Citation
Pregnancy recognition in cattle

Material Information

Title:
Pregnancy recognition in cattle effects of conceptus products on uterine prostaglandin production
Creator:
Knickerbocker, Jeffrey John, 1955-
Publication Date:
Language:
English
Physical Description:
xi, 273 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Cattle ( jstor )
Estrogens ( jstor )
Estrus cycle ( jstor )
Metabolites ( jstor )
Plasmas ( jstor )
Pregnancy ( jstor )
Prostaglandins ( jstor )
Sheep ( jstor )
Steroids ( jstor )
Uterus ( jstor )
Animal Science thesis Ph. D
Cattle -- Fertility ( lcsh )
Cattle -- Reproduction ( lcsh )
Dissertations, Academic -- Animal Science -- UF
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1985.
Bibliography:
Includes bibliographical references (leaves 229-271).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jeffrey John Knickerbocker.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
000505078 ( ALEPH )
22770836 ( OCLC )
ACS5231 ( NOTIS )
AA00004878_00001 ( sobekcm )

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Full Text


PREGNANCY RECOGNITION IN CATTLE:
EFFECTS OF CONCEPTUS PRODUCTS ON
UTERINE PROSTAGLANDIN PRODUCTION
BY
JEFFREY JOHN KNICKERBOCKER
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
1985


ACKNOWLEDGMENTS
During the last several years, I have had the good
fortune of interacting with a host of generous and talented
individuals. The many contributions, insights and
friendships offered me by these individuals are acknowledged
greatfully.
I wish to extend my heartfelt thanks and appreciation
to Dr. William W. Thatcher, chairman of my supervisory
committee, for giving generously of his time, experience and
knowledge throughout my Ph.D. training. Dr. Thatcher's
insight and contributions with regard to my education and
research have made lasting impressions on my perception of
reproductive function and philosophy of basic reproductive
research. His friendship and interest in my professional
growth and personal well-being are deeply appreciated. Dr.
Thatcher will always be held in my highest esteem as a
scientist, teacher and friend.
Special thanks are given to Dr. Fuller W. Bazer,
cochairman, for his constant interest, availability and
input during the course of my research endeavors. His
substantial contributions to my research program and
understanding of reproductive physiology have been
invaluable. Dr. Bazer's friendship throughout the years and
ii


"support" during the final stages of this degree will always
be appreciated greatly.
Thanks are expressed to Dr. Michael J. Fields for our
numerous stimulating conversations, and for his guidance and
support during early aspects of ray research in his
laboratory. My association and friendship with Dr. Fields
over the years have been a pleasure. His contributions to
my research endeavors and education are sincerely
acknowledged.
Dr. R. Michael Roberts is acknowledged for his
important role in the development of technical and
conceptual insight relevant to conceptus protein
biochemistry and physiology. In this respect, Dr. Roberts
has been a tremendous asset in my research endeavors
relative to conceptus secretory protein function in
cattle. I thank Dr. Roberts for his interest and input
while serving on my supervisory committee.
Dr. Donald Catn is acknowledged greatfully for his
input, expert surgical training and assistance in numerous
surgeries at the 34th Street laboratory during our
characterization of uterine responses to estradiol. I am
indebted to Dr. Catn for his contributions while serving on
my supervisory committee and for his friendship.
Thanks are extended to Dr. Robert J. Collier for his
comments and critical evaluation of my dissertation. His
friendship and willingness to participate in my dissertation
iii


defense in the absence ox Dr
. Thatcher are appreciated
greatly.
I wish to extend my sincere appreciation and thanks to
Dr. Donald H. Barron and Dr. Maarten Drost for their
gracious and generous support, and valuable interactions and
insight throughout the course of my studies at the
University of Florida. It has been tremendously gratifying
for me to have worked with these gentlemen. Thank you.
Special thanks are given to fellow graduate students,
Dallas Foster, Dr. Louis Guilbault, Dr. Frank 'Skip' 3artol
and Kimberly Fincher for their valued friendships,
interactions and inestimable assistance over the years.
Friendships and support offered me by Dr. Charlie Wilcox,
Dr. Herb Head, Dr. Dave Beede, Dr. Dan Sharp, III, Dr. Ray
Roberts, Dr. David Wolfenson, Dr. Lee Fleeger, Dr. Wilfrid
Dubois, Dr. Rod Geisert, Dr. Randy Renegar, Dr. Jeff
Moffatt, Dr. Charlie Ducsay, Dr. Juaquim Moya, Harold
Fischer, John McDermott, Dr. Helton Saturino, Karen
McDowell, Marlin Dehoff, Lokenga Badinga, Deanne Morse, Fran
Romero, Sue Chiachimonsour, Jeff Valet, Kathy Hart, Dr.
George Baumbach and Leslie Smith are deeply appreciated.
Thank you.
I am greatful especially to Dr. Paul Schneider and son,
Jordan, for their generosity, patience, hospitality and
encouragement during these last 3 months.
iv


I thank Carol Underwood, Candy Stoner, Jesse Johnson
and Warren ClarK for their tolerance, technical assistance
and efficiency in the laboratory, and Cindy Zimmernan for
typing this manuscript so skillfully.
To special friends, Chuck and June Wallace, and Gail
and Bob Knight, most sincere thanks and love are extended
from my family and I. Keep in touch.
I wish to thank my parents, John and Jan, for their
continued love, support and confidence. This proud day
would not have been possible without the principles and
motivation instilled in me by them both.
Lastly, the major contributions and sacrifices made by
my wife, Holly, throughout the course of my studies are more
than greatfully acknowledged. Her constant love, support,
patience and understanding have given me the strength to
endure and reasons to succeed. My deepest love and
affections are hers always.
v


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
A3STRACT viii
CHAPTERS
1 REVIEW OF LITERATURE 1
Introduction 1
The Bovine Estrous Cycle 2
The Corpus Luteum Life Cycle 17
Uterine Control of CL Function 49
Conceptus-Associated Events During Early
Pregnancy 59
2 PATTERNS OF PROGESTERONE METABOLISM BY
DAYS 19-23 BOVINE CONCEPTUS AND ENDOMETRIAL
EXPLANTS 88
Introduction 88
Materials and Methods 90
Results 102
Discussion 129
3 UTERINE PROSTAGLANDIN AND BLOOD FLOW
RESPONSES TO ESTRADIOL-178 IN CYCLIC CATTLE...139
Introduction 139
Materials and Methods 141
Results 146
Discussion 159
4 PROTEINS SECRETED BY DAY 16 TO 18 BOVINE
CONCEPTUS EXTEND CORPUS LUTEUM FUNCTION IN
CATTLE 166
Introduction 166
Materials and Methods 168
Results 176
Discussion 185
vi


5 INHIBITION OF ESTRADIOL-173 INDUCED UTERINE
PR0STAGLANDIN-F2ct PRODUCTION BY BOVINE
CONCEPTUS SECRETORY PROTEINS 193
Introduction 193
Materials and Methods 195
Results 201
Discussion 209
6 GENERAL DISCUSSION 215
APPENDICES
A MANUFACTURE OF SEPHADEX LH20 COLUMNS 221
B STEROID ELUTION BY GAS/LIQUID
CHROMATOGRAPHY 222
C MASS CALCULATIONS FOR ESTROGENS 224
D ELUTION OF [3H]-CONCEPTUS METABOLITES AND
[14C]-MARKERS ON HPLC (ACETONITRILE:WATER,
54:46) 226
E ELUTION OF RADIOINERT STEROID STANDARDS
ON HPLC (ACETONITRILE : WATER, 54:46) 228
REFERENCES 229
BIOGRAPHICAL SKETCH 272
vii


