Characterization of bovine uterine and conceptus proteins

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Title:
Characterization of bovine uterine and conceptus proteins
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Bovine uterine and conceptus proteins
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xi, 292 leaves : ill. ; 28 cm.
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Bartol, Frank Fitzhugh, 1953-
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Cattle -- Reproduction   ( lcsh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1983.
Bibliography:
Includes bibliographical references (leaves 256-291).
Statement of Responsibility:
by Frank Fitzhugh Bartol.
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Typescript.
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Vita.

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University of Florida
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Full Text















CHARACTERIZATION OF BOVINE UTERINE AND
CONCEPTS 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











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.










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.


















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS . . .. ii

LIST OF TABLES. . .. vii

LIST OF FIGURES . . viii

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















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 (E1,
E2, E2SO4, P4) in uterine milk supernatant (UMS) and
peripheral plasma (P). . . 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
















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

2.4 Two-dimensional polyacrylamide gel electrophoresis
(10% acrylamide) of acidic (IEF) 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 3H-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


viii










Figure Page

3.6 Fluorograph of a 2D-PAGE (IEF) gel (10% acrylamide)
depicting major bovine endometrial-specific polypeptides
present in MEM. . ... ..... 179

3.7 Fluorographs prepared from 2D-PAGE (IEF) 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 concepts cultures
following 24 h incubation in vitro. . ... 208

4.3 Fluorographs prepared from representative 2D-PAGE (IEF)
gels (10% acrylamide) of polypeptides in dialyzed bovine
concepts culture MEM from days 16 (A), 19 (B), 22 (C),
and 24 (D) . . 210

4.4 Fluorographs of 2D-PAGE (IEF) gels (10% acrylamide)
showing concepts tissue polypeptides into which 3H-Leu
was incorporated in vitro . .. 214

4.5 Fluorographs prepared from 2D-PAGE (IEF) gels (10%
acrylamide) of polypeptides in dialyzed bovine concepts
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 concepts proteins. .. 222

4.8 Isolation of the high molecular weight bovine concepts
glycoprotein. . . .. 225

















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 CONCEPTS 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 ( SIE) 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











was enriched in basic proteins of less than 40,000 molecular weight

(Mr). 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 concepts

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

enriched in polypeptides in four M (x 10- )/pH classes (I, =14/>7.2;
r
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 wererelated to stage of development. Day 16, 19, 22 and

24 conceptus-MEM was enriched in low Mr, acidic polypeptides (LMrA;

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 =
r r
735 22), produced by all concepts 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.

















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 concepts

(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 concepts 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 concepts interacts

with its maternal environment to establish and maintain pregnancy is

imprecisely understood.






-2-


Throughout history innumerable theories have been suggested to

explain the relationship between concepts and maternal units. One

of the earliest recorded attempts to explain maternal support of the

developing concepts 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 concepts 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 (1485-1558) 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 in 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 Animalium (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 concepts was nourished

by substances within the womb (uterus) much as the neonate was nourished

by the products of the breasts.











Walter Needham (1631-1691) was described as the father of the

dynamic aspect of physico-chemical embryology (Needham, 1959). In

his major treatise, De Formatu 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 (1643-1679) 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 concepts 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 concepts 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 concepts 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

vascularization.







-5-


During this same period potential sources of nutrient materials

provided to the concepts in 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 concepts in 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

concepts 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











(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 in 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 concepts and its uterine environment is unique. Furthermore,

substances provided the concepts 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











emphasized importance of the uterine environment for maintenance of

concepts 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 concepts, 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 concepts 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 concepts 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 concepts "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 concepts.











