic E receptors was highest during proestrus
and metestrus, when E was the dominant ovarian steroid, in cattle
(Senior, 1975; Henricks and Harris, 1978), ewes (Koligian and Stormshak,
1977a; Miller et al. , 1977) and gilts (Deaver and Guthrie, 1980). Endo-
metrial E receptor levels were depressed throughout diestrus in cattle

-35-

(Henricks and Harris, 1978) and ewes (Koligian and Stormshak, 1977a, b;
Miller et al. , 1977), but not in gilts (Deaver and Guthrie, 1980) in
which receptor levels increased transiently during mid-diestrus. The
latter observation was consistent with current concepts of maternal
recognition of pregnancy in the pig in which uterine recognition of
conceptus produced E is critical (see Bazer et al. , 1982).

Bovine endometrial content of cytoplasmic P receptors was higher
during proestrus (day 0) than diestrus (day 12; Zelinski et al., 1982).
Data were generally consistent with those of Koligian and Stormshak
(1977a) for cyclic ewes. Estrogen induction and P, antagonism of endo-
metrial cytosol receptor synthesis was demonstrated in the ewe (Koligian
and Stormshak, 1977b) . Studies related directly to endometrial steroid
receptor regulation in cattle are unavailable. However, Atkins et al.
(1980) reported that concentrations of P • R (receptor sites/cell) were
higher in the uterine horn ipsilateral as compared to that contralateral
to the CL-bearing ovary on day 10 but not on days 4 or 18 postestrus in
cyclic heifers. Data suggested a local uteroovarian field-effect
associated with propagation of a maximally responsive embryotrophic
uterine environment.

Endometrial Histology During the Estrous Cycle and Early Pregnancy

Histological and cytological alterations in bovine endometrial
epithelial cells reflect tissue metabolic activity in response to
fluctuating endocrine and other stimuli. In cyclic cattle, mitotic
activity in both surface and glandular epithelium was noted to begin
at or around estrus and continue for approximately 6 days into metestrus
(Marinov and Lovell, 1968). Epithelial cell heights were lowest at
estrus but continued to develop into diestrus (Marinov and Lovel, 1968;

'"^'^W

-36-

Skjerven, 1956a) . Slight hemorrhage was observed in propria mucosa of
caruncles just before ovulation (Marinov and Lovell, 1968). This
microscopic hemorrhage is seen externally, 24h-A8h later, as metorr-
hagia or metestrus bleeding (Hansel and Asdell, 1951).

Irregularities in bovine uterine epithelial cell heights were
noted throughout the estrous cycle, but reached a maximum developmen-
tally (30-35 lim) during mid-diestrus (Skj erven, 1956a; Marinov and
Lovell, 1968; Wathes and Wooding, 1980). Characteristic of active
diestrus endometrial tissue are columnar and pseudostratif ied columnar
epithelial cells which contain numerous mitochondria, well developed
golgi apparati and rough endoplasmic reticulum (RER; Skjerven, 1956a;
Marinov and Lovell, 1968; Wathes and Wooding, 1980). Nuclei were
basally located, and cells contained many lipid vacuole inclusions just
beneath apical microvilli (Marinov and Lovell, 1968, Wathes and Wooding,
1980). Histochemical and histological evidence of apocrine-type
secretion was noted (Skjerven, 1956a; Marinov and Lovell, 1968).

During late diestrus, in absence of a conceptus, degenerative or
regressive changes occur in bovine endometrium. Epithelial cells were
observed to become shorter and glands regressed from submucosa becoming
gradually less active (Skjerven, 1956a; Marinov and Lovell, 1968;
Mossman, 1980). Agenesis of both apical microvilli and intracellular
organelles was noted (Skjerven, 1956a; Marinov and Lovell, 1968). Also
characteristic of this period was appearance of large aggregates of
smooth endoplasmic reticulum (SER) associated with very large mitochon-
dria, the function of which remains unclear (4 to 5 times normal;
Wathes and Wooding, 1980). Elevated levels of E characteristic of late
diestrus and proestrus were reflected histologically by continued

-37-

regression of epithelial cells (Marinov and Lovell, 1968). Estrogen-
associated hypermia appeared as excess fluid in intercellular space
of the stratum compactum (Marinov and Lovell, 1968). Leucocytes were
identified between epithelial cells and at the basal lamina throughout
the estrous cycle (Wathes and Wooding, 1980; King et al. , 1981).

Histological and cytological descriptions of bovine caruncular
and intercaruncular epithelium during the peri-attachment period of
early pregnancy between days 17 and 30 were incomplete prior to recent
publications of King et al. (1980, 1981) and Wathes and Wooding (1980).
These studies indicated that before days 17 to 18 of pregnancy endo-
metrial tissues developed in a manner indistinguishable, histologically,
from that of cyclic cattle. However, after this stage alterations in
endometrial histology were unique to pregnancy and directly related to
interactions of maternal tissues with the chorionic surface of the
trophoblast. King et al. (1981) divided the period of early pregnancy
between days 17 and 30 into three phases based upon relationship of
trophoblast to endometrial surface. Included were: the apposition
phase (== days 17 to 18); the adhesion phase (== days 18 to 20); and the
attachment phase {- days 21-30).

Day 17 endometrial surface epithelium consisted of relatively
uniform columnar and pseudostratif ied columnar cells, indistinguishable,
ultrastructurally, from those of nonpregnant cattle (King et al. , 1981).
Epithelium was more regular in appearance in pregnant than nonpregnant
cattle on day 18 (Wathes and Wooding, 1980). Cells were columnar, 20
to 25 lim in height and at least 3% contained two nuclei. Binucleate
cells were evenly distributed between gravid and nongravid uterine
horns (Wathes and Wooding, 1980). Between days 19 and 20 definite

-38-

areas of adhesion were described between trophoblastic and endometrial
epithelium (Wathes and Wooding; King et al., 1981). However, microvilli
were observed only on the maternal epithelial surface and no inter-
digitation of conceptus and endometrial tissues was yet apparent (Wathes
and Wooding, 1980). Also between days 19 and 20, two areas of maternal
epithelium were identified. Endometrium beneath uninucleate trophoblas-
tic cells consisted of tall (35-45 ym) columnar epithelial cells which
contained SER and large mitochondria. Other areas were scattered among
columnar cells and consisted of large, pale, multinucleated, giant
cells (mGC) . These mGC contained numerous, basally located, membrane-
bound granules indistinguishable morphologically from granules seen
in conceptus trophoblastic binucleate cells (Wathes and Wooding, 1980;
King et al. , 1981).

Consistent with observations of Leiser (1975), attachment, evi-
denced by interdigitation of apical microvilli between maternal and
conceptus surface epithelia, was observed throughout the gravid
uterine horn between days 21 and 22 (Wathes and Wooding, 1980; King
et al. , 1981). Maternal giant cells continued to increase in size
and number such that by day 24 mGC comprised 25% of total cell number
and 50% of cell area in the gravid uterine horn (Wathes and Wooding,
1980; King et al., 1981). At this stage mGC were as much as 100 ym in
width and contained up to eight nuclei (Wathes and Wooding, 1980).
Maternal giant cells replaced strips of regular columnar epithelium
and columnar cells adjacent to mGC appeared degenerated, with no SER
and small mitochondria (Wathes and Wooding, 1980). Dying or degenerated
columnar cells appeared to be phagocytosed by uninucleate cells of the
trophoblast (Wathes and Wooding, 1980). Such maternal cell debris may
serve as a source of histotrophe.

-39-

By days 26 to 27, areas of attachment were apparent in the non-
gravid uterine horn (Wathes and Wooding, 1980; King et al. , 1980, 1981),
In the gravid horn mGC accounted for 7 0% of uterine luminal epithelium
(Wathes and Wooding, 1980). Tall, narrow, columnar cells, adjacent
to mGC, appeared to divide marginally forming a low cuboidal epithelium
(Wathes and Wooding, 1980). Thus, although areas of unchanged endome-
trial epithelium persisted, two cell types predominated by day 28:
(1) long narrow strips of mGC and (2) large areas of uninucleated
cuboidal cells. Beyond day 28, mGC degenerated and appeared to be
phagocytosed by trophoblastic (chorionic) epithelial cells (Wathes
and Wooding, 1980). In contrast, cuboidal cells persisted as the pre-
dominant endometrial intercaruncular cell type throughout the remainder
of gestation (Wathes and Wooding, 1980; Wooding and Wathes, 1980).

Both origin and function of multinucleated bovine endometrial
giant cells remain unclear. Previous descriptions of these cells by
several investigators (Leiser, 1975; Bjorkman and Bloom, 1957; Bjork-
man, 1968a; King et al. , 1979, 1980, 1981) indicated that mGC were
strictly of maternal origin. However, Wathes and Wooding (1980)
presented convincing morphological and ultrastructural evidence that
mGC form as a consequence of fusion of maternal columnar epithelial
cells and conceptus trophoblastic binucleate cells. These workers
suggested that transient formation of mGC between days 19 and 28
occurred primarily to allow delivery of conceptus binucleate cell
products to maternal circulation. Such a process could provide a route
for delivery of conceptus products necessary for luteostasis. However,
absence of mGC before day 19 suggests that events associated with
formation of these cells may be more important for establishment of an

-40-

embryotrophic uterine environment. Additionally, fusion of maternal
and fetal cells may be important in immunological aspects of pregnancy
recognition (Beer and Billingham, 1976) .

Components of the Uterine Environment

Response of uterine tissues to agents of maternal and conceptus
origin leads to establishment and maintenance of a unique, embryotrophlc
uterine environment. The dynamic, complex nature of this environment
is attested to by inability of investigators to maintain conceptuses,
from both large domestic and laboratory species, to or beyond the
hatched blastocyst stage in vitro (Brackett, 1981; Wright and Bondioli,
1981). As reviewed above and by Amoroso (1952), the importance of
components of the mammalian uterine environment long has been recog-
nized. The following section will review aspects of the nature of the
uterine environment in domestic farm species during the peri-attachment
period, with emphasis on the cow. Coimnents will be confined largely
to observations of the diestrus cyclic and early pregnant uterine en-
vironment during the first 30 days of gestation.

Collection techniques. According to Amoroso (1952), "uterine
milk" was first analyzed by Prevast and Morin (1842) and later by
Schlossberger (1855) and Gamgee (1864). Since that time a multitude
of studies in as many species have been conducted to examine compo-
nents of the uterine environment. Methods employed for collection of
uterine fluids are equally numerous and were reviewed by Bazer et al.
(1978). Two basic approaches have been used, (1) chronic collection,
involving placement of catheters or cannulae in the reproductive tract
and collection of fluids in vessels maintained either intra- or extra-
abdominally; and (2) acute collection, involving recovery from the

-41-

reproductive tract following either hysterectomy or slaughter and
lavage of the uterine lumen with a defined medium to obtain flushings.
Both methods were proven effective in large domestic species. However,
Bazer et al. (1978) noted that the method of choice could influence
results and should be chosen with experimental objectives in mind.
Specifically, chronic collection techniques often produced genital
tract fluids heavily contaminated with serum components (Bazer et al.,
1978; Heap and Lamming, 1960, 1962; Bartol et al., 1981b). Conse-
quently, recent studies suggest that more reliable profiles may be
obtained following acute collection procedures (Bazer et al. , 1978;
Bartol et al. , 1981a, b; Roberts and Parker, 1976).

Regardless of technique employed, amount of material, particularly
protein, recoverable from uteri of sheep and cattle during the estrous
cycle and early pregnancy is small (3 to 20 mg total protein/uterine
flush; Roberts et al., 1976; Bartol et al., 1981a). Such difficulties
led several investigators to exploit early observations of Bond (1898)
which indicated that ligation of the oviduct or uterine horn in rabbits,
guinea pigs and sheep permitted accumulation of copious amounts of
luminal fluid. Harrison et al. (1976) demonstrated that large amounts
of protein-rich uterine fluid could be obtained from late pregnant and
postpartum ewes in which a blind pouch was created surgically in one
uterine horn. Later, Bazer et al. (1979a) recovered similar fluid from
late pregnant ewes (> 100 days) rendered unilaterally pregnant by liga-
tion of one uterine horn. In both cases, uterine fluid accumulated in
the nonpregnant uterine horn in response to endogenous physiological
cues characteristic of late ovine gestation. It was suggested (Bazer
et al., 1979a) that, although recovered during late gestation, compon-
ents of such "uterine milk" might include substances normally required

-42-

to provide an embryotrophic environment during early pregnancy. To
date, this approach has not been attempted in cattle.

Electrolytes, free amino acids and reducing sugars. Analyses of

electrolyte components of chronically collected uterine fluids from

several species indicated regular cyclic fluctuations. Sodium (Na)

and phosphorous (P) in bovine and potassium (K) and P in ovine uterine

fluids were elevated during diestrus (Heap, 1962; Heap and Lamming,

1960). Heap (1962) and Heap and Lamming (1960, 1962) detected only

traces of calcium (Ca) in bovine uterine flushings regardless of

estrous cycle stage. However, others (Lamonthe and Gray, 1970; Schultz

et al., 1971) reported cyclic fluctuations in bovine uterine fluid Ca,

K, chloride (CI), and inorganic phosphate (PC,). Both K and PC, were

^ 4

elevated during diestrus, while Ca and CI were elevated during proestrus
and estrus. Mean uterine fluid concentrations of K and PC were higher,
Ca lower and Na not significantly different from those determined for
serum from the same cattle (Schultz et al., 1971; Olds andVan Demark,
1957b). Such differences between uterine fluid and serum in readily
diffusable electrolytes suggested that these two fluid pools were not
in simple equilibrium. Furthermore, cyclic fluctuations in uterine
fluid electrolytes suggested that endometrial cell membrane potential
was influenced by hormonal state.

Rasmussen (1981) reviewed data indicating that most secretory
cells respond to chemical (endocrine) stimuli with changes in membrane
potential. Such changes are effected by alterations in ion currents
(flux) across cell membranes (Rasmussen, 1981). Rasmussen (1981) in-
dicated that, in general, hormones with opposite effects have opposite
influences on membrane ion currents. Hence, opposing effects of E and

-43-

^4 ^^^^^4 ^^^^°^ ™^y dictate electrophysiological state of endometrial
mucosa. Although not demonstrated in bovine endometrial tissue, such
relationships were demonstrated in porcine chorioallantoic membranes
(Bazer et al. , 1981a).

Electrolyte components of uterine fluid from early pregnant cattle
have not been characterized. However, it seems likely that prior to
day 17, when embryo transfer is still possible (Betteridge et al. ,
1980) , diestrus cyclic and early pregnant bovine uterine environments
are quite similar. Although little is known about inorganic salt
requirements of pre-attachment conceptuses of large domestic animals
(Wright and Bondioli, 1981), in vitro studies indicated absolute re-
quirements for both Ca and K in maintenance of preimplantation mouse
and rat embryos (Biggers and Borland, 1976; Brackett, 1981; Wright and
Bondioli, 1981). Sodium chloride (NaCL) was also identified as essen-
tial for maintenance of osmotic equilibrium (Wright and Bondioli, 1981).
Identification of Ca, K and Na in diestrus bovine uterine fluid (Olds
and Van Demark, 1957b; Schultz et al. , 1971) suggests essential roles
for these ions in support of the pre-attachment conceptus.

Fluctuation in uterine luminal electrolyte content, especially
Ca, may reflect endometrial secretory activity and capacity to respond
to conceptus signals. As compared to diestrous cyclic gilts at the
same stage postestrus, Geisert et al. (1982b) observed a dramatic but
transient increase in uterine flushing Ca content between days 11 and
12 in gilts containing tubular and early filamentous conceptuses.
Similar observations were made in the roe deer during the period of
rapid blastocyst elongation following termination of embryonic dia-
pause (Aitken, 1977). In both species, rapid transient increases in

-44-

uterine Ca content heralded immediate but more persistent increases in
uterine protein content, as well as PGF and PGE^ content in pregnant
pigs (Aitken, 1977; Geisert et al. , 1982b). Douglas (1968) indicated
that Ca played an essential role in coupling of cellular excitation
to response in secretory cells (stimulus-secretion coupling). It was
observed that the source of Ca for such events could be either intra-
cellular or extracellular (Douglas, 1978). Geisert et al. (1982b)
suggested that porcine blastocyst-induced endometrial release of free
Ca was necessary to provide synchronized release of endometrial
secretory proteins. It was further suggested that porcine blastocyst-
induced endometrial Ca flux potentiated events necessary for maintenance
of an embryotrophic uterine environment.

In this respect, Milutinovic et al. (1977), studying pancreatic
acinar cells in vitro, showed that if Ca concentration in medium was
raised to between 10~ and 2 x 10 M, pancreatic zymogen granules
aggregated with the plasma membrane. This interaction was shown to be
specific for the luminal plasma membrane (Milutinovic et al. , 1977).
It was noted that, since Ca concentrations employed in vitro were
similar to extracellular levels predicted jja vivo after gland stimu-
lation, a similar extracellular Ca-mediated, luminal-specif ic
stimulation of secretion might function in vivo (Rasmussen, 1981).
Rasmussen (1981) indicated that, in secretory cells, changes in either
intra- or extracellular Ca concentrations were met by immediate
adjustments in plasma membrane potential necessary to maintain Ca ion
concentrations at or near maximal responsive levels. Thus, alterations
in diestrus bovine uterine fluid electrolyte profiles may reflect
changes in endometrial epithelial electrophysiology required to estab-
lish a maximally responsive environment for conceptus interaction.

-45-

Free amino acids and sugars may serve as important energy sub-
strates to both uterine and conceptus tissues. Fahning et al. (1967)
identified 25 free amino acids and/or amino compounds in chronically
collected bovine uterine fluids versus only 23 in blood serum from
the same cyclic cattle. Quantitative changes in uterine fluid amino
acid content were correlated with stage of estrous cycle and occurred
independently of blood serum profiles (Fahning et al., 1967). Lowest
uterine fluid amino acid concentrations were found at estrus, while
highest concentrations occurred between days 8 and 10 of diestrus.
These changes occurred independently of uterine fluid volume and,
therefore, appeared to be hormonally mediated. Similar data are not
available for early pregnant cattle.

Glucose was described as the major free sugar in bovine (Suga and
Masaki, 1973), ovine, porcine and equine uterine flushings (Haynes and
Lamming, 1967; Zavy et al. , 1982a). Concentrations of reducing sugars
in uterine fluids collected chronically (Schultz et al., 1971) and
acutely (Olds and Van Demark, 1957a, b) from cyclic cattle were highest
during late diestrus (day 14; Schultz et al. , 1971). Unfortunately,
character izational studies of changes in bovine uterine luminal content
of specific sugars are unavailable. Recently, however, Zavy et al.
(1982a) described changes in glucose and fructose content of uterine
flushings from cyclic and early pregnant pigs and mares. Total recov-
erable glucose increased after day 12 in both pregnant and nonpregnant
pigs with high levels detected in uterine flushings from pregnant pigs.
In contrast, glucose content of uterine flushings was not affected by
day postestrus or pregnancy status in mares (Zavy et al., 1982a).

Both amino acids and glucose were suggested to be important energy
substrates of uterine and fetal-placental tissues during later stages

-46-

of pregnancy in cattle, sheep and pigs (Ferrell and Ford, 1980; Ferrell
et al., 1976; Battaglia and Meschia, 1978; Silver and Comline, 1975,
1976). Additionally, in vitro culture studies indicated that pre-
attachment stage bovine, ovine and porcine conceptuses required either
an amino acid or glucose source for energy maintenance (Wright and
Bondioli, 1981). Present data suggest that, in domestic species includ-
ing cattle, uterine luminal content of both free amino acids and sugars
increases during diestrus to meet metabolic demands of both uterine and,
if present, conceptus tissues. Conversion of maternal glucose to
fructose and metabolism of this sugar via the oxidative arm of the
phosphogluconate pathway may be important for generation of reducing
equivalents (NADPH) and ribose sugars necessary for biosjntithesis of
other conceptus products (Zavy et al. , 1982a). This pathway of fruc-
tose metabolism would require that the conceptus depend heavily on
amino acids for energy (Zavy et al. , 1982a; Reitzer et al., 1979).

Proteins. Protein components of the uterine environment are un-
doubtedly important for support of the conceptus. However, definitive
data are lacking relative to specific embryotrophic functions of these
macromolecules, particularly in cattle, sheep and mares. Whether of
serum or uterine origin, proteins present in the uterine lumen of
domestic farm species may serve as enzymes and carrier molecules for
steroids, prostaglandins, vitamins and minerals (Bazer, 1975; Ellin-
wood, 1979b). Additionally, certain uterine proteins may be important
in limiting trophoblast invasiveness and regulating local immunological
processes associated with maternal recognition of pregnancy (Bazer
et al. , 1981b; Fazleabas et al. , 1982a; Segerson, 1981; Segerson et al. ,
1982; Wietsma et al. , 1982).

-47-

Quantitative and qualitative analyses of uterine luminal proteins
from cyclic cattle (Bartol et al. , 1981a; Mills, 1975; Roberts and
Parker, 1974a, 1976), sheep (Roberts et al., 1976), pigs (Murray et al. ,
1972; Squire et al. , 1972; Chen et al. , 1975) and mares (Zavy et al. ,
1979a,b, 1982b) revealed dynamic changes in protein profiles during
the estrous cycle with maximal stimulation observed during diestrus.
In these studies gel filtration and electrophoretic analyses of uterine
luminal proteins indicated that the majority of these proteins were of
serum origin or serum-like. However, increases in uterine protein
content during diestrus were accompanied by increases in complexity,
including appearance of proteins not characteristic of serum in cattle
(Bartol et al., 1981a; Roberts and Parker, 1976), ewes (Roberts et al. ,
1976a), pigs (Murray et al. , 1972; Squire et al. , 1972) and mares (Zavy
et al., 1979b, 1982b). Proteins characteristic of a maximally stimu-
lated diestrus uterine environment waned, both in amount and number,
during late diestrus in cyclic cattle (Bartol et al. , 1981a; Roberts
and Parker, 1 974a, b), sheep (Roberts et al., 1976a), pigs (Squire et al.,
1972; Geisert et al. , 1982b) and mares (Zavy et al., 1979b, 1982b),
but persisted or even increased in complexity if pregnancy was estab-
lished. Hence, while specific functions remain to be defined for many
of these macromolecules, data suggest that a complex array of uterine
luminal proteins is required for support of the conceptus and mainten-
ance of pregnancy.

Considering the economic importance of cattle, surprisingly few
studies have been conducted to investigate the qualitative array and
temporal pattern of change in bovine uterine luminal proteins. Prior
to the work of Roberts and Parker (1974a, b; 1976) virtually no informa-
tion of this type was available in the literature. Comparison of

-48-

electrophoretic, gel filtration and isoelectric focusing (lEF) profiles
of proteins in uterine flushings collected acutely from nonpregnant
and pregnant cattle from estrus through days 31 to 35 of gestation,
with those of serum proteins, indicated that not more than 2 to 3% of
total uterine luminal proteins were distinct from serum (uterine-
specific; Roberts and Parker, 1974a, 1976). However, electrophoresis
of proteins in bovine uterine washings toward the cathode at pH 4.5,
revealed three uterine-specific protein bands (Roberts and Parker,
1974a; Libby et al. , 1982). When bovine serum albumin (BSA) was
assigned the relative mobility value of 1 . 0 (Ma/b=1.0), the three
cathode migrating uterine-specific proteins (CMP) included one slow
CMP at Ma/b=1.2 and two faster CMP at Ma/b=1.3 and 1.42 (Roberts and
Parker, 1974a). All three CMP were detected electrophoretically in
uterine flushings from both nonpregnant and pregnant cattle. The slow
CMP (Ma/b=1.2) was observed in uterine flushings from both estrus and
day 7 pregnant cattle (Roberts and Parker, 1974a). The two faster
CMP (Ma/b=1.3 and 1/42) were detected as early as day 12 in pregnant
cattle but were also present, although less intensely, in nonpregnant
cattle on days 14 and 18 postestrus (Roberts and Parker, 1974a). In-
terestingly, a slower CMP (Ma/b=1.20), suggested to be different from
the protein band detected in uterine flushings from estrus and day 7
pregnant cattle, increased in intensity in flushings from day 16
pregnant cattle and persisted thereafter until day 35 when the study
was terminated (Roberts and Parker, 1974a). Further characterization
of these CMP proteins by gel filtration and lEF revealed two proteins
in a pH range of 8.4 to 8.6 and one in a pH range of 5.0 to 5.5. All
three fell in the 15,000 to 35,000 molecular weight (M ) range (Roberts

-49-

and Parker, 1974a). Additionally, three CMP were detected in uterine
flushings from a single ovariectomized cow treated with P (100 mg/day)
for two months (Roberts and Parker, 1974a), Similarly, Libby et al.
(1982) recently described three CMP proteins in uterine flushings from
both pregnant- and nonpregnant beef cows on day 17 postestrus. Amount
of each protein detected was correlated positively with CL wet weight
(Libby et al., 1982).

Fractionation of uterine flushing proteins from pregnant cattle
(days 7 through 31) on Sephadex G-lOO produced six fractions (F1-F6)
of which F3 and F5 were found to be enriched in uterine-specific pro-
teins (Roberts and Parker, 1976). Three uterine-specific F3 protein
bands were identified as distinct from serum by sodium-dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) . Molecular weight (xlO""^)
estimates of these proteins were 38, 40 and 48. A quantitative shift
in uterine content of F3 proteins was noted, with more of these products
present after day 17 of gestation (Roberts and Parker, 1976). Based
on SDS-PAGE all proteins in F5 were distinct from serum. Included were
four main bands at (M^ x 10 ) 22, 16, 11.5 and <8; and two faint bands
at 32 and 26 (Roberts and Parker, 1976). Isoelectric focusing of F5
proteins revealed two major intense bands at pH 7.9 and 8.1, two medium
stained bands at pH 5.5 and 5.7, and four faint bands between pH 4.9
and 5.5 (Roberts and Parker, 1976). Uterine proteins in F5 were seen
with greatest intensity and in highest numbers between days 14 and 15
of pregnancy (Roberts and Parker, 1976).

Studies of Roberts and Parker (1974a, 1976) indicated that at least
three and as many as 11 proteins were present in bovine uterine flush-
ings which were distinct from serum. These uterine-specific proteins

-50-

were generally less than 50,000 M and included two basic and as many
as six acidic products. The amount and number of these proteins in-
creased in late diestrus (after day 12) and many persisted with
pregnancy indicating a requirement for P . It was suggested that some
of the F3 proteins, which persisted until day 31 of gestation when the
study was terminated, might be conceptus products or cone eptus- induced
endometrial products (Roberts and Parker, 1976). In this respect, an
increasing quantitative shift in uterine flushing content of F3 proteins
was noted in pregnant cattle after day 17 (Roberts and Parker, 1976).
Additionally, identification of proteins in allantoic fluid with
similar electrophoretic character to F3 and F5 proteins suggested
conceptus uptake of uterine products (Roberts and Parker, 1976).

Analysis of protein in uterine flushings from cyclic cattle (days

4, 8, 12, 14, 16 and 19) by SDS-PAGE revealed protein bands in 32

_3
M^ (xlO ) categories between 18.7 and 292 (Bartol et al., 1981a).

Consistent with observations of Roberts and Parker (1974a, 1976), both

amount and number as well as frequency of appearance of proteins were

greatest during the late luteal phase (days 14 and 16; Bartol et al.,

1981a). Additionally, quantitative and qualitative changes observed in

SDS-PAGE uterine flushing protein profiles of cyclic cattle involved

primarily proteins of less than 150,000 M (Bartol et al., 1981a).

Uterine flushing protein SDS-PAGE profiles from day 19 pregnant cattle

were qualitatively and quantitatively similar to day 14 cyclic profiles.

Additionally, four protein bands were identified in uterine flushings

from day 19 pregnant cattle which were absent from flushings at all

stages of the estrous cycle examined (Bartol et al. , 1981a). Data

complemented those of Roberts and Parker (1974a, 1976), indicating

"5'^er

-51-

that the array of uterine luminal proteins, established during diestrus
in cyclic cattle, persisted or even increased in complexity if preg-
nancy was established. Data also provided further support for the
notion that conceptus-produced or induced products contributed to the
bovine uterine environment during early pregnancy (Bartol et al. ,1981a).

Laster (1977), using immunochemical techniques, detected a protein
in uterine endometrium and flushings from pregnant but not nonpregnant
cattle on day 15 postestrus. Based on gel filtration chromatography
(G-200) this protein had an estimated M (xlO ) of 50 to 60. This
antigen was suggested to comprise less than 1% of total unfractionated
endometrial protein. A second antigen, detected in uterine flushings
and endometrium from both pregnant and nonpregnant cows, was designated
uterine-associated since it could not be identified immunochemically
in plasma, liver, kidney, spleen or muscle from pregnant cows (Laster,
1977). Both proteins, detected by 15 days postmating, were still
present in cows at day 48 of gestation (Laster, 1972). It was suggested
that the protein(s) identified in this study was different from those
described by Roberts and Parker (1974a) although no definitive evidence
was presented (Laster, 1977).

Dixon and Gibbons (1979) recovered large amounts of proteins in
uterine secretions from three intact cattle that received 300 mg P,
daily to extend "diestrus" for 2 to 3 months (660 mg, 494 mg and 592
mg) . This represented a considerable increase in recoverable protein
compared to other studies in which only 3 to 20 mg of total protein
were recovered from untreated cyclic and early pregnant cattle (Roberts
and Parker, 1974a, 1976; Bartol et al. , 1981a, b). Fractionation of
proteins in bovine P,-induced uterine secretions by carboxymethyl

-52-

cellulose (CMC) cation exchange chromatography, gel filtration and disc
electrophoresis revealed at least nine nonserum proteins (Dixon and
Gibbons, 1979). Of these, seven appeared in small amounts and were not
characterized individually. Of the remaining two nonserum proteins
one was determined to be lactoferrin and the other an acid phosphatase.

Data are not available to indicate specific proteins which are
produced and secreted by bovine uterine endometrial epithelium. None
of the uterine-specific products described above (Roberts and Parker,
1974a, 1976; Laster, 1977; Dixon and Gibbons, 1979) have been shown to
result from de^ novo synthesis by endometrial tissue, although a require-
ment for P, is apparent. Wathes (1980) demonstrated that caruncular
and intercaruncular endometrial tissue from cattle on days 25, 34, 36
and 44 of gestation incorporated significant amounts of L - C
leucine ( C-Leu) and L- 4,5- H leucine ( H-Leu) during a 4 8h in vitro
culture. Endometrial H- and C-Leu incorporation was not affected
by stage of gestation. Addition of steroids (E, P^ or both) or placen-
tal tissues to culture medium had no systematic effect on overall

3 14
leucine incorporation or array of proteins into which H- or C-Leu

was incorporated (Wathes, 1980). Data indicated that bovine endometrial

tissue from early pregnancy could incorporate radiolabelled amino acids

into proteins in vitro. However, these labelled products, synthesized

de novo, in vitro , were not systematically characterized (Wathes, 1980).

Data of Wathes (1980) were consistent with earlier reports by Basha

et al. (1979) and Rice et al. (1981) in which no significant effect

of steroid treatment (E, P, or both) was detected on capacity of porcine

endometrial tissues to synthesize protein de novo from radiolabelled

amino acids ixi vitro. Together, data indicated that ability of bovine

-53-

and porcine endometrial tissues to sjmthesize proteins ±n vitro
depended upon in vivo hormonal state at the time they were removed.

A number of enzymes are present in bovine endometrivim and uterine
fluids. Histochemical techniques revealed alkaline phosphatase acti-
vity associated with the apical border of bovine uterine luminal and
glandular epithelium, as well as glandular secretions (Moss et al. ,
1954; Skjerven, 1956a, b; Kenney et al., 1965; Marinov and Lovell, 1968;
Leiser and Wille, 1975). Studies repeatedly demonstrated alkaline
phosphatase activity to be maximal during diestrus and depressed during
proestrus and estrus. An inverse relationship between uterine glycogen
content and alkaline phosphatase activity was recognized (Moss et al.,
1954; Mannov and Lovell, 1968). Larson et al. (1970) suggested that
alkaline phosphatase was important for bovine endometrial carbohydrate
metabolism and stimulation of secretory activity. In this regard,
Leiser and Wille (1975), using a histochemical approach, described
intense increases in endometrial surface alkaline phosphatase activity
associated with the apposition phase of bovine conceptus development.
Both alkaline and acid phosphatase activities in bovine uterine fluid
were maximal during diestrus (Schultz et al., 1971; Linford and losson,
1975) and increased following P treatment (Wordinger et al. , 1971).
A nonserum acid phosphatase (pl=9.7) was described by Dixon and Gibbons
(1979) as a major uterine specific component of P,-induced bovine
uterine fluid. Linford and losson (1975) showed that, in pregnant
cattle between days 25 to 30, endometrial acid phosphatase level in the
gravid uterine horn was twice that of the nongravid horn. Similar
relationships were not detected for either alkaline phosphatase or acid
cathepsin (Linford and losson, 1975). Both acid and alkaline phos-
phatases were more active in bovine uterine fluid than blood (Schultz

-54-

and Fahning, 1971). However, alkaline, but not acid phosphatase
activity in blood fluctuated with stage of the estrous cycle (Schultz
and Fahning, 1971). Hence, the conceptus-associated increase in
bovine endometrial acid phosphatase activity described by Linford and
losson (1975) may reflect a local c one eptus- induced increase in endo-
metrial enzyme activity associated with remodeling of the endometrium
to accomodate placentation.

Roberts and Parker (1974b) reported several glycosidases but no
proteinase or neuraminidase activity in bovine uterine flushings
collected from days 0 to 21 of pregnancy. Activities of a-L-fucosidase,
a-D-galactosidase, 3-D-galactosidase, a-D-glucosidase, a-N-acetylgalac-
tosaminidase and 3-N-acetylglucosaminidase were elevated in uterine
flushings as compared to serum. Of these, a-L-fucosidase activity was
greatest between days 13 to 15. Other enzymes reached peak activity
between days 19 to 21 of pregnancy (Roberts and Parker, 1974b). Gly-
cosidase activities in uterine flushings from five cyclic cattle
slaughtered between days 12 and 18 postestrus were similar to those
found for pregnant cattle between days 13 to 15 (Roberts and Parker,
1974b). However, glycosidase activities in uterine flushings from one
day 19 and two day 20 cyclic cows were much reduced when compared to
those from pregnant cattle of the same stage and more nearly resembled
the day 0 to 3 pregnant group (Roberts and Parker, 1974b). Data
suggested a pregnancy associated persistency in uterine luminal glyco-
sidase activity. In addition to glycosidases, antitrypsin activity
equal to approximately 50% of that found in serum was identified in
uterine flushings from pregnant cattle (Roberts and Parker, 1974b).
Glycosidase and antitrypsin activities may be involved in control of

-55-

blastocyst-endometrial adhesion and regulation of trophoblastic inva-
siveness (Roberts and Parker, 1974b; Mullins et al. , 1980a; Fazleabas,
1982a).

The amount of protein recovered in uterine flushings from pregnant
sheep increased after day 13 (Roberts et al. , 1976a) suggesting a
pregnancy associated quantitative shift similar to that described for
cattle (Roberts and Parker, 1974a). Roberts et al, (1976a) identified
a number of nonserum proteins in ovine uterine flushings. Eight such
proteins were revealed by PAGE at pH 8.9. Of these, five appeared as
faint bands in gels of proteins from days 7, 11 and 13, but two appeared
only in flushings from days 15 and 18 and one increased in intensity
from day 15 to 18 (Roberts et al. , 1976a). Electrophoresis at pH 4.5
revealed three nonserum bands. All were found in ovine uterine flush-
ings from day 7, 11, 13, 15 and 18 of pregnancy (Roberts et al., 1976a).
Isoelectric focusing (pH 3-10) of ovine uterine flushing proteins from
days 11, 13 and 15 of pregnancy revealed up to 32 protein bands (Roberts
et al., 1976a). This agreed with earlier reports of as many as 35
protein bands in lEF gels of bovine uterine flushings (Roberts and
Parker, 1974a). Using lEF, 10 nonserum protein bands were identified
in uterine flushings from a day 15 pregnant sheep. Five were also
identified in uterine flushings from ewes at earlier stages of pregnancy
and the estrous cycle (pi, 5.7 to 7.0) and five were seen only in day
15 pregnant ewes (pi, 4.3 to 5.6; Roberts et al., 1976a). Data
resembled those for cattle (Roberts and Parker, 1974a, 1976; Bartol
et al., 1981a) and suggested that ovine conceptus-produced or induced
proteins were present ±n utero by day 15.

Uterine milk recovered from the ligated uterine horns of unilater-
ally pregnant sheep on day 140 of gestation (Bazer et al. , 1979a)

-56-

contained an average (X ± SEM) of 13.4 ± 3.4g of total protein. This
fluid was recovered from unilaterally pregnant sheep as early as day
30 but did not appear in copious quantities until after day 100
(Moffatt et al., 1980; Bazer et al. , 1979a). Two basic polypeptides
with M (xlO ) of 57 and 59 were detected by SDS-PAGE as the major
protein species present in ovine uterine milk (Bazer et al. , 1979a) .
These two polypeptides were also present in uterine secretions from
ovariectomized ewes treated 120 days with P, alone (50 mg/day) or P,
plus estrone (E. , 5 yg/day) , with maximal stimulation in the latter
group (Moffatt et al. , 1980, 1981). Endometrial explants from pregnant

ewes and ovariectomized ewes treated with P. or P. + E secreted radio-

4 4

labelled uterine milk proteins into culture medium during incubation
with \-Leu (Moffatt, et al., 1980, 1981). Data indicated that the two
uterine milk proteins are the major secretory products of ovien endo-
metrium during pregnancy or chorionic P. treatment. The ovine uterine
milk proteins were not detected in uterine flushings from cyclic ewes
on days 0, 6, 10, 12 and 16 postestrus suggesting a requirement of

protracted P, /E, stimulation for induction of synthesis of these
4 1

proteins (Moffatt et al. , 1981).