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
PREGNANCY RECOGNITION IN CATTLE:
EFFECTS OF CONCEPTUS PRODUCTS ON
UTERINE PROSTAGLANDIN PRODUCTION
BY
JEFFREY JOHN KNICKERBOCKER
May, 1985
Chairman: William W. Thatcher
Cochairraan: Fuller W. Bazer
Major Department: Animal Science
In cattle, continued progesterone (P4) production by
the corpus luteum (CL) is required if pregnancy is to per
sist. Identification of putative conceptus-derived signals
and evaluation of their biological roles relative to CL
maintenance during early pregnancy were goals of this
research endeavor.
Using various chromatography systems, bovine con-
ceptuses (days 19 to 23) exhibited extensive metabolism of
tritiated P4 (90-98%), in vitro. A majority of conceptus
metabolites were 53-reduced pregnanes. A major conceptus
metabolite was 53-pregnan-3a-ol-20-one (5B-P). Conversely,
endometrial explant cultures metabolized 40 to 50% of P4
substrate to primarily 5a-reduced steroid products.
viii


An in vivo test system to evaluate uterine PGF2a pro
duction capacity was characterized in experiment two. Exo
genous estradiol-17B (E2; 3 mg I.V.) stimulated uterine
blood flow, and PGF£a production and metabolism. Concen
trations of the primary metabolite of PGF2a> 15-keto-13,14-
dihydro-PGF2a (PGFM) were significantly correlated (r=.66)
with the E2~induced uterine PGF2a production response. Use
of peripheral PGFM concentrations as an index of uterine
PGF2a production was supported.
In experiment three, CL function, interestrous inter
val, and spontaneous uterine PGF2 production were evaluated
in cyclic cows following intrauterine administration of
56-P, conceptus secretory proteins (CSP) or homologous serum
proteins (Control). Extensions in CL lifespan and
interestrous interval were detected in cows administered CSP
compared to 58-P and Control group responses. Lifespan of
CL and estrous cycle lengths were not different (P>.25)
between 5B-P and Control groups. Spontaneous PGF2a episodes
were depressed in CSP-treated cows but not in cows admini
stered 5B-P or serum proteins. In experiment four, E2-
induced uterine PGF2a production (peripheral PGFM response)
was evaluated in cyclic cattle following interuterine
administration of bovine CSP or day 18 pregnant serum
proteins (Control). Mean concentrations of PGFM in CSP-
treated cows were depressed (PC.01) compared to Control
responses. Estradiol injection failed to elicit any PGFM
ix


response in three of five CSP-treated cows. In contrast,
all Control animals exhibited PGFM responses during the
period of E2~induced PGF2a production. Results support a
role for CSP in suppression of uterine PGF2a production
during early pregnancy in cattle.
x


CHAPTER 1
REVIEW OF LITERATURE
Introduction
The recurrent nature of estrous cycles in cattle
centers around development and regression of the ovarian
corpus luteum (CL). Progesterone (P4)* the primary product
of CL, directs uterine development and secretory activity
such that an embryotropic uterine environment is established
during each estrous cycle. Luteal maintenance and P4
production are essential if successful pregnancy is to be
established. The process of CL maintenance during early
pregnancy involves numerous and complex biochemical inter
actions between the conceptus and its maternal host. Our
evaluation of mechanisms by which the conceptus and maternal
units interact during early pregnancy depend upon identifi
cation of putative conceptus-derived 'signals' and develop
ment of biological test systems through which conceptus
product function may be assessed. It was in this light that
research described herein was conducted.
1


The Bovine Estrous Cycle
General Considerations
The nature of the estrous cycle has been characterized
extensively in cattle. Early observations and documentation
of the recurrence of sexual behavior in animals provided the
first clue as to the cyclical nature of reproductive
processes in the female (Heape, 1900; Marshall, 1922;
Hammond, 1927; Asdell, 1969). Wallace in 1876 and
Ellenberger in 1892 (Hammond, 1927; as cited in Marshall,
1922) were among the earliest to report accurately the
interval between "sexual periods" in domestic female cattle.
The cyclical recurrence of estrous behavior follows a
periodicity of approximately 21 days with a range of 17 to
25 days considered normal (Hammond, 1922; Asdell et al.,
1949; Olds and Seath, 1951; Cupps et al., 1969).
Nulliparous heifers tend to exhibit slightly shorter
(approximately 1 day) interestrous intervals than
multiparous cows (Hammond, 1927; Olds and Seath, 1951).
Unlike sheep (Goodman and Karsch, 1981) and horses
(Sharp, 1980), photoperiod does not influence consistently
annual reproductive patterns in mature cattle (Rzepkowski et
al., 1982). Thus, reproductive activities in domestic
cattle are not restricted to specific seasons of the year
(Heape, 1900; Hammond, 1927). However, attainment of


3
puberty in cattle is enhanced by increased daily exposure to
light (Hansen et al., 1933).
Loss of body condition due to decreased nutrient
availability, lactational stress and parasitism has
detrimental effects on cyclicity and reproductive efficiency
in cattle (Lamond and Bindon, 1969; Dunn et al., 1969; Rakha
and Igboeli, 1971). Thus, wild cattle are known to exhibit
limited breeding and calving seasons under natural
conditions (Marshall, 1922; Heape, 1900). When such
stresses are reduced via management schemes in the confines
of a zoo, wild cattle are capable of breeding at all times
of the year (Heape, 1900).
High ambient temperature and humidity are other
important environmental influences which reduce expression
of estrus, blood flow to the reproductive tract, and may
alter the normal endocrine milieu in cattle (Thatcher and
Collier, 1985). Consequently, heat stress results in
decreased reproductive efficiency.
Historically, observations regarding cyclical
alterations in behavior and ovarian morphology led Walter
Heape (1900) to propose terminology which established the
first classification of these cyclical changes into four
recurrent phases. Thus, the estrous cycle ("diestrus cycle"
by Heape's terminology) in cattle consists of four phases:
proestrus (day 19 to estrus), estrus (day 0), metestrus (day
1-3) and diestrus (day 4-18).


4
Physiology and Endocrinology
Sexual receptivity of the female, or estrus, is a
convenient behavioral landmark which is commonly employed to
mark the start of an estrous cycle (day 0). Estrus is often
characterized by increased restlessness (Kiddy, 1976),
vocalizations, mounting activity and, most notably, standing
to be mounted by other cattle (Howes et al., 1960). Other
variables related to estrus are discussed by Lewis and
Newman (1984). Average length of behavioral estrus
approaches 18 to 20 hours in mature cattle (Trimberger and
Hansel, 1955; Schams et al., 1977); however, cattle in
subtropical regions generally experience shorter (10 to 13
hour) phases of estrus (Branton et al., 1957; Chenault et
al., 1975).
The temporal sequence of events which culminate in
behavioral estrus, and later in the preovulatory gonado
tropin surge, involve a synchronized and interdependent
cascade of physiological changes in the central nervous
system, hypothalamus, pituitary, ovaries and uterus. Char
acterization of endocrine profiles and study of mechanisms
controlling their changes have, at least partially,
unravelled the dynamics of various reproductive processes.
Approximately 2 to 4 days before behavioral estrus and
the preovulatory surge of gonadotropins, regression of the
corpus luteum (CL) is initiated, as determined by a
precipitous decline in plasma progesterone (P4)


9
1975; Peterson
)
concentrations (Chenault et al., 1975; Peterson et al
1975). In association with this decrease in P_p pulse
frequencies of LH (Rahe et al., 1980; Schallenberger et al.,
1984) and FSH (Schallenberger et al., 1984) increase, as do
basal concentrations of LH, but not FSH (Chenault et al.,
1975; Roche and Ireland, 1981; Milvae and Hansel, 19S3b;
Schallenberger et al., 1984).
Plasma concentrations of estradiol-17S (E2) begin to
rise during this period (Chenault et al., 1975; Walters and
Schallenberger, 1984; Schallenberger et al., 1984) as a
result of the initiation and accelerated growth of the
preovulatory follicle (Dufour et al., 1972; Merz et al.,
1981; Staigmiller et al., 1982; Ireland and Roche, 1982;
McNatty et al., 1984a,b). In fact, approximately ten times
more E2 was measured in ovarian vein plasma draining the
ovary containing the preovulatory follicle chan the contra
lateral ovary at estrus in cattle (McNatty et al., 1984a).
A significant correlation exists between LH and E2
pulse frequencies during all phases of the bovine estrous
cycle (Schallenberger et al., 1984; Walters and
Schallenberger, 1984; Walters et al., 1984). Prior to the
gonadotropin surge, LH pulsatile episodes occur every 30
minutes (high frequency) with E2 pulses being generated
responsively (Walters and Schallenberger, 1984). Addi
tionally, E2 pulse amplitudes gradually increase as estrus
approaches. However, these alterations in E2 pulse