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 concepts; 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










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 (E2) 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 P4 and increasing E2

(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 luteinization 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 E2 by 5h after

the LH peak and basal E2 concentrations (1 to 3 pg/ml) obtained by 14h

(Chenault et al., 1975). Luteinization results in formation of the

ovarian corpus luteum (CL) and initiation of P4 production. Histolo-

gical and cytological descriptions of luteinization 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 P4 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 P4 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 P4 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-F2a (PGF2 ), 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 E2 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

A5-pathway (conversion of pregnenalone to androstenedione through

A -3S-hydroxysteroids; 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 E2 concentration in follicular fluid. In vitro production

of E2 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 E2 (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. 1.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 (IUD), 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 E2 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 IUD'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 PGF2 Since the observations of Babcock (1966), and

Phariss and Wyngarden (1969), exogenous PGF2a 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 (Goding, 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 arachidonic 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 PGF2a (Shemesh and

Hansel, 1975b; Nancarrow, 1972) that occur after day 15. Furthermore,

evaluation of peripheral plasma concentrations of 13,14-dihydro-15-

keto-PGF2a (PPGFM), the inactive metabolite of PGF2a, revealed temporal

associations between elevated PPGFM and declining P4 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 PGF2 -specific binding sites were present in luteal

tissues. Hixon and Hansel (1974) observed that uterine infusion with

PGF2c per cervix on days 12 or 13 postestrus caused increases in PGF2a

concentrations in ovarian and carotid arterial and jugular venous

plasma. Collectively, these studies provided strong evidence favoring

PGF2, as the endogenous bovine uterine luteolysin.

Thatcher et al. (1979) reported that E2-170 (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-injection. Bartol et al. (1981b) demonstrated that the predictable

E2 induced 6h increase in PPGFM was accompanied by a concomitant in-

crease in uterine luminal content of the parent compound, PGF2a, 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 PGF2, and PGFM. These studies demonstrated that

bovine uterine PG synthesis and metabolism in response to E2 could be

accurately assessed by monitoring PPGFM.

The E2 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 E2 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),

15-hydroxyprostenoate dehydrogenase, and Al3-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 2. Data reviewed above are consistent with the concept that E2,

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 PGF2a from tissue stores of arachidonic acid. Luteolysis may

then result from binding of endometrially produced PGF2a 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 (P4 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 P4 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 P4 appears as the predominant maternal peripheral

plasma steroid.

Maintenance of peripheral plasma P4 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 P4.

In a recapitulation of the Born-Frankel theory, Hartman (1924) stated

that products) 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 concepts 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 concepts in utero. Consequently, luteal maintenance,

characteristic of pregnancy, reflects response of the CL to physiolo-

gical conditions initiated by the concepts and/or its products within

the uterine lumen. Short (1969) referred to the process by which the

peri-attachment concepts signaled its presence to the maternal unit,

as reflected by luteal maintenance, as maternal recognition of preg-

nancy. In cattle, the precise mechanisms) 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 concepts 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 concepts homogenates on days 15 through 17 extended

inter-estrus interval (Northey and French, 1980). Surgical transfer of







-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 concepts must be present in utero

at least from days 15 through 17 if luteal maintenance is to be ini-

tiated.

Location of the concepts 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 in 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

P4 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






-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 P4 by

dispersed bovine luteal cells in vitro. Similarly, Godkin et al.

(1978) detected a factor in day 13 to 15 ovine conceptuses which

stimulated in vitro production of P4 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

concepts induced CL maintenance. In cyclic cattle, uterine infusion

of concepts homogenates from days 15 through 17 delayed decline in

peripheral plasma P4, 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 concepts homogenates by radioreceptor assay, nor did

homogenates stimulate P4 synthesis 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 P4 levels were de-

clining in cyclic controls. Kindahl et al. (1981) suggested that, in

cattle, P4 might regulate duration of uterine release of PGF2a 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

PGF2a (Kindahl et al., 1981). Alternatively, maternal P products of

the concepts, 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 concepts could modify uterine response to an endocrine

stimulus. Plasma PGFM response of day 18 pregnant cattle given E2-17B

(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 E2-176-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