Progesterone stimulated and E suppressed acid and alkaline phos-
phatase activity in intercotyledonary ovine endometrium (Murdoch, 1972).
Histochemistry revealed increasingly strong acid phosphatase activity
associated with the apical uterine epithelial cell surface from day 14
to 16 in pregnant ewes, which gradually spread to subepithelial struc-
tures (Boshier, 1969). However, neither apical nor subepithelial
activity was seen in nonpregnant ewes 14 or 16 days postmating (Boshier,
1969). Acid phosphatase activity persisted into the fourth week of

-57-

gestation in intercaruncular, but not caruncular ovine uterine epithel-
ium. Alkaline phosphatase activity was intensely apparent at the
maternal-conceptus interface of sheep through the fourth week of ges-
tation (Boshier, 1969) . Glycosidase activity was highest between days
11 and 15 in uterine flushings from pregnant ewes (Roberts et al.,
1976a). These levels were substantially higher than similar values
obtained from cyclic ewes on days 13 and 15 postestrus (Roberts et al. ,
1976a). As in cattle, ovine uterine phosphatase and glycosidase
activities appeared to be enhanced by chronic P, and associated with
presence of the conceptus.

Among domestic farm species, porcine uterine proteins have been
characterized most extensively. As in other species, studies involving
comparison of porcine uterine flushing protein profiles with those of
serum indicated that uterine luminal proteins were, at least partially,
products of active endometrial synthesis and secretion (Roberts and
Bazer, 1980). Both quantity and heterogeneity of proteins identified
in porcine uterine flushings were greatest during diestrus, reaching
maximal levels on day 15 (Murray et al. , 1972; Squire et al. , 1972;
Bazer, 1975) and persisting or even increasing throughout early to
midpregnancy (Roberts and Bazer, 1980; Bazer, 1975; Geisert et al. ,
1982b). Quantitative and qualitative changes in porcine uterine flush-
ing protein profiles characteristic of diestrus were induced and
maintained in ovariectomized gilts treated with P, (Knight et al. ,
1974a). Knight et al. (1974a) reported a positive dose-response
relationship between amount of P, administered and total uterine protein
recovered. Estrogen alone had no effect on porcine uterine protein
content (Knight et al., 1974b). However, although ineffective alone.

-58-

E acted synergist ically when administered in lower doses (up to 25 Hg/kg
body weight/day) , but antagonistically when administered in higher doses
(50 Ug/kg body weight/day) with P (Knight et al., 1974b; Basha et al. ,
1980a; Roberts and Bazer, 1980). Basha et al. (1979) found that porcine
endometrial explants incorporated radiolabelled amino acids into radio-
labelled proteins, primarily lysozyme and a purple-colored phosphatase
(uteroferrin; Roberts and Bazer, 1980), de novo. Using this approach,
proteins synthesized by endometrium from pregnant and pseudopregnant
pigs were found to be qualitatively identical (Basha et al. , 1980a,b) .
However, explants of endometrium from pregnant gilts synthesized nearly
30 times as much uteroferrin as explants from diestrus gilts (Basha
et al. , 1979). Thus, the qualitative array of porcine endometrial pro-
teins produced in vitro appeared to be regulated by maternal endocrine
status and stimulated quantitatively by presence of a conceptus (Basha
et al., 1979; Geisert et al., 1982b).

The first porcine P -induced endometrial protein to be purified
and characterized was a purple-colored acid phosphatase found in maxi-
mal quantities in uterine flushings from cyclic gilts between days 12
and 16 postestrus (Squire et al. , 1972; Chen et al. , 1973; Schlosnagle

et al., 1974). This glycoprotein, now called uteroferrin (Utf; Roberts

_3
and Bazer, 1980), was shown to have a M (x 10 ) of 32 to 35, and pi

of 9.7. Each molecule of Utf is thought to bind one atom of ferric
iron (Fe ) , which gives the molecule its purple color (Roberts and
Bazer, 1980). Using immuno fluorescent microscopy, Chen et al. (1975)
and Renegar et al. (1982) found the site of synthesis and secretion of
Utf in both diestrus cyclic and pregnant pigs to be endometrial epi-
thelial cells. This protein was found to comprise 10 to 15% of total

-59-

proteins in P, -induced porcine uterine fluid, and was synthesized and

3
secreted as radiolabelled Utf by H-Leu- supplemented explants of

endometrium from diestrus cyclic, pregnant, pseudopregnant and P,-
treated pigs (Basha et al. , 1979, 1980a, b; Roberts and Bazer, 1980).
During gestation, maximum Utf synthesis occurred between days 35 and
75 when P,/E ratio was greatest (Bazer et al. , 1981b). Iron binding
properties of Utf (see Roberts and Bazer, 1980) suggested that this
protein might be important in supplying Fe to the porcine fetus
(Bazer, 1975). Recent studies of Renegar et al. (1982) indicated that
Utf, once synthesized and secreted by maternal uterine epithelium, was
taken up via chorionic areolae into placental chorioallantoic capil-
laries and delivered to the fetus via the umbilical vein. There, Utf
was suggested either to bind specifically to Kupfer and/or endothelial
cells in the fetal liver to provide iron necessary for hematopoesis,
or to be cleared by the fetal kidney and transported, in urine, to the
allantoic sac to serve as a temporary iron storage reservoir (Renegar
et al., 1982). Indeed, Utf was identified in porcine allantoic fluid,
during that time associated with maximal Utf synthesis (Bazer et al. ,
1975, 1981b). Here it was suggested to give up its Fe to transferrin
(Buhi et al., 1982). Thus, Utf is the first uterine-specific protein
to be definitively associated with an embryotrophic role in any of the
domestic farm species.

An inhibitor (s) of plasminogen activator was detected in uterine
flushings of diestrus cyclic gilts during that period associated with
maximal Utf synthesis (Mullins et al. , 1980a). Subsequently, P,-
induced porcine uterine secretions were shown to contain a group of
basic, low M serine-protease inhibitors (Fazleabas et al., 1982a).

-60-

One of these proteins, purified via a series of gel filtration and
cation exchange steps (M^ - 14,500), inhibited trypsin, plasmin and
chymotrypsin, but not elastase, subtilisin or thermolysin (Fazleabas
et al. , 1982a). Immunohistochemical data showed this serine-protease
inhibitor to be associated only with surface and glandular porcine

uterine epithelium. Endometrial explants from pseudopregnant gilts,

3
cultured with H-Leu, produced radiolabelled inhibitor, indicating

that this protein was synthesized de novo and was, therefore, a
uterine-specific product (Fazleabas et al. , 1982a). Antiserum to the
purified inhibitor cross-reacted with at least three other basic, low
M^ plasmin/trypsin inhibitors in porcine uterine secretions suggesting
a family of isoinhibitors constituting up to 15% of total recovered
uterine protein (Fazleabas et al., 1982a). In early pregnant pigs,
inhibitor activity in uterine flushings increased systematically, in
a manner related to stage of blastocyst elongation, with an abrupt
increase associated with conceptus-induced uterine Ca release (Fazlea-
bas et al., 1982b; Geisert et al. , 1982b). Similar abrupt increases
in uterine luminal plasminogen activator inhibitor activity were absent
in cyclic gilts, but could be induced by a single injection of E -173
(5 mg IM) on day 11 postestrus (Fazleabas et al. , 1982b). Thus, E,
acting on a P, dominated uterus, was stimulatory to inhibitor sjmthesis.
Fazleabas et al. (1982a) observed that the inhibitor (s) appeared to
coat the elongating blastocyst. It was suggested that these proteins
might protect the uterus from proteases known to be released by the
porcine trophoblast (Mullins et al., 1980a). Additionally, P, -induced
uterine serine-protease inhibitors were suggested to be important in
prevention of degradation of other components of the uterine environment

-61-

such as Utf . Although Roberts and Parker (1974b) detected antitrypsin

activity in uterine flushings from early pregnant cattle, the origin

of this activity (uterine vs. serum) was not determined.

Adams et al. (1981) described a retinol binding protein (RBP)

found in uterine flushings from diestrus (day 15) cyclic gilts. This

protein was also found in uterine flushings from ovariectomized gilts

treated with P, alone or P, + E, but not E alone or corn oil vehicle

(Adams et al. , 1981) . Evidence for a P,-induced retinoic acid binding

protein was also presented (Adams et al., 1981). The P, -induced RBP

4
_3
had an estimated M (xlO ) of 17 to 20 and a K^ for retinoic acid of
r d

1.93 X 10~ M (Adams et al,, 1981). As indicated by Bazer et al.
(1981b) , data are not available to explain whether RBP moves selectively
into the P,-dominated uterus from serum or is sjmthesized and secreted
by uterine tissues. However, detection of similar proteins in porcine
allantoic fluid suggested that such molecules might be important in
transport of Vitamin A from maternal to fetal units.

Activities of several enzymes in porcine uterine flushings in-
creased during diestrus or other periods associated with chronically
elevated P, levels or presence of a conceptus. Notably, acid phos-
phatase activity, associated primarily with Utf, was elevated during
diestrus, early to midpregnancy and in P, -induced uterine fluid as
reviewed above. Interestingly, the pi of this porcine acid phosphatase

(Utf, 9.7) is identical to that described for P, -induced bovine uterine

4

fluid-associated acid phosphatase by Dixon and Gibbons (1979). Glucose

phosphate isomerase (GPI) allows for interconversion of glucose-6-PO,

4

and fructose-6-PO, . Over all days examined (6, 8, 10, 12, 14, 15, 16
and 18) , GPI activity in uterine flushings was higher in pregnant than

-62-

nonpregnant gilts (Zavy et al., 1982a). Both total and specific activi-
ties of GPI in uterine flushings were greatest during early diestrus
(days 6 and 8) and proestrus (day 18) in nonpregnant gilts, and between
days 12 and 18 in pregnant gilts (Zavy et al., 1982a). Periods of
elevated GPI activity in pregnant gilts were temporally associated with
similar periods of elevated uterine E content of either conceptus or
maternal origin (Zavy et al., 1982a). Administration of estradiol
valerate (EV; 5 mg/day) to cyclic gilts on days 11 through 15 stimulated
uterine luminal GPI activity on days 15, 17 and 19 (Zavy et al., 1982a).
Similar responses, noted in early pregnant or EV treated gilts, for
uterine luminal, Ca, total protein, PGF, PGE and serine-protease
inhibitor activities (Geisert et al. , 1982a, c; Fazleabas, 1982a, b)
provided strong support for the notion that conceptus produced estro-
gens stimulated an acute luminally directed surge of embryotrophe in
the porcine uterus (Geisert et al., 1982b).

Although their functional roles are unclear, Roberts et al. (1976b)
detected lysozyme, leucine aminopeptidase and cathepsin A, B , C, D and
E activities in uterine flushings from ovariectomized P, -treated gilts.
Activities of cathepsin B, D and E did not reach detectable levels
before day 60 of chronic daily P, treatment, suggesting a requirement
of prolonged P, support for induction of these enzymes (Roberts et al.,
1976b). Similarly, Linford and losson (1975) failed to detect any
increases in bovine endometrial acid cathepsin activity prior to days
50 to 70 of gestation.

In addition to those proteins and enzymes described above, Basha
et al. (1980a, b) identified four low M acidic and two basic polypep-
tides in uterine flushings from pseudopregnant and unilaterally
pregnant gilts for which no definitive roles were suggested. Riboflavin

-63-

content was elevated in uterine flushings from gilts between days 6 and
8 of the estrous cycle and early pregnancy (Moffatt et al. , 1980).
Accumulation of riboflavin in the uteri of ovariectomized gilts was
stimulated by administration of P and E (Moffatt et al., 1980).

In cyclic mares, total uterine luminal protein content increased
after day 4 to maximal levels between days 12 and 16 and declined to
minimal levels by day 20 (Zavy et al., 1978a, 1979a, 1982b). In con-
trast, uterine protein content in pregnant mares was depressed between
days 8 to 14, but increased thereafter to at least day 20 (Zavy et al. ,
1982b). Among the nonserum proteins identified in equine uterine
flushings Zavy et al. (1979a) observed a lavender-colored component of
uterine flushings from a pseudopregnant mare. This basic protein was
shown to possess acid phosphatase activity and cross-react with antiserum
directed against porcine Utf (Zavy et al. , 1979a). This protein was
detected immunochemically in uterine flushings from day 16 cyclic mares
(Zavy et al. , 1979a). Additionally, 2D-PAGE of basic polypeptides in
uterine flushings from diestrus cyclic (days 12 to 16), early pregnant
and P -treated ovariectomized mares revealed a component with virtually
identical electrophoretic character to porcine Utf (Zavy et al., 1982b).
Similarity in placental type between horses and pigs (diffuse epithelio-
chorial; Stevens, 1975) suggested that a Utf-like molecule might also
be important in delivery of iron to the equine fetus (Zavy et al. ,
1979a; 1982b). However, this has not been demonstrated to date.
In addition to the Utf-like molecule, several other nonserum

polypeptides were identified in equine uterine flushings by 2D-PAGE.

_3
At least eight low M , acidic polypeptides (M x 10 )/pI range -

<21/5. 9-7.0) were identified in uterine flushings from cyclic mares

-64-

(Zavy et al., 1982b). These polypeptides appeared with greatest
intensity on day 12, but persisted through day 18 postestrus. The same
array of polypeptides was maintained beyond day 20 in uterine flushings
from pregnant mares and was detectable in flushings from day 45 as
well as in uterine fluids from pseudopregnant mares (Zavy et al. ,
1982b) . Examination of the more basic array of polypeptides revealed
six (19-24/7.0-7.5) which appeared in uterine flushings from cyclic
mares between days 12 and 16, but persisted with pregnancy, pseudo-
pregnancy or P, therapy (Zavy et al., 1982b). It was suggested that
some of these polypeptides might be the same as those identified on
more acidic 2D-PAGE gels (Zavy et al., 1982b). Another basic poly-
peptide (17/8.0), designated U , was detected in uterine flushings from
cyclic mares between days 3 and 14 and was maintained until day 20 in
pregnant mares (Zavy et al. , 1982b). However, this protein was not

induced by either P, or E„ and was not found in uterine flushings from

4 2

a day 45 pseudopregnant mare (Zavy et al., 1982b).

In addition to the P, -induced polypeptides identified in equine
uterine flushings, an E- induced component was also identified. In
ovariectomized mares, treated with E -173 alone (10 mg/day for 2 days),
two major proteins were identified in uterine flushings (Zavy et al.,
1982b). One was serum albumin and the other, designated D , was a
nonserum polypeptide with a M (x 10 ) of 70 and pi of 5.3 (Zavy
et al., 1982b). This product was sometimes detected faintly in
uterine flushings from diestrus and early pregnant mares (Zavy et al.,
1982b) . Estrogen induced uterine proteins have not been described in
pigs, cattle or sheep. Identification of this polypeptide (D ) , as
well as the other nonserum P -induced polypeptides, in uterine

-65-

flushings from nonpregnant, ovariectomized, hormone treated mares,
indicated maternal origin for these products. To date, however, data
relative to de novo synthesis of equine uterine proteins are un-
available.

Total acid phosphatase activity was higher in uterine flushings
from early pregnant (days 14, 16, 18 and 20) than cyclic pony mares
(Zavy et al., 1979b). Results were generally consistent with those
described above for bovine, ovine and porcine uterine phosphatase
activities. Uterine liminal leucine aminopeptidase (LAP) activity,
shown to be under P, control in pigs (Roberts et al. , 1976b), was
highest during diestrus in cyclic mares (Zavy et al., 1978a). Patterns
of GPA activity in uterine flushings were similar between cyclic and
pregnant gilts and mares (Zavy et al., 1982b). Both total and specific
GPI activities were elevated during late diestrus and persisted with
pregnancy (Zavy et al. , 1982b).

In this section proteins and enzymes characteristic of the uterine
environment of domestic farm animals were reviewed. Emphasis was
placed on those macromolecules shown to be distinct from serum proteins
and considered, or known, to be products of uterine endometrial tis-
sues. It is important to remember, however, that serum or serum-
identical proteins were consistently identified as the major protein
compounds in uterine fluids from all domestic species. Such serum
proteins should not be ignored. Transudation of serum proteins into
the uterine lumen occurs irrespective of molecular size (Beier, 1980).
For example, disc gel electrophoresis revealed prealbumins, albumins,
postalbumins, pretransferrins, transferrins and a-globulins in uterine
flushings from cyclic and early pregnant cattle (Roberts and Parker,

-66-

1974a). Additionally, there is a general qualitative disparity between
those serum- identical proteins identified in uterine flushings and in
serum (Beier, 1980; Roberts and Parker, 1976; Roberts et al., 1976a;
Basha et al. , 1979; Zavy et al., 1979a, b, 1982a). Such observations
suggest selective uptake of serum proteins into the uterine lumen. As
is the case for most uterine-specific proteins and enzymes described
above, functional roles of serum proteins in the uterine lumen of
domestic species are not known. However, it is undoubtedly the entire
array, rather than any single macromolecular component, that is essen-
tial to establish a complete embryotrophic uterine environment.

Prostaglandins. Like other components of the uterine environment,
prostaglandins (PG) appear in the uterine lumen and tissues in a manner
related to endocrine and pregnancy status. Interest in PGF„ as the
presumptive endogenous uterine luteolysin led to intense investigation
of PG in uterine flushings and tissues in cyclic and early pregnant
domestic animals. Content of PGF„ in uterine flushings from cyclic
cattle (Bartol et al. , 1981a; Lamonthe et al. , 1977), sheep (Inskeep
et al., 1980), pigs (Zavy et al., 1980) and mares (Zavy et al. , 1978b)
was elevated during mid- to late diestrus and associated with onset
of luteolysis as described above. Prostaglandin E was also detected
in uterine flushings from diestrus cyclic cattle (days 16 and 19; Lewis
et al., 1982), as well as sheep (Ellinwood et al. , 1979b) and pigs
(Geisert et al., 1982b). In sheep uterine flushing PGE^ content did
not vary between days 13, 15 and 17 (Ellinwood et al., 1979b). In
contrast, PGE content of porcine uterine flushings increased between
days 11.5 and 14 (Geisert et al., 1982b). Paradoxically, as compared
to cyclic controls, PGF„ content was higher in uterine flushings from

-67-

early pregnant cattle (Bartol et al., 1981a), sheep (Ellinwood et al.,
1979b; Inskeep et al., 1980) and pigs (Zavy et al, , 1980). The same
relationship was noted for PGE^ (Lewis et al. , 1982; Ellinwood et al. ,
1979b; Geisert et al., 1982b). Pattern of change in uterine luminal
PG content in pregnant animals generally paralelled that of cyclic
animals with major quantitative increases detected during the period
associated with blastocyst elongation and apposition to the endo-
metrium.

A major source of uterine luminal PG and the primary source in

cyclic animals is the endometrium. In vitro production of PG by

endometrial tissue was demonstrated for pregnant cattle (Lewis et al. ,

1982), and cyclic and pregnant sheep (Ellinwood et al., 1979; Findlay

et al., 1981; Inskeep et al., 1980), pigs (Guthrie et al. , 1978;

Guthrie and Revroad, 1981; Patck and Watson, 1976; Watson and Patek,

1979) and mares (Vernon et al. , 1981). Though production data are

unavailable for cyclic cattle, studies indicated that, in general,

endometrial content and Jji vitro production of PGF , increased from

mid- to late diestrus (Shemesh and Hansel, 1975a, b; Inskeep et al.,

1980; Patek and Watson, 1976; Vernon et al. , 1981). The condition of

pregnancy does not appear to suppress potential for endometrial PG

sjmthesis. Lewis et al. (1982) showed that endometrial slices from

day 16 and 19 pregnant cattle produced radiolabelled PGF„ , PGE„, PGFM,

2a 2

and at least four other unidentified metabolites, when incubated with

3
H -arachidonic acid. Quantities of PG produced were unaffected by

day postmating or location of uterine horn relative to CL-bearing ovary

(Lewis et al., 1982). Neither concentration nor content of PGF„ or

2a

PGFM in endometrial tissue differed between cyclic and pregnant cattle

-68-

on day 17 postestrus; although tissues from cyclic cattle synthesized
greater quantities of PG (J. Curl and W.W. Thatcher, personal communi-
cation) .

Overall, data from sheep suggested that endometrial tissue PG
content and production capacity was similar for pregnant and nonpreg-
nant ewes and might even be enhanced by the condition of pregnancy and
presence of a conceptus (Findley, 1981; Findley et al. , 1981; Hyland
et al., 1982; Inskeep et al. , 1980). Endometrial tissue concentration
and content, and in vitro production of PGF was higher in pregnant
than nonpregnant ewes on day 15 postestrus (Ellinwood et al. , 1979b;
Findlay et al. , 1981), Although Lewis et al. (1978) found no difference
in endometrial content of PGE between pregnant and nonpregnant ewes on
days 15 and 16, Ellinwood et al. (1979b) detected higher levels in
pregnant than nonpregnant ewes on days 15 and 17 postestrus (Ellinwood
et al., 1979b). Data indicated that endometrial and uterine luminal
content of both PGF„ and PGE were consistently higher in pregnant
than nonpregnant ewes on days 13, 15 and 17 postestrus, with an increase
in the PGE :PGF„ ratio seen in pregnant animals (Ellinwood et al.,
1979b; Findlay, 1981). The observation of Henderson et al. (1977)
that, in ewes, PGE was antagonistic to the luteolytic effect of PGF„
in vivo, led several investigators to propose that PGE might be the
antiluteolytic signal in pregnant sheep (Ellinwood et al. , 1979b;
Huie et al. , 1981; Reynolds et al. , 1981). In this regard, both PGI
(prostacyclin; Milvae and Hansel, 1980) and PGE (Kimball and Lauder-
dale, 1975) stimulated plasma P, when administered to cattle in vivo.
However, the possibility that PGE might be involved in bovine luteo-
stasis has not been examined carefully.

'""'^^T'

-69-

The role(s) of uterine PG, particularly PGF , in control of
luteal lifespan in domestic species, made discovery of elevated levels
of these substances in uteri of early pregnant animals especially un-
tenable. Requirement of luteostasis for maintenance of pregnancy led,
necessarily, to intense investigation of phenomena which might help to
explain how pregnancy is "recognized" in the face of this apparent
threat to luteal function (see Bazer et al, , 1981c). However, if
the goal of the reproductive process is production of viable offspring
and not luteolysis, then enhanced uterine PG responses characteristic
of early pregnancy must be consistent with this goal. Though this may
seem a true paradox, it is interesting to note that in cattle, sheep
and pigs, marked pregnancy-related increases in uterine PG responses
occur at a time when removal of the conceptus(es) will result in pro-
longation of the estrous cycle (Bartol et al., 1981; Northey and French,
1980; Rowson and Moor, 1967; Inskeep et al., 1980; Dhindsa and Dziuk,
1968; Zavy et al. , 1980; Bazer et al. , 1982). Thus, at a time when
uterine PG dynamics are most dramatically affected by pregnancy the
embryotrophic path has already been taken.

The period associated with increased uterine luminal content of
both PGF and PGE^ in early pregnant cattle (days 16-19; Bartol et al.,
1981a; Lewis et al., 1982) was also associated with persistency in
number and amount of uterine luminal proteins and conceptus related
stimulation of endometrial enzymes (Roberts and Parker, 1974a, b, 1976;
Leiser and Wille, 1975; Linford and losson, 1975; Bartol et al., 1981a).
Bartol et al. (1981a) reported positive correlations between uterine
lumenal content of PGF and total protein in both cyclic (r=0.37,
P < .02; days 4, 8, 12, 14, 16 and 19) and pregnant (r=0.42, P < .05;

'' .'■V>-'"'!Pt'^

-70-

days 8, 12, 14, 16 and 19) cattle. Findlay et al. (1981) presented
evidence for a discrete effect of the day 15 ovine conceptus on car-
uncular and, to a lesser extent, intercaruncular tissue. As assessed
±n vitro, rate of protein synthesis was higher in endometrial tissue
from pregnant than cyclic ewes on day 15 (Findlay et al. , 1981).
Increased uterine flushing content of both PGF and PGE in pregnant
as compared to nonpregnant ewes between days 13 and 17 was associated
with increased protein content and complexity (Ellinwood et al. , 1979b;
Roberts et al., 1976a). Porcine conceptus-induced increases in uterine
luminal content of PGF and PGE were associated with persistent in-
creases in amount and complexity of uterine flushing proteins (Geisert
et al. , 1982b). Similar responses were elicited in cyclic gilts by
administration of EV (5 mg on day 11 or 11-13; Geisert et al. , 1982b).
In both cases (conceptus-induced and EV-induced) PGE response preceded
PGF^^ response and was accompanied by a rapid transient increase in
uterine flushing Ca content (Geisert et al. , 1982b, c). These events
were suggested to be important for S3mchronized release of uterine
histotrophe (Geisert et al. , 1982b). Collectively, these data suggest
that pregnancy-associated alterations in uterine PG dynamics reflect
endometrial response to secretagogues, likely of conceptus origin,
necessary for maintenance of an embryotrophic environment.

It was observed that, in secretory cells (cells of the adrenal
cortex or exocrine pancreas) , Ca-dependent turnover (metabolism) of
arachidonic acid within the endogenous phosphatidylinositol (PI) pool
of cell membranes was an obligatory response to secretagogic stimula-
tion (Rasmussen, 1981, Rubin, 1982). Rubin (1982) observed that
turnover of membrane PI was a necessary event in the secretory process

-71-

and that one of the products of this reaction including arachidonic
acid (AAc) , PG or lysophospholipids, might participate in cellular

processes accompanying discharge of secretory product. When pancreatic

14
acinar cell membranes were labelled with C -AAc and exposed to a

secretagogue., amylase secretion increased and PI breakdown was seen
(Marshall et al. , 1980). Labelled AAc, incorporated into membrane PI,
disappeared rapidly. Approximately half of the C -AAc appeared as
phosphatidic acid and the other half as PGE (Marshall et al. , 1980).
Furthermore, PGE stimulated amylase secretion which was blocked by
inhibition of PG synthesis (Marshall et al., 1980), Explanations of
these data included possibilities that: (1) PGE might serve as a
coordinate messenger with Ca, or (2) secretagogue- induced PI turnover
leads to release of AAc and increased synthesis of PGE which acts
to open Ca channels and may possibly increase Ca conductance of the
plasma membrane (Rasmussen, .1981), Thus, response of certain secre-
tory cells to secretogogue stimuli results in production of agents
and/or conditions which may potentiate continued secretory response.

In light of these data, continued production and elevated uterine
content of PG during early pregnancy is far from untenable with
respect to establishment of an embryotrophic environment and mainten-
ance of pregnancy. Indeed, elevated levels of PG in uterine tissues
and flushings from pregnant as compared to cyclic animals (after time
associated with onset of maternal "recognition") may not only reflect
endometrial secretory activity, but be necessary for its maintenance.
In this respect, it is interesting that in both sheep and pigs, PGE :
PGF^^ ratios were higher during early pregnancy (Ellinwood et al, ,
1979b; Geisert et al, , 1982b). Alterations in amount and ratio of PG

-72-

in uterine flushings and tissues undoubtedly occur as a consequence of
altered endometrial membrane dynamics associated with maintenance of
an actively secretor epithelium and conceptus secretion of these
products.

Data from laboratory species indicated that, as components of an
embryotrophic uterine environment, PG may affect several other pro-
cesses important for establishment and maintenance of pregnancy
including (I) local uterine blood flow (Janson et al. , 1975); (2)
endometrial vascular permeability (Kennedy, 1980); (3) angiogenesis
(Ezra, 1979); (4) fluid and electrolyte transport across epithelia
(Biggers et al. , 1978); (5) cellular proliferation (MacManus and
Whitfield, 1974) and (6) steroid biosynthesis (Batta, 1975) (see
Bazer et al., 1982). Data are lacking relative to such PG-mediated
processes associated with reproduction in large domestic species.

Steroid metabolism. Progesterone and estrogens, in addition to
integrating uterine metabolic activities including protein and PG
synthesis, may be metabolized by endometrial tissue. Eley et al. (1983)

incubated endometrial slices from pregnant (days 16, 19, 23 and 27) and

3 3
nonpregnant (days 16 and 19) cattle with either H-P^ or H-androstene-

3
dione ( H-A^) in vitro. Both labelled substrates were extensively

metabolized during the Ih to 3h incubation. When data were adjusted

3
for tissue wet weight, amount of H-P, metabolized was similar in

explants from days 16 and 19 irrespective of reproductive status (preg-
nant vs nonpregnant) or location of uterine horn. Endometrium from day

3
27 of pregnancy metabolized more H-P than that from days 16 or 19

(59.1 ± 2.6% vs 44.0 ± 2.3%; P < .01), but a uterine horn effect was

still undetected (Eley et al. , 1983). Collectively, data indicated

-73-

that bovine endometrial metabolism of P and A, was affected by stage

of gestation but was independent of local conceptus effects, Chroma-

tographic analyses of radiolabelled metabolites indicated that H-P

4
3
was metabolized more extensively than H-A, , but that the type of

• metabolism was similar. Major metabolites were 5a-reduced base steroids

3
including, primarily, 3,20 dihydroxy-5a-pregnane from H-P and

3
3,17 dihydroxy-5a-androstane from H-A, (Eley et al. , 1983), No C19

3 3

metabolites were generated from H-P,, nor C21 from H-A,. Addition-

H 4

ally, neither testosterone (T) , E nor E were found (Eley et al. ,
1983). Similar results were reported for bovine endometrium by Knicker-
bocker et al. (1980) .

Estrogen sulfoconjugates, particularly E SO , are primary estrogens
in maternal peripheral plasma during later stages of gestation in
cattle (after day 70; Robertson and King, 1979; Hoffman et al. , 1976;
Thatcher et al. , 1982; Eley et al. , 1979a). However, detection of E SO
in both maternal plasma and fetal allantoic fluid as early as day 27
and 33 respectively suggested a requirement for sulfotransf erase
enzyme activity in either or both maternal and fetal tissues very
early in bovine gestation. Data from late bovine gestation (days 247
and 273) suggested that E conjugation was compartmentalized, with
maternal components of the placentome (caruncular tissue) responsible
for production of E,SO and E SO, detected in peripheral plasma (Hoff-
man et al., 197 6). To date, however, data relative to presence of
sulfotransferase activity in uterine tissues from either cyclic or
pregnant cattle are unavailable. Similarly, data are unavailable rela-
tive to bovine uterine sulfatase activity. However, Eley et al. (1979b)
demonstrated that E SO, was luteolytic when administered chronically

-74-

(28 or 56 mg E^ SO, /day S.C. from day 10 until onset of estrus) to
cyclic cattle. Thus the possibility of sulfatase activity in diestrus
bovine reproductive tissues exists.

Unlike bovine tissues, endometrium from pregnant sheep (days 14,

3 3
16, 18, 20, 60 and 140) converted H-P to H-A, , and both of these

substrates to T, E and E in vitro (Willis et al. , 1979a, b, 1981).
Conjugated estrogens (primarily E SO.) were also detected (Willis
et al., 1981). Findlay et al. (1981) detected trace amounts of radio-
labelled phenolic steroids in medium following incubation of endometrium

3
from pregnant and nonpregnant ewes (day 15 postestrus) with H-A,.

3 3
Additionally H-E and H-E SO, were metabolized to aqueous and ether

soluble forms respectively, indicating both sulfotransferase and

sulfatase activities (Findlay et al. , 1981). Data relative to the

possibility of a local conceptus effect on ovine endometrial steroid

metabolism are equivocal. Willis et al. (1981) reported that endome-

3 3
trial metabolism of both H-P, and H-A, was reduced in tissue from day

4 4

14 cyclic ewes as compared to that from pregnant ewes. However,

comparison of tissues from pregnant and nonpregnant ewes on day 15

3
revealed no pregnancy related effect on ±n vitro metabolism of H-A^,

3 3
H-E or H-E, SO,, although effects were detected on tissue PGF content
1 14

and rate of in vitro protein synthesis (Findlay et al. , 1981). Addi-
tionally, metabolism of H-P, by ovine endometrium from days 20, 60
and 140 of gestation was not affected by stage of gestation or presence
of conceptus membranes in culture (Willis et al., 197 9b). Though far
from definitive, data suggest that, like the cow, ovine endometrial
steroid metabolism is a maternally regulated process that may be
affected by duration of pregnancy.

-75-

3
Both porcine and equine endometrial tissues converted H-P, to E

In vitro (Dueben et al. , 1977, 1979; Fischer et al., 1981; Seamans

3 3
et al., 1979). Porcine endometrium converted both H-P, and H-A, to

T, E E and conjugated E and E (SO.) in vitro (Bazer et al. , 1982).

Steroid sulfates and glucuronides were shown to be major porcine

endometrial metabolites of P. (Bazer et al., 1982; Tindall and Henricks,

4

1971). Porcine endometrium from days 16 and 25 of pregnancy produced

3 3

H-E 173 (not 17a) from H-P, and metabolism increased with stage of

gestation (Fischer et al. , 1981). Endometrium from pseudopregnant

3
gilts (day 25) also produced H-estrogens, but products did not co-

chromatograph with E standards suggesting a modulating role for

conceptus tissues or products (Fischer et al. , 1981). Like the cow,

porcine endometrium was shown to have 5a-reductase activity (Henricks

and Tindall, 1971). Histochemical evidence indicated that equine

endometrium directly apposed to the conceptus (as well as the conceptus

itself) was a potential source of E (Flood and Marrable, 1975; Flood

3 3
et al., 1979). In vitro conversion of H-P, to H-E by endometrium

from pregnant (day 18) and cyclic (days 12 and 18) pony mares was

enhanced by co-incubation with conceptus tissues (Seamans et al. , 1979).

Present data indicate that, in contrast to porcine, equine and,

debatably (see Findlay, 1981) ovine endometrial tissues, bovine

3 3

endometrium produced neither E (E or E ) nor T from H-P^ or H-A^

in vitro (Eley et al., 1983; Knickerbocker, 1980). Therefore, bovine
endometrium is possibly lacking aromatase, as well as 17B-dehydrogenase

(173-HSD) and 173-oxidoreductase activities (Heap et al. , 1979). Fur-

3 3

thermore, primary products of bovine endometrial H-P, and H-A^

metabolism were 5a-reduced base steroids (Eley et al., 1983; Knicker-
bocker et al., 1980) which cannot be aromatized to estrogens (Wilson,

-76-

1972). Data are consistent with the fact that estrogens (E -17B and

E,SO,) are luteolytic when administered to cattle during diestrus
14

(Brunner et al. , 1969; Wiltbank et al., 1961; Eley et al., 1979a),
presumably via stimulation of endometrial PG synthesis (see above and:
Bartol et al., 1981a, b). Thus, direct conversion of circulating P,
to E, or aromatizable steroids, would be counter to the goal of
pregnancy maintenance. In sheep, in which E -17B is also luteolytic
(Stormshak et al. , 1969; Coding, 1974), evidence for aromatase and
173-HSD activity in endometrial tissue from pregnant ewes (Willis
et al. , 1979a, b, 1981) is contrary to this notion. However, Findlay

(1981) and Findlay et al, (1981) suggested that ovine endometrial

3
conversion of H-A, to either E„ or E was neglibible.
4 2 1

Since 5a-reduced steroids are, essentially, biologically inactive
(Holzbauer, 1976; Kubli-Garf iar et al., 1979, 1980), bovine endometrial
5a-reduction of P, may potentiate effectiveness of conceptus products
(Eley et al. , 1983). Additionally, 5a-reduction of P, may enhance
endometrial responsiveness to E by reducing P, suppression of E receptor

synthesis and E-R retention time (Clark et al., 1977; Katzenellenbogen

3
et al., 1980). In vitro metabolism of H-A, by endometrium from dies-
trus cyclic (days 16 and 19) and early pregnant (days 16, 19, 23 and
27) cattle was unaffected by presence of a conceptus (days 16 and 19),
but increased with stage of gestation (Eley et al- , 1983) . Considering
the importance of bovine endometrial responsiveness to the integrative
action of steroid hormones in establishment and maintenance of an
embryotrophic uterine environment, data suggest that at least partial
endometrial autonomy with respect to steroid metabolic activity may
represent an important uterine autoregulatory mechanism.

-77-

Endometrial sulphotransferase and sulphatase activities during
early pregnancy appear to be related to steroidogenic activity of the
conceptus. Conceptus produced E is considered essential for recog-
nition of pregnancy in pigs (Bazer et al., 1982). Both free and
conjugated E (E E , E E,SO,, E SO, and E SO^) content was increased
in uterine flushings from pregnant as compared to nonpregnant gilts
between days 10.5 to 14 (Geiserr et al. , 1982b), Uterine flushing E
content in pregnant gilts, particularly E E SO^ and E^SO^ increased
in a manner related to stage of conceptus development, and were markedly
elevated between days 11 and 14 (Geisert et al., 1982b). High levels
of endometrial sulphotransferase relative to sulphatase activity in
pregnant gilts prior to day 30 (Dwyer and Robertson, 1979) were sugges-
ted to provide readil> metabolizable substrate to the conceptus for
production of E, while preventing large amounts of unconjugated E from
entering maternal circulation (Bazer et al. , 1982; Heap and Perry,
1974). In sheep, conceptus production of E is negligible (Gadsby et al.,
1980; Willis et al., 1981). However, in contrast to pigs, ovine
endometrial sulphotransferase activity was suppressed and sulphatase
activity enhanced prior to day 20 of gestation (Dwyer and Robertson,
1979). Assuming a requirement for E at the conceptus-maternal inter-
face for initiation of pregnancy, Dickman and coworkers (see Dickman
et al., 1976; Dickman, 1979) suggested that species differences in
endometrial steroid metabolism, such as those described above, were
necessary to insure optimal levels of biologically active steroid
locally, at the level of the endometrium.