6
amplitude are not reflected by similar amplitude elevations
in LH episodes (Walters and Schallenberger, 1984). Inis may
suggest that LH pulse frequency and follicle sensitivity to
LH are major regulatory factors for follicular E2 production
in the cow.
Before reviewing data in support of such a claim, a
brief description of follicular anatomy and the two-cell
mechanism of follicular estrogen biosynthesis is in order.
The ovarian follicle consists of two steroidogenica1ly
active cellular compartments: the vascularized theca
interna and the avascular granulosa, which is separated from
the theca interna by an acellular basal laminae (see:
McNatty et al., 1984b, for references). According to the
two-cell mechanism of follicular estrogen production
proposed by Falck (1959), the theca compartment metabolizes
C-21 steroids to androgens which are then utilized by the
granulosa compartment to synthesize estrogens. More recent
evaluation of steroidogenic pathways utilized by bovine
follicular compartments (Lacroix et al., 1974) support this
concept. The theca cell layer utilizes, almost exclusively,
the A^-pathway (pregnenolone and 17ct-hydroxy-pregnenolone)
to synthesize androgens, of which androstanedione (A^) is
the major product. In vivo evaluation of A^ concentrations
in ovarian vein plasma and A^ production by the ovary
throughout the estrous cycle in cattle (Wise et al., 1982)
agree with in vitro appraisals of thecal androgen


7
biosynthesis by Lacroix and coworkers (1974). Metabolism of
A4 to estrogens by the granulosa is very efficient.
Conversely, only small amounts of estrogen are synthesizeu
by the theca compartment. Lacroix and coworkers (1974)
observed greater yields of estrogen with combined
incubations of theca and granulosa cells than yields with
incubations of either cell type separately, indicative of a
synergism between the two follicle compartments (Falck,
1959).
The capacity of follicles to synthesize estrogens
depends upon their ability to respond to gonadotropins. In
the cow, 85% of large (_>_ 8 mm diameter) follicles on both
ovaries bound FSH to granulosa cells and hCG (LH) to theca
interna cells, while only 38% of these follicles also bound
hCG to granulosa. Follicles with granulosa hCG binding
sites were associated with enhanced E2 concentrations in
follicular fluid (England et al., 1981; Merz et ai.,
1981). Sheep also exhibit a high positive correlation
between hCG binding to granulosa and follicular E2 content
and production (Webb and England, 1979, 1982). McNatty et
al. (1984a) suggested that there was at least one follicle
greater than 5 mm (range 1 to 3 follicles per cow) with
granulosa cells possessing aromatase activity on day -5
through estrus. However, most, if not all, healthy bovine
follicles greater than 2 mm and many atretic follicles were
capable of secreting A4 in response to LH. In no cases were


3
follicles with granulosa aromatase activity found in the
absence of an LH-responsive thecal compartment. Likewise,
Bartol et al. (1931) demonstrated that large follicles
capable of significant E£ production were present in ovaries
of cattle throughout the estrous cycle.
In the rat (Nimrod et al., 1977), pig (Channing, 1975)
and sheep (Weiss et al., 1978) ovarian follicle, granulosa
binding sites for hCG/LH are induced by FSH, thereby
enhancing granulosa cell responsiveness- to LH.
Additionally, P^, dihydroxytestosterone (DHT) and E£ plus
FSH synergistically stimulated granulosa cell responsiveness
to LH above levels found with FSH alone (Rani et al.,
1981). Low concentrations of E2 enhance production by
isolated bovine theca cells (Fortune and Hansel, 1979),
suggesting that E£ may also regulate theca sensitivity to
LH.
Thus, follicular development of granulosa aromatase
activity appears to occur secondarily to the thecal capacity
to synthesize androgens. Androgen biosynthesis by the theca
interna compartment is enhanced by exposure to LH, while FSH
may be important in sensitization of granulosa cells to
LH. Steroids, such as P^, DHT and E£ may also potentiate
this FSH effect.
One apparent consequence of granulosa-binding of LH is
activation of the aromatase enzyme complex. Mechanisms
involved in the selection of follicles which become


9
LH-responsive probably depend upon factors other than FSH
alone, perhaps intraovarian factors (Alexander et al., 1973;
Darga and Reichert, 1978; Scnomberg, 1979; Hsueh et al.,
1933; Hansel and Convey, 1983), since granulosa cells in a
majority of bovine follicles appear to bind FSH, yet in only
one to three large follicles do granulosa cells develop the
ability to bind LH and acquire aromatase activity (Merz et
al., 1981; Bartol et al., 1981; McNatty et al., 1984).
As mentioned, steroidogenic capacity of ovarian folli
cular compartments depend upon their ability to respond to
gonadotropins. Additionally, periodicity and magnitude of
gonadotropin release from the anterior pituitary play
important roles in ovarian function. Gonadotropin patterns
are, in turn, regulated by gonadal steroids via long loop
feedback mechanisms. In ovariectomized sheep (Goodman and
Karsch, 1980), frequency of tonic LH episodes is decreased
by with no apparent effect on pulse amplitude, while E2
reduces LH pulse amplitude and does not influence frequency
of LH release. Similar observations were made by Rahe et
al. (1980) following characterization of LH episodes during
mid-luteal and follicular phases of the estrous cycle in
intact cows. High amplitude, low frequency LH episodes
which occur during P^-dominated phases of the cycle are
thought to result from P^ negative feedback on the central
nervous system's (CNS) oscillator regulating episodic
secretion of gonadotropin releasing hormone (GnRH) (Knobil,


10
1980) and pituitary sensitivity to GnRH (Padmanabnan et al.,
1982). During estrogen-dominated phases, prior to she
preovulatory surge of gonadotropins, initially decreases
pituitary sensitivity to GnRH resulting in less LH releases
per GnRH pulse (Kesner et al., 1981; Kesner and Convey,
1982). However, by approximately 8 hours before the
gonadotropin surge, when highest preovulatory plasma
concentrations of occur (Walters and Schallenberger,
1984), pituitary sensitivity to GnRH reaches its maximum
(Kesner et al., 1981; Kesner and Convey, 1982; Padmanabhan
et al., 1982). Yet during this period, amplitude (but not
frequency) of gonadotropin episodes remains low (Walters and
Schallenberger, 1984). Walters and Schallenberger (1984)
suggested that a negative feedback effect of the elevated E£
on the hypothalamus may explain these endocrine patterns
during the preovulatory period.
Regulation of FSH secretion by gonadal steroids appears
to be more subtle than observed for LH. Concentrations of
FSH are considerably higher than LH during all phases of tne
estrous cycle in cattle (Schallenberger et al., 1984;
Walters and Schallenberger, 1984; Walters et al., 1984).
Interpulse frequencies for FSH range from approximately 25
minutes during the preovulatory phase to 30-50 minutes
during the mid-luteal phase of the estrous cycle (Walters
and Schallenberger, 1984; Walters et al., 1984) with pulse
amplitudes declining during the preovulatory period. Thus,