PGF2 observed during the normal period of luteolysis in cyclic cattle

(days 19-20), were absent in pregnant cattle at the same stage. A

concepts effect on uterine production and/or transport of the luteo-

lysin (PGF ) was suggested. Evidence that PG may be subject to active,

carrier-mediated 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 concepts

and underlying uterine tissues. This process undoubtedly involves

both luteotrophic and antiluteolytic phenomena. Products of the

concepts may act directly on the CL, either to stimulate luteal

function or antagonize the luteolysin. Additionally, interaction of

concepts 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 mechanisms) responsible for pregnancy recognition

in cattle will require further investigation of the nature and function

of substances produced by both maternal and concepts 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-atachment 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 concepts. 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 concepts 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

concepts does not appear to interact detectably with its uterine

environment prior to day 15 post-estrus. However, a strict requirement

for synchrony 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 concepts

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 P4. While Sauer (1979) indicated

that preovulatory ovarian E was important in potentiating uterine

response to this hormone (see below), P4 replacement therapy alone

maintained pregnancy in ovariectomized cattle (Hawk et al., 1963),

sheep (Foote et al., 1957; Cumming et al., 1974) and pigs (Gentry

et al., 1973). In cattle, several workers reported more successful

conception following estrous cycles in which plasma P4 was higher than

usual (Corah 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 concepts.

Perhaps the earliest physical response of the maternal unit to

presence of a concepts in the uterine lumen is an increase in uterine

blood flow and/or vascular permeability indicating initiation of inter-

action between the concepts 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 Christenson, 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 concepts,

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 concepts 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

concepts 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

estrouss) 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

concepts 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 concepts

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 concepts 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 pseudostratified 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 muscularis (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, 1976).

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

catecholaminess; 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 nonspecifically 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 P4 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 (Rc) forms an H-Rc complex which

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

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 10h 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-


enzyme 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 E2-178 (500 pg, I.M.) to diestrus ewes (Luebke et al., 1980).

Less immediately dramatic changes in protein and RNA synthesis

are seen following P4 exposure and nuclear translocation of P R
4 4 c
stimulation of specific mRNA occurs within 4h to 6h of P4 exposure,

with maximal stimulation not seen before 18h to 20h (Schrader and

O'Malley, 1978; Clark et al., 1977). Endometrial response to P4 re-

quires E priming (Walters and Clark, 1980). Since P4 may also regulate

tissue E receptor levels (Walters and Clark, 1980), temporal patterns

of appearance and relative amounts of each hormone (E and P4) 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

P4. Uterine sensitivity to ovarian steroids (E and P4) is modified

through the integrative action of E and P4. Estrogen-induced increases

in endometrial P4 cytosol receptors augment uterine response to P4

(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
n
et al., 1980). Consequently, P4 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 E2-178. Diminished responsiveness was restored

by administration of thyroid hormone (T ). Thyroid hormone alone did

not elicit a uterine response. However, T restored E2-170-induced

uterine responsiveness in a dose dependent manner. Thus, uterine

responsiveness to E may be modified by metabolic hormones.

In addition to its effect on uterine E receptor synthesis, P4

suppresses endometrial synthesis of its own cytosol receptor (Walters

and Clark, 1980; Schrader and O'Malley, 1978). A short term effect of

P4 on E-primed endometrial tissue included a rapid increase in trans-

location of P4 R to P 'R (Walters and Clark, 1980). Levels of P4 R
4 c 4n 4 n
were maximal at lh and returned to basal levels by 9h post-P4. Rapid

increase in P *R was accompanied by a depression in P. R followed by

replenishment within 12h post-P4. This rebound phenomenon was suggested

to indicate recycling of nuclear receptor to the cytosol (Walters and

Clark, 1980). However, following the 12h post-P4 replenishment of P4

cytosol receptors, levels decreased to those characteristic of unprimed

(E) tissue (Walters and Clark, 1980). Progestational reduction in P4

receptors was not irreversible and was overcome by administration of

E (Walters and Clark, 1980; Schrader and O'Malley, 1978). Tissue P4






-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 P4 depends upon temporal

pattern of appearance and amount of each hormone present (E/P4 ratio).