Like sheep, E production by peri-attachment bovine conceptuses
is minimal (Eley et al., 1983; Gadsby et al., 1980; Shemesh et al. ,

-78-

1979). Estrone and E,SO, concentrations in maternal peripheral plasma,

14

reflective of conceptus E production (Thatcher et al., 1982; Eley

et al., 1979a), were not elevated prior to day 60 of gestation (Eley

et al., 1979b; Robertson and King, 1979). Since neither E nor

aromatizable steroids are produced by diestrus bovine endometrial

metabolism of P, or A, (Eley et al., 1983), significant amounts of
4 4

biologically active E, if necessary for establishment of pregnancy,
must be provided by a maternal (ovarian) source (Hansel et al., 1973).
Absence of significant sulphotransf erase activity and/or enhanced
sulphatase activity in bovine endometrium during diestrus would facili-
tate action of unconjugated E at the maternal-conceptus interface. In
other species, notably the pig, biologically active E, in concert with
other hormones present in utero during the peri-attachment period,
was suggested to exert local effects on (1) uterine blood flow;

(2) water, electrolyte, carbohydrate and amino acid transport; and

(3) induction of histotrophe (Bazer et al. , 1979b; Findlay, 1981; Cook
and Hunter, 1978). Details of bovine endometrial metabolism of E
remain to be elucidated.

The Conceptus

Development

If the uterus is a major integrator of stimuli directed toward
achievement of reproductive success, the conceptus and its products
clearly represent primary effectors of uterine-mediated, embryotrophic
events. In cattle, as reviewed above, an embryotrophic uterine environ-
ment is established independently of presence of a conceptus prior to
day 16 postestrus. Once "the stage is set", however, events pursuant
to the embryotrophic path require uterine integration of both maternal

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and conceptus signals. Though data are unclear as to the nature of
such signals, their genesis appears to be related to developmental
stage of the conceptus. For this reason, a brief description of bovine
conceptus development is presented below. Emphasis is placed on
evolution of extraembryonic membranes during the peri-attachment -period
as it is these tissues that relate the conceptus to its uterine en-
vironment both physically and biochemically. Information for the
following section was obtained primarily from the works of Brackett
et al. (1980), Chang (1952), Flechon et al. (1978), Foley and Reece
(1953), Greenstein and Foley (1955, 1958a, b), Greenstein et al. (1958),
Hamilton and Laing (1946), Marion and Gier (1958), Melton et al.
(1951), Wathes and Wooding (1980), Winters et al. (1942), and Wooding
and Wathes (1980). Additionally, information was obtained from
reviews of Eckstein and Kelly (1977), Perry (1981), and Steven and
Morris (1975). For descriptions of bovine embryogenesis, as well as
growth and development of all components of the embryo and extra-
embryonic membranes the reader is referred to the works of Greenstein
and Foley (1958a, b), Eley et al. (1978), Hubbert et al. (1972), Swett
et al. (1948), Winters et al. (1942), and the classic treatise of
Hammond (1926). Developmental, morphological, histological and cyto-
logical descriptions of the bovine placentome were presented by
Bjorkman (1968a, b, 1973), King et al. (1980), Kingman (1948), and
Wooding and Wathes (1980).

In cattle, conception and initial cleavage stages occur in the
oviduct. Once formed, the zygote, enveloped in its mucopolysaccharide
coat, the zona pelucida (ZP) , requires approximately 96h to traverse
the oviduct. Consequently, on day 3-4 the 16 cell, ZP-encased morula

-80-

enters the uterine lumen. Cell divisions continue and, with rearrange-
ment of blastomeres, a unilaminar blastocyst and blastocoelic cavity
are formed by days 7-8. An area of one pole of the outermost layer
of the blastocyst (ectoderm) becomes thickened forming the inner cell
mass (ICM), or embryonic disc, which will give rise to the embryo
proper. The remainder of the ectoderm is the trophoblast (feeding or
nourishing layer; Perry, 1981), which will ultimately become the outer
layer (chorion) of the epitheliochorial placenta. Between days 9 and
10 a slit appears in the ZP and the blastocyst is extruded. This
process, referred to as hatching, signals the beginning of a more rapid
period of blastocyst development.

Following hatching (- days 10 to 12) blastocoelic ectoderm becomes
lined with a layer of endodermal cells derived by tangential division
of a few cells of the embryonic disc (Perry, 1981). Thus a rudimentary
yolk sac is formed and the trophoblast is bilaminar. Between days 12
and 15 a layer of mesoderm spreads outward from the ICM between the
trophoblastic ectoderm (trophectoderm) and endodermal layers forming
a trilaminar trophoblast. This somatic mesoderm splits, forming
avascular and vascular mesoderm. Avascular mesoderm fuses with troph-
ectoderm to yield trophoderm or somatopleure. Vascular mesoderm fuses
with the inner, endodermal layer forming the splanchnopleuric yolk
sac. Space between these two membranes is referred to as the exocoele
or exocoelom. The yolk sac and its contents serve as a primary source
of nutritional support to the developing embryo prior to establishment
of a functional chorioallantois. In addition to formation of a yolk
sac, establishment of the endoderm is prerequisite to elongation of the
trophoblast, which begins around days 13 to 15.

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The period of bovine conceptus development between day 16 and
attachment is characterized by rapid growth and development of both
embryo proper and extraembryonic membranes. Between days 16 and 17,
the trophoblast takes on a ribbon-like appearance (Chang, 1952).
Length of the trophoblastic vessicle is highly variable at this time,
with reports from 2 to over 500 mm (Hawk et al. , 1955; Betteridge
et al., 1980). Greenstein et al. (1958) observed that three cell types
were distinguishable in day 16 and 17 trophoblast membrane following
staining with Periodic-Ac id-Schiff (PAS; for glycoproteins), and Sudan
Black or Oil Red 0 (for lipids). Cell types included (1) polygonal or
cuboidal undifferentiated trophoblast or "stem" cells; (2) early stage
trophoblastic giant cells (tGC) , occasionally binucleated and PAS (+) ;
and (3) moderate columnar trophoblast cells with basally located lipid
vacuole inclusions (Greenstein et al. , 1958). Also at this stage, the
primitive streak is formed and embryonic organogenesis is underway.

By day 18 the bovine conceptus occupies nearly two-thirds of the
gravid uterine horn. Vitteline (yolk sac) circulation is well estab-
lished by this stage and thought to provide a major proportion of
embryonic nutrients. Histologically and cytologically the trophoblas-
tic surface at day 18 was shown to consist of cuboidal and columnar
cells interspersed between tGC, which accounted for approximately 6%
of the total cell population (Wathes and Wooding, 1980). Both cell
types contained large areas of lipid inclusions. However tGC were
often binucleated and always PAS (+) suggesting high glycoprotein
content (Greenstein et al. , 1958; Wathes and Wooding, 1980).

Between days 19 and 20, conceptus membranes extend throughout
the gravid and into the contralateral uterine horn. The amnion is

-82-

formed during this period by inversion of the trophoblast (chorion) on
the dorsal embryonic surface, and fusion of the mesodermal layers
(chorioamnion) . With establishment of embryonic heart primordia, the
yolk sac and choriovitelline circulation reach peak development and
begin to regress. The trophoblastic (chorionic) surface at days 19
to 20 consisted of both uninucleate, columnar/cuboidal cells and
binucleate tGC, which constituted as much as 20% of total cell popula-
tion (Greenstein et al. , 1958; Wathes and Wooding, 1980). By day 20
tGC were described to contain numerous membrane bound granules; large
active Golgi, confluent with developing granules; well developed RER
and numerous lipid inclusions (Wathes and Wooding, 1980).

Following establishment of the amnion and initiation of embryonic
heart-beat between days 20 to 22, the allantois emerges from embryonic
hindgut as a outgrowth of extraembryonic splanchnopleure. Growth of
the allantois proceeds rapidly. Its expansion into the exocoele on a
scaffolding of fluid and apposition with the trophoderm (chorion) is
coordinated with regression of the yolk sac and choriovitelline circu-
lation. Thus begins a coordinated transfer of function as the embryo
shifts its dependence from self contained nutrients, provided by the
choriovitelline circulation, to nutrients supplied via the chorioallan-
toic circulation of the true placenta. Vascularization of the chorion
occurs via vessels of the mesoderm of allantoic, splanchnopleure, which
are continuous with those of the embryo. Binucleate tGC remain promin-
ent components of the trophoblastic membrane at this stage and, as
reviewed above, interdigitation of apical microvilli occurs between
maternal and conceptus (chorionic) surface epithelia.

Rapid expansion of extraembryonic membranes continues such that,
by days 24 to 25, membranes extend to the tip of the nongravid uterine

-83-

born. Regression of yolk sac and choriovitelline circulation is
complete by days 23 to 24. Consequently, the burden of embryonic
support depends entirely upon establishment of a functional placenta.
Expansion of the allantois and its associated vascular bed is contin-
uous during this period. Fusion of allantoic splanchnopleure and
chorionic somatopleure occurs via villus projections on respective
mesodermal surfaces. Vascularization of the allantois is completed
between days 26 and 30. Thus, a functional chorioallantoic membrane
is completely established. Attachment of discrete areas of the
chorion and maternal caruncles, reported to occur as early as day 27
(King et al., 1980), initiates formation of the characteristic bovine
cotyledonary epitheliochorial placenta.

Con ceptus- induced events associated with maternal recognition of
pregnancy and reflected by dynamic alteration in the bovine uterus
and uterine environment, as described above, are temporally related to
several key events in conceptus development including (1) establish-
ment of extraembryonic endoderm, prerequisite to; (2) trophoblastic
elongation, and (3) differentiation of trophoblastic cell types (Green-
stein et al., 1958). Although endoderm is established and initial
elongation is underway by days 12 to 14 (Greenstein et al. , 1958;
Chang, 1952), uninucleate columnar and binucleate tGC were not de-
scribed prior to day 16 (Greenstein et al. , 1958). Bearing in mind
that embryo removal after this stage (day 16) results in prolonged
diestrus (Northey and French, 1980) , data suggest that this compliment
of cells and tissues is required to generate appropriate stimuli for
pregnancy maintenance. Relationship between these three developmental
events and genesis of biologically active conceptus products is unclear.

-84-

However, Boime et al. (1982) suggested that stage of trophoblast differ-
entiation may affect transcription and/or translation of message (mRNA)
necessary for synthesis of such products. Bovine trophoblast elonga-
tion, which is rapid after day 16, is essential (1) to insure
increased surface area for nutrient uptake during transition from yolk
sac to chorioallantoic nutrition; and (2) to increase uterine area
affected by conceptus products. Substances produced by the conceptus
in utero may act in a paracrine fashion to exert effects locally on
endometrial or other uterine tissues and/or in an endocrine fashion
where products enter the maternal bloodstream to affect distant target
tissues (Krieger, 1982). Differentiation of trophoblastic cell types
may facilitate such diverse modes of action. Wathes and Wooding (1980)
suggested that the normal function of bovine tGC at all stages of
gestation was to migrate into maternal uterine epithelium and release
their granular contents. A similar role was suggested for ovine tGC
(Steven et al. , 1978). Wooding and Wathes (1980) suggested that this
process might be important for transfer of large, nondiff usable mole-
cules across the conceptus-endometrial microvillar junction.

Conceptus Products

Evidence that components and/or products of peri-attachment
conceptuses are required to initiate luteostatic events in cattle,
sheep and pigs was reviewed previously. In addition, such substances
may be important for induction of histotrophe and establishment of
immunological privilege for the conceptus (Cook and Hunter, 1978; Beer
and Billingham, 1976, 1979; Beer and Sio, 1982). Surprisingly little
is known of the specific nature of products of peri-attachment-stage
conceptuses.

-85-

Protelns. With the exception of luteotrophic activity (suggested
to be proteinacious) reported for aqueous extracts of day 18 bovine
conceptuses (Beal et al. , 1981) , studies of protein components of bovine
extraembryonic membranes have been performed on tissues obtained after
definitive attachment. Avivi et al. (1982) isolated and characterized
a 94,000 dalton protein with thyrotropic (TSH) activity from bovine
placental tissues obtained from days 40 to 120 of gestation. Krieger
(1982) suggested that placental production of such pituitary-like
polypeptides might be necessary to compensate for hormonal effects
of pregnancy on maternal physiology. This TSH-like molecule has not
been identified in conceptus tissues from earlier periods in gestation.
Butler et al. (1982) described two very acidic proteins in extracts
of bovine placental membranes from days 28 through 270 of gestation.

One of these proteins (M x 10 - 47 to 53; pi 4.0 to 4.4) was uniden-

_3

tified. The second (M x 10 = 65 to 70; pi 4.6 to 4.8) was suggested

to be alpha^ -fetoprotein (a^-FP).

Alpha -fetoprotein is a fetus (conceptus) -specif ic serum protein
found in virtually all mammalian embryos (Gitlin and Boseman, 1967;
Lai et al. , 1978a; Tomasi, 1978), and produced primarily by yolk sac
and fetal liver. Lai et al. (1978b) showed a.-FP to be the most pre-
dominant of three bovine fetus-specific serum proteins including '^2~^^
and B-FP. None of these proteins were detected in maternal serum at
11 weeks of gestation (Lai et al. , 1978b). Smith et al. (1979) reported
maximal a.-FP concentrations in bovine fetal fluids and tissues during
the third to fourth month of gestation. Allantoic fluid levels of
a-FP exceeded those in amniotic fluid but remained very low in maternal
serum (Smith et al. , 1979) . During peak periods of elevated a-FP in

-86-

allantoic fluid levels in maternal serum were not different from
nonpregnant cattle (Smith et al. , 1979). Such results suggest an
intrauterine site of action for this protein. Recently, Janzen et al.
(1982) detected a^-FP in tissues of day 14 bovine trophoblasts (n=2)
and in allantoic fluid of one day 16 conceptus. Incubation of bovine
conceptus tissues (day 17, n=2; day 27, n=l) with "^^S-methionine
indicated primary sites of synthesis to be yolk sac and fetal liver
(Janzen et al. , 1982). Analysis of tissues and fluids from day 14 to
46 bovine conceptuses indicated increasing concentrations of a -FP
with stage of gestation. Concentrations of a -FP in maternal serum
were consistently and markedly lower (550 to 1.5 x 10^ times) than
those in conceptus tissues and fluids (Janzen et al. , 1982). Addition-
ally Uj^-FP concentrations in uterine fluid consistently exceeded those
of maternal serxim by a factor of at least 1 to 2 x 10 between days 14
and 46 (Janzen et al., 1982). Collectively, data indicated that syn-
thesis of bovine a^-FP begins well in advance of attachment (day 14) ,
and the protein appears to be sequestered in significant amounts within

the uterine lumen. ■ Failure of isolated day 27 allantois to synthesize

35 35

S-a^-FP from S-methionine suggested that appearance of a -FP

in utero occurred via transudation across tropho blast membranes early
in gestation (Janzen et al. , 1982).

The role of a^^-FP remains unclear. Presently, controversy exists
as to whether a -FP is an immunosuppressive or immuno stimulating pro-
tein (Tomasi, 1978; Suzuki and Tomasi, 1980). Clearly, the former role
(immunosuppressant) would be consistent with necessity for establish-
ment of immunological privelege for the conceptus (Beer and Sio, 1982).
Additionally, a^^-FP may regulate availability of uterin luminal

-87-

steroids. Rat a-FP was reported to bind both T and E with equal
affinity (Kd == 1 x 10"^°M; Clark et al., 1977).

Placental lactogens were identified and/or isolated from placentae
of cows (bPL; Buttle and Forsyth, 1976; Bolander and Fellows, 1976;
Murthey et al. , 1982) and ewes (oPL; Chan et al. , 1976; Fellows et al.,

1976), but not pigs (Forsyth, 1974). Recent reports suggest bPL to

_3
have a M^ (x 10 ) of 32-35 and pi in the range of 5.5-5.7 (Murthey

et al., 1982; Kensinger et al. , 1982). Kensinger et al. (1982)
demonstrated synthesis of bPL by near term fetal cotyledonary tissue
in vitro. However, the principle site of action of this polypeptide
in pregnant cattle is unclear. Bolander et al. (1976) reported peri-
pheral plasma bPL concentrations in excess of 1100 ng/ml during late
gestation in dairy cattle. In contrast, both Buttle and Forsyth (1976)
and Schellenberg and Friesen (1982) reported low to undetectable levels
of plasma bPL in late pregnant cattle. Concentrations of bPL were
highest in fetal serum (Schellenberg and Friesen, 1982) suggesting a
local (intrauterine or fetal) site of action for this polypeptide.
Such effects might be particularly important during early pregnancy.

Using a radioreceptor assay, Flint et al. (1979) detected bovine
placental lactogen (bPL) in homogenates of bovine conceptuses from
days 17, 24 and 25. It was suggested that presence of bPL in day 17
bovine conceptus homogenates was related to appearance of binucleate
tGC, shown to differentiate in bovine trophectoderm by day 16 (Flint
et al., 1979; Greenstein et al. , 1958; Staples et al. , 1961). This
concept was based on immunocytochemical, immunof luorescent and radio-
receptor assay studies in sheep which indicated that oPL was localized
in and, therefore, produced by ovine trophectodermal binucleate cells

-88-

(Martal et al., 1977; Reddy and Watkins, 1978). Watkins and Reddy
(1980) proposed the existance of two types of ovine trophoblastic
binucleate cells: (1) those responsible for production of oPL; and
(2) those responsible for elaboration of other products. Recently,
however, oPL was detected by immunof luorescent techniques in areas
surrounding lipid droplets in day 14 uninucleate trophoblastic cells,
prior to differentiation of binucleate cells (Carnegie et al., 1982).
Ovine chorionic binucleate cells, present at later stages of conceptus
development (days 28 and 55) , failed to bind antisera to oPL. Fluor-
escence was confined to areas surrounding lipid droplets of specific
uninucleate cells (Carnegie et al. , 1982). Considering these con-
flicting data in sheep and absence of any similar data in cattle,
assumption of a tGC source for bPL is unwarrented at present.

Whatever the source, detection of PL in peri-attachment-stage
bovine and ovine conceptuses raises questions as to the functional role
of this polypeptide. Appearance of bPL in day 17 bovine conceptuses
suggested a role for this protein in pregnancy recognition (Flint
et al., 1979), although evidence to this effect remains circumstantial.
The an tiluteo lytic action of ovine conceptuses is exerted by day 12
(Moor and Rowson, 1966a, b,c), at least two days before oPL was detected
in trophoblastic binucleate cells (Carnegie et al. , 1982). Thus oPL
does not appear to be the primary ovine luteostatic agent. Radio-
immunoassay of oPL in endometrial tissues from pregnant ewes (days 16,
18, 20 and 22) revealed a striking increase in both caruncular and
intercaruncular tissues after day 16 (Carnegie et al., 1982). While
data may reflect migration of oPL-rich trophoblastic cells into maternal
tissues (Martal et al., 1977; Watkins and Reddy, 1980), potential for

■ ■-■■*•■ ■■■-'v'l:

-89-

a local (paracrine) effect exists. Porter (1980) reviewed data indi-
cating that human PL (hPL) has a glucose-sparing, free fatty acid
mobilizing effect on maternal tissues. This effect was suggested to
be important for enhancing fetal (conceptus) glucose availability
(Porter, 1980).

In addition to effects on the maternal system, PL may have impor-
tant functions within the conceptus itself. Carnegie et al. (1982)
observed that appearance of oPL only after day 12, at the time of
blastocyst transformation from spherical to filamentous form, might
be related to blastocyst elongation and water movement across extra-
embryonic membranes. Indeed, hPL elicited changes in both short-circuit
current and potential difference of porcine chorioallantoic membranes
in vitro, which were consistent with prevention of water movement from
fetal to maternal compartments (Bazer et al. , 1981a). Recent observa-
tions that oPL, but not growth hormone or prolactin, stimulated amino
acid transport in fetal rat diaphragm (Freemark and Handwerger, 1983)
and ornithine decarboxylase activity in fetal and neonatal rat hepato-
cytes (Hurley et al., 1980) suggests that these polypeptides might play
important roles as fetal (conceptus) "growth hormones" (Freemark and
Handwerger, 1983).

Incubation of conceptus tissues in vitro, in the presence of
radiolabelled amino acids, and analysis of labelled products in tissues
and medium post incubation has proven to be an excellent method for
assessing the variety of proteins and polypeptides synthesized by
peri-attachment and later stage conceptuses. Wathes (1980) examined
effects of steroids (E and P ) and endometrial co-culture on ability
of bovine conceptus tissues from days 24 to 44 to incorporate

-90-

radiolabelled ( H and C) leucine In vitro. Leucine was incorporated
by conceptus tissues from all stages examined. Isolated allantois
from days 25, 26 and 37, incorporated more leucine than chorion from
the same stages, and incorporation was maximized in combined cultures
(allantochorion) . It was suggested that enhanced uptake of labelled
leucine by allantoic tissues was related to the rapid expansion and
fusion occurring during this period (Wathes, 1980). Incorporation of
leucine by bovine conceptus tissues was not affected significantly by
co-culture with uterine tissues (Wathes, 1980). Addition of steroids
^^4' ^2 °^ ^4"'"^2^ tended to depress leucine incorporation, but no
consistent change in protein profiles was seen following electrophore-
sis. Electrophoresis of chorionic tissue proteins in presence of SDS
revealed a broad band of nondialyzable radioactivity in the 85,000
M^ range. Labelled proteins in tissue and medium were not thoroughly
characterized.

Lewis et al. (1979) demonstrated continuous increase in uptake of

3 ^

several radiolabelled amino acids including H-glucosamine ( H-gIc),

14 14 35 35

C-leucine ( C-leu) , and S-methionine ( S-met) ; by day 19 bovine

and day 16 and 19 porcine conceptuses over a 24h period in vitro.
Post incubation analyses of nondialyzable radiolabelled components of
medium by 2D-PAGE and fluorography indicated presence of labelled
proteins synthesized de novo during culture (Lewis et al., 1979).
Additionally, several products, found to be enriched in medium were
present in only trace amounts in tissues. Data suggested that major
peri-attachment stage conceptus secretory products might not be found
in significant amounts in tissue extracts.

Masters et al. (1983) described a high molecular weight (M x 10~^
> 660) glycoprotein as the major H-glc-labelled product of bovine

-91-

(day 19), ovine (days 14-16) and porcine (day 16) conceptuses following

24h incubation in vitro. In each specie the glycoprotein appeared as

3
an early eluting DEAE (+) peak of H-glc. Each high M product con-
sisted of at least 50% carbohydrate and did not possess biochemical
characteristics typical of mucins. Careful analysis of the ovine gly-
coprotein showed it to be composed of N- rather than 0-linked
oligosaccharide chains containing D-galactose, N-acetylglucosamine,
D-mannose and L-fucose. No sialic acid was detected (Masters et al.,
1983) . These high M conceptus products were shown to be produced
in vitro by ovine conceptuses from days 13 to 23 (Godkin et al. , 1982b)
and porcine conceptuses from days 13 to 30 (Godkin et al., 1982a).
Production of these high M products was not apparent until elongation
was underway (Godkin et al., 1982a, b). Guillomot et al. (1982)
demonstrated a glycoprotein coat on the outer surface of ovine endo-
metrial and trophoblastic cells between days 13 and 15. Ruthenium red
and cationized ferritin reaction sites and concanavalin-A receptors
became more homogeneously distributed over the trophoblastic surface
from day 13 to day 15 (Guillomot et al., 1982). Similar changes were
not observed on endometrial cell surfaces suggesting a biochemical
change in the composition or distribution of ovine trophoblastic cell
glycoprotein coat associated with elongation and attachment (Guillomot
et al., 1982). Alterations in ovine trophoblastic surface glycoprotein
coating observed by Guillomot et al. (1982) were consistent with ob-
servations of Godkin et al. (1982b) relative to onset of production of
high M ovine glycoprotein. Masters et al. (1983) suggested that such
products might normally be deposited between trophoblastic cells or on
the chorionic surface to provide a protease resistant or immunologically

-92-

tolerant coating for the conceptus. Alternatively, these high M^
products may be associated with control of cell movement and rearrange-
ment during elongation (Geisert et al. , 1982a). Additionally, surface
glycoproteins may be important for adhesion of conceptus and endo-
metrial surfaces (Guillomot et al., 1982; Oppenheimer, 1978).

Godkin et al. (1982b) incubated individual ovine conceptuses
from days 13 to 23 with H-leu in vitro. Analysis of nondialyzable,
radiolabelled products in medium postincubation by 2D-PAGE and
fluorography indicated that day 13 ovine conceptuses produced only one
major polypeptide de novo. This product, called Protein-X, consisted
of three similar isoelectric species (pi = 5.4-5.7) and had a M^
(x lO""^) of 17 to 21 as determined by SDS-PAGE and gel filtration
(Godkin et al. , 1982b). Pattern of proteins produced de novo increased
in complexity with developmental stage of conceptuses. As observed by
Lewis et al. (1979), labelled products enriched in medium were absent
or present in only trace amounts in conceptus tissues. Protein-X
remained the primary product of ovine conceptuses from days 13 through
21. However, this polypeptide was no longer produced in detectable
amounts by conceptus tissues from days 23 or chorion from day 30
(Godkin et al., 1982b). Continued production of other polypeptides
including the high M glycoprotein (Masters et al., 1983) suggested
that Protein-X was transiently produced by conceptus membranes during
early pregnancy (Godkin et al., 1982b). Pattern of production of Pro-
tein-X (days 13 to 21) was similar to that of Trophoblastin (Martal
et al., 1979) detected in day 14-16 but not day 21-23 ovine conceptuses.
Trophoblastin, suggested to be a protein, was antiluteolytic when
infused into uteri of cyclic ewes (Martal et al., 1979). Thus a

-93-

potential role for Protein-X was established, but not verified (Godkin
et al., 1982b). Godkin et al. (1981) presented immunocytochemical
evidence for uptake of Protein-X by endometrial tissues from intact,
pregnant sheep. Recent in vitro studies (J.D. Godkin, personal communi-
cation) suggest that this association may stimulate synthesis and/or
release of specific endometrial proteins. Findlay et al. (1981) showed
that rate of protein synthesis in both caruncular and intercaruncular
tissues was greater in pregnant than nonpregnant ewes on day 15 post-
estrus. Observations are consistent with ovine conceptus production of
a histotrophic molecule (Godkin et al. , 1982b; Findlay, 1981).

In addition to the high M glycoprotein (Masters et al. , 1983),
porcine conceptuses were shown to produce a variety of polypeptides
de novo from radiolabelled amino acid substrate. Rice et al. (1981)
identified four major bands of radioactivity in SDS-PAGE gels of protein

in medium following explant culture of day 16 porcine conceptuses with

3 -3

H-leu (M^ X 10 ^ 75, 40, 32 and 28). Godkin et al. (1982a) demon-
strated that, like ovine conceptuses, array of polypeptides produced
de novo by porcine conceptuses was related to developmental stage.
Spherical (4.9 mm), tubular (10-50 mm) and early filamentous porcine
blastocysts (> 100 mm), recovered between days 10.5 and 12 of pregnancy,
all released a group of low M , acidic polypeptides (M x 10 = 20-25;
pi - 5.6-6.2) into medium as their principle products (Godkin et al, ,
1982a). The array of polypeptides synthesized by day 13 and 16 porcine
conceptuses was more complex. Production of the low M acidic proteins

continued, but predominant polypeptides produced were larger and more

_3
basic (M^ x 10 ^ 35-50; pi > 7.5) (Godkin et al. , 1982a). In day

18 porcine conceptus cultures, proteins described for earlier stages

-94-

persisted; however, their synthesis appeared depressed. Additionally,
several higher M^ embryonic products were detected (Godkin et al.,
1982a). These polypeptides were shown to have identical electrophoretic
properties to six major porcine fetal serum proteins including trans-
ferrin, fibrinogen (g-chain), a-FP, a -antitrypsin, a -ant ichymo trypsin
and fetuin (Godkin et al., 1982a; Buhi et al., 1982). Porcine concep-
tuses from days 25 and 30 no longer produced detectable amounts of
either the low M^ acidic polypeptides or slightly higher M , basic
polypeptides identified at earlier stages (Godkin et al., 1982a).

Instead, two acidic polypeptides (M x 10 - 30) and a major band of

_3
material (M^ x 10 - 50-60; pi 6.0-7.3) were identified as major

products at this stage (Godkin et al. , 1982a).

Definitive roles for ovine and porcine conceptus proteins remain

unclear. Observations that conceptuses of both species produce certain

polypeptides transiently, during elongation and attachment periods

(Godkin et al. , 1982a, b) suggests important roles for these products

early in gestation. Alterations in array of polypeptides produced by

ovine and porcine conceptuses during the peri-attachment period are

clearly related to dynamic developmental phenomena occurring at this

time (see Geisert et al. , 1982a; Bindon, 1971). However, dramatic

alterations in both quantity and quality of polypeptides produced by

post-attachment conceptus tissues suggests a physical and/or biochemical

effect of the maternal unit on conceptus protein production. Such

changes may also be related to transition of conceptus membranes from

a primarily secretory role, necessary to insure continued histotrophic

stimulation, to a primarily absorptive role, consistent with placental

function.

-95-

Prostaglandins ♦ Elevated levels of PG in uterine flushings from
pregnant cattle (Bartol et al. , 1981a; Lewis et al. , 1982), sheep
(Ellinwood et al. , 1979b) and pigs (Zavy et al. , 1980), primarily after
that period associated with maternal "recognition" of pregnancy and
blastocyst elongation, suggested a conceptus, as well as an endometrial
source for these ubiquitous molecules. Menezo et al. (1982) examined
fatty acid composition of conceptuses obtained from superovulated
cattle on days 7 through 14. Total fatty acid content increased
slightly from day 7 (1.5 Ug/conceptus) to 10 (4.2 ug/conceptus) and
more sharply thereafter reaching 13 yg/conceptus in day 13 tissues
(Menezo et al. , 1982). Qualitative analyses of bovine conceptus fatty
acids Indicated an increase in elongation and desaturation of these
molecules between days 11 and 13. Significant amounts of arachidonic
acid (precursor for PG synthesis) were not detected in bovine concep-
tuses obtained prior to day 14 (Menezo et al., 1982). Quantitative and
qualitative alterations in bovine conceptus tissue fatty acids were
clearly related to stage of development and were most dramatic following
"hatching" (loss of ZP) and initiation of trophoblastic elongation
(Menezo et al. , 1982).

Shemesh et al. (1979) collected bovine conceptuses from super-
ovulated cattle on days 13, 15 and 17 postestrus. Conceptuses were
placed immediately in 1 ml of defined culture medium and either
(1) frozen or (2) allowed to incubate 48h at 37C. All samples (tissue
+ medium) were then homogenized and analyzed for PGF and PGE by
radioimmunoassay (Shemesh et al. , 1979). Measurable amounts of both
PGF and PGE were detected in both incubated and unincubated homogenates
from all three stages. Prostaglandin content increased with conceptus

-96-

age in both groups, but was 4 to 17 times higher in homogenates of
incubated conceptuses suggesting active synthesis and release of PG
by conceptus tissues (Shemesh et al. , 1979). Data were consistent
with observations of Menezo et al. (1982) relative to availability of
arachidonic acid precursor by at least day 14.

Lewis et al. (1982) examined ability of bovine conceptuses from
days 16 and 19 to metabolize arachidonic acid and PGF in vitro.
Conceptuses from both stages produced PGF2a' ^^™» ^^^2 ^"^' ^^ l^^st,
four unidentified compounds as determined by Sephadex LH-20 chroma-
tography. Consistent with observationsof Shemesh et al. (1979),

conceptuses produced more PGF than PGE and total PG production

3
increased from day 16 to 19 (Lewis et al. , 1982). Conversion of H-

o

PGF to H-PGFM by bovine blastocysts suggested that, in addition
2a

to other enzymes essential for PG synthesis (see Ramwell et al. ,

1977), conceptuses also contained 15-hydroxy-prostaglandin dehydro-

13
genase and A -prostaglandin reductase (Anggard, 1971). However, as

compared to endometrial tissues from the same cattle, bovine conceptuses

3 3

produced considerably less PGFM (% of H-PGF converted to H-PGFM:

34.3 ± 1.5% vs 7.5 ± 1.6%); perhaps the uterus, not the conceptus, is
the major site of PGF metabolism.

Data relative to conceptus production and/or metabolism of PG in
other domestic species are limited. Recently, Hyland et al. (1982)
presented data indicating production of both PGF and PGE by ovine con-
ceptuses from days 14 and presence of both prostaglandins in conceptus
tissues from days 13 and 15. Ovine conceptuses contained and/or
released more PGF than PGE, in agreement with earlier reports for
bovine conceptuses (Shemesh et al., 1978; Lewis et al. , 1982). No

-97-

data were presented relative to metabolism of PG. Maule-Walker et al.
(1977) demonstrated metabolism of PGE to 13, 14-dlhydro-15-keto-PGE
by porcine conceptus tissues, and suggested that amniotic membranes
might be particularly important in conceptus conversion of both PGF„
and PGE to their respective metabolites. These observations were
generally consistent with those of Lewis et al. (1982) for bovine
conceptuses. However, no further data relative to PG synthesis or
metabolism by porcine conceptuses were presented.

Potential roles for PG in utero were reviewed above. Present data
clearly indicate that the bovine conceptus may contribute significantly
to the array of uterine luminal PG during early pregnancy. Preliminary
data in sheep and pigs suggest a similar possibility. Data are
entirely lacking relative to such information in the mare.

Steroids. Importance of steroid hormones in regulation of uterine
metabolic events suggests that their production and/or metabolism by
peri-attachment conceptus tissues may have dynamic consequences relative
to maintenance of an embryotrophic uterine environment. Evidence for
steroid production by bovine conceptuses was first presented by Shemesh
et al. (1979). Measurable amounts of P. were found in homogenates of
both incubated and unincubated bovine conceptuses from days 13, 15 and
16. Testosterone was also detected, but only in day 15 and 16 concep-
tuses (Shemesh et al. , 1979). Levels of both steroids (P^ and T) were
higher in incubated than unincubated conceptus homogenates (tissue +
medium), indicating production of these steroids by conceptus tissues
in vitro (Shemesh et al. , 1979). Data suggested a requirement for both
33-HSD and 173-HSD activities (see Heap et al. , 1979).

-98-

Bovine cbnceptus tissues were shown to metabolize neutral steroids
extensively to 53-reduced products (Chenault, 1980; Eley et al., 1983;
Knickerbocker et al. , 1980). Chenault (1980) reported that bovine
conceptuses from days 13, 14, 16 and 20, and trophoblast from day 24
converted H-A, , T and etiocholanolone (Et; 3a-hydroxy-53-androstan-
17-one) to 56-androstane products with 60 to 100% efficiency. Primary
products from H-A, were Et and 3a, 17a-dihydroxy-53-androstane. In

o

addition to these products, metabolism of H-T yielded 3a, 173-d ihydroxy-

3

53-androstane. Conceptus metabolism of H-Et produced 3a,17a-dihydroxy-

53-androstane (Chenault, 1980). Bovine conceptus steroid metabolic
activity was not related to stage of development (Chenault, 1980).
However, when day 24 conceptuses were separated into trophoblast, yolk
sac, embryo and allantois, and components incubated with radiolabelled
neutral steroid substrate, 98% of 53-reductase activity was associated
with the trophoblast (Chenault, 1980). Data suggested activity of
several steroid metabolizing enzymes in bovine conceptuses including
A^-steroid-53-reductase, 17a-HSD, 3a-HSD (Chenault, 1980) and 173-HSD
(Shemesh et al. , 1979). Results of Chenault (1980) were confirmed and
extended by work of Eley et al. (1983) and Knickerbocker et al. (1980).
Metabolism of P, is to primarily 56-reduced pregnanes by bovine concep-
tuses from days 16 to 27.

Bovine conceptus metabolism of neutral steroids to 53-reduced
products is in direct contrast to endometrial production of 5a-reduced
metabolites (Eley et al. , 1983, Knickerbocker et al. , 1980). Since
bioactivity of both 5a- and 53-reduced steroids is controversial
(Holzbauer, 1976; Kubli-Garf ias et al. , 1979, 1980), the possibility
exists that reduction of steroids by both tissues simply decreases

-99-

amount of biologically active steroids present in utero. However, as
observed by Eley et al. (1983), partitioning of isomeric forms of the
reductase enzyme in two such interdependent tissues suggests a more
important, functional role for one or both of these products. Chenault
(1980) cited studies implicating SB-reduced steroids in erythropoesis
and hemoglobin synthesis. It was suggested that such effects might be
important during genesis of the bovine circulatory system which begins
around days 17 to 18 (Chenault, 1980; Greenstein and Foley, 1958a, b).
The possibility that 56-reduced steroids are involved in regulation of
transcriptional events (see Chenault, 1980) suggests multiple sites
of action both in conceptus and maternal tissues. Kubli-Garf ias et al.
(1979, 1980) demonstrated inhibition of uterine motility in vitro
by 5B-, but not 5a-reduced steroids, suggesting an Important maternal
site of action for 5B-reduced conceptus products. At present data are
clearly inconclusive relative to the biological importance of 56-reduced
bovine conceptus steroid metabolites.