11
P4 appears to exert only slight reductions in F3H pulse
frequency in cattle. Evidence suggests that E exerts an
inhibitory effect on FSH release via the pituitary just
prior to the preovulatory gonadotropin surge (Kesner and
Convey, 1982; Walters and Schallenberger, 1984).
Additionally, exogenous E£ administered to ovariectomized
heifers reduced FSH concentrations to precastration levels
(Kesner and Convey, 1982). Follicular production of inhibin
may also regulate plasma FSH concentrations during periods
of follicular growth (De Jong, 1979; De Paolo et al., 1979;
Henderson and Franchimont, 1982; Padmanabhan et al., 1984),
possibly through decreasing pituitary release of FSH.
Low basal concentrations of P^ and peak follicular
production of E^ potentiate the onset of behavioral estrus
and trigger the preovulatory surge of LH and FSH from the
anterior pituitary (Scharas et al., 1977). Although estrous
behavior in cattle may be induced by estrogen treatment
alone (Melarapy et al., 1957), a period of P^ pre-exposure
enhances the sensitivity of brain centers responsible for
estrous behavior to E^ (Melampy et al., 1958). Therefore,
P4 priming, during the luteal phase of the cycle, may oe an
important aspect of estrous expression (see also: McEwen et
al., 1982; Pfaff and McEwen, 1983)- During physiological
states in which P^ priming is absent, as occurs in heifers
approaching puberty (Gonzalez-Padilla et al., 1975; Schams
et al., 1981) or in cows following postpartum anestrous


12
(Schams at al., 1978; Peters, 1934), behavioral estrus does
not accompany the first gonadotropin surge and ovulation.
Additionally, duration and magnitude of P4 exposure may
influence subsequent luteal function by modulating preovula
tory follicle development directly as was suggested Oy the
data of Snook et al. (1969) in heifers treated with bovine
LH antisera and McLeod and Haresign (1984) in the seasonally
anestrous ewe. Similarly, ovulations occurring witnout the
benefit of P4 priming in pubertal and postpartum cattle
often result in short-lived, less functional CL (Gonzalez-
Padilla et al., 1975; Schams et al., 1978; 1981; Peters,
1984).
Elevated P4 concentrations completely eliminate
induction of estrous behavior and the gonadotropin surge
(Hobson and Hansel, 1972; Short et al., 1979; Roche and
Ireland, 1981; Ireland and Roche, 1982; Padmanabhan et al.,
1982). With demise of the CL during the preovulatory
period, plasma P4 concentrations fall to below 1 ng/ml thus
removing the negative feedback on GnRH and gonadotropin
release rates. Follicular production of E£ provides
increasingly higher plasma E2 concentrations, the magnitude
of which is proportional to the size of the preovulatory
gonadotropin surge (Walters and Schallenberger, 1984).
Approximately 1 hour prior to the preovulatory surge of
gonadotropins, Walters and Schallenberger (1984) observed a
consistent decline in E^ concentrations and suggested that


13
this event removed E? negative feedback on the hypothalamus
and increased GnRH pulse amplitude which triggered one
gonadotropin surge from the maximally responsive pituitary
(Convey, 1973; Zolman et al., 1973; Kesner et al., 1961;
Kesner and Convey, 1982). The surge of LH and FSH occur
concomitantly near the onset of an 18 to 20 hour behavioral
estrus (Trimberger and Hansel, 1955; Schams et al., 1977)
and lasts 8 to 10 hours (Rahe et al., 1980; Kesner et al.,
1981; Kesner and Convey, 1982; Walters and Schallenberger,
1984). The gonadotropin surge is terminated due to the
refractoriness of the pituitary to GnRH (Kesner et al.,
1981; Kesner and Convey, 1982).
During and immediately following the preovulatory
gonadotropin surge, follicular (granulosa cell) production
of E£ and circulating levels of E£ decline rapidly as
follicular luteinization occurs (Chenault et al., 1975;
Ireland and Roche, 1982). Plasma concentrations of E£, P4
and LH remain low throughout a majority of the metestrous
phase. Pulses of LH are absent for 6 to 12 hours post
gonadotropin surge. Similarly, FSH pulse amplitudes are
low; however, pulse frequencies remain at a rate comparable
to that observed during the preovulatory gonadotropin surge
(Walters and Schallenberger, 1984). Prior to ovulation and
4 to 12 hours following the gonadotropin surge, FSH
concentrations become elevated as a result of an increase in
FSH pulse amplitude (Kazmer et al., 1981; Hansel and Convey,


14
1983; Walters and Schallenberger, 1934). The second rise in
FSH is of lower magnitude than the FSH surge and may be a
result of reduced follicular inhibin production during the
ovulatory process (De Jong, 1979; De Paolo et al., 1979;
Henderson and Franchimont, 1981; Padmanabhan et al.,
1984). The function of this secondary rise in FSH is
currently unknown, however, FSH may play a role in oocyte
maturation (Plachot and Mandelbaum, 1978) or in the
recruitment of preantral follicles (Hansel and Convey,
1983).
Ovulation occurs on day 1 of the estrous cycle,
approximately 24 to 30 hours after the preovulatory surge of
LH and FSH (Chenault et al., 1975; Scharas et al., 1977;
Hansel and Convey, 1983). Following ovulation, development
of CL function is reflected by the gradual increase in CL
weight (Shemesh et al., 1976; Bartol et al., 1981) and P4
content (Shemesh et al., 1976), plasma P4 concentrations
(Chenault et al., 1975; Peterson et al., 1976; Schams et
al., 1977; Wise et al., 1981; Hansel and Convey, 1933) and
P4 production (Wise et al., 1981), and ovarian blood flow
(Ford and Chenault, 1981; Wise et al., 1981). Follicular
growth and atresia occur continuously throughout the estrous
cycle (Rajakoski, 1960; Marion et al., 1968; Ireland and
Roche, 1983) with moderate rises in plasma E2 concentrations
occurring responsively (Hansel et al., 1973; Hansel and
Convey, 1983; Ireland and Roche, 1983).


15
During the early luteal phase, both LH and FSH secre
tion patterns may be characterized as high frequency-low
amplitude (Rahe et al., 1980; Walters et al.f 1984). The
majority of LH and FSH pulses occur simultaneously during
this period. Similarly, pulsatile episodes of £2 anci p4 are
high frequency. High amplitude pulses of E2 follow LH
pulses in 90 to 96% of the cases observed, while occurrence
of low amplitude episodes were more similar to FSH
pulsatile patterns (Walters et al., 1984). These
gonadotropin-steroid pulsatile relationships become more
obvious during the mid-luteal phase. By day 10 to 12 of the
estrous cycle, frequency of LH episodes are significantly
decreased (Rahe et al., 1980; Walters et al., 1984). Rahe
and coworkers (1980) also described increased LH pulse
amplitude during the mid-luteal phase, however, elevations
in LH pulse amplitude were not evident in the data set of
Walters et al. (1984). Pulse amplitude of FSH was similar
during the early and mid-luteal phases, although FSH pulse
frequency decreased during the mid-luteal phase.
Nevertheless, 41% more FSH than LH episodes were observed
during the mid-luteal phase. Low amplitude E2 episodes were
consistently preceded by pulses of LH and 92 to 100% of
concurrent FSH and LH pulses and 97% of separate FSH pulses
were followed by high amplitude episodes of P^. In no case
were separate FSH pulses associated with episodes of E2
(Walters et al., 1984).