Following E-induction of high levels of endometrial P4 receptors, P4

may eliminate or modify E-induced uterotrophic responses. However, as

P4 suppresses endometrial production of its own receptors, E-induced

events become less suppressed. Hence, P4 receptor levels dictate P4

response as reflected by E-induced uterotrophic events (Walters and

Clark, 1980). In this respect, the P4 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 P4 receptor popula-

tion throughout gestation. In cattle and other species, such stimuli

may be provided by the concepts. Recently, Findlay et al. (1982)

presented evidence that the ovine concepts 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 P4

in normally cyclic domestic farm species revealed predictable patterns

with respect to the review of receptor dynamics presented above. Endo-

metrial content of cytoplasmic E2 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 E2 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

concepts produced E is critical (see Bazer et al., 1982).

Bovine endometrial content of cytoplasmic P4 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 P4 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 4R (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;







-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-48h 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 pm) during mid-diestrus (Skjerven, 1956a; Marinov and

Lovell, 1968; Wathes and Wooding, 1980). Characteristic of active

diestrus endometrial tissue are columnar and pseudostratified 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 concepts, 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 pseudostratified 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 um 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 concepts 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 pm) 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 concepts 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

concepts 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 Pm 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 70% 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 chorionicc) 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 concepts trophoblastic binucleate cells. These workers

suggested that transient formation of mGC between days 19 and 28

occurred primarily to allow delivery of concepts binucleate cell

products to maternal circulation. Such a process could provide a route

for delivery of concepts 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 concepts

origin leads to establishment and maintenance of a unique, embryotrophic

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. Comments 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 (2 100 days) rendered unilaterally pregnant by liga-

tion of one uterine horn. In both cases, uterine fluid accumulated in

the nonpregnantuterine 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 (Cl), and inorganic phosphate (PO ). Both K and PO4 were

elevated during diestrus, while Ca and Cl were elevated during proestrus

and estrus. Mean uterine fluid concentrations of K and PO4 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 secretary

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-


P4 (E/P4 ratio) may 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 VanDemark, 1957b; Schultz et al., 1971) suggests essential roles

for these ions in support of the pre-attachment concepts.

Fluctuation in uterine luminal electrolyte content, especially

Ca, may reflect endometrial secretary activity and capacity to respond

to concepts signals. As compared to diestous 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 PGE2 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 secretary 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

secretary 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 in vivo after gland stimu-

lation, a similar extracellular Ca-mediated, luminal-specific

stimulation of secretion might function in vivo (Rasmussen, 1981).

Rasmussen (1981) indicated that, in secretary 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 concepts interaction.






-45-


Free amino acids and sugars may serve as important energy sub-

strates to both uterine and concepts 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,

characterizational 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, concepts 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 biosynthesis of

other concepts products (Zavy et al., 1982a). This pathway of fruc-

tose metabolism would require that the concepts 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 concepts. 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, 1974a,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 concepts 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 (IEF) 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 IEF 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 (Mr) range (Roberts






-49-


and Parker, 1974a). Additionally, three CMP were detected in uterine

flushings from a single ovariectomized cow treated with P4 (100 mg/day)

for two months (Roberts and Parker, 1974a). Similarly, Libby et al.