Although 56-reduced metabolites predominated, Eley et al. (1983)
reported production of small amounts (< 200 pg) of both £^-176 and Ej^
from H-A, by bovine conceptuses from days 16 to 27. Neither E^-lla
or E were detected, nor was there any evidence for production of sul-
phoconjugated estrogens (Eley et al., 1983; Chenault, 1980). Although
produced in small amounts, E -176 was the predominant metabolite of A^
by peri-attachment bovine conceptus tissues (Chenault, 1980; Eley et al.,
1983). In contrast, E , E -17a and their sulphoconjugates were pre-
dominant estrogenic products of bovine placental tissues later in
gestation (Hoffman et al., 1979; Robertson and King, 1979; Eley et al. ,
1979). Data suggest a specific requirement for conceptus produced free

-100-

estrogens, especially E -17B, during the peri-attachment period,
although evidence remains purely correlative. Gadsby et al. (1980)
indicated that aromatose activity in bovine conceptuses was highest
between days 16 and 22. Production of even small amounts of E by
bovine conceptuses during this period may be important in initiation
of events associated with establishment of pregnancy; particularly if
endometrial tissues are hypersensitive to E stimulation during this

time as suggested above.

3
Ovine trophoblast tissue from days 14 to 18 converted both H-A,

and dehydroepiandrosterone (DHT) to other unidentified neutral steroid
metabolites with 90 to 95% efficiency (Gadsby et al. , 1980). Although
53-reduced products have not been described for peri-attachment stage
ovine conceptus tissues, Ainsworth and Ryan (1967) demonstrated 53-
reductase activity in near term ovine placental tissues. Like cattle,
conversion of neutral steroid precursors to phenolic compounds by ovine
conceptuses was low (Gadsby et al. , 1980). Ovine trophoblasts ob-
tained between days 16 and 18 did not produce significant amounts of

either E or E -17B from H-A, precursor (Gadsby et al. , 1980). How-

3 3
ever, Willis et al. (1981) reported metabolism of both H-A^ and H-P^

to E by ovine conceptuses from days 14, 16 and 18. Production of E

by ovine conceptus tissues remains controversial.

Production and metabolism of steroids by porcine peri-attachment

conceptuses have been reviewed extensively. The reader is referred to

Heap et al. (1979, 1981a, b) for details. While porcine conceptuses

3 3
(day 16) converted H-A and H-DHT to other neutral steroid metabolites

with 40% efficiency, phenolic steroids were, consistently, the pre-
dominant products of steroid metabolism (Gadsby et al. , 1980).

-101-

Initiation of porcine conceptus E production is associated with the
period of rapid blastocyst elongation (Heap et al. , 1981b) and is
reflected by dramatic increase in both free and conjugated estrogens
in uterine flushings obtained from pregnant gilts between days 10 and
14 (Geisert et al. , 1982b; Zavy et al., 1980). Heap et al. (1979)
summarized data suggesting that porcine blastocysts may be able to
produce estrogesn from acetate precursor. Additionally, sulfatase
activity was demonstrated in pig conceptus tissues (Heap and Perry,
1974). Thus, significant amounts of free estrogens may be produced by
porcine conceptuses via deconjugation of sulphated E provided by endo-
metrial tissues (see above and Bazer et al., 1982). Production of
large amounts of estrogens by peri-attachment porcine conceptuses is
consistent with current theories of pregnancy recognition and mainten-
ance in this species (see Bazer and Thatcher, 1977; Bazer et al. ,
1982). Furthermore, porcine conceptus estrogen production was suggested
to be critical for synchronization of endometrial release of histotrophe
during the peri-attachment period (Geisert et al., 1982b, c; Bazer
et al. , 1982).

Increases in uterine luminal content of both E and E in pregnant
mares between days 14 and 16 suggested a conceptus source for these
steroids (Zavy et al., 1979). Incubation of equine conceptus membranes
obtained from days 8 to 20 of pregnancy and radioimmunoassay of medium
indicated conceptus production of both E and E . Estrogen production
increased from day 12 to day 20 at a level comparable to that seen in
porcine conceptuses. This increase in E production was clearly associa-
ted with increasing tissue mass since production per mg of conceptus
tissue was constant during this period (Zavy et al. , 1979). Whether

-102-

equine conceptus produced estrogens are required for pregnancy recog-
nition in a manner similar to that suggested for pigs is debatable
(see Bazer et al., 1982; Sharp, 1980). However, production of these
steroids does occur at significant levels during that period associated
with recognition in this specie (see Allen, 1979),

Conclusion

As is apparent from the preceeding review, the interdependent
relationship between the conceptus and its uterine environment is
complex. Considerable data have been amassed pertaining to the nature
of components of this system, a system designed by nature to maximize
chances of reproductive success. Temporal patterns of appearance of
these components, whether contributed by maternal or conceptus units,
have led to considerable speculation relative to their functional roles
in establishment and maintenance of an embryotrophic uterine environ-
ment. However, definitive information in this regard is clearly
lacking. Theories of function derived from factual correlative
evidence are valuable in attempting to understand the dynamics of such
a complex system. As facts continue to accumulate, however, perhaps
the most propitious attitude to take relative to their organization is
that suggested by Claude Bernard (1865; p. 168): "We must have robust
faith and not believe,"

CHAPTER II
UTERINE PROTEINS IN UNILATERALLY PREGNANT CATTLE

Introduction
Over 300 years ago Walter Needham coined the term "uterine milk"
to describe fluid present in the uterine lumen which might serve as a
source of nutrition to the developing fetus (Amoroso, 1952). The
concept that the uterus must provide a complete medium for support of
the developing conceptus (embryo and extraembryonic membranes) has,
therefore, been long recognized. This concept is particularly impor-
tant in domestic species including the horse, pig, sheep and cow in
which time from conception to placental attachment is prolonged (Cook
and Hunter, 1978; Steven and Morris, 1975; Wathes and Wooding, 1980).
Studies designed to examine components of uterine fluid from domestic
species have been hampered by small amounts of material recovered in
uterine flushings, particularly from sheep and cattle (Heap and Lamming,
1960; Iritani et al., 1969; Roberts and Parker, 1974a, 1976; Bartol
et al., 1981a). Observations of Bond (1898), that ligation of the
oviduct or uterine horn in rabbits, guinea pigs and sheep resulted in
accumulation of luminal fluid, set the stage for more recent studies of
a similar nature. Large volumes of protein-rich uterine fluid were
obtained from pregnant sheep in which a pouch was surgically created in
one uterine horn (Harrison et al., 1976). Later, Bazer and coworkers
(1979a) recovered large amounts of protein-rich fluid from the ligated
uterine horn of unilaterally pregnant sheep.

■103-

-104-

Procedures similar to those reported for sheep (Harrison et al.,
1976; Bazer et al., 1979a) have not been attempted in cattle. However,
Dixon and Gibbons (1979) showed that protein-rich uterine fluid could
be obtained from nonpregnant cattle following chronic progesterone
therapy. While obviously a useful technique, components of such fluid
may not be quantitatively or qualitatively representative of the array
of macromolecules or other substances induced by normal endocrine and
metabolic conditions of pregnancy. Objectives of the present study
were to (1) establish a technique for creation of unilateral pregnancy
in cattle in order to recover uterine milk (UM) from the ligated non-
gravid uterine horn, and (2) to characterize the composition of this
fluid pool.

Materials and Methods
Preparation of Unilaterally Pregnant Cattle

' Fifteen (15) cross-bred beef heifers were subjected to mid-ventral
laparotomy after induction of deep analgesia with xylazine (Rompun;
Haver -Lockhart, Shawnee, Kansas; Clark and Hall, 1969; Pippi and Lumb,
1979). For each animal, the reproductive tract was exposed and the ovary
containing the corpus luteum removed. An umbilical tape ligature was
tied securely around the base of the ipsilateral uterine horn as near
the external bifurcation as possible (Figure 2.1A). After placement of
the ligature, the uterus was returned to the abdominal cavity and sub-
cutaneous tissues and skin closed with suture materials. Cattle were
maintained on pasture and bred by natural service during the first
estrous period (day of onset of estrus = day 0) postsurgery with the
goal of obtaining unilaterally pregnant cattle (Figure 2. IB).

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-106-

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-107-

Detection and Recovery of Uterine Milk

After insemination cattle were palpated per rectum once each month
for 4 months and once each week thereafter until day 270. Pregnancy
status and presence or absence of UM in the ligated uterine horn were
noted. On days 180 (n=5) , 210 (n=6) and 240 (n=4) cattle were sub-
jected to standing flank laparotomy under local knesthesia (lidocaine
-HCL, 2%; Generix Drug Corp., Hollywood, FL) . At surgery the ligated
uterine horn was exteriorized and, if possible, a sample of UM withdraw
into a sterile syringe through a 16 gauge hypodermic needle. On day
270, pregnant cattle were killed at the University of Florida Meats
Laboratory and reproductive tracts recovered. Uterine milk, present in
ligated horns of all cattle on day 270, was collected into sterile
hypodermic syringes and the total volume recovered was recorded.

Other Fluids and Tissues

Jugular venous blood was collected into heparinized syringes from
all cattle on days 180, 210 and 240. Mixed peripheral blood was
collected in heparinized containers from each animal at slaughter on
day 270. Additionally on day 270, placental and fetal wet weights,
amniotic and allantoic fluid volumes and fetal sex were recorded.
Samples of day 270 endometrium from the intercaruncular areas in both
ligated and pregnant uterine horns were obtained, fixed in Bouins
fixative, embedded in paraffin and prepared for examination by light
microscopy for general histological integrity.

Processing of Uterine Milk and Plasma

At the time of collection, an aliquot of each UM sample was applied
to sterile bovine blood-agar bacteriological plates and incubated 24 to

a

-108-

48h at 37C to evaluate the possibility of bacterial contamination.
Whole UM samples were stored in aliquots of 50 ml or less at -20C until
needed for analyses. In each case UM samples were thawed, centrifuged

t 4C for 20 min. at 6,000g, and supernatant (UMS) subjected to
analyses described below. Peripheral blood samples were centrifuged
as described above for UM, and individual plasma samples stored at
-20C prior to analyses.

Radioimmunoassays

Progestin concentrations in all plasma and UMS samples were
determined by radioimmunoassay (RIA) according to general procedures
described by Abraham et al. (1971). Description and precision of this
assay were reported by Chenault et al. (1975). Validation of the
progestin isolation and assay procedures was reported by Knight et al.
(1977). Antiserum, a gift of Dr. J.L. Fleeger, Texas A and M Univer-
sity, was prepared in a rabbit against 11-a-hydroxy-progesterone
hemisuccinate conjugated to bovine serum albumin. Specificity of the
RIA was such that the assay essentially measured progesterone (pregn-4-
ene-3,20 dione; P^) . Sensitivity of this assay with respect to the
standard curve was 25 pg. Inter- and intraassay coefficients of
variation were 16.0% and 19.2%.

Estrone (E^^) and estradiol (E ) concentrations were measured by
RIA in all UMS samples and in all plasma samples collected simultan-
eously with UM (days 180, 210, 240 and 270). Estrogens (E and E )
were separated by LH-20 column chromatography following extraction with
diethyl ether. The antibody, provided by Dr. V.L. Estergreen, Wash-
ington State University, was developed in sheep against estra-1,3,5
(10)-triene-3,173-diol,17-succinyl bovine serum albumin. Description

-109-

and validation of the assay procedure was reported previously (Chenault
et al., 1975, 1976). Inter- and intraassay coefficients of variation
for E^ were 11.7% and 13.8% and for E 17.4% and 16.6%.

Estrone sulfate (E^SO^) concentrations in UMS and plasma were
determined by RIA after extraction of free estrogens (E and E ) and
hydrolysis with a sulfatase enzyme (sulfatase type H-II, Sigma Chemical
Co., St. Louis, MO). Estrone liberated during hydrolysis was extracted
with diethyl ether and quantified as described above. Validation of
this procedure in our laboratory was published previously (Eley et al.,
1981a). Inter- and intraassay coefficients of variation were 8,43% and
8.66%.

Concentrations of unextracted prostaglandin F (PGF) were determined
directly in all UMS samples with a double-antibody RIA system described
by Cornette et al. (1972). First antibody, donated by Dr. K.T. Kirton
of the UpJohn Company (Kalamazoo, MI), was generated in rabbits against
PGF2a conjugated to bovine serum albumin at the C carbon. Second
antibody was generated in goats against both heavy and light chains of
rabbit IgG (Cappel Laboratories, Inc., Downington, PA). This assay was
validated in our laboratory for measurement of immunoreactive PGF in
bovine uterine flushings (Bartol et al., 1981b). Inter- and intraassay
coefficients of variation were 7.58% and 13.68%. Ligated uterine horn
content of PGF (yg/horn) was calculated as the product of PGF concen-
tration (ng/ml) and UM recovery volume (ml) .

Assay for Total Protein and Sugars

All protein concentrations were estimated according to procedures
of Lowry et al. (1951) using purified bovine serum albumin (BSA;
fraction V, Sigma Chemical Co., St. Louis, MO) as a standard. Each

-no-

sample of UMS was assayed in duplicate. Total protein content (g/
ligated uterine horn) was calculated as the product of UMS protein
concentration (mg/ml) and recovery volume (ml) .

Concentrations of glucose in UMS were determined according to
methods previously described by Keilin and Hartee (1952). Fructose
concentrations in UMS were determined by the method of Roe (1934).
Glucose content (mg/ligated uterine horn) was calculated as described
above for both PGF and total protein contents.

Analysis of Calcium

Calcium (Ca) concentrations in UMS were determined with a Calcette
(Precision Systems Inc., Sudbury, MA). This apparatus employed EGTA
for fluorometric titration of Ca in aqueous solutions as described by
Alexander (1971). Calcium was measured in all undialyzed UMS samples
obtained on day 270. Aliquots of each sample were then dialyzed
extensively against several thousand volumes of 10 mM tris (tris hydrox-
ymethylaminomethane)-HCL buffer, pH 8.2 at 4C for 24h in 3500 M^ cut-off
dialysis tubing (Spectrapor 3; Spectrum Medical Ind., Los Angeles, CA) .
Following dialysis, nondialyzable Ca was measured in each dialyzed UMS
sample. Percentage of Ca retained was calculated as the ratio of post-
and predialysis Ca concentrations for each sample.

Gel Filtration Chromatography

An aliquot of each day 270 UMS sample was subjected to gel filtra-
tion chromatography on Sephacryl S-200 (Pharmacia Fine Chemicals,
Uppsala, Sweden) in a 90 X 1 . 5 cm column at 4C in 10 mM tris-HCL
buffer, pH 8.2, containing 0.33M NaCl. The K for each protein peak
was calculated as described by Reiland (1971) and compared with K

3. V

-Ill-

values of the protein standards aldolase (M - 158,000), ovalbumin
(M - 45,000), and ribonuclease A (M - 13,700) used to calibrate the
column. Column void volume was determined using Blue Dextran (M -
2 X 10 , Pharmacia Fine Chemicals, Uppsala, Sweden). Elution profiles
were generated by determining absorbance of a 500 Ul aliquot of each
fraction at 750 nm following Lowry protein assay reaction (Lowry et al.,
1951).

Anion Exchange Chromatography

Aliquots of UMS , rennet precipitated bovine colostral whey, and

bovine serum were titrated with ammonium sulfate (NH,)„SO, to a final

4 2 4

concentration of 41%, centrifuged at 1500g for 15 min. , the resultant
pellet resuspended in distilled water and the ammonium sulfate precipi-
tation procedure repeated. The final pellet was dialyzed against
several thousand volumes of 10 mM sodium phosphate buffer, pH 7.4, at
4C for 4h, centrifuged as described above and the supernatant subjected
to anion exchange chromatography. Anion exchange chromatography was
performed on DEAK-cellulose (DE-52; Whatman, Inc., Clifton, NJ)
according to methods described by Williams et al. (1975) for gross
fractionation of bovine immunoglobulin classes. Proteins not binding
to DE-52 were eluted with a 10 mM phosphate buffer (pH 7.4). Remaining
DE-52 positive proteins were eluted with a stepwise increase in NaCl
concentration through 0.04M, 0.08M, O.IOM and 0.15M. Absorbance at
280 nm (Model 35 spectrophotometer; Beckman, Palo Alto, CA) was deter-
mined for each eluted fraction for evaluation of elution profiles.
Aliquots of UMS from each major DE-52 fraction area were subjected to
double immunodiffusion (Ouchterlony, 1958) against rabbit anti-bovine
IgA, IgG and IgM (Miles Research Products Div. , Elkhart, IN).

-112-

Two Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)

Uterine milk supernatant and plasma samples were dialyzed exten-
sively in Spectrapor 1 tubing (M cut-off = 6-8000; Spectrum Medical
Ind., Los Angeles, CA) at 4C against 10 mM tris-HCL buffer, pH 8.2.
Individual samples were then lyophilized and this product dissolved in
a volume of 5 mM K Co containing 9.4M urea, 2% (^/v) Nonidet P40 (BDH
Chemicals Ltd., Poole, England), and 0.5% (*'/v) dithiothreitol (Clelands
reagent; Sigma Chemical Co., St. Louis, MO) appropriate to allow 50 to
100 Mg of total protein to be loaded on each gel in a volume less than
or equal to 100 yl (Horst and Roberts, 1979). Separation of both acidic
and basic proteins by 2D-PAGE was accomplished according to methods
described by Horst and Roberts (1979) and Basha et al. (1980a).

Crossed Immunoelectrophoresis (C-IEP)

Samples of UMS and plasma from the same cattle were subjected to
C-IEP following procedures of Weeke (1973) and Eckersall and Beeley
(1980). Electrophoretic procedures were performed using an LKB 2117
Multiphor horizontal bed apparatus (LKB, Stockholm, Sweden) attached
to a Forma Scientific Model 2095 Bath and Circulator (Mallinckrodt Inc.,
Marietta, OH) adjusted to 14C. Current for electrophoresis was
provided by an EC-400 completely regulated power source (E-C Apparatus
Corp., St. Petersburg, FL) . A homogeneous barbital buffer system
consisting of 0.06M Sodium barbital, O.OIM barbituric acid, 0.37M tris
and 0.75M glycine (pH 8.68) was used. All separations were performed
in 1% C/v) agarose (Sigma type II, EEC; Sigma Chemical Co., St. Louis,
MO) in buffer.

For initial separation, 3.5 ml of molten agarose was poured onto

2
warmed 5.0 cm glass plates (1 mm thick) and allowed to solidify at room

-113-

temperature. A 0.25 cm well was punched in one corner of each gel 1 cm
from an edge and 0.5 cm from the base. Gel plates were then placed onto
the LKB 2117 Multiphor with wells oriented near the cathode. Buffer
chambers were filled with 700 mis each of barbital buffer and gels
bridged to each chamber with wicks consisting of three thicknesses of
paper soaked in buffer (5 X 15 cm Whatman 1; Whatman Inc., Clifton, NJ) .
Samples (3 to 7 yl) were loaded into wells and electrophoretic separa-
tion of sample proteins accomplished by application of 2 v/cm of gel
(10 v) for 2h at 14C. Voltage applied was measured directly through
the gel by insertion of voltmeter probes.

Following initial separation, the upper 4 cm of each gel, not con-
taining sample proteins, was removed and replaced with 2.8 ml of 1%
agarose homogeneously mixed with 200 yl of specific antiserum diluted to
working concentration in barbital buffer (total secondary gel volume =
3.0 ml). Secondary electrophoresis was performed at 1 v/cm of gel (5 v)
for 15 to 18h, at right angles to initial separation, toward the annode,
under identical buffer and temperature conditions. Comparison of UMS
and plasma protein components by C-IEP involved use of several antisera
including goat anti-bovine whole serum (Lot No. 28475) and rabbit anti-
bovine albumin (Lot No. 28476; U.S. Biochemical Corp., Cleveland, OH)
as well as rabbit anti-bovine IgA (Lot No. 23, Control No. R054) , IgG
(Lot No. 31, Control No. R074) and IgM (Lot No. 31, Control No. R034;
Miles Research Products Div. , Elkhart, IN).

After secondary electrophoresis, background protein was removed

by pressing each gel under several layers of filter paper (Whatman 1;

2
Whatman Inc., Clifton, NJ) and a 5 cm glass plate for 15 min. at

90g/cm^. Gels were then rinsed 30 min. in 0.9% C/v) NaCl followed by

-114-

distilled HO and pressed a second time. Deproteinized gels were dried
onto 5 X 7.5 cm glass slides at 60C for 20 min. and stained 3h with

0.5% Coomassie brilliant blue R-250 (Sigma Chemical Co., St. Louis, MO)

w V

/v in an ethanol: acetic acidrwater solution ( /v, 9:2:2). Gels were

destained in ethanol: acetic acid:water solution ( /v, 9:2:2) for 5 to
15 min.

Crossed immunoelectrophoresis allowed both quantitative and quali-
tative assessment of immunochemically similar proteins in UMS and
plasma. The system employed allowed as many as four gels to be run
simultaneously and, where possible, comparisons were made among gels
within a run.

Statistical Analysis

Where appropriate, data were subjected to analysis of variance
using procedures available in the General Linear Models program of the
Statistical Analysis System (SAS; Barr et al. , 1979). Statistical
models considered variability associated with day, fluid pool (UMS vs.
plasma) and cow. Day comparisons were made using orthogonal contrasts.

Results
General

Twelve of 15 cattle subjected to the unilateral ligation procedure
were pregnant within two estrous cycles postsurgery. Of the three
remaining cows, one never cycled and two failed to conceive and were
culled. Earliest detection of UM by rectal palpation occurred in one
cow on day 166. Uterine milk was detectable by rectal palpation in 11
of 12 cows by day 180. Bovine UM was recovered successfully at flank
laparatomy from cattle on days 180 (5/6), 210 (6/6) and 240 (4/6).

-115-

Recovery volumes at laparotomies ranged from 3.5 to 50 ml (maximal
volume attempted) . In three instances adhesions prevented exteriori-
zation of the ligated uterine horn required for UM recovery. Three of
12 unilaterally pregnant cattle gave birth to live calves between days
245 and 260. Uterine milk was recovered from all nine cows remaining
pregnant at day 270 (time of slaughter) .

At all stages recovered, UM was golden-brown and odorless. Absence
of growth on bovine blood agar bacteriological plates after 24 to 48 h
at 37C suggested that UM was bacteria free, and its presence was not
bacterially induced. Arithmetic means (X ± SEM) and ranges of observa-
tions for day 270 UM recovery volumes as well as fetal weights,
placental fluid volumes, and membrane weights are presented in Table
2.1.

Gross morphology of day 270 placentae and fetal-maternal attach-
ments appeared normal in every respect. In two cases in which uterine
ligation was incomplete and the ligated uterine horn was patent with
the pregnant horn, placental membranes did not pass through the opening
into the ligated horn. However, UM was found in both compartments.
Maternal caruncles in the day 270 ligated uterine horns were small and
unstimulated. General appraisal of intercaruncular endometrial his-
tology did not reveal any striking abnormalities or dissimilarities
between pregnant and ligated uterine horns.

Components of Uterine Milk

Presented in Table 2.2 are arithmetic means (± SEM) of concentra-
tions of E , E , E SO, and P, in UMS and plasma obtained on days 180,
210, 240 and 270. Comparison of steroid concentrations between fluid
pools (UM vs. plasma) only involved data where paired samples of UM and

-116-

Table 2.1. Arithmetic Means (± SEM) and ranges for uterine and fetal-
placental physical responses measured on day 270 of
gestation for nine cows.

ITEM MEAN RANGE

UM volume, ml 205.3 ± 68.2 20.0 - 635.0

Fetal wt.. Kg 32.5 ± 1.51 25.8 - 38.1

Allantoic fluid, L 11.73 ± 2.14 6.94 - 27.14

Amniotic fluid, L 3.27 ± 0.45 1.30 - 6.23

Placentome number 82 ± 5 62-112

Placentome wt., g 4335.6 ± 593.2 2555.0 - 6203.0

Intercotyledonary memb.

wt., g 1548.4 ± 215.7 806.0 - 2883.0

Total placental wt., g 6053.9 ± 371.5 3615.0 - 7816.0

-117-

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-119-

plasma were obtained. Overall, mean concentrations of E and E were
not different (P > .10) between UMS (E , 80.9 ± 12.0 pg/ml; E 88.7 ±
15.7 pg/ml) and plasma (E , 186.5 ± 32.66 pg/ml; E , 103.3 ± 10.72
pg/ml) . Additionally, neither E nor E concentrations were affected
significantly by day (P > .10). In contrast, overall EiSO, concentra-
tions (X ± SEM) were higher in plasma than in UMS (2953.2 ± 234.9 pg/ml
vs. 100.00 ± 10.24 pg/ml; P < .0001). Concentrations of E SO, in UMS
varied among days (P < .076; Table 2.2), increasing after day 210
(P < .02) to highest concentrations on day 240 (P < .09). A more
systematic effect of day was detected for plasma E SO (P < .02; Table
2.2) which increased after day 180 (P < .01) to highest concentrations
on days 240 and 270 (P < .07). Progesterone concentrations (X ± SEM)
were nearly ten times higher in plasma than UMS (P < .0001; 14.7 ± 1.06
ng/ml vs. 1.9 ± 1.2 ng/ml) . Concentrations of P, in UMS were not
affected by day (P > .10), but plasma P, concentrations were higher on
day 210 than later (P < .03; Table 2.2). Significant day X fluid inter-
actions were not detected for any steroids measured.

Presented in Table 2.3 are significant (P < .10) gross and within-
animal partial correlations detected between steroids measured in
peripheral plasma and UMS. Positive correlations were detected between
plasma E^ , E and E SO, concentrations. A negative correlation was
detected between plasma E SO, and P.. When data were adjusted for the
effect of cow, significant within-animal partial correlations were
detected only between plasma E and E (r = .97, P < .05) and P, and
the two unconjugated estrogens, E and E (r = .99, P < .01; r = .98,
P < .01). Of steroids measured in UMS, significant gross and partial
correlations were only detected between E and E (gross: r = .69,

-120-

Table 2.3. Gross (upper) and partial within-cow (lower) correlations
of steroids in peripheral plasma and uterine milk
supernatant.

Plasma

UMS

h S h^% \

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+ * **

significant at P < .10; significant at P < .05; significant at

P < .01.

-121-

P < .05; partial: r = . 92, P < . 10) . Positive correlations were

detected between plasma and UMS unconjugated estrogens (Table 2.3).

Negative correlations were found between plasma E and E SO and UMS

2 14

E^SO^, as well as between plasma P^ and UMS E (Table 2.3). Neither
positive nor negative gross correlations remained significant when
adjusted for the effects of cow. When data were adjusted for cow,
however, significant partial within-animal correlations were detected
between UMS P^ and plasma E^ (r = .97, P < .01), E^ (r = .97, P < .05)
and P^ (r = .99, P < .01). Reduction in the number of significant
correlations found following adjustment of data for variability asso-
ciated with cow reflected the high within-animal variation present in
the data set.

Concentrations and total amounts (X ± SEM) , as well as range values,
of selected constituents of day 270 bovine UMS are presented in Table
2.4. Day 270 UMS contained large amounts of immunoreactive PGF (267.30
± 86.62 yg/ligated horn) and total protein (11.84 ± 3.49 g/ligated
horn). Mean (± SEM) protein concentration of UMS did not differ signif-
icantly among days (63.36 ± 8.37 mg/ml) . Mean (± SEM) glucose content
was 32.95 ± 10.78 mg/ligated horn, while fructose was undetectable in
1 ml of UMS. Calcium concentration in undialyzed UMS was 28.35 ± 8.52
mg% with a range of 5,83 to 66.19 mg%. An average of 1.63 ± 0.89% of
total UMS Ca was nondialyzable indicating that 93.8 to 99.9% of total
UMS Ca was present as free Ca in utero. Uterine milk predialysis Ca
concentration was positively correlated with both UMS protein concentra-
tion (r = .68, P < ,09) and content of PGF (r = .92, P < ,08).

Quantitative Analyses of Proteins

Depicted in Figure 2.2 is a typical S-200 elution profile of UMS
from day 270, Four primary fraction areas were identified with

-122-

Table 2.4. Concentration and content (X ± SEM and range) of selected
constituents of bovine uterine milk supernatant recovered
on day 270 of gestation.

ITEM

N

MEAN

RANGE

PGF, ng/ml

Total PGF, yg

Protein, mg/ml

Total protein, g

Glucose, mg/ml

Total glucose, mg

Fructose, mg/ml

Ca , mg/100 ml

Nondialyzable Ca ,
mg/100 ml

I I

% Ca nondialyzable

7

3049.5 ± 831.51

1279.9

- 5681.9

7

267.30 ± 86.62

130.7

- 588.4

9

67.97 ± 11.96

20.6

- 133.4

9

11.84 ± 3.49

0.114

- 29.08

7

0.127 ± 0,004

0.105

- 0.140

7

32.95 ± 10.78

2.99

- 85.70

7

N.D.^

~

7

28.35 ± 8.52

5.83

- 66.19

7

0.21 ± 0.07

0.04

- 0.43

7

1.63 ± 0.89

0.10

- 6.20

No fructose detectable in 1 ml of sample.

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-124-

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-125-

approximate molecular weight estimates (M X 10 ) at their peak of I,
> 150; II, 82; III, 43; and IV, 14. The majority of UMS protein
consistently eluted in fraction IV suggesting that major components of
UMS were of lower molecular weight.

Representative Coomassie brilliant blue R-250-stained 2D-PAGE gels
(12.5% acrylamide) of UMS from two cows are shown in Figure 2.3. Acidic
proteins are seen in panel A (lEF) and basic proteins in panel B
(NEPHGE) . The 2D-PAGE technique revealed a complex polypeptide array
in UMS. Proteins were present across the molecular weight range des-
cribed by S-200 gel filtration (Figure 2.2). Those most likely
representative of S-200 fraction area IV (Figure 2.2) were seen
primarily on NEPHGE gels (Figure 2.3B). Polypeptides in this category
had molecular weights of less than 35,000 and were present in the 6.8
to 8.6 pH range.

Within-animal comparisons of 2D-PAGE (lEF) gels of UMS and periph-
eral plasma proteins revealed striking similarities in array of
polypeptides present in these two fluid pools. Representative gels
are shown in Figure 2.4. The dominant protein seen in gels of both UMS
and plasma was bovine serum albumin (Figure 2.4). Numerous other poly-
peptides were common to both fluids as shown (Figure 2.4). Of note,
however, were several polypeptides present in UMS which were consist-
ently absent from 2D-PAGE (lEF) gels of peripheral plasma proteins.
One group of UMS-specific polypeptides was found in the 25 to 30,000
molecular range with isoelectric points (P ) between 6.6 and 7.3
(Figure 2.4A). Two other polypeptides (Figure 2.4A, UMl and UM2) had
molecular weights of 20 to 21,000 and P of approximately 6.3 and 6.1,
respectively.

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Figure 2,4. Two-dimensional polyacrylamide gel electrophoresis (10%
acrylamide) of acidic (lEF) proteins characteristic of
UMS (A) and peripheral plasma (B) . BSA (shovm in A) was
dominant protein in both fluids. UMS-specific proteins
(A) included one group in the molecular weight (M^ x 10-3)/
pH range of 25-30/6.6-7.3 (indicated by large arrow); and
two other polypeptides (UMl and UM2) at approximately
20-21/6.3 and 6.1 respectively.

-129-

A.

S
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- 31

- 24

- 20

- 16

:

t.14

1

IE F

-> +.

-130-

Crossed Immunoelectrophoresis was used to examine immunochemical
similarities between protein components of UMS and plasma. When equal
amounts of UHS and plasma protein were run under identical conditions
using goat anti-whole bovine serum (U.S. Biochemical Corp., Cleveland,
OH), a large number of immunochemical identities were seen in both
fluids (Figure 2.5). The largest most prominant peak seen in both UMS
and plasma (Figure 2.5) was electrophoretically and Immunochemically
identical with a commercial preparation of BSA (Sigma fraction V; Sigma
Chemical Co., St. Louis, MO). This protein was consistently present
in UMS as the major serum protein. As shown in Figure 2.5, BSA was
present In approximately equal proportions in UMS and plasma. Compari-
son of the array of proteins identified Immunochemically in UMS and
plasma by C-IEP Indicated that not all proteins present in plasma were
consistently present in UMS. Quantitatively, many UMS postalbumln
proteins, migrating more slowly than BSA, were present in proportion-
ately lower amounts compared to peripheral plasma as evidenced by
smaller rockets in UMS gels (Figure 2.5). In these studies, where
equal amounts of UMS or plasma proteins were loaded and electrophoresed
under identical conditions, the proportionate reduction in amount of
some postalbumln proteins suggested that a significant proportion of
total UMS protein was not immunochemically identical with plasma.

Shown in Figure 2.6 are ion-exchange chromatograms of ammonium
sulfate (41%) precipitable proteins from UMS (panel A) , bovine serum
(panel B) and bovine colostral whey (panel C) . Fraction areas desig-
nated I, H, III, IV and V were eluted from DE-52 either with 10 mM
sodium phosphate buffer (pH 7.4) or with 0.04M, 0.08M, O.IOM or 0.15M
NaCl, respectively. Williams and coworkers (1975) demonstrated that

Figure 2.5. Crossed-immunoelectrophoresis indicating serum- identical
proteins in UMS (top) and peripheral plasma (bottom) from
a day 270 unilaterally pregnant cow. Presence of rockets
in both gels indicates positive immunochemical response
to goat anti-whole bovine serum (Lot No. 28475, U.S. Bio-
chemical Corp., Cleveland, OH; final dilution 1:75).
(*) Indicates largest rocket in both UMS and plasma which
was electrophoretically and immunochemically identical
with a commercial preparation of BSA (fraction V; Sigma
Chemical Co., St. Louis, MO). Postalbumin proteins are
to left of BSA. Equal amounts (15 Ug) of UMS and plasma
protein were electrophoresed. Separations were performed
toward the annode in 1% agarose (^/v) in barbital-tris-
glycine buffer (pH 8.68).

UMS

-132-

■ ^' '■■'A.

.n:

■.'i€^ /

VV-'-.

. *.^' ■■

ri.

>

'I' ■-

r '/m-

■Id

£ii>;^^.M::-S':

.^

Plasm.q__ _

Figure 2.6. DEAE-ion-exchange chromatograms of ammonium-sulfate (41%)
precipitable proteins in UMS (A), bovine serum (B) and
bovine colostral whey (C) . DEAE (-) proteins (area I)
were eluted with 10 mM sodium-phosphate buffer (pH 7.4).
Remaining DEAE (+) proteins were eluted by stepwise in-
creases in NaCl concentration through 0.04M (area II),
0.08M (area III), O.IOM (area IV) and 0.15M (area V).
Absorbance at 280 nm for each fraction (Y axis) is
expressed relative to peak absorbance, which was assigned
an A-280r value of 1.0. Fractionation was performed
according to methods of Williams et al. (1975) which
showed bovine serum fraction areas I, II, HI and V to be
enriched in IgG^, IgG^, IgA and IgM respectively.

-13A-

1.0

A..

NnCI (Mi Q.QQ

n.OA noH 0.10

a6

<

a2-

NofI IM) Q.QQ

A '

nnA a.nn o.io — (U5_
II 1 III I IV I V

Serum

-135-

fractions I, II, III and V, obtained from bovine serum by identical
methods, were enriched in immunoglobulins (Ig) IgG., IgG , IgA and IgM,
respectively. Present data (Figure 2.6) suggested that UMS contained
significant proportions of Ig in all classes described by Williams
et al. (1975). Double immunodiffusion (Ouchterlony, 1958) revealed
precipitin lines of identity when UI-IS fractions I, II and III (Figure
2.6A) were tested against rabbit antibovine IgG and when UMS fractions
III and V (Figure 2.6A) were tested against rabbit anti-bovine IgA and
IgM, respectively.

Immunoglobulins were immunochemically identified in whole UMS by
C-IEP (Figure 2.7). Whole UMS (200 Jig total protein) was subjected to
C-IEP with goat anti-bovine whole seriom to intensify the postalbumin area
shown in panel A (Figure 2.7). Precipitin line rockets in panel B
(Figure 2.7) illustrate positive immunochemical response between UMS
proteins and rabbit anti-bovine IgG. Presence of three separate rockets
indicates electrophoretically distinguishable antigens with immunochem-
ical identities and, based on earlier double immunodiffusion results,
reveal both IgG (1 and 2; Williams et al., 1975) and IgA. The single
rocket in panel C (Figure 2.7) illustrates the electrophoretic mobility
of a UMS antigen which reacted positively against rabbit anti-bovine
IgA. Data suggest that IgG and IgA as well as IgM may be among the UMS
antigens recognized by goat anti-bovine whole serum in the postalbumin
area (Figure 2.7A).

Discussion
In the present study it was demonstrated that unilateral ovariec-
tomy and ligation of the ipsilateral uterine horn permitted
establishment of unilateral pregnancy in 12 of 15 cattle. A similar

Figure 2.7. Crossed-inununoelectrophoresis (C-IEP) revealing serum- and
immunoglobulin- identical components of day 270 bovine UMS.

(A) Whole UMS (200 Ug total protein) subjected to C-IEP
with goat anti-bovine whole serum (Lot No. 28475, U.S.
Biochemical Corp., Cleveland, OH; final dilution 1:75).

(B) positive immunochemical response between UMS proteins
and rabbit anti-bovine IgG (Lot No. 31, Control No. R047;
Miles Research Products, Elkhart, IN; final dilution 1:75).
Presence of three separate rockets indicates electro-
phoretically distinguishable, immunochemically identical
antigens which may include IgG (1 and 2; Williams et al. ,
1975) and IgA. (C) Positive immunochemical response and
characteristic position of a UMS antigen which reacted
positively with rabbit anti-bovine IgA (Lot No. 23, Control
No. R054; Miles Research Products, Elkhart, IN; final
dilution 1:90). In each case (A, B, and C) whole, un-
fractionated UMS protein was electrophoresed. Separations
were performed toward the annode in 1% agarose (*'/v) in
barbital-tris-glycine buffer (pH 8.68).