16
The dynamics of endocrine changes during the luteal
phase support previously described roles for F3H ana LH in
follicular steroid production. Additionally, it would
appear that FSH, in conjunction with LH, regulates
secretion patterns. The major site of P^ production is the
CL (Shemesh et al., 1976). The bovine CL possesses both FSH
(Mann and Niswender, 1983) and LH (Rao et al., 1979, 1983)
receptors and responds to these gonadotropins, in vitro,
with elevated P^ production (Romanoff, 1966; Hixon and
Hansel, 1979; Milvae et al., 1983).
In cyclic cattle, lifespan of the CL is regulated, to a
large extent, by interactions between the ovary and
uterus. During late diestrus-early proestrus phases of the
cycle, estrogens produced by the developing, large antral
follicle(s) are thought to initiate the process of luteal
regression. Exogenous estrogens are known to shorten the
lifespan of CL in cattle (Greenstein et al., 1958; tfiltbank,
1966; Eley et al., 1979). Luteolytic activity of estrogen
is thought to be mediated through stimulation of uterine
prostaglandin (PG)-F2a synthesis and release (Thatcher et
al., 1984b). The luteolytic activity of exogenous PGFpa in
cattle has been documented by several groups (Hansel et al.,
1973; Hafs et al., 1974; Lauderdale, 1974; Thatcher and
Chenault, 1976). Furthermore, episodic pulses of uterine
venous PGF2a (Nancarrow et al., 1973), and peripheral
measurements of its primary metabolite,


17
15-keto-13,14-dihydro-PGF2a (PGFri; Granstrom and Kindahl,
ly82), are always associated with spontaneous luteal
regression in cattle (.Peterson et al., 1978; Kindahl et ai.,
1976; Betteridge et al., 1984). Uterine PGF2a production
requires a period of P^-priming which increases the tissue's
potential to synthesize PGF2a while suppressing copious
secretion of the luteolysin (Hansel et al., 1973; Horton and
Poyser, 197b; Rothchild, 1981).
An indepth discussion of CL characteristics, function
and mechanisms of regression will be reserved for a
subsequent section of this chapter.
The Corpus Luteum Life Cycle
Origin and Development of Corpus Luteum Cells
Cells which make up the corpus luteum (CL) are derived
from the preovulatory follicle. Two distinct luteal cell
types have been described in CL of cattle (Donaldson and
Hansel, 19b5b; Ursely and Leymarie, 1979a,b; Koos and
Hansel, 1981; Weber et al., 1984; Alila and Hansel, 1984),
sheep (O'Shea et al., 1979 1980; Fitz et al., 1982; Rogers
et al., 1983, 1984), swine (Lemon and Loir, 1977; Lemon and
Mauleon, 1982), rat (Kenny and Robinson, 1982), rabbit (Yuh
et al., 1982) and human (Gaukroger et al., 1979). These
cell types are described generally as small and large luteal
cells. Current dogma suggests that small luteal cells are


13
derived from the theca interna and large luteal cells from
the granulosa of the preovulatory follicle.
In the ewe, large luteal cells of the mature CL are
approximately six-times larger than small luteal cells and
occupy 25% or more of the CL volume (Niswender et al., 1976;
Rodgers et al., 1984) as compared to approximately 18% by
small luteal cells (Rodgers et al., 1984). Small luteal
cells outnumber large luteal cells by nearly five to one.
By far, the most numerous cell type within the mature ovine
CL is endothelium (Rodgers et al., 1984), which attests to
the high vascular density of this tissue.
Cellular mitotic activity in the CL is thought
generally to be at a minimum (Donaldson and Hansel, 1965;
Rodgers et al., 1984). Histological examination and DNA
determinations of the bovine preovulatory follicle (6 hours
following the onset of estrus) and CL throughout the estrous
cycle (Donaldson and Hansel, 1965b) indicated that
granulosa-luteal cell mitotic activity was reduced following
the preovulatory surge of LH and ceased altogether by
approximately day 4. The theca-derived luteal cells,
however, mi tosed freely until approximately day 7.
Neovascularization continues in CL until a maximum metabolic
potential is reached around days 10 to 12. In vivo exposure
of developing bovine CL to hCG or LH (Donaldson et al.,
1965a) stimulated mitotic activity in small luteal cells,
vascular endothelium and stromal cells, whereas mitotic


19
figures were rarely observed in large luteal cells
(Donaldson and Hansel, 1965). Thus, during CL development,
LH appears to limit granulosa cell division and enhance
theca-luteal cell mitosis (Donaldson and Hansel, 1965;
Friedrich et al., 1975; McNatty and Sawers, 1975).
In addition, estimates of granulosa cell numbers in
mature preovulatory follicles compare favorably with numbers
of granulosa-luteal cells in the CL (McNatty, 1979; McNatty
et al., 1984a; Rogers et al., 1984), suggesting that large
luteal cells are derived solely from follicular granulosa
cells. Furthermore, increases in follicular size are
correlated positively with follicular cell numbers (McNatty,
1979; McNatty et al., 1982; Ireland and Roche, 1983). Thus,
it may be assumed logically that events which regulate
viability, proliferation and biosynthetic potential of
preovulatory follicular cells markedly influence the
functionality of the CL subsequently formed post-ovulation
(see: McNatty, 1979; Richards, 1979, 1980; di Zerega and
Hodgen, 1981; Murdoch et al., 1983).
Observations in the cow (Donaldson and Hansel, 1965;
Alila and Hansel, 1984) and ewe (Fitz et al., 1981) support
the hypothesis that CL growth occurs initially via
hypertrophy and hyperplasia by both theca- and granulosa-
derived luteal cells. However, these authors suggest that
as the CL matures, theca-derived lutein cells enlarge to
form large luteal cells. It has further been suggested that


20
the large luteal cells (granulosa- or tneca-derived), which
possess receptors for PGF£a, play a key role in CL
regression (Fitz et al., 1932; Alila and Hansel, 1934; Hoyar
et al., 1984).
Factors Regulating CL Steroidogenesis
The formation of luteal tissue is begun prior to
ovulation as the preovulatory rise and surge release of LH
promote several biochemical (Savard, 1973) and cytological
(Donaldson and Hansel, 1965b; Blanchette, 1966a,b;
Priedkalns and Weber, 198; Enders, 1973; Van Blerkom and
Motta, 1978; 1979) changes in both theca and granulosa cells
of the preovulatory follicle.
Unlike the follicle, cells of the bovine CL lack the
endoplasmic reticulum-associated 17-hydroxylase and 19-
hydroxy lase-aroma tase enzyme systems (Savard and Telegdy,
1965; Savard, 1973). The obvious physiological
manifestations of these biochemical alterations are the
sudden loss of androgen and estrogen biosynthetic capacity
by luteal cells (Savard and Telegdy, 1965; Ireland and
Roche, 1982; Walters and Schallenberger, 1984). Thus,
bovine CL exhibit a very abbreviated steroidogenic pathway
from which P4 is the primary end-product, with lesser
amounts of 20B-hydroxy-4-pregnen-3-one (2O8-P4),
pregnenolone (P5), and 5a-reduced progestins (5a-P) being
generated (Wise and Fields, 1978; Albert et al., 1982).
Corpus luteum development and steroidogenic capacity may be


21
grossly assessed by monitoring plasma concentra cions.
Bovine CL mass increases rapidly from day 3 (_<_ 1 gram) to
day 7 (4 gram) and is maintained at a weight of 3 to grams
from days 10 through 17. Likewise, plasma concentrations of
P4 are low (_<_ 1 ng/ml) on day 3, but rise rapidly to levels
of approximately 5 ng/ml by day 7 and are maintained between
6 and 10 ng/ml from days 10 through 17 (Donaldson and
Hansel, 1965b; Erb et al., 1971; Rao et al., 1979; Milvae
and Hansel, 1983a). During regression of the CL, a loss in
function (reduced plasma P^ concentrations) consistently
precedes any decline in CL weight.
Luteal steroidogenic potential is regulated by several
factors. Luteinizing hormone is considered the primary
luteotropin in cattle. During the past 20 years, a
tremendous number of studies have been performed which
support this concept. Administration of exogenous LH
(Donaldson and Hansel, 1965a), hCG (i/iiltbank et al., 1961),
and GnRH-agonists, to induce elevated endogenous LH
concentrations (Milvae et al., 1984), increased plasma P4
concentrations and prolonged CL function in cattle. Several
in vitro studies demonstrated increased P4 production by
dispersed luteal cells or luteal slices when incubated with
LH or hCG (Hansel and Seifart, 1967; Hansel et al., 1973;
Hixson and Hansel, 1979; Milvae and Hansel, 1983a; Tan and
Biggs, 1984; Milvae et al., 1985). Antiserum to LH
prevented this luteotropic effect of LH on luteal slices in