(1982) recently described three CMP proteins inuterine 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-100 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

-3
polyacrylamide gel electrophoresis (SDS-PAGE). Molecular weight (xl-3)

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
-3
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 P4. It was suggested that some

of the F3 proteins, which persisted until day 31 of gestation when the

study was terminated, might be concepts products or conceptus-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

concepts 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 (xl0-3 ) categories between 18.7 and 292 (Bartol et al., 1981a).
r
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 Mr (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






-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-3) 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 P4

daily to extend diestruss" 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 P4 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 -14C

leucine ( 14C-Leu) and L- 4,5-3 H leucine ( H-Leu) during a 48h in vitro

culture. Endometrial H- and 14C-Leu incorporation was not affected

by stage of gestation. Addition of steroids (E, P4 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 1C-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, P4 or both) was detected on capacity of porcine

endometrial tissues to synthesize protein de novo from radiolabelled

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






-53-


and porcine endometrial tissues to synthesize proteins in vitro

depended upon in vivo hormonal state at the time they were removed.

A number of enzymes are present in bovine endometrium 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 secretary 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 concepts 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 P4-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 conceptus-induced increase in endo-

metrial enzyme activity associated with remodeling of the endometrium

to accommodate 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 c-L-fucosidase,

a-D-galactosidase, a-D-galactosidase, a-D-glucosidase, a-N-acetylgalac-

tosaminidase and -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 IEF gels of bovine uterine flushings (Roberts and

Parker, 1974a). Using IEF, 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 in 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
-3
with M (xlO ) of 57 and 59 were detected by SDS-PAGE as the major
r
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 P4 alone (50 mg/day) or P4

plus estrone (E1, 5 Ig/day), with maximal stimulation in the latter

group (Moffatt et al., 1980, 1981). Endometrial explants from pregnant

ewes and ovariectomized ewes treated with P4 or P4 + E secreted radio-

labelled uterine milk proteins into culture medium during incubation

with 3H-Leu (Moffatt, et al., 1980, 1981). Data indicated that the two

uterine milk proteins are the major secretary products of ovien endo-

metrium during pregnancy or chorionic P4 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 /EI stimulation for induction of synthesis of these

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 P4 and associated with

presence of the concepts.

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 P4 (Knight et al.,

1974a). Knight et al. (1974a) reported a positive dose-response

relationship between amount of P4 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-


E2 acted synergistically when administered in lower doses (up to 25 pg/kg

body weight/day), but antagonistically when administered in higher doses

(50 pg/kg body weight/day) with P4 (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 concepts (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

and Bazer, 1980), was shown to have a M (x 10-3) 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 immunofluorescent 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 P4-induced porcine uterine fluid, and was synthesized and

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

endometrium from diestrus cyclic, pregnant, pseudopregnant and P4-

treated pigs (Basha et al., 1979, 1980a,b; Roberts and Bazer, 1980).

During gestation, maximum Utf synthesis occurred between days 35 and

75 when P4/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 transferring

(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, P4-

induced porcine uterine secretions were shown to contain a group of

basic, low M serine-protease inhibitors (Fazleabas et al., 1982a).
r







-60-


One of these proteins, purified via a series of gel filtration and

cation exchange steps (Mr = 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,

cultured with 3H-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 E2-178

(5 mg IM) on day 11 postestrus (Fazleabas et al., 1982b). Thus, E,

acting on a P4 dominated uterus, was stimulatory to inhibitor synthesis.

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, P4-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 P4 alone or P4 + E, but not E alone or corn oil vehicle

(Adams et al., 1981). Evidence for a P4-induced retinoic acid binding

protein was also presented (Adams et al., 1981). The P -induced RBP
_3
had an estimated M (xl3 ) of 17 to 20 and a Kd for retinoic acid of
r d
-6
1.93 x 10-6 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 synthesized 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 P4 levels or presence of a concepts. 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

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

phosphate isomerase (GPI) allows for interconversion of glucose-6-PO4

and fructose-6-PO4. 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 concepts 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 concepts 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, BI, C, D and

E activities in uterine flushings from ovariectomized P4-treated gilts.