-137-

^a;^-:

B

• 0 "-^^i©

^•. »*c>" ■ J -

V* •«:--i. :i

.: I'tJ^^^fe

•;,:.v;'*^

.W4.

:1^'

-138-

preparation was described for sheep by Bazer and coworkers (1979a).
Fetal weights and placental weights and fluid volumes measured in nine
cattle on day 270 (Table 2.1) were consistent with similar values
reported for cattle by Eley et al. (1978), Ferrell et al. (1976) and
Winters et al. (1942).

In cattle, the point at which significant retardation of pheno-
typic expression of the conceptus (embryo and extraembryonic membranes)
occurs as a consequence of limitations in the maternal component might
be defined as uterine capacity. In the present study, confinement of
the bovine conceptus largely to one uterine horn was not sufficient to
compromise uterine capacity as reflected by physical or endocrinological
responses measured. It was interesting, however, that three of 12 uni-
laterally pregnant cattle delivered calves, live at birth, prior to day
270. While every attempt was made to standardize placement of uterine
ligatures, it is conceivable that fetal-placental confinement in three
cows which delivered prematurely was sufficient to compromise uterine
capacity. Unilateral pregnancy can be established in both cattle
(present study) and sheep (Bazer et al., 1979a). These preparations
present intriguing models for study of uterine capacity in animals which
are generally monotocous and possess a cotyledonary placenta.

Development and general morphology of the bovine placenta was the
subject of numerous investigations (Bjorkman, 1968a, b; Greenstein et al.,
1958; Hafez and Rajakoski, 1964; King et al. , 1980, 1981; Kingman, 1948).
In the present study, morphology of placentae and placental attachments
(placentomes) appeared normal. Alexander and Williams (1966) suggested
that P, was required for normal caruncular development in sheep. Bovine
uterine arterial progesterone concentrations were shown to increase

-139-

systematically during gestation (Ferrell and Ford, 1980). In the
present study P, was detected in UMS (Table 2.2). Despite the apparent
potential endocrine support for maternal caruncular development, as
evidenced by detection of both free estrogen and P in UMS, caruncles
in the ligated uterine horn of unilaterally pregnant cattle were not
developed significantly and were perhaps only slightly more prominent
than those normally seen in the nonpregnant bovine uterus. These ob-
servations support the concept that a specific trophoblastic/chorionic
stimulus and/or chorion-endometrial interaction is required for normal
placentome development.

Observations in unilaterally pregnant sheep indicated that fluid
accumulated in the ligated uterine horn (Bazer et al. , 1979a) or uterine
pouch (Harrison et al. , 1976) during gestation. Moffatt and coworkers
(1980), following procedures of Bazer et al. (1979a), found ovine
uterine fluid within the ligated uterine horn as early as day 30 of
pregnancy. Copious amounts of fluid did not appear, however, until
after day 100 (Bazer et al., 1979a).

In the present study, UM was palpably detectable by day 166 and,
in one case, as much as 635 ml accumulated in the ligated uterine horn
by day 270 (Table 2.1). This period of rapid accumulation of bovine UM
(> day 166) corresponds well developmentally with a similar period
described above for sheep (Bazer et al., 1979a). In cattle, this period
of gestation was preceeded by a rapid increase in uterine arterial and
venous estrogen concentrations followed by increases in uterine blood
flow (Ferrell and Ford, 1980) and increased net nitrogen accretion by
the gravid uterus (Ferrell and Ford, 1980; Ferrell et al. , 1976). This
period of gestation also corresponds to the time of maximal fetal

-140-

growth rate (Eley et al., 1978; Winters at al. , 1942) and may be
associated with increasing circulating levels of bovine placental
lactogen (Bolander and Fellows, 1976). While no causal relationships
may be determined from the present study, observations agree with
those of Bazer et al. (1979a) and support the concept that circulating
agents of gonadal and/or placental origin may be important in the
integration of uterine secretory activity.

Sustenance of the conceptus during gestation depends largely upon
nutrients supplied by uterine endometrial epithelium, whereas sustenance
of the neonate, postpartum, depends upon nutrients supplied by mammary
gland epithelium. Transfer of function between these two tissues must
be coordinated to insure maximal reproductive success. As reviewed
above, bovine UM began to accumulate in significant quantities during
a period of gestation associated with dynamic changes in uterine hemo-
dynamics, fetal growth rate and maternal endocrine status. Onset of
accumulation of bovine UM in the ligated uterine horn after day 166
could well reflect responses of a secretory epithelium to circulating
agents present during late gestation. Additionally, however, this
phenomenon may reflect a more general response of maternal secretory
epithelia associated with integration of early events necessary not
only to insure maximal nutrient support to a growing conceptus ^Jl utero,
but also to insure smooth transition of nutritional support from uterus
to mammary gland at birth.

Concentrations of peripheral plasma steroids presented in Table
2.2 are similar to those reported by Collier et al. (1982), Erb et al .
(1968), Eley et al. (1981a, b) and Robertson and King (1979) for circu-
lating levels of plasma steroids in late pregnant cattle. Overall mean

.£t^

-141-

concentrations of unconjugated estrogens (E and E ) in UMS and plasma
were not different significantly, while E SO, and P, concentrations
were nearly 30 and ten times higher respectively in plasma than UMS
(Table 2.2).

Several lines of evidence suggest that the bovine placentome is
the major site of estrogen sjmthesis in late pregnancy (Thatcher et al. ,
1982). Hoffman and coworkers (1976, 1979) suggested that fetal and
maternal components of the placentome secreted estrogenic products into
their respective fluid compartments. In this respect, steroids found
in bovine UMS might be expected to reflect circulating plasma steroid
profiles since no fetal membranes were present in the ligated uterine
horn. Differences in steroid concentrations between UMS and plasma
could reflect metabolism of steroids by uterine tissues.

Eley (1980) observed that P, (pregn-4-ene-3, 20 dione) was not
converted to estrogens isi vitro by bovine endometrium but was shown to
be converted to predominantly 5a-pregnane base steroids (Knickerbocker
et al., 1980; Eley, 1980). Estrone sulfate was luteolytic when chroni-
cally administered to cyclic beef heifers suggesting the potential of
sulfatase activity in bovine reproductive tissues (Eley et al., 1979b).
Endometrial sulfatase activity was shown to increase during late gesta-
tion in both pigs and sheep (Dwyer and Robertson, 1979). From these
studies it seems unlikely that UMS concentrations of free estrogens
were affected by uterine metabolism of P, . If present, deconjugation
of E SO by a uterine sulfatase could account for E SO, concentration
differences between UMS and plasma (Table 2.2) and might account, in
part, for similar levels of free estrogens in the two fluid pools.
Similarly, endometrial conversion of P. to 5a-pregnanes would reduce

■,-ii

-142-

UMS P, concentrations. In fact P, in IMS was indeed less than in

"4 4

plasma. Alternatively, steroids might be in simple equilibrium between

UM and plasma. Heap et al. (1971) observed that ratios of P, :E

4

affected their relative solubilities in aqueous solutions and liposomes.
Lipid solubility of E was enhanced by P, , whereas P, solubility was
retarded by E .

Prostaglandin F concentrations and content in day 270 bovine UMS
(Table 2.4) agreed with values reported for similar uterine fluids
obtained from sheep (Bazer et al. , 1979a; Harrison et al. , 1972, 1976).
However, these values were several hundred to several thousand-fold
higher than those reported for uterine flushings collected either in
vivo or post-mortem from cyclic or early pregnant cattle (Bartol et al.,
1981a, b). Concentrations of PGF in day 270 UMS were several hundred-
fold higher than peak values reported by Knickerbocker et al. (1982) in
uterine venous plasma from diestrus cattle 6h following administration
of E (3 mg, I, v.). The presence of a concentrated pool of PGF in UM
supports the idea that specific endocrine conditions may permit intra-
uterine sequestering of prostaglandins (Bazer et al. , 1979a; Bazer and
Thatcher, 1977). Observations suggest the possibility of an active
mechanism associated with bovine uterine accumulation of PGF. A
mechanism for active transport of PGF in rabbit vaginal tissue was
described by Bito and Spellane (1974).

Overall, total protein concentrations and content in UMS (Table
2.4) were equal to and generally greater than values reported for ovine
uterine fluids obtained in a similar manner (Bazer et al. , 1979a;Harri-
son et al. , 1976) and bovine uterine fluid obtained following chronic P,
therapy (Dixon and Gibbons, 1979). Uterine milk protein content greatly

^^•'

-143-

exceeded those reported for bovine uterine flushings collected either
in vivo (Bartol et al. , 1981b; Schultz et al., 1971) or post-mortem
(Bartol et al . , 1981a, b; Roberts and Parker, 1974a, 1976; Olds and
Van Demark, 1957a,b) and establish bovine UM as a rich source of such
material.

Glucose was shown to be an important metabolite in uterine as well
as fetal and placental tissues in cattle, sheep, pigs and horses
(Battaglla and Meschia, 1978; Silver and Comline, 1975, 1976). Mean
uterine glucose uptake during late gestation in cattle was reported as
9.0 ± 0.8 mg/kg/min. with total utero-placental tissue uptake of 14.7
mg/kg/min. (Silver and Comline, 1975) . Glucose concentrations in bovine
uterine arterial and venous plasma were relatively constant throughout
gestation with no significant changes in uterine glucose A-V difference
between days 31 and 258 (Ferrell and Ford, 1980). The observed
increase in uterine blood flow noted between gestational days 92 and
199, however, (Ferrell and Ford, 1980) suggested potential for in-
creased utero-placental metabolism of glucose as gestation proceeded.

In the present study mean (± SEM) day 270 UMS glucose concentration
(0.127 ± 0.004 mg/ml; Table 2.4) was approximately five times lower than
similar values reported for uterine arterial or peripheral plasma from
cattle during late gestation (Ferrell and Ford, 1980; Grigsby et al.,
1974) , supporting the possibility of uterine metabolism of glucose.
Bovine UMS glucose concentrations were similar to those reported for
ovine uterine fluid (Bazer et al., 1979a). Also as reported for sheep
(Bazer et al. , 1979a) , fructose was not detected in bovine UMS (Table
2.4) although it appears as the principle carbohydrate of fetal blood
in cattle, sheep, pigs and horses (Battaglia and Meschia, 1978). This
further supports the nonplacental or conceptus source of UM .

-144-

Mean (± SEM) Ca concentration (28.35 ± 8.52 mg%) of day 270
bovine UMS (Table 2.4) was considerably higher than that reported for
peripheral plasma (9.0 - 10.3 mg%) or uterine flushings (0.0 - 5.3 mg%)
obtained from cyclic cattle (Heap, 1962; Schultz et al., 1971). As
much as 99% of total UMS Ca was dialyzable (Table 2.4) and likely
available as free Ca J^ utero. Uterine milk Ca concentration correla-
ted positively with both UMS protein concentration (r = .68, P < .09)
and content of PGF (r = .92, P < .08) suggesting an interrelationship
among these UM components. A rise in intracellular Ca concentration is
a key feature in the sequence of events referred to by Douglas (1968)
as stimulus-secretion coupling. The source of Ca may be either intra-
cellular or extracellular (Douglas, 1978). Extracellular Ca concentra-

-3
tions are generally higher than intracellular concentrations (10 M vs.

10 M; Rasmussen and Goodman, 1977). Concentrations of Ca in UMS (Table
2.4) fell in or slightly above the normal extracellular range. Douglas
(1978) observed that in exocrine cells, influx of extracellular Ca
could provide an intense stimulus for secretion. It was further ob-
served that such cells became increasingly dependent on extracellular
Ca as stimulation was prolonged (Douglas, 1978). The essential role of
Ca in activation of phospholipase A , essential for prostaglandin
synthesis, and accompanying alterations in plasma membrane phospholipid
turnover were identified as critical to the chain of events leading to
discharge of secretory product (exocytosis; Rubin, 1982). It was noted
that agents which increase Ca availability would be expected to stimu-
late prostaglandin synthesis and were associated with increased
exocytotic activity (Rubin, 1982) . Energy dependent mechanisms asso-
ciated with intracellular Ca efflux also were associated with regulation

-145-

of permeability of intercellular gap junctions (Rasmussen and Goodman,
1977). Correlations noted above between UMS Ca, PGF and protein may,
in this light, reflect the dynamic roles of these substances or their
precursors in establishment of this rich fluid pool.

Bovine UMS was enriched in proteins of less than 40,000 M (Figure
2.2). Gel filtration profiles were similar to those reported for
equine (Zavy et al. , 1979a, b) and bovine (Mills, 1975; Roberts and
Parker, 1974a) uterine flushings, but did not reveal a major peak
eluting in the 60 to 65,000 M range reported for ovine uterine fluid
(Bazer et al. , 1979a). Two dimensional-PAGE (Figure 2.3) of UMS pro-
teins revealed the majority of polypeptides less than 40,000 M to be
basic in nature, migrating on NEPHGE (Figure 2.2B) between pH 6.8 and
8.6. Observations of several investigators indicated that uterine-
specific proteins identified either in bovine uterine flushings (Roberts

and Parker, 1974a, 1976) or P, induced bovine uterine fluid (Dixon and

4

Gibbons, 1979) were less than 50,000 M and migrated well electrophore-
tically at pH 4.5 or below indicating products of a basic nature.
Additionally, major uterine-specific or P, induced uterine proteins
from the pig, sheep and horse were shown to be basic polypeptides on
2D-PAGE (NEPHGE; Bazer et al. , 1981b). The 2D-PAGE (NEPHGE) profiles
of bovine UMS (Figure 2.3B), however, were distinct from those of other
domestic species and did not reveal either a uteroferrin-like polypep-
tide, as described both for the pig and mare, or either of the two major

-3
polypeptides found in ovine uterine fluid (M X 10 = 57 and 59, lEP

- 8.4 - 8.6; Bazer et al. , 1979a, 1981b).

Comparison of 2D-PAGE (lEF) profiles of UMS and plasma (Figure

2.4) revealed several polypeptides apparently unique to UMS. One

-146-

group appeared in the 25 to 30,000 M range with isoelectric points
between 6.6 and 7.3, consistent with observations cited above. Two
other polypeptides (Figure 2.4A, UMl and UM2) had nearly identical
M (X 10 ) of 20 to 21, and approximate isoelectric points of 6.3 and
6.2, respectively. These polypeptides were nearly identical electro-
phoretically to those identified in porcine uterine secretions and
endometrial explant medium (Basha et al. , 1980a, b). Such electro-
phoretic properties were similar to those expected for retinol and
retinoic acid binding proteins (Adams et al., 1981). These progester-
one-induceable proteins were identified in uterine secretions of
luteal phase gilts and are assumed to be involved in transport of
Vitamin-A to the fetal-placental unit (Adams et al., 1981),

As shown in Figures 2.4 and 2.5, a large number of proteins in
bovine UMS were physically and immunochemically identical to proteins
found in bovine serum, Roberts and Parker (1974a) noted that no more
than 2% of total protein in bovine uterine flushings might be expected
to be uterine-specific. The predominance of serum-like macromolecules
in bovine uterine fluids should not be ignored. Such macromolecules
may play important roles in maintenance of intrauterine osmotic or
oncotic equilibrium with plasma and lymph and may be involved either
specifically or nonspecif ically in binding and/or transport of various
hormones and nutrients. Bovine serum albumin, identified in this and
other studies as a major component of bovine uterine fluid (Bartol
et al. , 1981a; Roberts and Parker, 1974a) may, for example, be present
in sufficient quantities in extracellular space to significantly alter
equilibrium distribution of steroids in cells (Clark and Peck, 1981) .
The observations of fewer serum-like proteins in bovine UMS than plasma

-147-

(Figure 2.5) were consistent with those of Roberts and Parker (1974a)
and support the possibility that certain serum components of UMS may
arrive through selective transport mechanisms.

Immunochemical identification of IgA, IgG and IgM in bovine UMS
(Figures 2.6 and 2.7) was consistent with identification of these
macromolecules in reproductive tract secretions of numerous domestic
and laboratory species as well as in humans (Corbeil et al. , 1981;
Wira and Sandoe, 1980; Vaerman and Ferin, 1974). These macromolecules
are thought to participate in the secretory immune system (Vaerman and
Ferin, 1974) as a first line of defense against bacterial infection.
The source of Ig in genital tract secretions was suggested to be
primarily fixed plasma cells which populate the lamina propria of
endometrial submucosa and produce specific Ig's (Vaerman and Ferin,
1974) . Transepithelial transport of diraeric IgA, IgG or pentameric
IgM was shown to require J-chain, sjmthesized by monocytes of the
lamina propria (Brandtzaeg and Baklien, 1977) , and secretory component
(SC) , synthesized by epithelial cells (Brandtzaeg and Baklien, 1977;
Vaerman and Ferin, 1974). Halsey et al. (1980) noted that even trans-
port of circulating Ig's into secretory fluids was mediated by SC
availability at the epithelial cell border. Epithelial synthesis of
SC was shown to require estrogens (Wira and Sandoe, 1980; Sullivan and
Wira, 1981). Uterine fluid content of Ig's would be affected, there-
fore, by relative local steroid levels (Wira and Sandoe, 1980; Vaerman
and Ferin, 1974). Though no specific roles other than those associated
with local immune response have been demonstrated for reproductive
tract Ig's, Ngah and coworkers (1982) recently reported bovine IgM to
be a major factor contributing to serum- induced head-to-head agglutina-
tion in bovine spermatozoa.

-148-

As previously observed, study of normal bovine uterine luminal
fluids has been hampered by availability of only small amounts of
material for analysis. In this respect, fluids suggested to contain
larger amounts of uterine-specific components, including allantoic
fluid (Roberts and Parker, 1976) and P induced uterine fluid (Dixon
and Gibbons, 1979), may provide reasonable alternative sources for
study. From the present study, however, it is apparent that copious
amounts of protein-rich UM can be obtained from unilaterally pregnant
cattle after days 166 to 180 of gestation. Components of this fluid
are likely representative of substances which would appear ±n_ utero
under normal physiological and endocrinological conditions of late
pregnancy. Constituents of bovine UM appeared to include substances of
serum origin as well as those either synthesized and secreted or pos-
sibly metabolized by uterine tissue, and were not directly contaminated
by products of the fetal-placental unit. Evaluation of polypeptides
produced by bovine endometrium using 2D-PAGE and fluorography (Horst and
Roberts, 1979; Laskey and Mills, 1975) would reveal those macromolecular
components contributed to UM specifically by endometrial S3mthesis and
release. Developmen tally, the pattern of bovine UM accumulation was
similar to that described for sheep (Bazer et al., 1979a; Harrison
et al. , 1976) suggesting common inductive mechanisms. Qualitatively,
however, major polypeptide components of bovine UM were unique and did
not reflect patterns seen in either porcine or ovine uterine fluids.
In addition to providing a rich source of uterine fluid, unilaterally
pregnant cattle present an intriguing model for study of bovine uterine
capacity, placental development and epithelial response to physiological
conditions of pregnancy.

CHAPTER III
CHARACTERIZATION OF BOVINE ENDOMETRIAL PROTEINS

Introduction

Recently, Mossman (1980) observed that, in placental mammals,
the burden of sustenance of the conceptus from the early morula or
blastocyst stage until parturition is borne largely by the uterine
endometrium. This is especially true of domestic farm species such as
the cow, sheep, horse and pig which possess chorioallantoic epithelio-
chorial placentae (Steven and Morriss, 1975; Wathes and Wooding, 1980)
that do not invade or erode maternal endometrium, and in which ectopic
(extrauterine) pregnancy does not occur. In these species, the uterus
might be considered a preferred site for conceptus development. In
this respect, components of the intrauterine environment of these
species must serve, collectively, as a unique, permissive, non-hostile,
complete medium in which the conceptus may develop. '

What little is known of protein components of the bovine intra-
uterine environment has come primarily from analyses of uterine
flushings (Bartol et al., 1981a; Roberts and Parker, 1974a, b, 1976),
progesterone- induced uterine fluid (Dixon and Gibbons, 1979) and uterine
milk obtained from unilaterally pregnant cattle (Chapterll) . In addi-
tion, some studies dealt with biochemical and/or iimnunochemical analyses
of bovine endometrial tissue homogenates (Laster, 1977) or explant
medium following incubation of bovine uterine tissues with radio-
labelled amino acids (Wathes, 1980). Consistent with observations in

-149-

-150-

other domestic species (Bazer et al. , 1981b), these studies indicated
that, while serum or serum-like proteins comprise a major proportion
of the total intrauterine protein milieu, a small but important pro-
portion of these proteins appear to be uterine-specific.

The array of polypeptides contributed to the bovine uterine
environment specifically by endometrial tissue has not been char-
acterized using high resolution techniques. Chromatographic and
electrophoretic techniques including two dimensional polyacrylamide
gel electrophoresis (2D-PAGE; Horst and Roberts, 1979; Horst et al.,
1980) and fluorography (Laskey and Mills, 1975) have permitted identi-
fication and characterization of both major and minor uterine-specific
polypeptides produced in vitro by both porcine and ovine endometrial
tissues (Bazer et al. , 1981b; Moffatt et al. , 1980; Roberts and Bazer,
1980).

It was the purpose of this study to characterize proteins (poly-
peptides) produced ^^ vitro by bovine endometrial tissue obtained from
pregnant and nonpregnant cattle.

Materials and Methods
Materials

Tissue culture supplies were purchased from Grand Island Biological

3 3

Co., Grand Island, NY; L-4,5- H-leucine ( H-Leu; Sp. act. 55 Ci/mmol)

L-1- C-leucine ( C-Leu; Sp. act. 50 mCi/mmol) and D-6- H-gluco-

samine hydrochloride ( H-Glc; Sp. act. 10-25 Ci/mmol) were obtained

from the Radiochemical Centre, Amersham, England; acrylamide, re-

crystalized according to procedures described by Loening (1967), N,N,

N' ,N'-tetramethylenediamine and X-Omat RP film XRP-1 were products of

-151-

Eastman Kodak, Rochester, NY; sodium dodecyl sulfate (SDS) and Nonidet
P-40 were from BDH chemicals Ltd., Poole, England; N,N'-diallyltartar-
diamide and Coomassie brilliant blue R-250 were from Bio-Rad
Laboratories, Richmond, CA; ampholines were from LKB, Uppsala, Sweden;
urea was from Pierce Chemicals, Rockford, XL; insulin was from Eli
Lilly; Lilly Research Laboratories, Indianapolis, IN; amino acids,
dithiothreitol, protein standards, 2-mercaptoethanol, trishydroxymethyl-
aminomethane (tris), glycine, glucose, sodium chloride (NaCl) and
agarose were from Sigma Chemical Co., St. Louis, MO; diethylaminoethyl
(DE-52; DEAE) cellulose and carboxymethyl cellulose (CM-52; CMS) were
products of Whatman Inc., Clifton, NJ; Sephacryl S-200 and Sepharose
CL-6B were purchased from Pharmacia Fine Chemicals, Piscataway, NJ. All
inorganic chemicals were reagent grade or better.

Preparation of Medium

Eagle's minimum essential medium (MEM) was prepared according to

procedures of Basha et al. (1979). Content of L-leucine was limited to

3
5.2 Ug/ml (1/10 normal) to enhance uptake of H-Leu added to endometrial

explants. Medium was supplemented with penicillin (200 units/ml) ,

streptomycin (200 pg/ml) , fungizone (0.5 Ug/ml) , insulin (0.2 units/ml),

nonessential amino acids (1%, /v) and glucose (5 mg/ml) . This medium

was filter sterilized (0.22 y) and stored at 4C until used.

Animals

Twenty-three cross-bred beef heifers were observed twice daily for
behavioral estrus and bred by natural service on the day of standing
estrus (day 0) . Cattle were then assigned to be slaughtered on day
16, 19, 22 or 24 post-mating. At slaughter each reproductive tract was

-152-

excised within 15 min. of exsanguination, trinmed of excess connective
tissue, the ovary containing luteal tissue noted, the cervix clamped,
and the tract transported to the laboratory on ice. Contents of each
uterus were flushed out with 20 to 80 ml of sterile isotonic saline
under a sterile laminar flow hood according to procedures similar to
those described by Bazer et al. (1978). Pregnancy was confirmed by
identification of conceptus tissues in the saline flush which, if
present, were transferred immediately to petri dishes containing 15 ml
sterile MEM and used in a separate study (Chapter IV) . Reproductive
tracts not containing conceptus tissues were designated nonpregnant.
Distribution of early pregnant and nonpregnant cattle is shown in Table
3.1. Additionally, uterine tissues were obtained from one cyclic (non-
bred) cow on day 4, one pregnant cow on day 69 of gestation, and from
both pregnant and ligated (nonpregnant) uterine horns of three unila-
terally pregnant cattle on day 270 of gestation (Chapter II) .

Preparation of Endometrial Explants

Uteri, obtained as described above, were opened along their anti-
mesometrial border to expose the uterine mucosa. Endometrial tissue
was then dissected carefully away from underlying muscular and connec-
tive tissues using sterile forceps and number 10 scalpel blades.
Tissue was recovered from both uterine horns and placed immediately into

sterile MEM. Aliquots of endometrial tissue from each uterine horn were

3
sliced into small pieces (approximately 2-4 mm ) using number 10

scalpel blades, blotted quickly on sterile gauze, weighed and approxi-
mately 500 mg of wet tissue placed into individual petri dishes

3

containing 15 ml of sterile MEM. Between 5 and 50 yCi of H-Leu was

then added to each explant dish. Explants of endometrium from

-153-

Table 3.1. Distribution of cattle.

No. Cattle

Day Post-estrus Pregnant Nonpregnant

16 4 4

19 6 2

22 3

24 4

total: 17

a „

estrus = day 0.

-154-

14 3

unilaterally pregnant cattle received 100 yCi C-Leu and 50 uCi H-Glc.

Procedures were carried out under a sterile laminar flow hood.

Explants, prepared as described, were placed into a controlled
atmosphere chamber (Bellco Biological Glassware, Vineland, NJ) , the
chamber flooded for 2-3 min. with a gas mixture consisting of 45% 0 :
50% N : 5% CO ( /v) and tissues incubated on a rocking platform
(Bellco Biological Glassware, Vineland, NJ) at 6 cycles/min. and 37C
for 24h. This procedure is essentially the same as that described by
Basha et al. (1979, 1980a) for explant culture of porcine endometrium.
The protocol insures that tissue will be exposed alternately to MEM and
the gaseous atmosphere. This technique was used successfully in several
different tissue explant systems (Trump et al., 1980). Lewis and
coworkers (1982) successfully employed an identical system to study
in vitro metabolism of arachidonic acid by bovine endometrium. This
system also was used to study synthesis and release of proteins in vitro
by ovine endometrium (Mof fatt et al. , 1980) and bovine fetal cotyledon-
ary tissue (Kensinger et al. , 1982), as well as, ovine and porcine,
peri-attachment conceptuses (Godkin et al. , 1982a, b).

At the end of the incubation period tissue and medium were
separated by centrifugation at 40 and 20,000g for 20 min. Medium

(supernatant) was dialyzed extensively against several thousand volumes

-3
of 10 mM tris-HCL buffer, pH 8.2 in Spectrapor 1 dialysis tubing (M xlO

cut-off- 6-8000; Spectrum Medical Industries, Los Angeles, CA) .

Comparison of content of radioactivity pre- and post-dialysis permitted

calculation of percentage of total DPM radiolabelled amino acid added

that was retained as nondialyzable material. Tissue and medium samples

were stored in separate glass vials at -20C until analyzed.

■155-

Synthesis and Release of Protein

To determine viability of the explant system for study of in vitro
production and release of polypeptides by bovine endometrium, 12
endometrial explant dishes were prepared from a single luteal phase
bovine uterus as described above. Each explant contained approximately

500 mg of endometrial tissue (X ± SEM = 498 ± 5.30 mg wet weight; n=12)

3
and received 10 \lC± H-Leu. Sample aliquots of meditam (225 yl) were

taken under aseptic conditions at 12h intervals for 48h (0, 12, 24, 36

and 48h) when the incubation was terminated. Sample aliquots of medium

were mixed with distilled water (1:9.5, /v) and dialyzed extensively

against 10 mM tris-HCL buffer, pH 8.2 as described above. Percent

3
retention of H-Leu as nondialyzable product as well as total DPM

3
H-Leu/ml of medium post-dialysis were determined for each explant at

each time period.

Gel Filtration Chromatography

Aliquots of dialyzed endometrial explant medium were subjected to
gel filtration chromatography on Sephacryl S-200 and selected S-200 void
volume (Vo) fractions applied to Sepharose CL-6B. Gel filtration was
conducted at 4C in 90 X 1.5 cm glass columns and 10 mM tris-HCL buffer,
pH 8.2 containing 0.33M NaCl. The K for each peak of nondialyzable
radioactivity was calculated as described by Reiland (1971) , and com-
pared with K values obtained for protein standards aldolase (M =:
av r

158,000), bovine serum albumin (BSA; M ^ 66,000), ovalbumin (M =
45,000) and ribonuclease A (M - 13,700) for S-200; and porcine IgM
(M =: 900,000), thyroglobulin (M ~ 669,000), ferritin (M - 440,000),
catalase (M - 232,000) and BSA (M - 66,000) for CL-6B. Void volumes
of both columns were determined using Blue dextran (M - 2 X 10 ;

-156-

Pharmacia Fine Chemicals, Uppsala, Sweden). Elution profiles were
generated by determining content of radioactivity in 100 yl aliquots
of each effluent fraction (Beckman LS-8000 Liquid scintillation spec-
trometer; Beckman, Palo Alto, CA) . Absorbance at 280 nM or, following
a Lowry protein assay reaction (Lowry et al., 1951), at 750 nM also was
determined for an aliquot of each fraction (Model 35 Spectrophotometer;
Beckman, Palo Alto, CA) .

Ion Exchange Chromatography

Materials retained in endometrial explant medium post-dialysis
which were not subjected to gel filtration or electrophoresis (dialyzed,
unfractionated products) were pooled by day post-estrus (pregnancy,
days 16, 19, 22 and 24; nonpregnant, days 16 and 19) and adsorbed onto
a 5 X 1 . 5 cm carboxymethyl cellulose (CMC) column equilibrated at 4C
in 10 mM tris-HCL buffer, pH 8.2. Proteins which did not bind to CMC
(CMC-) were eluted with a thorough wash of equilibration buffer. Bound
proteins (CMC+) were eluted with a 150 ml linear salt gradient (0.0 -
0.5M NaCl) . Fractions (2.6 ml) were collected throughout the elution
process. Content of radioactivity was determined for an aliquot (100 yl)
of each effluent fraction in order to generate elution profiles. Pro-
teins eluting in the CMC (-) peak were adsorbed onto a 10 X 1.5 cm DEAE
cellulose column equilibrated as described above for CMC. Elution of
DEAE (+) proteins was accomplished with a 200 ml linear salt gradient
(0.0 - 0.5M NaCl). Effluent fractions were collected and elution pro-
files generated as described for CMC.

Two Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) and Fluoro-
graphy ~~~ ~

Aliquots of unfractionated, dialyzed explant medium were lyophili-
zed and individual dried samples dissolved in a volume of 5 mM K CO

-157-

containing 9.4M urea, 2% C /v) Nonldet P-40 and 0.5% (^/v) dithio-
threitol, sufficient to allow 100 to 500 yg total protein, or 50,000 to
150,000 cpm nondialyzable radioactivity to be loaded onto each gel in
a total volume of not more than 150 jil. Endometrial tissue to be
examined by 2D-PAGE was suspended in 0.5 - 1 , 0 ml of 5 mM K CO and
9.4m urea and sonicated on ice at 20 to 50% probe intensity (Bronwill
Biosonic; Bronwill Scientific, Rochester, NY) for six 15 sec. periods
at 1 min. intervals. Sonicated endometrial tissue was brought to 2%
Nonidet P-40 ( /v) and 0.5% C/v) dithiothreitol, centrifuged at 18C
and 20,000g for 30 min. and an aliquot of the resultant supernatant
subjected to 2D-PAGE. Sample preparation, tissue solubilization and
electrophoretic separation of acidic and basic polypeptides by 2D-PAGE
were accomplished according to methods described by Horst and Roberts
(1979) and Horst et al. (1980). In the first dimension, polypeptides
of a more basic nature were separated by nonequilibrium pH gradient
electrophoresis (NEPHGE) , whereas polypeptides with isoelectric points
(lEP) in a more acidic range between 4.0 and 9.0 were separated by
isoelectric focusing. These procedures were described by Basha et al.
(1980a).

Following electrophoresis, Coomassie blue R-250 stained polyacry-
lamide slab gels were photographed, impregnated with sodium salicylate
(IM, 30 min.; Chamberlain, 1979), dried (Model 224 Gel Slab Dryer; Bio-
Rad Laboratories, Richmond, CA) and fluorographs prepared from Kodak
XRP-1 x-ray film according to procedures of Laskey and Mills (1975).
Fluorography of dried Coomassie blue R-250 stained 2D-PAGE gels per-
mitted visualization and identification of radiolabelled polypeptides
separated by 2D-PAGE. Fluorographs of endometrial tissue homogenates

-158-

primarily revealed structural and functional polypeptides into which
radiolabelled amino acids were incorporated during the incubation period
(24h) ; whereas fluorographs of dialyzed explant medium primarily re-
vealed polypeptides which were synthesized during the incubation period
and released into the medium. The latter group would represent the
array of polypeptides potentially synthesized and secreted by bovine
endometrium.

Protein Assay

Total protein concentrations in explant medium were determined by
the method of Lowry et al. (1951) using BSA (Fraction V; Sigma Chemical
Co., St. Louis, MO) as a standard.

Statistical Analyses

Where appropriate, data were subjected to analysis of variance
according to procedures available in the General Linear Models section
of S.A.S. (Barr et al. , 1979). Analyses of endometrial explant time
trial data considered variability due to individual explants with time

(h) as a continuous independent variable. Analysis of data from

3
thirty-four 50 yCi H-Leu-supplemented endometrial explants obtained

from seventeen pregnant cows on days 16, 19, 22 and 24 post-mating

(Table 3.1) considered variability due to day and cow. Comparisons of

similar explants from pregnant (n=20) and nonpregnant cattle (n=12;

days 16 and 19; Table 3.1) also considered variability due to pregnancy

status and appropriate interactions. Explant tissue wet weight was

included in analyses as a covariate.

-159-

Results
Synthesis and Release of Protein

The explant system described for this study has been used success-
fully by several investigators to examine the qualitative array of
macromolecules, presumably proteins, produced and released by both
endometrial and conceptus tissues (Basha et al. , 1979, 1980a; Moffatt
et al., 1980; Kensinger et al., 1982; Godkin et al., 1982a, b). In the
present study, dialyzed samples of MEM, obtained aseptically at 12h
intervals between 0 and 48h of culture from 12 bovine endometrial ex-
plants, contained increasing amounts of nondialyzable radiolabelled

3
macromolecules (Figure 3.1). Nondialyzable radioactivity ( H-Leu) in

MEM increased steadily during the first 24h and even more rapidly

thereafter until 48h when incubations were terminated. Data (Figure

3.1) were best described by a second order polynomial equation (Y =

2602.8 - 9.2190X + 6.5551X^; R^ = .98; P < .0001; Y = DPM of "^H-Leu;

3
X = hours of incubation). Percent retention of total H-Leu added as

nondialyzable product followed an identical curvilinear trend (Y =

0.6288 = 0.0008X + 0.0016X^; R^ = .97; P < ,0001; Y = Percentage of

3
total H-Leu; X = hours of incubation). Data indicate that bovine

endometrial explants, prepared as described above, can be successfully

maintained for periods in excess of 24h for recovery of macromolecular

(protein) products from explant medium. Tissue appeared viable

throughout the 48h incubation period.

Listed in Table 3.2 are responses (arithmetic means ± SEM) of 24h

endometrial explants prepared from early pregnant (n=34 explants) and

nonpregnant (n=12 explants) cattle (Table 3.1) which received 50 yCi

3

H-Leu. Mean (± SEM) tissue wet weight (TW) for all explants (n=46)

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-164-

was 524 ±4.93 mg. When tissue weight was considered as a covariate
and orthogonal contrasts made among days post-mating, overall total
protein (TP) concentrations (yg/ml) in dialyzed MEM were lower for day
19 than for day 22 and 24 explants from pregnant cattle (P < ,05; Table
3.2), and higher in explants from nonpregnant than pregnant cattle
(days 16 and 19 only; P < .01; 982.6 ± 60.68 vs. 771.8 ± 14.96 yg/ml) .
Additionally, a pregnancy-status by day post-mating (status X day)

interaction (P < .01) was detected (Table 3.2). Specific activity of

3 3

unfractionated, dialyzed proteins in MEM (DPM H-Leu/yg and DPM H-Leu/

yg/mg) was significantly affected by day post-mating in explants from

both pregnant (P < .10) and nonpregnant (P < .05) cattle, tending to

increase after day 16 (Table 3.2). Percent retention (%r) of total

3
H-Leu added (50 yCi) as nondialyzable radiolabelled molecules was not

affected (P > .10) by day post-mating in explants of pregnant endome-
trium. In contrast, %r was affected by day in explants of endometrium
from nonpregnant cattle (days 16 < 19; P < .01; Table 3.2), and a status
X day interaction was detected (P < .01). Percent retention was highest

in explants of endometrium from day 19 nonpregnant cattle (n=2; X ± SEM

3
%r = 10.7 ± 1.08%). Percent retention of H-Leu (50 yCi) in six endo-
metrial explants (X ± SEM of TW = 508.3 ±4.77 mg) from a single day

4 cyclic (non-bred) cow(X ± SEM) was 14.69 ± 0.402%.

14 3

Mean (± SEM) %r of C-Leu (100 yCi total added) and H-Glc (50

yCi total added) in explants (n=12; = TW = 500 mg) of endometrium from

ligated (n=6 explants) and pregnant (n=6 explants) uterine horns of

three unilaterally pregnant cows (day 270; Chapter 2) were: ligated,

6.4 ± 0.7% and 6.1 ± 0.6%; and pregnant, 6.4 ± 0.7% and 6.2 ± 0.7%.