22
vitro (Hansel and Seifart, 1967) and induced a decline in CL
weight and content of CL in intact heifers (Snook et al.,
1969). In hysterectomized heifers, LH treatment increased
P4 content of CL (Brunner et al., 1969) while hysterectomy
plus hypophysectomy or hypophysectomy alone caused a decline
in content of CL, lower plasma P4 concentrations, and a
reduction in CL weight in cattle (Henricks et al., 1989).
Lastly, oxytocin and PGF2a induced CL regression was
circumvented by administration of LH or GnRH to cyclic
cattle (Hansel and Seifart, 1967; Thatcher and Chenault,
1976). Therefore, maximal function of the bovine CL is
contingent upon LH stimulation.
Direct binding studies, using radiolabelled ligands,
revealed the presence of specific, high affinity-low
capacity receptors for LH/hCG in bovine luteal cell
membranes (Rao et al., 1979, 1983) and intracellular
organelles (Rao et al., 1983). Furthermore, luteal
concentrations of LH receptor sites are correlated with P^
secretion during the estrous cycle. Other ligands (to be
discussed later) which bind specifically to luteal tissue
include FSH (Mann and Niswender, 1983), PGF2a (Kimball and
Lauderdale, 1975; Rao et al.,1979; Bartol et al., 1981; Fitz
et al., 1982), PGE-| and PGE2 (Kimball and Lauderdale, 1975;
Fitz et al., 1982), and E2 (Glass et al., 1984).
Recent application of cell separation techniques have
enabled researchers to better characterize and evaluate the


23
function of specific luteal ceil populations with regard to
their roles in production and luteolysis. As described
previously, small luteal cell numbers exceed large luteal
cells by approximately five-fold although large luteal cells
comprise more of the CL volume. In cattle (Ursely and
Leymarie, 1979a,b; Koos and Hansel, 1981; Weber et al.,
1984), sheep (Fitz et al., 1982; Rodgers and O'Shea, 1982;
Rodgers et al., 1983), and swine (Lemon and Loir, 1977;
Lemon and Mauleon, 1982) incubations enriched in large
luteal cells produce consistently more tnan equivalent
numbers of small luteal cells. However, only small luteal
cells were capable of responding to LH/hCG stimulation, as
determined by a significant increase in P^ production over
nonstimulated cells. This observation suggested, as was
demonstrated by Fitz and coworkers (1982), that the
luteotropic effect of LH/hCG was due to the interaction of
LH/hCG with specific receptor sites on small luteal cells.
Small cell steroidogenesis was also stimulated by cyclic AMP
and PGEp. The deficiency of LH/hCG receptors in the large
luteal cell population and a constant secretion of P¡_ in the
presence or absence of secretagogues (Fitz et al., 1982)
indicate that peak steroidogenic activity in large luteal
cells does not require receptor-mediated cyclic AMP
generation as it does in small luteal cells (Hoyer et al.,
1984). Instead, maximum potential for P4 production by


24
large lutein cells appears to be dependent upon precursor
(primarily cholesterol) availability.
Lemon and Mauleon (1962) demonstrated an interaction
between small and large luteal cell types in the porcine CL
with regard to production. These authors utilized a
superfusion system in which medium was pumped from one
chamber, containing small luteal cells, through a second
chamber, containing large luteal cells, and vise-versa.
Progesterone production by the small cell to large cell 'in
series' superfusion exceeded (20%) the sum total of P^
produced by each cell type alone. No such increase in P^
production was detected when the superfusion was conducted
with cell populations in a reversed order. Thus, products
derived from small luteal cells stimulated P^ production by
large luteal cells. When steroid precursors were added to
superfusions of either small or large luteal cells, P^ was
metabolized to P^ equally well by both cell types. In
contrast, only large luteal cells responded to exogenous
cholesterol with increased P^_ production compared to control
cells superfused without cholesterol. Metabolism of
cholesterol by large cells was not stimulated by exogenous
LH. Pulses of exogenous LH to small luteal cell
superfusions stimulated P^ production responsively.
However, P^ production in response to LH was not affected by
the addition of cholesterol to small luteal cells. Lemon
and Mauleon (1982) suggested that local transfer of


25
cholesterol from small to large luteal cells was responsible
for enhanced P^ production by large luteal cells.
Furthermore, LH-stiraulated P^ production and cnolesoerol
mobilization in small luteal cells would provide additional
substrate to large luteal cells. The end result would be a
coordinated increase in P^ production by both cell types.
The following discussion will summarize some of the
important processes involved in cellular cholesterol
regulation. Pertinent references may be found in reviews by
Savard, 1973; Brown et al., 1981; Kaplan, 1981; Savion et
al., 1982; Strauss et al., 1982.
Steroidogenic cells, and extrahepatic cells in general,
synthesize very little cholesterol. Instead, the bulk of
cholesterol required for steroidogenesis and membrane
synthesis is derived from plasma and stored as cholesteryl
esters within the cell.
Approximately 70% of the total plasma cholesterol pool
resides within low density lipoprotein (LDL) particles in an
esterified form. These LDL particles are approximately 220
o
A in diameter and contain a core of nearly pure cholesteryl
ester surrounded by two copies of the 250,000 Dalton
glycoprotein, apoprotein B. Cellular uptake of cholesterol
involves binding of LDL to specific, high affinity receptors
which become localized on specialized regions of the plasma
membrane called coated pits (Pastan and Willingham,
1981a,b). Receptor recognition of specific regions of


26
apoprotein B ensures the selective uptake of LDL. Receptor
binding affinity to LDL is Ca+2-dependent, the importance of
which will become obvious. Receptor-LDL complexes are
rapidly internalized enmass via receptor mediated
endocytosis (Pastan and Dillingham, 1981b). Coated pits,
containing numerous receptor-LDL complexes, invaginate and
pinch off to form endocytotic coated vesicles which
ultimately fuse with lysosomal membranes in the cytoplasm.
Receptor-ligand complexes dissociate allowing the LDL
receptors to be recycled for further use. Dissociation is
thought to occur as a result of low Ca+2 concentrations
within the endocytotic vesicle and lysosome. Hydrolysis of
LDL within the lysosome frees cholesterol for use or storage
within the cell.
Intracellular cholesterol concentrations are highly
regulated by the cell. As intracellular cholesterol
concentrations rise following hydrolysis of LDL in the
lysosomes, cellular cholesterol biosynthesis is suppressed
via a decrease in the activity of a pivitol enzyme in the
cholesterol biosynthetic pathway, HMG-CoA reductase.
Secondly, acyl-CoA:cholesterol acyltransferase activity is
enhanced, thus catalyzing the re-esterification of
cholesterol for storage. Most importantly, LDL receptor
biosynthesis is suppressed, preventing further uptake of
cholesteryl ester-rich LDL from the plasma.


27
These data suggest that small and large luteal ceils
also adjust to intracellular cholesterol concentrations in
accordance with changing cellular requirements. Thus, large
lutein cells, which synthesize approximately 80% of the
total P4 in unstimulated CL (Hoyer et al., 1984), would be
expected to maintain a higher cholesterol requirement than
small luteal cells. Furthermore, it may be suggested that
large luteal cells in the pig (Lemon and Mauleon, 1932) were
in a cholesterol-deficient state, with regard to their
maximum steroidogenic capacity, since P^ production was
elevated following exposure to exogenous cholesterol.
Conversely, small luteal cells, which utilize cholesterol
less rapidly, may possess larger stores of intracellular
cholesterol and would not be expected to exhibit elevations
in P4 production in response to exogenous cholesterol (Lemon
and Mauleon, 1932). Demand for cholesterol by LH-sensitive
small luteal cells increases following stimulation with LH
or cyclic AMP. However, intracellular stores are apparently
sufficient to accomodate this demand (Lemon and Mauleon,
1982).
Luteinizing hormone and cyclic AMP increase cholesterol
esterase and cholesterol side chain cleavage (SCC) enzyme
activities, lipoprotein uptake and P^ production (Savard,
1973; Bisgaier et al., 1979; Caffrey et al., 1979a; Strauss
et al., 1982). However, specific mechanisms of LH action on
luteal cells are not defined clearly.