Activities of cathepsin B, D and E did not reach detectable levels

before day 60 of chronic daily P4 treatment, suggesting a requirement

of prolonged P4 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 Mr 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 P4 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 P4-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 )/pl range =
<21/5.9-7.0) were identified in uterine flushing from cyclic mares
<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 P4 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 U1, 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 P4 or E2 and was not found in uterine flushings from

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 E2-178 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 D1, was a
-3
nonserum polypeptide with a M (x 10-3) 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 (D1), 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 luminal leucine aminopeptidase (LAP) activity,

shown to be under P4 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, transferring 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 PGF2 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 PGF2a 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 E2 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 PGE2 content did

not vary between days 13, 15 and 17 (Ellinwood et al., 1979b). In

contrast, PGE2 content of porcine uterine flushings increased between

days 11.5 and 14 (Geisert et al., 1982b). Paradoxically, as compared

to cyclic controls, PGF2a 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 PGE2 (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 in vitro production of PGF26 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

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

day 16 and 19 pregnant cattle produced radiolabelled PGF2 PGE2, PGFM,

and at least four other unidentified metabolites, when incubated with

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 PGF2a or

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 concepts (Findley, 1981; Findley et al., 1981; Hyland

et al., 1982; Inskeep et al., 1980). Endometrial tissue concentration

and content, and in vitro production of PGF2a 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 PGE2 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 PGF2, and PGE2 were consistently higher in pregnant

than nonpregnant ewes on days 13, 15 and 17 postestrus, with an increase

in the PGE2:PGF2- ratio seen in pregnant animals (Ellinwood et al.,

1979b; Findlay, 1981). The observation of Henderson et al. (1977)

that, in ewes, PGE2 was antagonistic to the luteolytic effect of PGF2a

in vivo, led several investigators to propose that PGE2 might be the

antiluteolytic signal in pregnant sheep (Ellinwood et al., 1979b;

Huie et al., 1981; Reynolds et al., 1981). In this regard, both PGI2

(prostacyclin; Milvae and Hansel, 1980) and PGEI (KLmball and Lauder-

dale, 1975) stimulated plasma P4 when administered to cattle in vivo.

However, the possibility that PGE2 might be involved in bovine luteo-

stasis has not been examined carefully.







-69-


The role(s) of uterine PG, particularly PGF2a, 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 PGE2 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 concepts 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;






-70-


days 8, 12, 14, 16 and 19) cattle. Findlay et al. (1981) presented

evidence for a discrete effect of the day 15 ovine concepts on car-

uncular and, to a lesser extent, intercaruncular tissue. As assessed

in 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 PGF2a and PGE2 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 PGE2 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) PGE2 response preceded

PGF2, 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 synchronized 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 concepts origin,

necessary for maintenance of an embryotrophic environment.

It was observed that, in secretary 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 secretary 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 secretary product. When pancreatic

acinar cell membranes were labelled with 14C -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 1C -AAc appeared as

phosphatidic acid and the other half as PGE2 (Marshall et al., 1980).

Furthermore, PGE2 stimulated amylase secretion which was blocked by

inhibition of PG synthesis (Marshall et al., 1980). Explanations of

these data included possibilities that: (1) PGE2 might serve as a

coordinate messenger with Ca, or (2) secretagogue-induced PI turnover

leads to release of AAc and increased synthesis of PGE2 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 secretogogic stimuli results in production of agents

and/or conditions which may potentiate continued secretary 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 secretary activity, but be necessary for its maintenance.

In this respect, it is interesting that in both sheep and pigs, PGE2:

PGF2. 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 concepts 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 (1) 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

nonpregnant (days 16 and 19) cattle with either 3H-P4 or H-androstene-

dione (3H-A4) in vitro. Both labelled substrates were extensively

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

for tissue wet weight, amount of 3H-P4 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

27 of pregnancy metabolized more 3H-P4 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 P4 and A4 was affected by stage

of gestation but was independent of local concepts effects. Chroma-

tographic analyses of radiolabelled metabolites indicated that H-P4

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

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

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

3,17 dihydroxy-5c-androstane from 3H-A4 (Eley et al., 1983). No C19
3,13
metabolites were generated from 3H-P nor C21 from 3H-A4. Addition-

ally, neither testosterone (T), E1 nor E2 were found (Eley et al.,

1983). Similar results were reported for bovine endometrium by Knicker-

bocker et al. (1980).