14 3

Overall mean (± SEM) %r for C-Leu and H-Glc in all endometrial

-165-

explants (ligated and pregnant uterine horns) from day 270 unilaterally
pregnant cows were 6.4 ± 0.5% and 6.1 ± 0.5%. Data indicated that
endometrium from both ligated and pregnant uterine horns were equally
able to incorporate radiolabelled amino acids into nondialyzable pro-
ducts in vitro. Mean (X ± SEM) TP (yg/ml) in MEM from 12 explants was
538.2 ± 50.3.

Gel Filtration

Shown in Figure 3.2A is a typical S-200 gel filtration elution
profile obtained following chromatography of dialyzed bovine endometrial

explant medium. Absorbance of each fraction at 750 nM is shown by the

3
solid line and DPM H-Leu per fraction (100 yl) by connected symbols.

Three major peak areas of nondialyzable radioactivity were consistently

3 3

obtained including area I, M > 200 X 10 ; area II, 160 X 10 > M >

r r

3 3

45 X 10 ; and area III, M < 35 X 10 . Nearly identical S-200 elution

profiles were obtained following chromatography of MEM from all stages
of pregnancy. This is illustrated by comparison of Figure 3.2A with
a similar elution profile obtained from day 270 endometrial explant
medium shown in Figure 3.2B (absorbance of each fraction at 280 nM
shown in solid line) . Additionally, MEM from explants of endometrium
obtained from both ligated and pregnant uterine horns of unilaterally
pregnant cattle (Chapter 2) produced similar S-200 gel filtration elu-
tion profiles.

A representative elution profile obtained following application of
S-200 area I fractions to Sepharose CL-6B is presented in Figure 3.3.
A peak of nondialyzable radioactivity eluted ahead of the porcine IgM
standard (pIgM; M =: 900,000), for a molecular weight estimate in excess
of 1 . 0 to 1. 5 X 10 . A second area of high molecular weight endometrial

Figure 3.2. Comparison of representative Sephacryl S-200 gel filtra-
tion elution profiles of dialyzed MEM from day 19 pregnant
(A) and day 270 unilaterally pregnant (ligated uterine
horn) endometrial explants (B) . Absorbance of each
fraction (left axis) at 750 nM (A) or 280 nM (B) is
depicted by solid line. DPM (x 10 ^) -^H-Leu (A) or
%-Glc (triangles) and ^^C-Leu (squares) (B) per 100 yl
of each fraction are shown by connected symbols. Non-
dialyzable radioactivity consistently eluted in three
areas (1, II and III). M^. (x 10"^) estimates were: I,
Mj. > 200; II, 160 > M^. > 45; III, M^. < 35.

-167-

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protein eluted after the thyroglobulln standard (M - 669,000) but ahead
of catalase (M - 232,000), in the range (M X lO"^) of 340 to 669.

Together, gel filtration data indicated that, as assessed by gross
partitioning by M categories, bovine endometrium from all stages of
gestation (days 16, 19, 22, 24 and 270) produced similar arrays of

macromolecules ±n vitro. A fraction of these endometrial products

3
(S-200 area I) was of extremely high molecular weight (> 340 X 10 ) and

would likely not enter standard polyacrylamide gels for electrophoresis.

Ion Exchange Chromatography

Ion exchange chromatography (CMC and DEAE) of dialyzed endometrial
explant medium from cattle during early pregnancy (days 16, 19, 22 and
24) , as well as from nonpregnant cattle (days 16 and 19) produced nearly

identical elution profiles. As shown in Figure 3.4A, the majority of

3
nondialyzable H-Leu (solid line) as well as total protein (connected

sjmibols; assessed by absorbance of an aliquot of each fraction at 750

nM following Lowry protein assay; Lowry et al., 1951) was CMC (-) at

pH 8.2, eluting during the tris-HCL buffer wash prior to beginning of

the linear salt (NaCl) gradient. Only one minor CMC (+) peak was

consistently eluted during the gradient as shown on the expanded DPM

scale in Figure 3.4A.

When CMC (-) proteins were adsorbed to DEAE and eluted as described

3
above, elution profiles of nondialyzable H-Leu similar to that shown

in Figure 3.4B were consistently obtained. The majority of CMC (-)

proteins bound to DEAE at pH 8.2 and were eluted during the linear salt

gradient. Data suggested that bovine endometrial explants from early

pregnant and nonpregnant cattle produced primarily acidic (DEAE +; pH

8.2) macromolecules in vitro. Data were consistent with results of gel

Figure 3.4. Representative ion exchange chromatograms of dialyzed
bovine endometrial explant medium. (A) CMC-cation-
exchange profile of unfractionated explant medium.
CMC (-) proteins were eluted via an extensive wash with
10 mM tris-HCL buffer (pH 8.2; indicated by horizontal
line extending from origin to fraction 15). CMC (+)
proteins were then eluted with a 150 ml linear salt gra-
dient (O.OM - 0.5M NaCl; indicated by the diagonal line).
Absorbance at 750 nM (connected symbols) and DPM (x 10" )
^H-Leu/lOO yl of each fraction (solid line) are shown.
Note expanded DPM scale (inset) necessary to demonstrate
minor CMC (+) endometrial products. (B) DEAE-anion
exchange profile of CMC (-) , nondialyzable, bovine endo-
metrial proteins.

-172-

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BUCTtON NQ

-173-

filtration studies in that no major qualitative differences were de-
tected in ion exchange profiles of explant medium associated with
pregnancy status or stage of gestation.

2D-PAGE

Analysis of polypeptides present in bovine endometrial explant
medium and tissue homogenates was accomplished by 2D-PAGE (Horst and
Roberts, 1979; Horst et al. , 1980) to assess both basic (NEPHGE) and
acidic (lEF) components (Basha et al., 1980a). Fluorographs prepared
from dried, Coomassie brilliant blue R-250-stained 10% 2D-PAGE poly-
acrylamide slab gels revealed polypeptides into which radiolabelled
amino acids were incorporated during the 24h incubation period. Hence,
fluorographs prepared from 2D-PAGE gels of unfractionated, dialyzed
explant medium revealed those polypeptides which were synthesized and
released during incubation. In contrast, fluorographs prepared from
2D-PAGE gels of endometrial tissue homogenates revealed those structural
and functional proteins or polypeptides into which labelled amino acids
were incorporated. Comparison of fluorographs prepared from MEM and
tissue homogenates, respectively, permitted assessment of major and minor
polypeptides which were not retained within tissue, but released into
MEM (secreted) during incubation. This array of polypeptides should
include major bovine endometrial secretory products.

Shown in Figure 3.5A and B are representative Coomassie brilliant
blue R-250-stained 2D-PAGE gels depicting the array of basic (NEPHGE;
Figure 3.5A) and acidic (lEF; Figure 3.5B) polypeptides seen in dialyzed
bovine endometrial explant medium. Patterns were similar to those
described for bovine uterine milk and peripheral plasma (Chapter 2) and
included BSA as a major component on lEF gels (Figure 3.5B). Such

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serum components were likely leached from tissues during incubation.
Fluorographs prepared from gels shovm in Figure 3.5A and B are presented
in Figure 3.5C and D, and may be compared with fluorographs of basic
(NEPHGE) and acidic (lEF) polypeptide components of the same endometrial
tissue homogenate shown in Figure 3.5E and F respectively.

As shown (Figure 3.5C and D) , bovine endometrium was capable of
producing a complex array of polypeptides in vitro. The array of
radiolabelled polypeptides identified on fluorographs of unfractionated,
dialyzed explant medium (Figure 3.5C and D) was consistent with results
of gel filtration and ion exchange chromatography studies. Products in
MEM were present across the entire molecular weight range resolvable by
10% polyacrylamide gels (approximately 14,000 to 400,000), and the
majority of labelled polypeptides were seen on fluorographs of lEF
2D-PAGE gels (Figure 3.5D) indicating production of primarily acidic
type proteins. Additionally, some very high molecular weight polypep-

tides (M X 10 > 400) were identified on fluorographs of unfractionated

r

MEM (lEF and NEPHGE; Top, Figure 3.5C and D) . These macromolecules were
seen at the interface of 4% stacking and 10% running gels, or a streaks
in the stacking gel. Hence, radiolabelled endometrial products were
present in MEM which were not resolvable by 2D-PAGE.

Obvious absence of BSA from fluorographs of 2D-PAGE (lEF) gels of
polypeptides in MEM (Figure 3.5B vs. 3.5D) supports the contention that,
while significant amounts of serum proteins may have leached from
tissues during incubation, products identified on fluorographs represent
polypeptides which were synthesized during culture and released
(secreted) into MEM. Polypeptides with 2D-electrophoretic characteris-
tics typical of immunoglobulin (Ig) heavy and light chains (Anderson

-177-

and Anderson, 1977; Latner et al., 1980) were also prominent on
Coomassie-stained 2D-PAGE (lEF) gels of MEM proteins (Figure 3.5B),
but absent on fluorographs (Figure 3.5D). If produced locally, Ig are
thought to be synthesized by fixed plasma cells in the lamina propria
of endometrial submucosa (Vaerman and Ferin, 1974). Such cells may have
constituted an insignificant proportion of the total tissue mass in
culture. Alternatively, recent evidence (Sullivan and Wira, 1983)
indicated that, in mice, rapid increases in uterine tissue content of
IgA, IgG and IgM occurred primarily as a consequence of serum transuda-
tion. Hence, like BSA, Ig's may have simply leached from tissues during
culture.

Careful comparison of fluorographs of unfractionated MEM (Figure
3.5C and D) with those prepared from tissue homogenates (Figure 3.5E
and F) indicated that polypeptides in at least four molecular weight
(M )/pH (lEP) classes were absent or present in only trace amounts in
tissue, but enriched in explant medium. Products of a more basic nature
in this category (class I) included two polypeptides, identified at the
dye front on both NEPHGE and lEF 2D-PAGE fluorographs (M - 14,000) in
a pH range greater than 7.2 (Figure 3.5C and D) . Molecular weight
(M X 10 )/pH (lEP) ranges of other endometrial polypeptide classes
included: class II, 19-24/5.4-6.3; class III, 28-31/6.9-7.3; and class
IV, > 150/< 5.1. These areas (classes I, II, III and IV) are identified
on the fluorograph of polypeptides in MEM shown in Figure 3.6.

Polypeptides in class I (Figure 3.6) were of a similar size, al-
though somewhat less basic, than several progesterone induced porcine
endometrial-specif ic serine protease inhibitors (Mullins et al., 1980a;
Fazleabas et al., 1 982a, b). Bovine endometrial-specif ic polypeptides in

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-180-

classes II and III (Figure 3.6) displayed electrophoretic mobility
similar to several uterine-specific polypeptides identified in UM from
unilaterally pregnant cattle (Chapter II) . Products in class IV
resembled polypeptides thought to be associated with the porcine endo-
metrial epithelial cell brush border.

Fluorographs prepared from 2D-PAGE lEF gels of endometrial explant
medium from a day 16 nonpregnant cow (Figure 3.7A) and day 24 and 69
pregnant cattle (Figure 3.7B and C) are presented for comparison in
Figure 3.7. Comparison of individual fluorographs prepared from 2D-PAGE
gels (lEF and NEPHGE) of endometrial explant medium from early pregnant
and nonpregnant cattle (Table 3.1), one day 4 cyclic and one day 69
pregnant cow, and from pregnant and ligated uterine horns of three day
270 unilaterally pregnant cattle (Chapter II) failed to reveal any
obvious qualitative differences in array of polypeptides synthesized by
bovine endometrium associated with pregnancy status or stage of gesta-
tion. Polypeptides in all classes described above were produced and
released in vitro during the 24h incubation period by endometrial
tissues from cyclic cattle on days 4, 16 and 19, and pregnant cattle on
days 16, 19, 22, 24 and 69, as well as by tissue from both ligated and
pregnant uterine horns of day 270 unilaterally pregnant cattle. Methods
employed in the present study did not permit quantitative assessment of
polypeptides produced in vitro by bovine endometrium. Data suggested,
however, that quantitative rather than major qualitative alterations
might be associated with changing reproductive status.

Discussion

In the present study it was demonstrated that bovine endometrial
tissue explants could be maintained in vitro for periods of at least

Figure 3.7. Fluorographs prepared from 2D-PAGE (lEF) gels of poly-
peptides in bovine endometrial explant MEM. Shown are
polypeptides synthesized d_e novo from H-Leu substrate,
and released into MEM by endometrial tissues from: (A)
a day 16 nonpregnant cow; (B) a day 24 pregnant cow; and
(C) a day 69 pregnant cow.

-182-

PH

8.9 8.4 7.9 7.3 6.8 6.4 6.2 5.8 5.5 5.1 4.4 3.6

IE F

■»- +

-183-

24 to 48h as assessed by incorporation of radiolabelled amino acids
into nondialyzable product (Figure 3.1). The procedures employed were
essentially the same as those described by Basha et al. (1979, 1980a)
for explant culture of porcine endometrium and were used previously to
study bovine endometrial metabolism of arachidonic acid (Lewis et al.,
1982) . This system was also successfully employed to examine synthesis
and release of proteins in vitro by ovine endometrium (Moffatt et al.,
1980), bovine fetal cotyledonary tissue (Kensinger et al., 1982), and
ovine and porcine peri-attachment conceptuses (Godkin et al., 1982a, b).
These studies consistently demonstrated that explant medium was enriched
in radiolabelled macromolecules which were absent, or present in only
trace amounts in tissue homogenates. This suggests that methods em-
ployed in the present study provide excellent means for obtaining and
identifying major bovine endometrial secretory products Jji vitro.

Medium from endometrial explants prepared from early pregnant

3
cattle tended to contain TP of higher specific activity (DPM H -Leu/

3

yg TP and DPM H -Leu/yg TP/mg TW) if tissues were recovered on day 24

(Table 3.2). This observation is consistent with earlier biochemical
and histochemical data, generated from bovine uterine flushings and
tissues from cattle of similar reproductive status, which indicated
general stimulation and persistency of accumulation and/or production
of uterine-specific luminal proteins during periods of chronic proges-
terone (P,) exposure such as occur during diestrus and early pregnancy
(Schultz et al., 1971; Roberts and Parker, 1974a, b, 1976; Leiser and
Wille, 1975; Linford and losson, 1975; Wathes, 1980; Bartol et al. ,
1981a; Libby et al. , 1982). Absence of significant effects of day
post-mating on %r for explants of endometrium from early pregnant cattle

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(Table 3.2) may reflect metabolic stability of these tissues. Such
stability would be important during early pregnancy to insure consistent
production of histotrophe and other substances required for support and
maintenance of a conceptus.

In contrast to relatively stable responses of endometrial explants
from early pregnant cattle, explants from nonpregnant cattle (days 16
and 19; Table 3.2) differed in response depending upon day of tissue
recovery. Both specific activity of dialyzed MEM proteins and %r were
higher in day 19 than day 16 explants (Table 3.2), and significant
(P < .01) pregnancy status X day post-mating interactions were detected
for both TP and %r. Interactions reflected elevated responses of ex-
plants from day 19 nonpregnant cattle whereas explants of tissue from
pregnant cattle maintained a stable but lower response (Table 3.2).

Endometrial tissues in short term explant culture appear to reflect
metabolic state of tissues as they existed jji vivo prior to excision.
Attempts to modify activity of cultured endometrium by addition of
steroids in vitro have not been successful. Basha et al. (1979) ob-
served that treatment of porcine endometrial explants with P and/or
estradiol-176 (E ) failed to alter qualitative patterns of proteins
produced in vitro. Wathes (1980) reported that neither steroid treat-
ment (P/> E or both) nor coincubation of bovine uterine tissues with
fetal-placental tissues altered the qualitative array of uterine pro-
teins produced in culture. Similarly, Lewis et al. (1982) reported no
significant alteration in production or metabolism of prostaglandins
(PG) by explants of bovine endometrium following addition of E to
cultures.

Since uteri of cattle designated nonpregnant did not contain con-
ceptus tissues at the time of slaughter, these animals could be

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considered cyclic. Hence, by day 19 post-estrus, nonpregnant cattle
would be in either late diestrus or early proestrus (Hansel et al. ,
1973; Ford et al., 1979). Ford et al. (1979) reported that in cyclic
cattle, peripheral plasma concentrations of P, declined after day 17
(estrus = day 0) as E concentrations increased; while in pregnant
cattle, P, concentrations were consistently higher than E concentra-
tions after day 16 until day 30, the latest day considered. The

3
increase in production of labelled proteins and %r of H-Leu by explants

of endometrial tissues from day 19 as compared with day 16 nonpregnant

or day 16 and 19 early pregnant cattle (Table 3.2) may, therefore,

reflect 2£. vitro responses of tissues which were exposed to a higher

E„/P, ratio in vivo.
2 4

Increased E./P, ratio is associated with increased uterine meta-
2 4

bolic activity. Bovine and ovine endometrial epithelial cells from
late diestrus or proestrus were observed to contain larger amounts of
smooth endoplasmic reticulum and larger mitochondria when compared with
cells from earlier in the estrous cycle (Kojima and Selander, 1970;
Wathes and Wooding, 1980; Hoyes, 1972). Administration of exogenous E
during diestrus stimulated uterine metabolic activity as reflected by
both increased uterine blood flow and PG synthesis in cattle (Roman-
Ponce et al., 1978; Bartol et al., 1981a; Knickerbocker et al. , 1982),
sheep (Huckabee et al., 1970; Warren et al., 1973) and pigs (Dickson
et al., 1969; Frank et al. , 1978).

In cattle, several studies suggested that endocrine conditions
which enhanced uterine PG production, a calcium (Ca) dependent process
involving arachidonic acid (AA) as principle substrate (Ramwell et al.,
197 7), were related if not coupled to production and/or accretion of

-186-

uterlne proteins (Bartol et al. , 1981a, b; Knickerbocker et al., 1982).
Increases in endometrial concentrations of AA and PGF (Hansel et al. ,
1975; Shemesh and Hansel, 1975b) , followed by increased uterine luminal
content of PGF were associated with increases in amount and frequency
of appearance of uterine luminal proteins in cyclic cattle (Bartol
et al., 1981a).

Rubin (1982) suggested that products generated as a consequence of
Ca-dependent turnover of AA within the cellular phosphatidyl inositol
pool might be involved with cellular processes governing discharge of
secretory products. Volpi et al. (1980) indicated that metabolites of
AA might participate in mechanisms regulating cellular Ca gating by
acting as endogenous Ca ionophores. Agents which increase Ca availa-
bility were associated with increased exocytotic activity (Rubin, 1982).
Metz et al. (1982) recently proposed that metabolism of AA could yield
a feed-forward intermediate(s) involved in the sequence of events
leading to release or secretion of cellular products.

In light of these observations it does not seem unreasonable to
suggest that endometrial tissues obtained from nonpregnant cattle in
late diestrus, a period associated with increasing E and decreasing
P., might well possess an increased capacity for synthesis and/or re-
lease of macromolecular products ^^1 vitro. By comparison, the systemic
effect of chronic peripheral plasma P, combined with the local effect
of the conceptus itself appears to have a stabilizing effect on uterine
tissues which may be necessary to insure continued secretion of endome-
trial products required for support of the conceptus.

Percent retention of radiolabelled amino acids was nearly identical
in explants of endometrium from ligated and pregnant uterine horns of

-187-

day 270 unilaterally pregnant cattle. This observation supports the
contention that the ligation procedure and subsequent accumulation of
UM did not adversely effect metabolic capacity of endometrial tissues
as assessed in vitro by the present study. Results were generally
consistent with those of Moffatt et al. (1980) from unilaterally
pregnant sheep. Bovine endometrial explants from all reproductive
statuses examined possessed the capacity to incorporate radiolabelled
amino acids into nondialyzable products.

To date, aittempts to identify bovine uterine-specific proteins
have involved primarily comparisons of chromatographic and electro-
phoretic profiles of proteins in uterine flushings or fluids with those
in peripheral plasma or serum (Roberts and Parker, 1974a,b, 1976;
Laster, 1977; Dixon and Gibbons, 1979; Libby et al., 1982; Chapterll) .
Such studies consistently demonstrated that the majority of bovine
uterine luminal proteins were of serum origin or serum-like. Uterine-
specific proteins were defined as those consistently absent from serum,
but present in uterine fluids. Therefore, uterine-specific products
with similar chromatographic properties to serum proteins might have
been overlooked. Additionally, since bovine uterine-specific proteins
were suggested to comprise less than 3% of total recoverable uterine
luminal proteins (Roberts and Parker, 1974b, 1976; Laster, 1977),
amounts of uterine-specific proteins recovered in uterine flushings may
have been insufficient to permit identification of the complete array
of uterine products.

In the present study, short term (24h) explant culture of endo-
metrial tissue from cyclic, pregnant, nonpregnant and unilaterally
pregnant cattle in presence of radiolabelled amino acids permitted

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recovery and characterization of radiolabelled macromolecules produced
by tissues and released into MEM. This approach allowed a relatively
concentrated pool of proteins to accumulate in explant medium during
the culture period. Serum proteins, which may have leached from
tissues but were not produced by them, were radio inert, as demonstrated
by the absence of BSA on fluorographs of explant medium (Figure 3.5).
Radiolabelled proteins and polypeptides identified in dialyzed MEM
therefore, clearly represented bovine uterine endometrial products
produced d_e novo in culture.

Chromatographic techniques including gel filtration (Figure 3.2)
and ion exchange (Figure 3.3), as well as the high resolution technique
of 2D-PAGE and fluorography (Figures 3.5, 3.6 and 3.7) revealed consist-
ently repeatable patterns of radiolabelled proteins and polypeptides in
MEM. Data indicated that bovine endometrial tissue is capable of
producing a much more complex array of macromolecules than was pre-
viously believed. Additionally, no obvious differences were noted in
the qualitative array of proteins or polypeptides associated with
pregnancy status (pregnant vs. nonpregnant) or stage of gestation (days
16, 19, 22, 24, 69 and 270). Observations suggest that alterations in
endometrial-specif ic components of the bovine uterine environment may
reflect quantitative rather than qualitative changes in uterine pro-
teins. Data are generally consistent with observations from other
domestic species including pigs, sheep and mares which indicated that
a relatively constant array of proteins is produced by or present in
the uterine lumen throughout gestation or during prolonged periods of
P treatment (Bazer, 1975; Bazer et al., 1981b).

Gel filtration chromatography (S-200 and CL-6B; Figures 3.2 and
3.3) of proteins in endometrial explant medium revealed several very

-189-

high molecular weight proteins which were not resolvable by 2D-PAGE.

These products eluted in the void volume on S-200 (area I M > 200 X

r
3
10 ) and, when applied to CL-6B, eluted as two broad areas with weight

estimates of greater than 1.0 to 1.5 X 10 and between 340 and 669 X 10
respectively. Gel filtration (G-200) of uterine flushings from early
pregnant and cyclic cows revealed trace amounts of a very high molecular
weight, slightly hexose-rich component (Roberts and Parker, 1974b).
Proteins eluting in or near the void volume of G-200 (M X 10 > 200)
were also reported for porcine (Murray et al., 1972) and equine uterine
flushings (Zavy et al. , 1979a, b),as well as for bovine UM (Chapterll) .
Hence, very high molecular weight proteins and/or glycoproteins may
comprise a significant proportion of uterine- specific products in
several domestic species.

The majority of radiolabelled proteins in dialyzed explant medium
were CMC (-) at pH 8.2, but bound to DEAE at this pH (Figure 3.4) in-
dicating production of primarily acidic macromolecules. Consistent
with these findings, fluorography of 2D-PAGE gels (lEF and NEPHGE) of
proteins in explant medium (Figures 3.5 and 3.7) revealed the majority
of uterine-specific polypeptides to be separated over a wider more
acidic pH range on lEF gels. Polypeptides identified on fluorographs
of NEPHGE gels were present only on the least basic side (pH < 7.6)
and were, in fact, resolvable by lEF 2D-PAGE (Figure 3.5). Noticeably
absent from all NEPHGE fluorographs, including those prepared from
2D-PAGE gels of day 270 MEM from both ligated and pregnant uterine
tissues, were the very basic (pH > 7.3) low M polypeptides identified
as major components of bovine UM (Chapter 2; Figure 2.3). These data
suggest that low M basic polypeptides in UM were either enzymatic

-190-

degradation products, or were delivered to UM from serum. Bovine serum
albumin and Ig heavy and light chain polypeptides, as well as other
serum-like proteins (Anderson and Anderson, 1977), were prominent
components of dialyzed MEM as assessed by Coomassie-blue-stained 2D-PAGE
(lEF) gels (Figure 3.5B). Patterns were similar to those described for
bovine UM (Chapter II; Figure 2.3) in which IgA, IgG and IgM were iden-
tified immunochemically (Chapter II; Figures 2.6 and 2.7). However,
neither BSA nor the Ig polypeptide chains (prominent serum components
in MEM) appeared as prominent radiolabelled components of MEM as
assessed by fluorography of 2D-PAGE (lEF) gels of polypeptides from day
270 endometrial explants (ligated and pregnant uterine horn tissues)
or explants from any earlier stage or pregnancy status (Figures 3.5,
3.6, 3.7). Data suggest that such proteins appear in utero, and as
components of bovine UM, primarily via transudation from serum and do
not represent major uterine tissue products. Bovine endometrial explants
did not produce polypeptides similar to either uteroferrin, a major
progesterone- induced porcine endometrial specific glycoprotein (Roberts
and Bazer, 1980), or the 50,000 to 55,000 M basic protein identified
as the major ovine endometrial product (Moffatt et al. , 1980; Bazer
et al. , 1981b). In this respect the cow would appear to be functionally
unique from the gilt, ewe and mare in that these species were all shown
to produce or possess major basic proteins as uterine-specific products
(Bazer et al. , 1981b), while the bovine uterus appears to synthesize
primarily more acidic polypeptides.

In the present study comparison of fluorographs of unfractionated

MEM with those prepared from tissue homogenates (Figure 3.5) revealed

_3
polypeptides in at least four M (X 10 )/pH classes (I, II, III and IV)

-191-

which were enriched in MEM (Figure 3.6). Two distinct polypeptides in
class I (- 14/> 7.2) were of similar size although somewhat less basic
than a series of low M , basic, serine-protease inhibitors. These were
identified as P, induced products of porcine endometrium and thought to
be involved in limiting invasiveness of the peri-attachment trophoblast
(Mullins et al., 1980a; Fazleabas et al. , 1982a, b). Polypeptides in this
size class were also described as uterine-specific components of bovine
uterine flushings (Roberts and Parker, 1976) . Polypeptides in class II
(19-24/5.4-6.3) included two products with similar electrophoretic
properties to retinol and retinoic acid binding proteins (Adams et al. ,
1981; Bazer et al., 1981b). These binding proteins were shown to be P,
induced products of porcine endometrium and were suggested to be in-
volved in transport of Vitamin A to the porcine conceptus (Adams et al. ,
1981). Two polypeptides with nearly identical electrophoretic charac-
teristics to the porcine Vitamin A binding proteins were also identified
as major UM-specific polypeptides (Chapterll; Figure 2.4, UMl and UM2) .
Several polypeptides with electrophoretic properties similar to those
of class III uterine-specific polypeptides (28-31/6.9-7.3) were also
found to be UM-specific (Chapterll, Figure 2.4). Identification of
polypeptides in classes II and III on fluorographs of MEM gels from all
reproductive statuses, including those from day 270 ligated and pregnant
uterine tissue explants, indirectly supports the concept that endome-
trial tissues of unilaterally pregnant cattle were involved in active
synthesis and secretion of uterine-specific products. Bovine UM may
therefore provide a rich source of uterine-specific proteins for future
inve s t iga t ion s .

The array of polypeptides identified in class IV (> 150/< 5.1;
Figure 3.6) displayed similar characteristics to those described for a

-192-

plasma membrane-enriched fraction from porcine endometrium (Mull ins
et al., 1980b). This membrane fraction was enriched in several brush
border marker enzymes including alkaline phosphatase, leucine amino-
peptidase and 5 '-nucleotidase (Mullins et al., 1980b). These porcine
endometrial brush border -associated polypeptides were lee tin-binding
glycoproteins and, like bovine class IV polypeptides, were present in
cyclic and pregnant as well as pseudopregnant gilts. Moss et al. (1954)
detected alkaline phosphatase activity associated with the distal
border of bovine uterine epithelial cell surfaces throughout the
estrous cycle. Similar activity was detected in glandular epithelium
and in glandular secretions (Moss et al. , 1954). Histochemical studies
indicated intense increases in endometrial surface alkaline phosphatase
activity associated with the precontact stage of bovine conceptus
attachment (Leiser and Wille, 1975) .

Surface of brush border associated glycoproteins such as alkaline
phosphatase were suggested to be involved in cellular processes impor-
tant to establishment and maintenance of pregnancy. Considerable
evidence exists to support involvement of such molecules in cell-cell
adhesion and fusion (Frazier and Glaser, 1979). Cell fusion may be
important during early pregnancy when functional fetal-maternal asso-
ciations are first established. Fusion of trophoblast with endometrial
cells was described for the cow (Wathes and Wooding, 1980), goat (Dent,
1973) and sheep (Wooding et al., 1980). Alkaline phosphatase was
suggested to be involved with endometrial carbohydrate metabolism
(Larson et al. , 1970; Boshier, 1969) and stimulation of uterine
secretory activity (Larson et al. , 1970; Murdoch, 1970, 1972) necessary
to insure continued production of histotrophe (Leiser and Wille, 1975).

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The wide range of molecular weight and pH over which bovine
uterine-specific polypeptides were identified on fluorographs of
2D-PAGE gels (Figures 3.5, 3.6 and 3.7) was not, in general, incon-
sistent with earlier reports of bovine uterine-specific proteins,
identified by various single dimensional techniques, in uterine flush-
ings, fluids, tissues or explant medium (Roberts and Parker, 1974a, b,
1976; Libby et al., 1982; Bartol et al . , 1981a; Dixon and Gibbons,
1979; Laster, 1977; Wathes, 1980). While many uterine-specific poly-
peptides identified in the present study possessed physiochemical
characteristics similar to proteins of known biological function, no
specific functions have been definitively demonstrated for any isolated
bovine uterine-specific protein. Potential functional roles for such
molecules, however, are many and, as noted above, may include
(1) control of trophoblast invasiveness, (2) binding and transport of
nutrients and hormones, (3) stimulation of uterine secretory activity,
(4) mediation of conceptus-matemal tissue interactions, and (5) regu-
lation of maternal immune response to the conceptus, as well as serving
as part of the general histotrophe (Bazer, 1975; Bazer et al., 1981b;
Beer and Billingham, 1979; Segerson et al. , 1982). Characterization of
the diverse array of polypeptides produced by bovine endometrial tissue
in vitro sets the stage for future investigations directed toward
isolation and identification of function of the major bovine uterine-
specific macromolecules.

Absence of obvious differences in qualitative array of polypeptides
produced in vitro by endometrial tissues from pregnant, nonpregnant and
cyclic cattle (Figure 3.7) suggests that reports of pregnancy-specific
bovine uterine proteins (Laster, 1977; Bartol et al. , 1981a) reflect

-194-

contributions made to the uterus or uterine environment by the con-
ceptus. Conceptus contributions to the uterine environment are, no
doubt, critical for establishment and maintenance of pregnancy. As
observed by Beier and Mootz (1979) , it is the balance of interactions
between the complete array of both maternal and conceptus products
in utero that ultimately determines success or failure of a pregnancy.
In order to begin to understand dynamics of the intrauterine environ-
ment that permit establishment and maintenance of pregnancy it will
be important to identify and characterize contributions of conceptus
as well as maternal units.

CHAPTER IV

CHARACTERIZATION OF PROTEINS PRODUCED IN VITRO BY

PERI-ATTACHMENT BOVINE CONCEPTUSES

Introduction
Amoroso and Perry (1975) noted that, from an evolutionary
standpoint, adaptation of eutherian embryos to prolonged uterine ges-
tation must have required development of self-sufficiency with respect
to production and secretion of complex biologically active molecules
by the conceptus (embryo and extraembryonic membranes) . In large
domestic species including cattle, sheep and pigs, in which ectopic
pregnancy does not occur, presence of conceptus tissues in utero prior
to (estrus = day 0) days 17 (Northey and French, 1980; Sreenan, 1978;
Betteridge et al., 1978), 12 (Moore and Rowson, 1966a, b,c) and 13,
respectively (Dhindsa and Dziuk, 1968; Bazer et al. , 1982), results in
prolongation of the estrous cycle as a consequence of maintenance of
functional ovarian corpora lutea (luteostasis) . A central phenomenon
associated with maternal recognition of pregnancy, luteostasis is only
one of several conceptus induced or mediated events. Others include
establishment of immunological privilege and induction of histotrophe,
which must occur if pregnancy is to be established and maintained (Cook
and Hunter, 1978; Beer and Billingham, 1979). Clearly, therefore,
conceptuses of these species must acquire the ability to produce and
secrete biologically active molecules very early in development which
effect a myriad of physiological processes essential to their sustenance
throughout gestation.

-195-

-196-

Of the biologically active substances known to be produced by
peri-attachment conceptuses of large domestic animals, steroids,
especially estrogens, play a major role in establishment of pregnancy
in the pig (Bazer et al., 1982). Production of steroids by conceptus
tissues was investigated in sheep (Heap et al. , 1981; Gadsby et al.,
1980) and to a greater extent in cattle (Shemesh et al. , 1979; Eley
et al., 1979a; Eley et al. , 1983; Gadsby et al., 1980; Knickerbocker
et al., 1980; Chenault, 1980; Thatcher et al. , 1980), but no
definitive roles for these conceptus products have been established.
Production of prostaglandins (PG) of both F and E series was demon-
strated in pre-attachment bovine conceptuses (Lewis et al., 1982;
Shemesh et al., 1979). The latter (PGE ) was suggested to be involved
with conceptus induced antiluteolytic effects in sheep (Pratt et al. ,
1977; Ellinwood et al. , 1979b;Huie et al., 1981; Reynolds et al. , 1981).

Conceptus produced proteins were suggested to play important roles
in maternal recognition of pregnancy in both sheep (Martal et al., 1979)
and cattle (Beal et al. , 1981) and protein extracts from pig conceptuses
were shown to bind to LH receptors in porcine luteal tissue (Saunders
et al., 1980). Recently, Godkin and coworkers (1982a, b) reported that
peri-attachment stage porcine and ovine conceptuses were capable of
producing complex arrays of polypeptides Ji vitro. Additionally,
Masters et al. (1983) identified high molecular weight (M ) glycopro-
teins released by peri-attachment ovine, porcine and bovine blastocysts
in vitro. The approach in these studies was to culture peri-attachment
conceptuses in presence of radiolabelled amino acid substrate and, after
approximately 24h, to examine medium for nondialyzable radiolabelled
polypeptides. Such polypeptides were interpreted to represent

-197-

macromolecular products of the type presumably released in utero by
conceptuses. Production of polypeptides appeared related to stage of
conceptus development (Godkin at al. , 1982a, b).

While recent data reveal a rather complete picture of polypeptides
produced by peri-attachment porcine and ovine conceptuses (Godkin
et al., 1982a, b; Masters et al., 1983), data of a similar nature are
not available for the cow. Considering the potential physiological
importance of such macromolecules, the objective of the present study
was to characterize polypeptides produced by bovine peri-attachment
stage conceptuses.

Materials and Methods
Materials

Tissue culture supplies were purchased from Grand Island Biological

3 3

Co., Grand Island, NY; L- 4,5- H -leucine ( H-Leu; Sp. act. 55 Ci/mmol)

and D- 6- C -glucosamine hydrochloride ( C-Glc; Sp. act. Ci/mmol) were

from the Radiochemical Centre, Amersham, England; acrylamide,

recrystalized according to procedures described by Loening (1967), N,N,

N' ,N'-tetramethylenediamine and X-Omat RP film XRP-1 x-ray film were

products of Eastman Kodak, Rochester, NY; sodium dodecyl sulfate (SDS)

and Nonidet P-40 were from BDH Chemicals Ltd., Poole, England; N,N'-

diallyltartardiamide and Coomassie brilliant blue R-250 were from

Bio-Rad Laboratories, Richmond, CA; ampholines were from LKB, Uppsala,

Sweden; urea was from Pierce Chemicals, Rockford, IL; insulin was from

Eli Lilly, Lilly Research Laboratories, Indianapolis, IN; amino acids,

dithiothreitol, protein standards, 2-mercaptoethanol, trishydroxymethyl-

aminomethane (tris, glycine, glucose, sodium chloride, polyethylene

glycol, and agarose were from Sigma Chemical Company, St. Louis, MO;

-198-

diethylaminoethyl (DE-52; DEAE) cellulose was purchased from Whatman
Inc., Clifton, NJ; Sephacryl S-200, Sephadex G-75 (superfine) and
Sepharose CL-6B were from Pharmacia Fine Chemicals, Piscataway, NJ.
All inorganic chemicals were reagent grade or better.

Preparation of Medium

Eagle's minimum essential medium (MEM) was prepared according to
procedures described by Basha et al. (1979) and was identical to that
described by Godkin et al. (1982a, b) . The same MEM preparation was
used also for explant culture of bovine endometrium (Chapter III) . As
in these studies, content of L-leucine was limited to 5.2 yg/ml (0.1
normal) to enhance tissue uptake of H-Leu.