23
Intracellular Ca+^ and cyclic AMP concentrations, and
cyclic AMP-dependent protein kinase activation are known tso
play essential roles in transduction of receptor-mediated
signals (see: Rasmussen, 1981). General concepts regarding
the mechanism of action of peptide hormones on target cells
have centered around intracellular accumulation of Ca+^ and
cyclic AMP, and activation of cyclic AMP-dependent protein
kinase(s) (Catt and Dufau, 1976; Marsh, 1976). This enzyme
mediates the effects of cyclic AMP via phosphorylation of
specific substrates which in turn regulate cell function.
Cyclic AMP-dependent protein kinase(s) has been localized in
the cytoplasm and numerous cellular oganelles (plasma
membrane, mitochondria, ribosomes, nucleus and endoplasmic
reticulum) suggesting that diversity of cellular responses
to cyclic AMP may depend on availability of specific
substrates within compartmentalized regions of the cell
(Langan, 1973).
Recent evaluation of membrane structure and function
has expanded our understanding of mechanisms involved in
receptor-mediated information flow into the cell. Over the
past decade, it has become apparent that the organization of
phospholipids and proteins within membranes is more complex
than described by the "fluid mosaic" membrane model of
Singer and Nicolson (1972). Several components of
biological membranes (i.e., receptors, enzymes,
phospholipids) are asymmetrically distributed between the


29
inner and outer bilayer (Rothman and Lenard, 1977; Lodisn
and Rothman, 1979). Furthermore, membrane components may
aggregate preferentially in specific domains of the Dilayar
structure (Chapman et al., 1979; Karnovsky et al., 1932).
Assembly of phospholipid and protein domains facilitate
events such as intercellular communications (Loewenstein,
1970; Garfield et al., 1979; Hertzberg et al., 1931),
endocytosis (Pearse, 1980; Schlessinger, 1980; Pastan and
Willingham, 1981a,b), ion movement and enzymatic activity
(Savard, 1973; Strauss et al., 1982; Lentz et al., 1933).
Membrane components are in a state of constant
reorganization, synthesis and breakdown in response to
external stimuli. Current data suggest that, in addition to
Ca+2 and cyclic AMP messenger systems, rapid and transient
alterations in membrane phospholipid composition,
biosynthesis and metabolism are essential in stimulus-
response coupling in cells (Hirata and Axelrod, 1980; Davis
et al., 1931; Crews, 1982; Farese, 1983; Milvae et al.,
1983). Phospholipid methylation (Hirata and Axelrod, 1980;
Crews, 1982; Milvae et al., 1983) and phosphotidylinositol
(PI) metabolism (Davis et al., 1981; Crews, 1982; Farese,
1983; Nishizuka, 1934b) are two mechanisms by which membrane
phospholipids are transiently altered in response to
receptor-ligand binding.
Methylation of phospholipids is dependent upon two
phospholipid methy1transferase (PMT) enzymes which are


30
distributed on the inner (PMT-1) and outer (PiVlT-2) aspects
of the cell membrane bilayer. Likewise, the phospholipid
substrates for PMT-1 (phosphatidylethanoiamine, PE) ana PMT-
2 (phosphatidyl-N-monomethylethanolamine, PME) are
asymmetrically distributed within the membrane. Formation
of PME requires the PMT-1 catalyzed transfer of a single
methyl group to PE. Conversion of PME to PC requires two
successive methyl transfers by PMT-2. Successive
methylations of PE by this system result in translocation of
methylated phospholipids (PME and phosphatidylcholine, PC)
toward the outer aspects of the membrane bilayer.
As a result of these chemical alterations in
phospholipid composition, the membrane becomes less
viscous. Enhanced membrane fluidity is thought to result
from the accumulation of deeply embedded PME within the
phospholipid bilayer. With the increase in membrane
fluidity, lateral mobility of membrane proteins is
potentiated thereby enhancing the coupling of surface
receptors with adenylate cyclase and cyclic AMP formation
(Rimon et al., 1978; Axelrod, 1983). Additionally,
formation and deposition of PC in the outer phospholipid
layer of the membrane promotes the unmasking of cryptic
membrane receptors. Thus, receptor availability is enhanced
(Hirata and Axelrod, 1930; Crews, 1982).
Milvae et al. (1983) demonstrated the importance of
phospholipid methylation in expression of LH-stimulated P^


31
production by dispersed bovine luteal cells. Incorpora cion
of [5H] -methyl-groups into PME, PC and PI was stimulated by
LH, in vitro. An endogenous methyl-donor, 3-aaenosy1-L-
methionine (SAM), enhanced LH-induced P^ production while
methylation inhibitors, 3-deazaadenosine (DZA) and S-
adenosyl-L-homocysteine (SAH), prevented the luteotropic
action of LH on dispersed luteal cells. Cyclic AMP effects
on P^ production were not influenced by DZA or SAH,
supporting the view that inhibition of phospholipid
methylation prevents coupling of ligand-receptor complexes
to adenylate cyclase, cyclase activation and cyclic AMP
generation in the bovine CL.
The degree of membrane phospholipid methylation also
appears to influence cellular Ca+2 fluxes, possibly via
regulation of calcium ion channels and Ca+^-dependent
adenosinetriphosphatase (ATPase) activity (Hirata and
Axelrod, 1980; Crews, 1982). Regulation of cell function by
calcium ion concentrations and intracellular Ca+^ binding
proteins, such as calmodulin, have been reviewed by several
authors (Cheung, 1979; Means, 1981; Rasmussen, 1981).
Receptor mediated Ca+^ influx occurs secondarily to
methylation of phospholipids and, like cyclic AMP
generation, is prevented by methylation inhibitors.
Activation of Ca+^-dependent phospholipase A£ parallels the
influx of Ca+2, as demonstrated by elevated free arachidonic
acid and lysolecitnin (lysophosphatidylcholine)


32
concentrations following receptor activation (Crews,
1932). These observations suggest that hydrolysis of newly
synthesized methylated phospholipids by Ca+^-dependent
phospholipases serve to regulate methylated phospholipid-
induced membrane effects, viz., increased fluidity, Ca+"~
influx, receptor availability. It is interesting to note
that Ca+^ is not required for membrane phospholipid
methylation or cyclic AMP generation in several cell
systems, although many of the physiological responses to
cyclic AMP are Ca+^-dependent (Rasmussen, 1970).
In addition to phospholipid methylation, considerable
interest has been directed toward receptor-mediated
alterations of the phosphatidate-inositide cycle (Crews,
1982; Farese, 1983; Nishizuka, 1984b). There are two known
mechanisms by which the phosphatidate-inositide cycle may be
affected by receptor activation. The first involves the
hydrolysis of phosphatidylinositides to diacylglyceride (DG)
and inositol phosphates via phospholipase C catalyzed
removal of the phosphorylated inositide head group. This
mechanism is thought to regulate plasma membrane Ca+^ fluxes
and intracellular Ca+2 distribution. The phosphatidylino-
sitides are localized primarily in the inner phospholipid
layer of the plasma membrane bilayer, and membranes of
endoplasmic reticulum and mitochondria as mono-, di- and
triphosphorylated inositol phospholipids (PI, PIP and PIP2>
respectively). These phospholipids bind Ca+
avidly and