Estrogen sulfoconjugates, particularly E1 S4, 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 SO4

in both maternal plasma and fetal allantoic fluid as early as day 27

and 33 respectively suggested a requirement for sulfotransferase

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 SO4 and E2SO4 detected in peripheral plasma (Hoff-

man et al., 1976). 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 SO4 was luteolytic when administered chronically






-74-


(28 or 56 mg E SO4/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-P4 to H-A4, and both of these

substrates to T, E1 and E2 in vitro (Willis et al., 1979a,b, 1981).

Conjugated estrogens (primarily E1SO4) 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

from pregnant and nonpregnant ewes (day 15 postestrus) with 3H-A4.
3 3
Additionally H-E and H-E SO4 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 concepts 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-A4 was reduced in tissue from day

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

comparison of tissues from pregnant and nonpregnant ewes on day 15

revealed no pregnancy related effect on in vitro metabolism of 3H-A4,

H-E or H-E SO4, although effects were detected on tissue PGF content

and rate of in vitro protein synthesis (Findlay et al., 1981). Addi-

tionally, metabolism of H-P4 by ovine endometrium from days 20, 60

and 140 of gestation was not affected by stage of gestation or presence

of concepts membranes in culture (Willis et al., 1979b). 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-


Both porcine and equine endometrial tissues converted 3H-P4 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 3H-A4 to

T, El, E2 and conjugated El and E2 (SO4) in vitro (Bazer et al., 1982).

Steroid sulfates and glucuronides were shown to be major porcine

endometrial metabolites of P4 (Bazer et al., 1982; Tindall and Henricks,

1971). Porcine endometrium from days 16 and 25 of pregnancy produced
3 3
H-E2178 (not 17a) from H-P and metabolism increased with stage of

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

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

chromatograph with E standards suggesting a modulating role for

concepts 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 concepts (as well as the concepts

itself) was a potential source of E (Flood and Marrable, 1975; Flood
3 3
et al., 1979). In vitro conversion of H-P4 to H-E1 by endometrium

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

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

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

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

endometrium produced neither E (E1 or E2) nor T from H-P or H-A4

in vitro (Eley et al., 1983; Knickerbocker, 1980). Therefore, bovine

endometrium is possibly lacking aromatase, as well as 178-dehydrogenase

(178-HSD) and 178-oxidoreductase activities (Heap et al., 1979). Fur-
3 3
thermore, primary products of bovine endometrial 3H-P and 3H-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 SO4) are luteolytic when administered to cattle during diestrus

(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 P4

to E, or aromatizable steroids, would be counter to the goal of

pregnancy maintenance. In sheep, in which E2-17 is also luteolytic

(Stormshak et al., 1969; Goding, 1974), evidence for aromatase and

178-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

conversion of 3H-A4 to either E2 or E was neglibible.

Since 5a-reduced steroids are, essentially, biologically inactive

(Holzbauer, 1976; Kubli-Garfiar et al., 1979, 1980), bovine endometrial

Sa-reduction of P4 may potentiate effectiveness of concepts products

(Eley et al., 1983). Additionally, 5t-reduction of P4 may enhance

endometrial responsiveness to E by reducing P4 suppression of E receptor

synthesis and E-R retention time (Clark et al., 1977; Katzenellenbogen
3
et al., 1980). In vitro metabolism of H-A4 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 concepts (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