Animals

Normally cyclic cross-bred beef heifers were bred by natural
service on the day of estrus (day 0) . Individual conceptuses were
recovered from pregnant cattle on days 16 (n=4) , 19 (n=6) , 22 (n=3)
and 24 (n=4) as described previously (Chapter III) . This range of
developmental stages was chosen since it encompassed that period of
early pregnancy in cattle associated with maternal recognition of
pregnancy (Northey and French, 1980; Sreenan, 1978) as well as with
early stages of trophoblast-endometrial contact and attachment pre-
requisite to establishment of a functional placenta (Wathes and Wooding,
1980) . Additional day 19 conceptuses were recovered from two cows
which were superovulated with FSH-p (Armour-Baldwin Laboratories,
Omaha, NE; Bellows et al., 1969). Based upon total number of corpora
lutea on ovaries from FSH-p treated cattle, a maximum of 13 conceptuses
(13 Cp) were recovered from one and 17 (17 Cp) from the other cow.

-199-

Conceptuses appeared normal if not somewhat less expanded than single
day 19 tissues. Conceptus tissues also were recovered from a day 29
and a day 69 pregnant cow. In these instances uteri, recovered as
previously described (Chapter III), were placed in a sterile laminar
flow hood and carefully opened along their antimesometrial surfaces to
expose conceptus tissues. The entire day 29 conceptus was removed
aseptically and representative pieces of day 69 chorion dissected free
of underlying allantols. Tissues were incubated as described below.

In Vitro Culture of Conceptuses

Individual bovine conceptuses or conceptus tissues, recovered as
described, were placed immediately into sterile MEM. Since conceptuses
increased in size with increasing gestational age^ those from days 16
and 19 were incubated in 15 ml MEM, while those from later stages (days
22, 24 and 29) were incubated in 40-50 ml. Day 19 conceptuses recovered
from superovulated cattle (n=2) were incubated en mass in 50 ml MEM
since they tended to be tangled and difficult to separate. Two pieces
of isolated day 69 bovine chorion (3.8g and 3.6g wet weight) were in-
cubated in separate 50 ml aliquots of MEM. Individual conceptus culture

3
dishes were supplemented with 50 to 150 iaCi H-Leu. Cultures of day

3
19 conceptuses from superovulated cattle were supplemented with H-Leu

3 14

alone (100 uCi; n=l; 17 Cp) or both H-Leu (100 yCi) and C-Glc

(20 uCi; n=l; 13 Cp) . Each day 69 chorion explant received 300 UCi

\-Leu. All procedures were conducted under a sterile laminar flow

hood.

Conceptus explants were incubated under physical and atmospheric
conditions identical to those previously described for short term ex-
plant culture of bovine endometrial tissue (Chapter III) . Identical

-200-

procedures were used previously by Godkln et al. (1982a,b) for in vitro
culture of porcine and ovine conceptuses. Additionally, nondialyzable
radioactivity in explant medium increased significantly over a 24h
period during ±n vitro culture of porcine, ovine and bovine conceptuses
maintained under essentially identical conditions to those described
above (Masters et al. , 1983).

At the end of the 24h incubation period, tissue and MEM were
separated by centrifugation at 4C and 20,000g for 20 min. Medium
(supernatant) was dialyzed extensively against several thousand volumes
of 10 mM tris-HCL buffer, pH 8.2, in Spectrapor 1 dialysis tubing (M
cut-off - 6-8000; Spectrum Medical Industries, Los Angeles, CA) . Com-
parison of content of radioactivity in MEM pre- and post-dialysis
permitted calculation of percentage of total DPM radiolabelled amino
acid added that was retained as nondialyzable product (%r) . Tissue and
MEM samples were stored in separate vials at -20C until used.

Ion Exchange Chromatography

An aliquot (3-5 ml) of dialyzed, unfractionated MEM from each
individual conceptus explant was saved for electrophoresis and materials
in the remaining volume adsorbed to a 10 X 1.5 cm DEAE cellulose column
equilibrated in 10 mM tris-HCL buffer, pH 8.2, at 4C. After the entire
volume of each MEM sample was loaded, columns were thoroughly washed
with at least two bed volumes (approximately 40 ml) of equilibration
buffer to elute DEAE (-) proteins. Bound proteins (DEAE +) were eluted
with a 300 ml linear salt gradient (0.0 - 0.5M NaCl in 10 mM tris-HCL,
pH 8.2). Fractions {- 2.6 ml) were collected throughout the loading and
elution periods. Elution profiles for each explant were generated by
plotting content of radioactivity in a 100 yl aliquot of each fraction

-201-

versus fraction number (elution volume). Peaks of radioactive proteins
identified in this manner were pooled. Specific peaks of pooled DEAE
(+) conceptus proteins were concentrated either by exchanging material
back onto 0.5 X 1.0 cm DEAE columns and elating again with 3-5 ml of
0.5M NaCl (Godkin et al. , 1982b) or by dehydration of pooled DEAE (+)
fractions with polyethylene glycol (M > 20,000) through Spectrapor 3
dialysis tubing (M cut-off - 3500; Spectrum Medical Ind., Los Angeles,
CA) . Concentrated DEAE (+) fractions could then be subjected to gel
filtration.

Gel Filtration Chromatography

Pooled, concentrated DEAE (+) bovine conceptus proteins were
subjected to gel filtration chromatography on Sephacryl S-200, Sephadex
G-75 (superfine) or Sepharose CL-6B as appropriate. Gel filtration was
conducted at 4C in 90 X 1.5 cm glass columns and 10 mM tris-HCL buffer,
pH 8.2, containing 0.33M NaCl. The K for each peak of nondialyzable
radioactivity was calculated as described by Reiland (1971) and compared
with similar values of protein standards. Protein standards used to
calibrate S-200 and CL-6B columns were described previously (Chapter
III). The G-75 column was calibrated with conalbumin (M - 77,000),
ovalbumin (M = 43,000) and lysozyme (M - 13,900). Void volumes of
all columns were determined using blue dextran (M - 2 X 10 ; Pharmacia
Fine Chemicals, Uppsala, Sweden) and elution profiles generated as
described for ion exchange chromatography.

Two Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) and Fluoro-
graphy

Aliquots (3-5 ml) of unfractionated, dialyzed explant medium from
each individual conceptus culture were lyophilized and individual dried

-202-

samples dissolved in a volume of 5 mM K CO. containing 9.4M urea, 2%
( /v) Nonidet P-40 and 0,5% (^/v) dithiothreitol, sufficient to allow
100,000 to 150,000 cpm of nondialyzable radioactivity to be loaded onto
each gel in a total volume of not more than 100 yl. Conceptus tissues
to be examined by 2D-PAGE were suspended in 0.5 - 1 . 0 ml of 5 mM K CO
with 9.4M urea and sonicated on ice at 20 to 30% probe intensity (Bron-
will Biosonic; Bronwill Scientific, Rochester, NY) for six 15 sec.
periods of 1 min. intervals. Sonicated conceptus tissue was brought to
2% Nonidet P-40 C /v) and 0.5% dithiothreitol, centrifuged at 18C and
20,000 g for 30 min. and an aliquot of resultant supernatant subjected
to 2D-PAGE. Methods of sample preparation, tissue solubilization and
electrophoretic separation of polypeptides by 2D-PAGE were those des-
cribed previously by Horst and Roberts (1979) and Horst et al. (1980),
and were essentially identical to procedures described previously both
for bovine endometrial polypeptides (Chapter III) and by Godkin et al.
(1982a, b) for porcine and ovine conceptus polypeptides. Following
electrophoresis, Coomassie blue R-250 stained 10% polyacrylamide slab
gels were photographed and fluorographs were prepared as described
previously (Chapter III; Chamberlain, 1979; Laskey and Mills, 1975).

Protein Assay

Total protein concentrations in dialyzed MEM were determined by
the method of Lowry et al. (1951) using bovine serum albumin as a
standard (BSA; Fraction V; Sigma Chemical Co., St. Louis, MO).

Statistical Analyses

Where appropriate, data were subjected to analysis of variance
according to procedures available in the General Linear Models section

-203-

of SAS (Barr et ai., 1979), Analyses considered variability due to
conceptus age (day) and comparison among days 16, 19, 22 and 24 were
made by orthogonal contrasts.

Results

Incorporation of Labelled Amino Acids

Least square means (± SEM) for percent retention of ^H-Leu (%r)
labelled nondialyzable product in 24h cultures of bovine conceptuses
from days 16, 19, 22 and 24 are presented graphically in Figure 4.1.
Percent retention was lowest in day 16 cultures (P < .01; 1.16 ± 4.10%),
increased in day 19 (16.8 ± 3.67%) and day 22 cultures (20.9 ± 5.80%),
and decreased (P < .07) in day 24 cultures (6.87 ± 4.10%). Mean
(X ± SEM) content of total protein (yg/ml X ml of MEM) in MEM from days
16, 19, 22 and 24 conceptus cultures (N=17) was not significantly
effected by conceptus age (day; 7.40 ± 1.23 mg/culture) . Since content
of total protein was similar for conceptus cultures of all early stages,
but %r increased after day 16 (Figure 4.1), data suggested a general
increase in production of labelled polypeptides by bovine conceptuses
from later stages of early pregnancy.

Percent retention of radiolabelled amino acids by day 19 concep-
tuses from superovulated cattle was 7.21% for those which received only
3
H-Leu (17 Cp). Percent retention in the day 19 culture MEM to which

3 14

both H-Leu and C-Glc were added (13 Cp) was 4.16% and 7.11% respec-
tively. Content of total protein in these two cultures was 10.2 mg and
10.8 mg respectively. Hence, comparatively more total protein was
recovered in MEM from cultures of day 19 conceptuses from superovulated
cattle (X ± SEM = 10.5 ± 0.3 mg) , than from cultures of individual
bovine conceptuses (7.4 ± 1.2 mg) . However, %r data suggested that

(0

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-206-

conceptuses from superovulated cattle, cultured en mass, were less
active in incorporating radiolabelled amino acids into nondialyzable
product in MEM. Further study is warranted to determine comparative
efficacy of these two procedures for obtaining bovine conceptus-specif ic

products in vitro. The single day 29 and two day 69 cultures retained

3
16.2% and 20.4 ± 0.6% of total H-Leu added as nondialyzable product.

DEAE Anion Exchange Chromatography

Representative DEAE anion exchange chromatograms of labelled pro-
teins in MEM from day 16, 19, 22 and 24 conceptus cultures are shown
in Figure 4.2. Although the proportionate amount of various labelled
proteins produced by conceptuses in vitro may have changed with
gestational age, qualitative patterns of DEAE chromatograms were
reproducible and fairly constant. Anion exchange chromatography
consistently revealed labelled conceptus proteins in three distinct
areas (Figure 4.2; 1, 2 and 3) including a small DEAE (-) area (1),
an early eluting DEAE (+) area (2) and a later eluting DEAE (+) area
(3) . The majority of nondialyzable radiolabelled proteins bound to
DEAE at pH 8.2 indicating conceptus production of primarily acidic
polypeptides.

2D-PAGE of Bovine Conceptus Proteins

Fluorographs prepared from representative 2D-PAGE (lEF) gels of
polypeptides in dialyzed bovine conceptus culture medium from days 16,

19, 22 and 24 are shown in Figure 4.3. Primary polypeptides produced

_3

by day 16 conceptuses (Figure 4.3A) had M (X 10 ) of 22-26 based on

electrophoretic mobility in presence of SDS , and migrated in an iso-
electric point (lEP) range of 6.5 - 5.6 under denaturing conditions in

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-210-

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-211-

presence of urea. Other less prominent polypeptides identified at this

_3
stage included several in the M (X 10 ) /lEP range of 40-50/6.2-7.4.

The array of polypeptides shown in Figure 4.3A was typical nf the two

day 16 conceptuses which were at least 5-6 cm in length. These con-

3
ceptuses incorporated 1.5% and 2.7% respectively of the total H-Leu

added to MEM (50 yCi) . The same array of polypeptides was less prom-
inent or undetectable on fluorographs of MEM proteins from the two day

16 conceptuses which were less than 2.0 cm in length. These conceptuses

3
incorporated 0.02% and 0.40% of total H-Leu added to culture medium

(50 yCi) . Data suggest that the ability of peri-attachment stage
bovine conceptuses to incorporate amino acids and synthesize and release
proteins is related to the process of elongation of the trophoblast or
their developmental stage.

Pattern of polypeptides synthesized by day 19 and later stage
conceptuses (Figure 4.3B, C and D) was somewhat more complex than that
of day 16 conceptuses and suggested a general qualitative and quanti-
tative increase in protein production. The lower M acidic polypeptides

_3
(M X 10 22-26/6.5-5.6) remained as prominent products in MEM through

day 24 (Figure 4. 3D). Two even more acidic lower M polypeptides were

particularly evident on fluorographs from days 19 and 22 although they

were also present in day 24 culture medium. These products included

_3
one polypeptide in M (X 10 )/IEP ranges of 20-26/5.5-5.4 and a second

of 16-20/5.0-4.5 (Figure 4.3B, C) . Higher M products included several

polypeptides in the 40-70/7.4-5.5 range and a very large acidic product

in the >400/< 5.0 range.

Fluorographs of 2D-PAGE (lEF) gels revealing the array of conceptus

3

tissue polypeptides into which H-Leu was incorporated are shown for

-212-

representative day 16 and 22 conceptus homogenates in Figure 4.4.
Primary polypeptides identified in day 16 conceptus tissue homogenates

are seen on the central right side of Figure 4.4A and migrated in a

_3
M (X 10 )/IEP range of 46-60/6.3-4.5. Fluorographs of conceptus

homogenates from later stages of gestation (days 19, 22 and 24; Figure

4.4B) were virtually identical but more complex than those from day 16,

and did not include the distinct array of polypeptides shown in Figure

4.4A. Data indicated a significant alteration in type and diversity of

structural and functional bovine conceptus polypeptides into which

3
H-Leu was incorporated. This may be associated with the process of

trophoblast elongation and increased secretory activity.

The array of bovine conceptus tissue polypeptides into which

3

H-Leu was incorporated (Figure 4.4) was distinct from that of poly-
peptides identified in dialyzed MEM at all stages (Figure 4.3).
Therefore, the majority of polypeptides identified by fluorography in
dialyzed MEM represent proteins produced and released (secreted) by
bovine conceptuses in vitro, and are likely most representative of the
array of macromolecules secreted by peri-attachment bovine conceptuses
in utero.

To determine if the low M acidic array of polypeptides continued
to represent a major product of bovine conceptus tissues later in gesta-
tion when fetal -maternal attachments were more intimate and placentomes
were formed, fluorographs prepared from early stage (day 16, 19, 22 and
24) conceptus MEM (Figure 4.5A) were compared with those from day 29
(Figure 4.5B) conceptus and day 69 chorionic MEM (Figure 4.5C). While
the total array of polypeptides produced by bovine conceptus tissues
tended to increase in complexity with gestational age, low M acidic

Figure 4.4, Fluorographs of 2D-PAGE (lEF) gels (10% acrylamide)

showing conceptus tissue polypeptides into which -'H-Leu
was incorporated in vitro. (A) Day 16 bovine conceptus
tissue homogenate. (B) Day 22 bovine conceptus tissue

homogenate.

-214-

PH

8.9 8.4 7.9-Z3 6.8 6.4 6.2 5.8 5.5 5.1 4.4 3.6
I ' ' ■ > t i I I t i I 1 . » ■ t . t_t_U-|

A.

CO

r4oob

P-280X
-140 1^
^76g

- 562

- 47^

- 31 <

- 16-J
tt 14 s

lEF

■> +

Figure A. 5. Fluorographs prepared from 2D-PAGE (lEF) gels (10% acry-
lamide) of polypeptides in dialyzed bovine conceptus
culture MEM from days 19 (A) and 29 (B) , and isolated
chorionic culture MEM from day 69 (C) . Arrow indicates
general area of major low molecular weight, acidic
polypeptides.

-216-

PH

8.9 8.4 7.9 7.3 6.8 6.4 6.2 5.8 5.5 5.1 4.4 3.6
I I I I I I I I I I I I ' ■ I ■ I ■ I ■ I . I

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lEF

-217-

products (22-26/6.5-5.6) in MEM, prominent on fluorographs from early
stage bovine conceptuses (days 16, 19, 22 and 24; Figure 4.3), were
absent or present in only trace amounts on fluorographs from day 29 and
69 conceptus tissue cultures (Figure 4.5A, B, C) . Polypeptides with

extremely similar electrophoretic character to the major low M acidic

r

products (22-26/6.5-5.6) of early stage conceptuses were detected on
fluorographs from day 69 MEM (Figure 4.5C). It is possible that the
larger mass of chorionic tissue incubated in these cultures (> 3.0g/
50 ml) produced sufficient low M acidic product for detection on
fluorographs. Clearly, however, these polypeptides were no longer
major products of the bovine conceptus by day 29 of gestation.

Characterization of Major Bovine Conceptus Proteins

As noted above (Figure 4.2), anion exchange chromatography of
proteins in dialyzed MEM from conceptus cultures revealed two major
DEAE (+) fraction areas (2 and 3). When fractions from the later
eluting DEAE (+) area 3 were pooled, as shown in Figure 4.6A, concen-
trated, and proteins subjected to S-200 gel filtration chromatography,
the majority of nondialyzable radioactivity consistently eluted as a
major peak with a K slightly less than that of chymotrypsinogen-A

(K - 0.44; M = 27,500), for a M estimate of 26,750 ± 1030 (K
av r r av

.40; Figure 4.6B). The two step fractionation procedure described
above and illustrated in Figure 4.6 involved identical conditions to
those used to purify a major low M (17,000) acidic protein produced by
ovine trophoblast between days 13 and 21 of gestation (Godkin et al. ,
1982b).

When low M acidic bovine conceptus product, prepared as shown in
Figure 4.6, was subjected to 2D-PAGE (lEF) , fluorographs similar to

Figure 4.6. Fractionation procedure for partial purification of major
low molecular weight, acidic polypeptides produced
in vitro by bovine conceptuses. (A) Unfractionated,
dialyzed MEM is subjected to DEAE anion exchange chroma-
tography and late eluting DEAE (+) area (3) fractions
pooled as indicated by stippled area. (B) Representative
Sephacryl S-200 gel filtration elution profile obtained
following chromatography of DEAE (+) area (3) bovine con-
ceptus proteins. Majority of nondialyzable radioactivity
appears as a major broad based peak eluting just ahead of
chymotrypsinogen-A (Chym-A; M - 27,500).

-219-

I30t

>0

90t

10

30 50

FRACTION NO,

Gtym-A

30 50

FRACTION NO.

-220-

that shown in Figure 4 . 7A were obtained consistently. Primary labelled
polypeptides were those in the 22-26/6.5-5.6 range identified as major
low M products of peri-attachment bovine conceptuses (Figure 4.3).

However, traces of labelled product were also seen just above the major

_3
group in the same IE? range with M (X 10 ) of 50-55 and 75-80. When

aliquots of DEAE (3) proteins, purified' through S-200, were applied to
Sephadex G-75 three symmetric peaks of nondialyzable radioactivity were
obtained (Figure 4.7B) with M (X 10 ) estimates of approximately 22,
49 and 77. It appeared, therefore, that labelled DEAE (+) area 3
bovine conceptus proteins, fractionated through S-200, approached
radiochemical purity as assessed by fluorography of 2D-PAGE (lEF) gels.
However, identification of trace amounts of higher M^ polypeptides of
the same lEP as the major low M polypeptides on fluorographs of frac-
tionated material (Figure 4.7A), together with the fact that S-200
purified product produced three distinct peaks on G-75, (two with M^
weight estimates at roughly twice and three times that determined for
the primary product 22-26 X 10 by gel filtration and 2D-PAGE;
Figure 4.7B) suggested that this bovine conceptus protein had strong
tendencies to aggregate under condition used in the present study.
Additionally, these fractionation procedures were apparently inadequate
for separation of the major grouping of low M^ polypeptides from the
even smaller more acidic product seen in the bottom right of Figure
4.7A (see Figure 4.3B and C) .

In addition to the prominent group of low M^ acidic polypeptides
produced by peri-attachment bovine conceptuses (days 16, 19, 22 and 24)
a second major product was identified in the early eluting DEAE (+)
area (2) fractions of dialyzed conceptus MEM from all stages examined

Figure 4.7. Characteristics of partially purified low molecular weight,
acidic bovine conceptus proteins. (A) Fluorograph prepared
from a 2D-PAGE (lEF) gel (10% acrylamide) of DEAE (+) area
(3) bovine conceptus proteins following fractionation on
Sephacryl S-200 (see: Figure 4.6). (B) Sephadex G-75
elution profile of DEAE (+) area (3) bovine conceptus
proteins (shown as DPM (x 10" ) ^H-Leu/lOO Ml of each
fraction) following fractionation on Sephacryl S-200.
Three peaks of nondialyzable radioactivity are shown rela-
tive to elution positions of molecular weight standards
conalbumin (Con; Mj. - 77,000), ovalbumin (Oval; M^. -
43,000) and lysozyme (Lys; M^. - 13,900).

-222-

A,

S
D
S

PH

8.9 8.4 7.9 7.3-6.8 6.4 6.2 5.8 5.5 5.1 4.4 3.6
I ■ > ' i t I I 1 I i I ' . » ■ t ■J_t_Ljq

lEF

.VW"

p-400O

l280x
-140^1

- 562

- 47^

- 31 <

- 24 3

- 16 -J

■^ <A O

"^ 14 5:

■> 4-

B.

40 60

FRACTION NO.

80

-223-

(days 16, 19, 22, 24 and 69). When day 19 conceptuses from a super-
ovulated cow were incubated in presence of both H-Leu and C-Glc,
proteins in DEAE (+) area (2) were heavily labelled with C-Glc
(Figure 4.8A). When early eluting (area 2) DEAE (+) fractions from
conceptus MEM from each stage (days 16, 19, 22, 24 and 69) were con-
centrated and subjected individually to CL-6B gel filtration, a
symmetric peak of nondialyzable radioactivity was consistently obtained

as shown in Figure 4.8B. This high M bovine conceptus glycoprotein

_3
had elution properties intermediate to porcine IgM (M X 10 ~ 900)

-3 -3

and thyroglobulin (M X 10 - 669) for an estimated M (X 10 :

r r

X ± SEM, n=10) of 735 ± 22. This high M glycoprotein was previously
described by Masters et al. (1983) as a major product of day 19 bovine
conceptuses. In the present study fractionation of day 16, 19, 22, 24
and 69 DEAE (+) area (2) proteins through CL-6B (Figure 4.8) indicated
that this high M bovine conceptus glycoprotein was produced throughout
the peri-attachment period as well as by explants of chorion from day
69 of gestation.

Discussion
In the present study, polypeptides produced ±n vitro by peri-
attachment stage bovine conceptuses were characterized. Using an
identical explant system, Lewis et al. (1979) first described uptake of
radiolabelled amino acids and ^e novo synthesis of labelled proteins by
day 19 bovine conceptuses. Later, Masters et al. (1983) used this
system to obtain high M glycoproteins from cultured bovine, porcine
and ovine conceptuses. Methods employed in all studies were essentially
identical to those described by Godkin et al. (1982a,b) for character-
ization of polypeptides produced by peri-attachment stage porcine and
ovine conceptuses.

Figure 4.8. Isolation of the high molecular weight bovine conceptus
glycoprotein. (A) DEAE anion exchange chromatogram of
radiolabelled proteins in MEM from a day 19 bovine con-
ceptus culture. Conceptuses (from a single superovulated
cow; 13 cp) were incubated with both -^H-Leu (solid line;
DPM (x 10"^) ^H-Leu/100 \il of_each fraction) and C-Glc
(connected symbols; DPM (x 10 ) ^'^C-Glc/100 Ul of each
fraction). Stippled area (A) indicates heavily gluco-
samine-labelled early eluting DEAE (+) area (2) fractions
which were pooled and applied to Sepharose CL-6B. (B)
Sepharose CL-6B gel filtration elution profile of DEAE (+)
area (2) bovine conceptus proteins. Major peak of
nondialyzable radioactivity is shown relative to elution
positions of molecular weight standards porcine IgM
(pIgM; M - 900,000) and thyroglobulin (Thyg; M ==669,000)

-225-

40t

B

26.0t

20*

§ '5.6+

5 10.^'

5^0- ■

K)

10

30 50 70

FRACTION NO.

pIgM Thyg

30 50

FRACTION NO.

-226-

To date, few studies have dealt with proteins associated with or
produced by bovine conceptus tissues during the peri-attachment stage
of early pregnancy. Several studies of bovine conceptus related pro-
teins involved examination of extracts of placental tissues. Butler
et al. (1982) described two very acidic proteins in. extracts of bovine

placental membranes from days 25 through 270 of gestation. One had an

_3
estimated M^ (X 10 ) of 65-70 and an lEP of 4.6-4.8. This placental

component was suggested to be a -fetoprotein. The second was somewhat

_3
smaller (M^ X 10 - 47-53) and more acidic (lEP - 4.0-4.4). Avivi

et al. (1982) isolated and characterized a 94,000 dalton protein with

thyrotropic activity from bovine placentae obtained from days 40 to

120 of gestation. Products described in those studies were associated

with conceptus tissues from later stages of pregnancy than were of

interest in the present study. Regardless, these products did not

appear as singularly unique, prominent labelled components of either

MEM or conceptus tissues as assessed by fluorography of 2D-PAGE (lEF)

gels in the current study. Clearly, a variety of products were seen in

both tissue and MEM.

Flint et al. (1979) detected bovine placental lactogen (bPL) in

homogenates of bovine conceptuses from days 17, 24 and 25, but other

conceptus products were not investigated. Wathes (1980) examined

effects of steroids and co-culture of conceptus and uterine tissues on

ability of bovine chorion from days 24-44 of gestation to incorporate

radiolabelled leucine in vitro. However, products detected in medium

were not subjected to high resolution chromatographic and electrophore-

tic techniques. In this light, present results represent a first

attempt to identify and characterize proteins produced by bovine

-227-

"°" °f Pr.gn.nc, and e.tabl • .„ "" "'""^' "=°^"^-

percentage of ,„,,! 3„_,^„ ^
-Uures „..c. „a3 .nc„.p„.a.e. .nt " ^ '"""' =°""^'-

"- -. .„ .a, . C.L J ;rr ~ ~ -W ..,

V^ ^ .01; 1.15 ± 4 1071 ,„j .
™^-^a.l.aS.S.3...).„,,,,,,^^^; ^^ — -

Which bovine conceptuses convert^H u
steroid and prosta^land • -bstrates for both

P ostaglandm synthesis increased w.>.
(Chenault. 1980- El. gestational age

' '^^y-'^al.. 1983; Lewis etal I9«.i
^^ < -07) in ,r noted in day 27 i ^^•'^^«2)- The decrease

^^ 2^ cultures (6.87 ± 4 107^ n,

"— - -s=ue ..a..a, a, a c„nae,„ance „. ^ . ^

- -^ - -^p.u3 .... ..„.. .1^:: ; :"""^ ---^•

—^ "^^=.en. ...n c.ca.. an. j „ r ^T ^^

begun (Wathes and W d" "tissues had

"«e neca.a. . Z^^ ^^ ^"""^ ^^^^"^ "— -
- -e .acn 3n„.c.n. Jl '''''' '"' ''' "-"- -» -

-""^"-. ..3 a.: : c™^'^ ^'""'^ "-^°"""= "—

- -^ -^ =^^ -eana.. :TZr ^^ ""^^"' ---°"
-'^".^ ..e auan..3 .3 no. ,„, ' "' ""^ ^^^^ »"' •

— ™ :„ : ■ 7"^^ ^"" ^" -- -^-. ^.3

-^" and Foley, igsgal r

- ^= rag.e33a. an. a .. • ' '°""^"'""- ^^ ^- " -e ,oX.

a rud^entary functional chorloallan, •
-- .3 p„3an.. Oa„aa3cd .nco.po„»on of ^ ^^^"^

"-^- -^— into

"" ^' ""'^°" «"-' » "an3.„o„

extraembryonic membranes i-^r a

~ ^eaa 3ac.to. to. ... ..3. c ^ ^^^ ^^^"^"^ ^^

respect to macromolecules.

-228-

Anion exchange (DEAE) profiles of dialyzed proteins in bovine
conceptus MEM (Figure 4.2) were similar to those reported for both
porcine and ovine conceptus proteins (Godkin et al. , 1982a, b). Bovine
conceptuses, however, did not produce a major DEAE (-) basic protein
such as that produced by porcine conceptuses (Godkin et al. , 1982a),
and in this respect DEAE profiles more nearly resembled those presented
for peri-attachment ovine conceptuses (Godkin et al. , 1982b). The
majority of nondialyzable radiolabelled proteins in bovine conceptus
MEM from all stages examined bound to DEAE at pH 8,2 indicating that,
like ovine conceptuses (Godkin et al., 1982b), peri-attachment bovine
conceptuses produced primarily acidic proteins. No major qualitative
changes in DEAE profiles of bovine conceptus proteins were,
associated with stage of gestation (Figure 4.2). These profiles sug-
gested that major alterations in conceptus protein production after day

16 were primarily quantitative.

3
Uptake of H-Leu and production of polypeptides by day 16 bovine

conceptuses appeared related to stage or extent of development of the
trophoblast. Those day 16 conceptuses which had expanded to some
degree were apparently more active in incorporation of labelled amino
acid substrate and sjmthesis and release of labelled polypeptides.
Consistent with present observations, several studies indicated day 16
bovine conceptus lengths to be highly variable, extending over a range
of less than 2 mm to over 200 mm (Hawk et al., 1955; Staples et al.,
1961; Betteridge et al., 1980). Elongation of bovine trophoblast begins
after establishment of endoderm, progenitor of both yolk sac and allan-
toic membranes (Eckstein and Kelly, 1977; Greenstein and Foley, 1958a),
and is accompanied by appearance of extraembryonic somatopleuric

-229-

mesoderm, source of trophoblastic/chorionic tissues (Greenstein and
Foley, 1958a). Therefore, ability of elongated day 16 bovine concep-
tuses to metabolize amino acid substrate and produce detectable amounts
of labelled polypeptides suggests that these processes are initiated
following establishment of embryonic endoderm, and are associated with
appearance of somatopleuric mesoderm which ultimately gives rise to
the chorionic layer of the chorioallantois.

Further indirect evidence of a relationship between stage of
conceptus development and metabolic capacity was revealed by fluoro-
graphy of 2D-PAGE (lEF) gels of polypeptides in MEM and conceptus
tissue homogenates. As illustrated in Figure A. 3, the number and/or
amount of polypeptides produced in vitro and released into MEM by
bovine conceptuses increased after day 16. These changes were related

to or accompanied by alterations in the array of structural and func-

3
tional conceptus tissue polypeptides into which H-Leu was incorporated.

This pattern became qualitatively different and more complex in later

stage conceptuses (day 16 vs. 19, 22 and 24; Figure 4.4).

The time between days 16 and 19 of bovine gestation constitutes a

dynamic period of rapid elongation of the trophoblast, initial expansion

of allantois, establishment of a functional but temporary yolk sac and

vitteline circulation, and appearance of large binucleate cells on the

external trophoblastic surface (Greenstein and Foley, 1958a; Greenstein

et al., 1958; Chang, 1952; Wathes and Wooding, 1980; King et al., 1980).

Consequently, the array of tissues and cell types which may contribute

to the compliment of proteins produced ±n vitro by conceptuses is

established during this period. With the exception of regression of

the yolk sac and continued expansion of the allantois, this array of

-230-

t issues is relatively constant throughout the peri-attachment period
after day 16 until placentome formation begins. The dramatic change

observed after day 16 (Figure 4.4) in array of conceptus tissue-

3
associated polypeptides into which H-Leu was incorporated undoubtedly

reflected these dynamic developmental events. Similarly, products

identified by fluorography in dialyzed MEM from later stage conceptus

cultures may represent contributions of the yolk sac as well as the

chorion and chorioallantois. Labelled polypeptides released by isolated

peri-attachment stage ovine yolk sac and porcine embryo-amnion units

were shown to be distinct from corresponding trophoblastic (chorionic)

products (Godkin et al., 1982a,b).

As evidenced by comparison of Figure 4.4 with Figure 4.3, the

3
array of bovine conceptus tissue polypeptides into which H-Leu was

incorporated was distinct from that identified in MEM. Therefore,
polypeptides identified as being enriched in MEM, but absent or present
in only trace amounts in tissue, appeared to be selectively released by
conceptuses during culture. Consequently, these polypeptides are most
likely representative of macromolecular products contributed to the
uterine environment by peri-attachment stage bovine conceptuses. Addi-
tionally, observations suggest that it would be difficult to extract
significant amounts of these conceptus products from tissues themselves.

Present data indicated that bovine conceptuses were capable of
producing a variety of polypeptides in vitro when recovered from as
early as day 16 of gestation (Figure 4.3 and 4.4). Of the many poly-
peptides produced by conceptus tissues and resolved by fluorography of
2D-PAGE (lEF) gels, a family of low M acidic polypeptides was espe-
cially enriched in MEM from day 16 conceptus cultures, and remained a

-231-

prominent component of day 19, 22 and 24 cultures. Synthesis and
release of these polypeptides by early expanded day 16 conceptuses
suggested a trophoblastic source for this product as discussed above.

The major group of low M acidic polypeptides in MEM was found in
a M (X 10 )/IEP range of 22-26/6.5-5.6. Associated with this primary
group were two more acidic polypeptides at 22-26/5.6-5.5 and 16-20/
5.0-4.5 (Figure 4.3). Comparison of 2D-PAGE (lEF) fluorographic
patterns of polypeptides in MEM from peri-attachment stage (day 16, 19,
22 and 24) bovine conceptuses with that of a day 29 conceptus (Figure
4.5) indicated that, by day 29, the low M acidic family of polypeptides
were no longer major conceptus products. In MEM of day 69 explants,
trace amounts of polypeptides were detected with identical electro-
phoretic character to several of the low M acidic products on
fluorographs of polypeptides in MEM from peri-attachment preparations.
This suggested that production of some of these proteins may not be
terminated later in gestation, even after placentome formation.

Identification of a major group of low M acidic polypeptides
(22-26/6.5-5.6) produced in vitro by peri-attachment stage bovine
conceptuses was consistent with reports of similar but not identical
polypeptide groupings produced ix\_ vitro by porcine and ovine tropho-
blasts (Godkin et al. , 1982a, b). These polypeptides were also prominent
but transient products, produced by ovine conceptuses from days 13-21
and by porcine conceptuses from days 10.5-16. The temporal pattern of
appearance and transient nature of production of such low M acidic
polypeptides by conceptuses of three major domestic species allows one
to speculate that such products might serve important functional roles
during the peri-attachment period associated with establishment of
pregnancy.

-232-

Evidence for the role of a conceptus produced protein in maternal
recognition of pregnancy is perhaps strongest, although still circum-
stantial, in sheep. In cyclic ewes intrauterine infusion of homogenates
of day 14-16 ovine conceptuses was luteostatic. This effect was ablated
in heat treated conceptus homogenates and not reproducible when homo-
genates of day 21-23 conceptuses were infused (Rowson and Moor, 1967;
Martal et al. , 1979). Martal et al. (1979) presented evidence for a
heat labile, protease intolerant component of day 14-16 ovine conceptus
homogenates (trophoblastin) which was antiluteolytic when infused into
uteri of cyclic ewes. The period of time during which the ovine con-
ceptus possessed antiluteolytic properties in these studies coincided
well with the period of production of ovine trophoblastic protein-X
(Godkin et al. , 1982b). This low M acidic protein is produced in vitro
by sheep conceptuses from days 13-21. However, a definitive role for
this protein has yet to be established.

Evidence that the bovine conceptus can alter corpus luteum (CL)
function early in gestation was presented by Northey and French (1980).
In this study inter estrous interval was extended in cattle from which
conceptuses were removed on day 17 (25 ± 1.2 days) as compared with
either non-mated controls (21 days) or cattle from which conceptuses
were removed on day 15 (20.2 ± 0.8 days). Additionally, intrauterine
infusions of conceptus homogenates on days 15 through 17 extended inter-
estrous interval and delayed decline in peripheral plasma progesterone
associated with luteal regression. Results indicated that a conceptus
related component must be present Jji utero from at least days 15 to 17
in order to exert an antiluteolytic effect, and that this component is
either not present, or present in insufficient quantity to elicit an

-233-

effect on or before day 15. Beal et al. (1981) demonstrated that
homogenates and extracts of day 18 bovine blastocysts stimulated pro-
gesterone synthesis by dispersed luteal cells in vitro. Luteotrophic
activity appeared to be due to a heat labile substance of less than
12,000 M^, but authors did not exclude the possibility that nonprotein
molecules such as steroids or prostaglandins might be involved.
Poffenbarger et al. (1982) indicated that luteinizing hormone activity
was not present in day 16-20 bovine conceptus homogenates or explant
medium as assessed by mouse Leydig cell bioassay.

Available evidence suggests that both antiluteolytic and luteo-
trophic mechanisms may be involved in maternal recognition of pregnancy
in the cow as defined by luteostasis. Data also suggest that conceptus
products involved in initiating these processes may act both at the
level of the uterus and directly at the level of the CL. Present data
indicate that, in addition to a variety of steroids and prostaglandins
of both E and F series produced by bovine conceptuses from as early as
day 13 (Shemesh et al. , 1979; Chenault, 1980; Lewis et al., 1982; Eley
et al., 1983), a group of low M acidic polypeptides appear as major
products of the bovine conceptus from day 16 to at least day 24. Like
steroid and prostaglandin products, these polypeptides are present
during a critical period associated with maternal recognition of preg-
nancy. At present it is only the transient nature of production of
detectable amounts of these polypeptides that suggests a functional
relationship. No definitive roles related to luteostatis have yet been
established for any bovine peri-attachment conceptus products.