concepts. Conceptus produced E is considered essential for recog-

nition of pregnancy in pigs (Bazer et al., 1982). Both free and

conjugated E (E1, E2, E3, E SO4, E2SO4 and E3SO4) content was increased

in uterine flushings from pregnant as compared to nonpregnant gilts

between days 10.5 to 14 (Geisert et al., 1982b). Uterine flushing E

content in pregnant gilts, particularly E2, E2SO4 and E SO4 increased

in a manner related to stage of concepts 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 readily metabolizable substrate to the concepts 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, concepts 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 SO4 concentrations in maternal peripheral plasma,

reflective of concepts 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 P4 or A4 (Eley et al., 1983), significant amounts of

biologically active E, if necessary for establishment of pregnancy,

must be provided by a maternal (ovarian) source (Hansel et al., 1973).

Absence of significant sulphotransferase 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 concepts 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 concepts prior to

day 16 postestrus. Once "the stage is set", however, events pursuant

to the embryotrophic path require uterine integration of both maternal






-79-


and concepts signals. Though data are unclear as to the nature of

such signals, their genesis appears to be related to developmental

stage of the concepts. For this reason, a brief description of bovine

concepts 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 concepts 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 concepts 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-Acid-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 concepts 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, concepts 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 chorionicc) 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 concepts chorionicc) 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.

Conceptus-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 concepts 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 concepts 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 concepts products. Substances produced by the concepts

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, nondiffusable 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 concepts (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.






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Proteins. 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 concepts 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.
-3
One of these proteins (M x 103 = 47 to 53; pI 4.0 to 4.4) was uniden-
r
-3
tified. The second (M x 103 = 65 to 70; pi 4.6 to 4.8) was suggested

to be alphal-fetoprotein (a1-FP).

Alphal-fetoprotein is a fetus (conceptus)-specific 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 al-FP to be the most pre-

dominant of three bovine fetus-specific serum proteins including a2-FP

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 a1-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






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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 al-FP in tissues of day 14 bovine trophoblasts (n=2)

and in al-lantoic fluid of one day 16 concepts. Incubation of bovine

concepts tissues (day 17, n=2; day 27, n=l) with 35S-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 al-FP

with stage of gestation. Concentrations of al-FP in maternal serum

were consistently and markedly lower (550 to 1.5 x 106 times) than

those in concepts tissues and fluids (Janzen et al., 1982). Addition-

ally ca-FP concentrations in uterine fluid consistently exceeded those

of maternal serum by a factor of at least 1 to 2 x 106 between days 14

and 46 (Janzen et al., 1982). Collectively, data indicated that syn-

thesis of bovine aI-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

35S-al-FP from 35S-methionine suggested that appearance of al-FP

in utero occurred via transudation across trophoblast membranes early

in gestation (Janzen et al., 1982).

The role of a1-FP remains unclear. Presently, controversy exists

as to whether a1-FP is an immunosuppressive or immunostimulating pro-

tein (Tomasi, 1978; Suzuki and Tomasi, 1980). Clearly, the former role

immunosuppressantt) would be consistent with necessity for establish-

ment of immunological privelege for the concepts (Beer and Sio, 1982).

Additionally, al-FP may regulate availability of uterin luminal







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steroids. Rat c-FP was reported to bind both T and E2 with equal

affinity (Kd = 1 x 10-1; 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 10-3 ) of 32-35 and pi in the range of 5.5-5.7 (Murthey
r
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 concepts 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, immunofluorescent 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 existence 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 immunofluorescent 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 laterstages of concepts

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 antiluteolytic 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






-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 conceptsu) glucose availability

(Porter, 1980).

In addition to effects on the maternal system, PL may have impor-

tant functions within the concepts 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 conceptsu) "growth hormones" (Freemark and

Handwerger, 1983).

Incubation of concepts tissues in vitro, in the presence of

radiolabelled amino acids, and analysis of labelled products in tissues

and medium postincubation 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 (E2 and P ) and endometrial co-culture on ability

of bovine concepts tissues from days 24 to 44 to incorporate