Another potential but as yet unconfirmed role for transiently
produced low M acidic conceptus polypeptides may be related to stimula-
tion of histotrophe production. The period of time during which bovine

-234-

as well as ovine and porcine low M acidic products are produced
coincides developmentally with rapid expansion of the trophoblast ,
regression of the yolk sac and establishment of a functional chorio-
allantois (Heuser, 1927; Green and Winters, 1945; Winters et al., 1942;
Greenstein et al. , 1958). All three species produce miolecithal eggs
(Mossman, 1980), and transient production of low M acidic polypeptides
by each species occurs during a critical period of transition from
embryonic dependence on yolk nutrients from the temporary vitteline
circulation, to dependence on histotrophic nutrients provided from the
uterine environment by the chorioallantoic circulation. Godkin et al.
(1981) presented immunocytochemical evidence for uptake of ovine
trophoblast protein-X (Godkin et al., 1982b) by uterine endometrium in
intact pregnant sheep. Recent evidence indicated that this association
may effect selective stimulation of specific uterine proteins j^ vitro
(J.D. Godkin, personal communication). Production of polypeptides
during the peri-attachment period which might selectively stimulate
uterine histotrophe production would be particularly beneficial to the
conceptus during this period of transition in nutrient source dependence.

Fractionation of DEAE (+) area (3) bovine conceptus proteins
through S-200 (Figure 4.6) produced a relatively broad based peak of
nondialyzable radioactivity with an average M estimate (X ± SEM) of
26,750 ± 1030. When proteins from such pooled S-200 peaks were sub-
jected to 2D-PAGE (lEF) and f luorography, this protein fraction was found
to consist primarily of the major low M acidic polypeptides (22-26/
6.5-5.6), but also included the two minor acidic polypeptides (22-26/
5.5-5.4; 16-20/5.0-4.5; Figure 4.7A). Evidence of aggregation was seen
on fluorographs above the major products in the same lEP range. Further

-235-

fractionation of this S-200 purified material indicated that the major
low M acidic polypeptides had a tendency to aggregate in tris-HCL
buffer (pH 8.2 with 0.33M NaCl) , both in presence and absence of IM
sucrose. Consequently these polypeptides were not definitively puri-
fied. Data indicated that purification of the major low M acidic
polypeptide products of peri-attachment bovine conceptuses may require
more elaborate fractionation procedures and altered buffer conditions
from those described above and by Godkin et al. (1982b) for purification

of ovine trophoblastic protein-X.

_3
The M (X 10 )/IEP estimate determined for the major group of low

M acidic polypeptides (22-26/6.5-5.6; Figure 4.3) was similar to that

reported for bPL (22.15/5.9) by Bolander and Fellows (1976). More

-3
recent estimates indicated purified bPL to have a M (X 10 ) in the

32-35 range (Kensinger et al., 1982; Murthey et al., 1982). Although

this placental protein was detected by radioreceptor assay in homogen-

ates of bovine conceptuses from days 17, 24 and 25 (Flint et al. , 1979)

it appears to be produced throughout gestation and not transiently

during the peri-attachment period (Bolander and Fellows, 1976; Buttle

and Forsyth, 1976; Murthey et al., 1982). Therefore, it seems unlikely

that the low M acidic polypeptides are bPL.

The high M glycoprotein identified as the major component of early

eluting DEAE (+) area (2) (Figure 4.8) was first described by Masters

et al. (1983) as the major glucosamine-labelled product released

in vitro by day 19 bovine blastocysts as well as by day 14-16 ovine and

day 16 porcine blastocysts. Present data extend the observations of

Masters et al. (1983) and indicate that this early eluting DEAE (+)

glycoprotein is a major product of peri-attachment stage bovine

-236-

conceptuses from days 16, 19, 22 and 24, as well as bovine chorion from

day 69 of gestation. Gel filtration of DEAE (+) area (2) glycoprotein

from each of these stages on CL-6B (Figure 4.8) indicated a M estimate

r
_3 _

(X 10 ) for this major bovine conceptus product (X ± SEM) of 735 ± 22.

Masters and coworkers (1983) reported that, in each species ex-
amined, the high M glycoprotein consisted of at least 50% carbohydrate.
These products did not possess biochemical characteristics typical of
mucins. The ovine glycoprotein was shown to be composed of N- rather
than 0-link.ed oligosaccharide chains containing D-galactose, N-acetyl-
glucosamine, D-mannose and L-fucose. No sialic acid was detected. None
of these species produced any sulfated proteoglycans or hyaluronic acid.

In contrast to the major low M acidic polypeptides, production of
the high M glycoprotein by peri-attachment conceptuses or chorionic
tissue suggested a less transient role for this macromolecule. The
high M glycoprotein may normally be more intimately associated with the
trophoblast. It was suggested that such products might not normally be
released in soluble form but, instead, deposited between trophoblast
cells or on the trophoblastic surface (Masters et al. , 1983). In this
way such macromolecules might provide a protease resistant or immuno-
logically tolerant coating for the blastocyst or chorion, or be
necessary for control of cell movement or cell-cell interaction during
the elongation process (Masters et al. , 1983). Cellular rearrangement
was reported to be a major mechanism associated with porcine trophoblast
elongation (Geisert et al., 1982a). Production of high M glycoprotein
by peri-attachment stage ovine conceptuses was not apparent until elon-
gation had begun (Godkin et al., 1982b).

Results of the present study indicated that peri-attachment stage
bovine conceptuses produced a complex array of proteins and polypeptides

-237-

In vitro. Production of these macromolecules appeared related to
developmental stage of the conceptus. Two major bovine conceptus
products were identified. These included a group of late eluting
DEAE (+) low M^ (X 10 ) acidic polypeptides (22-26/6,5-5.6), and an
earlier eluting DEAE (+) high M (735 ± 22) glycoprotein. The former
were detected as major products of bovine conceptuses from as late as
day 24, but were no longer prominent products of day 29 conceptus
tissues. These polypeptides were produced during a period of early
pregnancy associated with maternal recognition of pregnancy and estab-
lishment of a functional chorioallantois. The latter were prominent
products of conceptuses and conceptus tissues from all stages examined.
Results agreed generally with those reported for both ovine and porcine
conceptuses (Godkin et al. , 1982a, b) and suggest a commonality in nature
and pattern of production of polypeptides by peri-attachment conceptuses
by three major domestic species. Similarity among species in physico-
chemical properties and pattern of production of these products does not
necessarily imply similarity in function. However, identification of
these properties and subsequent isolation and purification of the
proteins themselves are necessary first steps in elucidation of specific
roles for this intriguing class of conceptus products.

CHAPTER V
GENERAL DISCUSSION

Establishment and maintenance of pregnancy in eutherian mammals
involves complex interactions between two interdependent systems, the
maternal uterus and the conceptus (Mossman, 1937). As observed by
Mossman (1980; p. 3) the purpose of the uterus is to "contain and sus-
tain the developing conceptus from the morula or early blastocyst
period until parturition". Consequently, maternal uterine support of
the conceptus is both physical and environmental. Such support is
particularly important in species (including cattle) which possess an
epitheliochorial placenta (Steven, 1975b; Wathes and Wooding, 1980).
Pregnancy in these species is not established ectopically, and con-
ceptuses undergo a characteristically prolonged free-living period
in utero (Cook and Hunter, 1978). During this period (which, in
cattle, may extend from days 3-4 until days 21-22; Greenstein et al. ,
1958; Leiser, 1975; Wathes and Wooding, 1980) the conceptus depends
upon the intralumenal uterine environment for both physico-chemical
and, presumably, nutritional support (Amoroso, 1952; Bazer et al. ,
1981b). An embryotrophic uterine environment is established early in
pregnancy as a consequence of uterine response to maternal endocrine
and other physiological signals (see Chapter I). Components of this
environment may originate from maternal serum (hemotrophic elements)
or as products of the uterine endometrium (histotrophic elements) .
Maintenance of this environment, pursuant to the embryotrophic rather

-238-

-239-

than the luteolytic path, is thought to require participation of the
conceptus with respect to production of biologically active agents
(Amoroso and Perry, 1974).

In cattle the period of early pregnancy, from initiation of tropho-
blast elongation (days 14-16) until definitive microvillar attachment
of the outer trophoblastic membrane (chorion) to maternal endometrium
(- days 22-24), is especially critical (Chang, 1952; Greenstein et al.,
1958; Leiser, 1975; Wathes and Wooding, 1980). During this period
uterine integration of appropriately timed maternal and conceptus
signals may result in "recognition" and establishment of pregnancy
(Short, 1969; Bazer et al. , 1981c). However, the consequence of
failure of either maternal or conceptus components is failure of preg-
nancy. The delicate nature of the relationship between maternal and
conceptus units during the peri-attachment period is illustrated by
the high incidence of embryonic mortality and fertility failure char-
acteristic of this stage of bovine gestation (Ayalon, 1978; Hawk, 1979;
Hawk et al., 1955). Knowledge of factors which may contribute to
successful intercommunication between maternal and conceptus units is
necessary if solutions to bovine fertility problems are to be obtained.
Additionally, information of this nature contributes to basic under-
standing of phenomena associated with establishment and maintenance of
pregnancy.

Analysis of uterine secretions from cyclic and early pregnant
cattle and sheep has been hampered by small amounts of material re-
coverable in uterine flushings (Bazer et al. , 1978). In sheep it was
demonstrated that copious amounts of protein-rich uterine fluid could
be obtained late in gestation (> day 100) . This was achieved by

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creating a pouch in one uterine horn via two different experimental
approaches (1) surgical anastomosis of apposing endometrial surfaces
(Harrison et al., 1976), or (2) unilateral ovariectomy and ligation of
the ipsllateral uterine horn (Bazer et al. , 1979a), so that the fetal-
placental unit was confined to the remaining intrauterine space.
Present research (Chapterll) demonstrated that unilateral ovariectomy
and ligation of the ipsilateral uterine horn permitted establishment
of unilateral pregnancy in cattle (Figure 2.1). The technique employed
was similar to that described for sheep by Bazer et al. (1979a). Fetal
weights, placental weights and fetal fluid volumes, measured in nine
unilaterally pregnant cows on day 270 of gestation (Table 2.1), were
consistent with similar values reported for normally pregnant cattle
by Eley et al. (1978), Ferrel et al. (1976) and Winters et al. (1942).
Thus, confinement of the bovine conceptus to one uterine horn was not,
in general, sufficient to compromise growth and development of the
fetal-placental unit. The observation that three of twelve unilater-
ally pregnant cattle delivered live calves prior to day 270 suggested
that uterine capacity in these cows may have been compromised by the
ligation procedure. It also was observed that, while placental morphol-
ogy was normal, caruncles in the ligated uterine horn of unilaterally
pregnant cattle were not developed significantly. This observation
demonstrated a clear requirement for a trophoblastic/chorionic stimulus
and/or chorion-endometrial interaction for normal placentome develop-
ment. Hence, in addition to providing a rich source of uterine fluid,
unilateral pregnancy presents an intriguing model for study of bovine
uterine capacity and chorion-endometrial interaction.

Bovine UM was palpated per rectum by day 166 of gestation and
accumulated rapidly thereafter. Developmentally, the pattern of bovine

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UM accumulation was similar to that described for unilaterally pregnant
sheep (Bazer et al. , 1979a; Harrison et al., 1976). This period of
bovine gestation is associated with the time of maximal fetal growth
rate (Eley et al., 1978; Winters et al., 1942) and is preceded by
dramatic increases in uterine arterial and venous E concentrations,
UBF and net uterine nitrogen accretion (Ferrell and Ford, 1980; Ferrell
et al., 1976). Additionally, this period may be associated with
elevated circulating levels of bPL (Bolander and Fellows, 1976). Thus,
while no causal relationships may be determined from the present study,
data support the notion that circulating agents of gonadal, placental
and/or fetal origin may be involved in stimulation of UM accumulation
(Bazer et al. , 1979a). Such uterine response may be necessary to meet
increased demands of the rapidly growing fetus (conceptus) . Increases
in amount, number, frequency and persistency of appearance of proteins
in uterine flushings also occur during the peri-attachment period in
cattle (Roberts and Parker, 1976; Bartol et al. , 1981a), as well as
sheep (Roberts et al., 1976a) and pigs (Geisert et al. , 1982b). Such
responses are associated with rapid expansion of the trophoblast and
accompanying transient increases in uterine blood flow (Ford et al.,
1979; Greiss and Anderson, 1970; Ford and Christenson, 1979). Increases
in uterine content of embryotrophe may, therefore, occur regularly in
response to both local and systemic stimuli produced or induced by
rapidly growing conceptus tissues.

In unilaterally pregnant sheep, UM was recovered as early as day
30 of gestation, but did not appear in copious quantities until after
day 100 (Moffatt et al. , 1980; Bazer et al. , 1979a). Proteins charac-
teristic of ovine UM were present in uterine flushings of ovariectomized

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ewes treated 120 days with P^ alone, or P, plus E . Maximal stimulation
occurred in the latter group (Moffatt et al., 1980, 1981). These pro-
teins were not detected in uterine flushings from cyclic ewes between
days 0 and 16 postestrus (Moffatt et al. , 1981). If mechanisms
responsible for induction of UM are similar in sheep and cattle, data
suggest that some protein components of this fluid may be characteristic
of those which require protracted P,/E. stimulation for synthesis and
secretion.

Differences in steroid concentrations between UMS and peripheral
plasma (Table 2.2) supported the notion that these two fluid pools are
not in simple equilibrium. Data suggested that differences noted
between UMS and plasma steroid concentrations might be explained, in
part, by uterine steroid metabolism (Eley et al., 1983; Knickerbocker
et al., 1980) and/or relative solubility of steroids in uterine cell
membranes (Heap et al., 1971). Uterine endometrial metabolism of
steroids was suggested to be important in (1) providing the conceptus
with more readily metabolizable steroid substrate, (2) decreasing the
amount of biologically active steroid present in utero, and (3) en-
hancing uterine responsiveness to conceptus-produced steroids or other
signals (Bazer et al. , 1982; Eley et al. , 1983; Heap and Perry, 1974).
During early pregnancy in both pigs (Geisert et al. , 1982b) and mares
(Sharp, 1980) dramatic increases in uterine luminal steroid content is
thought to reflect conceptus steroid production and metabolism of
available substrate. Since fetal membranes were absent from the
ligated uterine horn in unilaterally pregnant cattle, UM steroid pro-
files do not reflect conceptus contributions, but may be representative
of the normal substrate pool available to the conceptus.

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Detection of glucose but not fructose in bovine UMS (Table 2.4)
is consistent with earlier reports which described glucose as the
major free sugar in bovine (Suga and Masaki, 1973), ovine, porcine
and equine uterine flushings (Haynes and Lamming, 1967; Zavy et al.,
1982a). Renard et al. (1980) demonstrated that, while there was no
morphological difference between day 10-11 bovine blastocysts which
either did or did not take up glucose xn vitro, development in vivo
following embryo transfer was better for those which had taken up
glucose (% pregnant at day 50; 69.2% vs 14.2%). Thus, glucose may be
an important substrate during early bovine embryonic development.
Observations of Zavy et al. (1982a), suggesting a conceptus source
for uterine lumenal fructose, are consistent with present findings in
that levels of fructose were undetectable in bovine UM, since fetal
membranes were absent from the ligated uterine horn.

Day 270 bovine UM was enriched in PGF, protein and Ca (Table 2.4)
and consistent with observations in sheep (Bazer et al. , 1979a; Harri-
son et al. , 1976). Presence of a concentrated pool of PGF in UM
supports the idea that specific endocrine conditions may permit
sequestering of prostaglandins within the uterine lumen (Bazer et al. ,
1979a; Bazer and Thatcher, 1977). Indeed, evidence that PC may be
subject to active, carrier-mediated transport in reproductive tissues
of various species was presented by Bito and coworkers (Bito, 1972;
Bito and Spellane, 1974). Specific evidence to this effect is lacking
in cattle. However, data from the University of Florida (D. Wolfenson,
personal communication) indicated that increases in ovarian arterial
concentrations of PGF observed during the period of luteolysis in
cyclic cattle (days 19-20), were absent in pregnant cattle at the
same stage. Additionally, recent observations of Curl (J. Curl,

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personal communication) suggest an effect of pregnancy on bovine endo-
metrial uptake of PG. Such uterine responses are thought to be
effected locally by presence of the conceptus. However, elevated PGF
levels in bovine (and ovine; Bazer et al., 1979a) UM suggest potential
for a systemic effect of the conceptus as well.

Both concentration and content of protein in bovine UMS (Table
2.4) were equal to or greater than similar values reported for ovine
UM (Bazer et al. , 1979a; Harrison et al., 1976) and bovine uterine
fluid obtained following chronic P, therapy (Dixon and Gibbons, 1979).
Uterine milk protein content greatly exceeded similar reports for
bovine uterine flushings collected either in vivo (Bartol et al.,
1981b; Schultz et al. , 1971) or postmortem (Bartol et al. , 1981a, b;
Roberts and Parker, 1974a, 1976; Olds and Van Demark, 1957a, b) and
establish bovine UM as a rich source of this proteinaceous material.
Although mechanisms responsible for induction of bovine UM are unclear,
positive correlations were detected between Ca concentration of UMS
and both protein concentration (r=-68, P < .09) and PGF content (r=.92,
P < .08) of UMS suggesting a functional interrelationship among these
UM components. Positive correlations were reported between uterine
lumenal content of PGF and total protein in both cyclic and early
pregnant cattle (Bartol et al., 1981a), and similar relationships were
noted in sheep (Ellinwood et al., 1979b). Additionally, Geisert
et al. (1982b, c) reported that increases in uterine lumenal content of
both PGF and PGE , accompanied by rapid transient increases in Ca
content, preceded persistent increases in both amount and complexity
of proteins in porcine uterine flushings. These relationships occurred
whether induced locally by presence of a conceptus or systemically by

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administration of EV. Data reviewed by Rasmussen (1981) and Rubin
(1982) indicated that the process of secretion involves obligatory
Ca-dependent turnover of membrane-associated arachidonic acid and
synthesis of PG. Substances produced (PG) or released (Ca) as a
consequence of this process may further potentiate secretory events
and/or alter epithelial cell membrane electrophysiology (Rasmussen,
1981; Rubin, 1982). Consequently, intercellular transudative pro-
cesses may also be affected (Rasmussen, 1981). Thus, relationships
noted between UM Ca, protein and PGF may reflect active mechanisms
associated with luminally directed secretions and transudation of
proteins and other substances. Such mechanisms may be characteristic
of processes required to establish an embryotrophic uterine environment,

Qualitative analyses of proteins in bovine UM revealed a complex
array of polypeptides (Figures 2.2 and 2.3). Bovine UM was enriched
in proteins of less than 40,000 M (Figure 2.2), the majority of which
were basic in nature, migrating on NEPHGE between approximately pH
6.8 and 8.6 (Figure 2.3B). However, NEPHGE profiles of bovine UMS
polypeptides were distinct from similar profiles of uterine fluid
proteins from other species and revealed neither a Utf-like polypep-
tide, as described for both the pig and mare, nor either of the two
major polypeptides found in ovine UM (Bazer et al. , 1979a, 1981b) .

Two-dimensional PAGE (lEF) and C-IEP (Figures 2.4 and 2.5)
indicated that the majority of proteins in bovine UMS were physically
and immunochemically identical to bovine serum proteins. This obser-
vation is consistent with earlier reports indicating that no more
than 2 to 3% of total protein in bovine uterine flushings might be
expected to be uterine-specific (Roberts and Parker, 1974a, 1976).

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Beier (1980) noted that transudation of serum proteins into the uterine
lumen occurs irrespective of protein molecular size. Present data
indicated a general qualitative and slight quantitative disparity
between serum- identical proteins seen in UM and plasma (Figure 2.5).
Collectively, data support selective uptake of serum proteins into UM.
In this respect, selective uterine uptake of serum proteins is likely
as important as synthesis and secretion of uterine-specific products
in establishment of an embryo trophic environment. Immunoglobulins,
such as IgA, IgG and IgM, identified immunochemically in bovine UM
(Figures 2.6 and 2.7), may be important for maintenance of uterine
luminal sterility and bacteriocidal activity (Sullivan and Wira, 1983).
Serum proteins, in addition to serving as a source of hemotrophic
nutrition to the conceptus, may be involved functionally with mainten-
ance of dynamic equilibrium between the uterine lumen and peripheral
plasma (see Clark and Peck, 1981; Clark et al. , 1977).

Despite the preponderance of serum- identical proteins in bovine
UM, several polypeptides apparently unique to UMS were identified
(Figure 2.4). One group appeared in the 25 to 30,000 M range with
isoelectric points between 6.4 and 7.0. Two other polypeptides
(Figure 2.4A; UMl and UM2) had nearly identical M of 20 to 21,000
and approximate isoelectric points of 6.3 and 6.1, respectively.
These polypeptides were similar, electrophoretically, to porcine
uterine retinol and retinoic acid binding proteins (Adams et al. ,
1981; Bazer et al. , 1981b). Thus, as has been demonstrated for bovine
uterine flushings obtained from cyclic and early pregnant cattle
(Roberts and Parker, 1974a, b, 1976), bovine UM contained both uterine
luminal-specif ic, serum- identical proteins and uterine-specific pro-
teins as determined by absence of these components from plasma.

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Chromatographic and electrophoretic analyses of radiolabelled
proteins and polypeptides in dialyzed MEM from endometrial explants
(Chapter Ii:D indicated that bovine endometrial tissue may produce a
much more complex array of macromolecules than has been demonstrated
previously (Roberts and Parker, 1976; Laster, 1977; Dixon and Gibbons,
1979; Wathes, 1980). However, in contrast to other major domestic
species including the pig, sheep and mare (Bazer et al., 1981b),
bovine endometrium synthesized primarily acidic polypeptides in vitro
as assessed by ion exchange chromatography (Figure 3.4) and fluoro-
graphy of 2D-PAGE (lEF and NEPHGE) gels (Figures 3.5, 3.6 and 3.7).
Consistent with observations of bovine UM, bovine endometrial explants
did not produce polypeptides similar to either Utf (Roberts et al. ,
1980) or the 50 to 55,000 M , basic proteins identified as the major
ovine endometrial products (Moffatt et al., 1980; Bazer et al. , 1981b),
Also absent from all NEPHGE fluorographs, including those prepared
from 2D-PAGE gels of day 270 MEM proteins from both ligated and
pregnant uterine tissue explants, were the very basic (pH > 7.3) low
M polypeptides identified as major components of bovine UM (Figure
2.3). Data suggest that, if these low M polypeptides appear as normal
components of the uterine environment, they are either derived from
serum, or appear as enzymatic degradation products. Such proteolysis
may be necessary to make hemotrophic and histotrophic elements more
accessible to the conceptus.

Endometrial tissues from nonpregnant/cyclic (days 4, 16 and 19),
pregnant (days 16, 19, 22, 24 and 69) and unilaterally pregnant cattle
(day 270; ligated and pregnant uterine horns) produced virtually iden-
tical arrays of polypeptides from radiolabelled amino acid substrate

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ui vitro (Figures 3.2 and 3,7). As compared to tissue, MEM was en-
riched in polypeptides in at least four M (x 10~'^)/pH classes (I,
= 14/> 7.2; II, 19-24/5.4-6.3; III, 28-31/6.9-7.3 and IV, > 150/< 5.1;
Figures 3.5 and 3.6) suggesting that these polypeptides are major
endometrial secretory or cell surface products. Several polypeptides
in both classes II and III had electrophoretic characteristics similar
to nonserum-identical UM-specific proteins (Figure 2.4). Included
(class II) were polypeptides resembling UMl and UM2, suggested to be
similar electro phoretically to porcine retlnol and retinoic acid
binding proteins (Adams et al., 1981; Bazer et al. , 1981b). Thus, UM
may serve as a valuable reservoir of uterine-specific proteins.
Uterine-specific polypeptides in class I were observed to be similar,

although somewhat less basic, than a series of low M , P, -induced

r 4 '

serine-protease inhibitors shown to be products of porcine endometrium
(Fazleabas et al. , 1982a, b). Anti-trypsin activity was described in
uterine flushings from early pregnant cattle, although its origin
(uterine vs serum) was not determined (Roberts and Parker, 1974b).
Such uterine proteins were suggested to be involved in limiting invas-
iveness of the trophoblast (Fazleabas et al. , 1982a). Polypeptides
in class IV were suggested to be similar to endometrial surface or
brush border glycoproteins thought to be important in a number of
processes critical to establishment and maintenance of pregnancy in-
cluding (1) regulation of endometrial carbohydrate metabolism (Larson
et al., 1970; Boshier, 1969), (2) stimulation of uterine secretory
activity (Larson et al., 1970; Murdoch et al. , 1970), and (3) adhesion
and fusion of cells (Frazier and Glaser, 1979; Wathes and Wooding,
1980; Wooding et al. , 1980; Dent, 1973). Absence of obvious differences

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in the qualitative array of proteins produced by bovine endometrial
tissues associated with reproductive status (nonpregnant, pregnant or
unilaterally pregnant) or stage, suggests that alterations in endo-
met rial-specific components of the uterine environment reflect
quantitative rather than major qualitative changes.

Absence of obvious qualitative differences in array of polypeptides
produced de^ novo by bovine endometrial explants is consistent with
observations in other domestic species, including pigs, sheep and mares,
which indicated that a relatively constant array of proteins is pro-
duced by, or present in the uterus throughout gestation or during
prolonged periods of P treatment (Bazer, 1975; Roberts et al., 1980;
Bazer et al., 1981b; Zavy et al., 1982b). Maintenance of histotrophe
production is considered critical for pregnancy maintenance in these
species (Cook and Hunter, 1978). In this respect, relatively stable
responses of endometrial explants from early pregnant cattle (Table
3.2) reflected metabolic stability of tissues. Such stability may be
important to insure continued production of histotrophe. Comparison
of responses of endometrial explants from pregnant and nonpregnant
cattle (days 16 and 19 only; Table 3.2) suggested that stability in
protein synthetic capacity of tissues from early pregnant cattle was
related to presence of a conceptus. Pregnancy status x day postmating
interactions, detected for both TP and %r (P < .01), reflected elevated
responses of day 19 as compared to day 16 tissues from nonpregnant
cattle versus stable, lower responses of tissues from pregnant cattle
at the same stage (Table 3.2). Data suggested that the systemic effect
of chronic peripheral plasma P (Ford et al., 1979; Lukazewska and
Hansel, 1980), combined with the local effect of the conceptus itself,
had a stabilizing effect on uterine tissues.

-250-

Differences in metabolic capacity of endometrium between early
pregnant and cyclic cattle, particularly after days 16 to 17, may
reflect differences in physiological commitment; the former committed
to an embryotrophic and the latter to a luteolytic goal, Lewis et al.
(1982) showed that endometrial slices from day 16 and 19 pregnant
cattle produced equal quantities of PG from H-arachidonic acid
in vitro. Curl (J. Curl, personal communication) observed that, while
neither concentration nor content of PGF or PGFM in endometrial
tissues differed between cyclic and pregnant cattle on day 17 post-
estrus, PG synthetic capacity was greater in tissues from cyclic
cattle. Present data are generally consistent with these observations
and suggest that enhanced endometrial metabolic activity during late
diestrus in cyclic /nonpregnant cattle reflects events pursuant to uterine
mediated luteolysis. Such events are enhanced by increased E /P, ratio
characteristic of late diestrus (Ford et al., 1979; Bartol et al.,
1981b). Studies in sheep suggest that such E-induced effects may
involve responses of different areas (caruncular vs intercaruncular)
and/or cell types within the endometrium (Findlay et al., 1981; Huslig
et al., 1979).

If an embryotrophic uterine environment is to be established and
maintained, then the conceptus must contribute to the array of stimuli
integrated by uterine tissues. Although specific sites of action have
not been identified for any bovine conceptus products, ^ vitro pro-
duction of known biologically active agents, including both
prostaglandins and steroids, was documented for bovine conceptuses
from as early as day 13. Production increased in a manner related to
elongation of the trophoblast and increasing conceptus mass (Chenault,

-251-

1980; Eley et al., 1983; Shemesh et al., 1979; Lewis et al., 1982).
As shovm for both ovine and porcine peri-attachment conceptuses
(Godkin et al, , 1982a, b), present data (Chapter IV) indicate that
bovine peri-attachment conceptuses can also produce a variety of
complex polypeptide macromolecules de novo from radiolabelled amino
acid substrate.

Ability of bovine conceptuses to incorporate radiolabelled amino
acid substrate into nondialyzable product was clearly related to stage
of conceptus development. Among day 16 conceptuses, those which had
expanded to some degree were more active in incorporation of "^H-Leu
into labelled polypeptides. Overall, %r of H-Leu as nondialyzable
product in MEM was lowest in day 16 cultures (X ± SEM = 1.16 ± 4.11%,
P < .01), increased in day 19 (16.8 ± 3.67%) and 22 cultures (20.9 ±
5.80%) and decreased (P < .07) in day 24 cultures (6.87 ± 4.10%).
Marked increases in %r after day 16 were reflected by increases in
complexity of the array of polypeptides identified in both tissue and
MEM (Figures 4.3 and 4.4). Such striking changes are undoubtedly re-
lated to development and differentiation of tissues and cell types
required to produce the complete array of conceptus proteins. ^ vitro
production of proteins was related to stage of development in both
sheep and pig conceptuses (Godkin et al. , 1982a, b).

Polypeptides produced by bovine conceptuses were primarily DEAE (+)
acidic macromolecules (Figure 4.2). Anion exchange profiles of radio-
labelled macromolecules in dialyzed MEM were especially similar to
those reported for sheep (Godkin et al., 1982b). For each conceptus
stage examined (days 16, 19, 22 and 24), numerous polypeptides identi-
fied in MEM were absent, or present in only trace amounts in tissues

-252-

(Figures 4.3 and 4.4). Consequently, these polypeptides were considered
most likely representative of proteins contributed to the uterine
environment by peri-attachment stage bovine conceptuses. Observations
are consistent with those of Lewis et al. (1979) for day 19 bovine
conceptuses and with similar studies in both sheep and pigs (Godkin
et al., 1982a, b). Data indicate that explant medium following short
term (24-48h) culture of conceptus tissue , rather than tissue extracts,
may provide the richest source of these products for future study.

Medium from bovine conceptus explants was enriched in two major
types of protein products, including (1) a group of late eluting
DEAE (+) , low M acidic polypeptides (M x 10 /lEP - 22-26/6.5-5.6)
and, (2) an earlier eluting DEAE (+) , high M (x 10 ; 735 ± 22) glyco-
proteins. The low M polypeptides were principle products of day 16
conceptuses and remained major products of conceptuses from days 19,
22 and 24 (Figure 4.3). These polypeptides were no longer prominent
components of MEM from a day 29 conceptus explant, suggesting transient
production of these conceptus proteins. However, MEM from day 69
bovine chorionic explants contained trace amounts of polypeptides with

similar electrophoretic character to some of the low M proteins iden-

r

tified in earlier stage conceptus MEM (Figure 4.5). Hence, production
of certain of the low M acidic polypeptides may not be terminated,
but only depressed in chorionic tissues from later in gestation. Tran-
sient production of low M , acidic polypeptides was also reported for
peri-attachment stage porcine and ovine conceptuses (Godkin et al. ,
1982a, b).

Temporal pattern of appearance of the low M acidic polypeptides
suggests that these conceptus products may play important functional

-253-

roles in establishment and maintenance of pregnancy. Appearance of
these polypeptides as major products of bovine conceptuses from day
16, the point after which removal of the conceptus results in prolon-
gation of the estrone cycle (Northey and French, 1980), presents the
possibility that these proteins may be involved, either directly or
indirectly, with the process of luteostasis. Ginther (1981) presented
evidence suggesting the presence of a "luteotrophic factor" in uterine
venous effluent of the gravid uterine horn during the bovine peri-
attachment period. However, current evidence for a bovine conceptus-
produced, proteinaceous luteotrophin is equivocal (Beal et al., 1981;
Poffenberger et al. , 1982; Smith et al., 1982). As observed above, it
is difficult, if not impossible, to separate those conceptus-induced
maternal responses associated with luteostasis from those required for
maintenance of an embryotrophic uterine environment. Indeed, mechanisms
associated with one may well insure the other. Data reviewed above
suggested that low M acidic proteins, produced transiently by peri-
attachment stage conceptuses, may be required to stimulate endometrial
production and secretion of histotrophe during a critical period of
conceptus development. Such conceptus-induced changes in endometrial
protein synthesis and secretion may involve alterations in endometrial
arachidonic acid metabolism and prostaglandin production characteristic
of stimulus-secretion coupled events (Rubin, 1982; Rasmussen, 1981).
Consequently, endometrial responses to these conceptus products,
necessary to insure maintenance of an embryotrophic uterine environ-
ment, may have indirect effects on luteostasis as a result of altered
PG metabolism.

Identification of the early eluting DEAE (+) , high M glycoprotein
— _3
(X M^ X 10 ± SEM - 735 ± 22) as a major component of MEM from day

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16, 19, 22 and 24 bovine conceptus cultures, as well as from day 69
bovine chorion cultures, confirmed and extended earlier observations
of Masters et al. (1983). It was suggested that such molecules, shown
to be produced by bovine, ovine and porcine peri-attachment concep-
tuses, might normally provide an Immunologically tolerant or protease
resistant coating to the chorion (Masters et al., 1983). Guillomot
et al. (1982) reported that cytochemical alterations in the glycoprotein
surface coat of ovine conceptuses between days 13 and 15 were related
to initiation of conceptus attachment. It was suggested that such
alterations might involve de novo synthesis of glycoprotein and that
these products might be important in adhesion between conceptus (chor-
ionic) and endometrial surfaces (Guillomot et al., 1982).

Pattern of production of bovine conceptus proteins may reflect

functional necessity for these products. Proteins such as the low M

r

acidic polypeptides, produced transiently prior to day 29, may be
required specifically during the peri-attachment period to induce
conditions necessary for support of the conceptus prior to completed
vascularization of the chorioallantois and establishment of definitive
placental attachments (Greenstein et al. , 1958; King et al. , 1980).
Present data do not exclude the possibility that synthesis and secre-
tion of certain "transiently produced" polypeptides continue beyond
attachment at reduced rates. Other polypeptides may be involved in
processes less specific to the peri-attachment period including
(1) maintenance of immunological privelege for the conceptus, (2) com-
pensation for hormonal effects of pregnancy on maternal physiology,
(3) stimulation of local uterine responses required for continued
fetal -placental development, and (4) interaction with other components

-255-

of the extraplacental (uterine) environment so as to make them more
readily available to the conceptus (i.e., binding /carrier proteins
or proteases; see Krieger, 1982; Beer and Billingham, 1976). Clearly,
any or all of these processes may require the integrated action of
several conceptus products including polypeptides, steroids, prosta-
glandins and other as yet unidentified compounds.

Collectively, present data indicate that, in cattle, proteins
characteristic of an embryotrophic uterine environment may appear
in utero as a consequence of both selective uptake from serum and
de_ novo endometrial synthesis and secretion. Additionally, de novo
production of proteins by peri-attachment stage bovine conceptuses
may provide a third source of uterine lumenal proteins which, together
with other conceptus products, may be essential for maintenance of
pregnancy. Bovine uterine-specific proteins were primarily acidic
and, in this respect, unique from more basic porcine, ovine and equine
uterine-specific proteins (Bazer et al. , 1981b; Zavy et al. , 1982b).

In contrast, both nature and pattern of production of low M acidic

r

and high M glycoproteins by bovine conceptuses were similar to those
reported for ovine and porcine conceptuses from comparable developmental
stages (Godkin et al. , 1982a, b), suggesting a common requirement for
such products. Historically, study of form has preceded that of
function. In this light, present data provide a basis from which
elucidation of functional interrelationships of these maternal and
conceptus products may proceed.

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Adams, K.L., F.W. Bazer, and R.M. Roberts. 1981. Progesterone-induced
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Ainsworth, L, , and K.J. Ryan, 1967. Steroid hormone transformations
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Allen, W.R. 1979. Maternal recognition of pregnancy and immunological
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Amoroso, E.C, and J.S. Perry. 1975. The existence during gestation
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rPT?'

BIOGRAPHICAL SKETCH

Frank Fitzhugh Bartol, son of Dorothy Foster and the late Henry
Fitzhugh Bartol, was born in Arlington, Virginia, on April 15, 1953.
Attending Arlington County public schools throughout his primary and
secondary education, he graduated from Yorktown High School, Arlington
County, Virginia, in June, 1971. He received his Bachelor of Science
degree in dairy science from Virginia Polytechnic Institute and State
University, Blacksburg, Virginia, in June, 1975. Having a special
interest in reproductive physiology, he began graduate work in the
Department of Dairy Science at the University of Florida, Gainesville,
Florida, in fall of 1975. Under guidance of Dr. William W. Thatcher
of that department, he received the Master of Science degree in dairy
science in March, 1980. Remaining in the Department of Dairy Science
at the University of Florida, he continued study pursuant to the
Doctor of Philosophy degree in animal science, with emphasis in repro-
ductive biology. In June of 1981 he was elected as an associate member
of Sigma Xi. On August 22, 1981, he was married to Anne Ambrose Wiley.

Upon completion of the Doctor of Philosophy degree he will serve
in the position of assistant professor in the Department of Animal and
Dairy Science, Auburn University, Auburn, Alabama.

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I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

William W. Thatcher, Chairman
Professor of Animal Science and
Dairy Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

%^

Fuller W. Bazer

Professor of Animal Science

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

■y

Maarten Drost

Professor of Veterinary Medicine — IFAS

ll'«:-c

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

R. Michael Roberts

Professor of Biochemistry and Molecular

Biology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.

Daniel C. Sharp, III !J

Associate Professor of Animal Science

This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate School, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

April 1983

\lu.<i^ T. c:/v.

Dean, jC6llege of Agricul

Dean for Graduate Studies and Research

UNIVERSITY OF FLORIDA

3 1262 08554 0101

UNIVERSITY OF FLORIDA

3 1262 08554 0101