33
provide a releasable Ca+- pool upon hydrolysis. Recent
evidence, reviewed by Nishizuka (1984b), suggests that PIP^,
rather than PI or PIP, is rapidly degraded following
receptor activation. Once hydrolysis of PIP2 i3 initiated
in the plasma membrane the process is thought to self-
propagate as a result of elevated free Ca+^ concentrations,
increased Ca+^-dependent phospholipase activity, and
extended hydrolysis of phosphatidylinositides within the
plasma membrane and intracellular organelles. One
metabolite of PIP2 hydrolysis, inositol triphosphate, may
serve as a mediator for Ca+^ release from intracellular
stores (Nishizuka, 1984b). A second metabolite, DG, arises
following phospholipase C catalyzed hydrolysis of PIP2> PIP
or PI. Diacylglycerol may serve as a precursor for the
regeneration of phosphatidylinositides as well as PE and
PC. The phosphatidylinositides and DG contain primarily
arachidonate at fatty acid position -2. Thus, hydrolysis by
Ca+^-dependent phospholipase provides free arachidonic
acid for use in prostaglandin and hydroxyperoxylipid
biosynthetic pathways (Ramwell et al., 1977), and various
lysolecithins, many of which are required for membrane
fusion reactions during uptake and secretion of products
(Crews, 1982). Additionally, DG activates the Ca+^- and
phospholipid-dependent protein kinase C (Nishizuka,
1984a,b). Protein kinase C activation has been implicated
in the elicitation of cell proliferation (Nishizuka, 1984a)


34
and inhibition of gonadal steroidogenesis (Welsh et al.,
1984). It is interesting to note that protein Kinase C
serves as a receptor for tumor promoting phorbol esters
(Nishizuka, 1984afb) which are thought to irreversibly
activate the enzyme. Phorbol esters possess a similar
chemical structure to DG, the endogenous activator of
protein kinase C. Phorbol esters have been shown to innibit
gonadotropin-induced steroidogenesis, in vitro (Welsh et
al., 1984), supporting a similar role for endogenous DG in
the regulation of steroid production. Therefore,
steroidogenesis may be antagonized as a consequence of
phosphatidylinositide hydrolysis.
Conversely, de novo phosphatidate-inositide
biosynthesis may be important in the mediation of
gonadotropin effects on steroidogenesis (Davis et al., 1981;
Crews, 1982; Farese, 1984). Several studies have
demonstrated that LH and ACTH stimulate biosynthesis of
phosphatidylinositides and phosphatidic acid (PA) in gonadal
and adrenal cortex cells, respectively (Davis et al., 1981;
Strauss et al., 1982; Farese, 1984). Data reviewed by
Farese (1984) indicated that identical responses were
triggered by cyclic AMP, suggesting that phospholipid
biosynthesis occurred after cyclic AMP generation. Both
hormone- and cyclic AMP-induced phosphatidate-inositide
biosynthesis, and steroidogenesis are blocked by protein
synthesis inhibitors, puromycin and cyclohexiraide.


35
Apparently, the two enzymes responsible for PA biosynthesis,
viz., glycerol-3-phosphate acyltransferase and diglyceride
kinase, are substrate activated and require protein
synthesis. Hormone activation of these enzyme systems is
indirect and thought to occur by increasing glycerol
phosphate, glucose, fatty acid and diglyceride availability
through stimulation of glycogenolysis, glycolysis, glucose
uptake and lipolysis. Phosphatidylinositol kinase catalyzes
the conversion of PA plus ATP to PI, and subsequent
phosphorylations to PIP and PIP2.
Phosphatidate-inositol biosynthesis parallels
steroidogenic activation in adrenal and luteal tissues
following exposure to ACTH and LH, respectively.
Furthermore, addition of certain polyphosphorylated
phospholipids, viz., the phosphatidylinositides and
cardiolipin, to adrenal cortex and luteal cell cultures
stimulated steroid biosynthesis in a dose dependent manner
(Strauss et al., 1982; Farese, 1984). Effects of these
"steroidogenic" phospholipids are not prevented by
cycloheximide or puromycin.
The site of phospholipid action in both adrenal cortex
and luteal cells appears to be the mitochondria. Specifi
cally, steroidogenic phospholipids stimulate cholesterol
side chain cleavage (SCC) by intact cells and acetone
powders of luteal mitochondria (Strauss et al., 1982;


3b
Farese, 1984), although the mechanism through whicn this is
accomplished remains unknown.
At present, it is known that trophic hormones, such as
LH and ACTH, stimulate steroidogenesis by influencing
several biochemical steps. Free intracellular cholesterol
concentrations are elevated via enhanced cellular uptake of
cholesterol from plasma and mobilization of cholesterol from
intracellular stores following cholesterol esterase
activation. Apparently, hormone induced elevations in free
intracellular cholesterol are not affected by cycloheximide,
but are blocked by cytochalasin B suggesting that microfila
ments, but not protein synthetic events, are crucial in the
acquisition of substrata for steroid biosynthesis (Gemmel
and Stacy, 1977; Sawyer et al., 1979; Silavin et al., 1980;
Rasmussen, 1981; Gwynne and Condon, 1982). Microtubule
involvement in steroidogenesis is, however, less clear with
evidence reported both pro (Gemmel and Stacy, 1977; Sawyer
et al., 1979) and con (Gwynne and Condon, 1982).
In addition to elevating intracellular substrate
concentrations, LH and ACTH increase mitochondrial content
of cholesterol, presumably as a result of enhanced
cholesterol transfer from cytosol to mitochondria (see:
Rasmussen, 1981, for references). This event requires
protein synthesis and microfilament action. Hormone- and
cyclic AMP-induced biosynthesis of the "steroidogenic"
phospholipids are also dependent on protein synthetic


37
events, although their stimulatory effects on mitochondrial
SCC activity are not (Strauss et al., 1982; Farese, 1984).
Furthermore, mitochondrial SCC activity is substrate- and
Ca+--dependent. Thus, it appears as though hormone-induced
steroidogenic activity involves primarily processes which
increase intracellular cholesterol content and transfer of
cholesterol from the cytosol to the SCC enzyme complex,
viz., sterol carrier proteins (see: Savard, 1973, for
references). Translocation of cholesterol across the
mitochondrial membranes may be regulated by the phospha-
tidylinositides and other "steroidogenic" phospholipids
(Strauss et al., 1982; Farese, 1984), the synthesis of which
is dependent on some, as yet undetermined, protein synthetic
event. Increased SCC activity may also involve structural
changes in the enzyme-phospholipid domain (Savard, 1973;
Strauss et al., 1982), and altered mitochondrial Ca+^
concentrations (Crews, 1982; Farese, 1984).
It is becoming more apparent that membrane phospholipid
dynamics, in addition to cyclic AMP and Ca+2 second
messenger effects, are of paramount importance in cellular
information flow and stimulus-response coupling. The reader
is referred to Rasmussen (1981) for further discussions on
this broad and rapidly developing area of cell physiology.
Following side chain cleavage of cholesterol, newly
synthesized P5 is transferred out of the mitochondria to the
endoplasmic reticulum. Here, the membrane bound


33
3p-hydroxy-A^-steroid dehydrogenase/a^-a^ isomerase enzyme
complex (H3D) catalyzes the rapid conversion of zo P,
the primary steroid end product of the CL. These latter
steps in the biosynthesis of do not appear to be
regulated by trophic hormones. However, luteal P^
production may be modified by local and systemic mechanisms
involving steroids, arachidonic acid metabolites, and
ovarian peptide hormones.
Intraovarian control of CL function
The dependency of CL production on LH has been
extensively documented in the literature, as has the
importance ox uterine-derived PGF2a in luteolytic events.
However, the level and duration of luteal P^ production may
also be determined by the local ovarian environment in which
the CL resides.
A large body of evidence supports a role for steroids
in the regulation of steroidogenic enzyme activities in the
ovary, testes, adrenal and placenta (see: Rothchild, 1981;
Gower and Cooke, 1983). Caffrey et al. (1979b) demonstrated
that the affinity of HSD for its product, P^, was
substantially greater than for P^ substrate and that P^ (IQ-
50 ug) inhibited HSD activity in ovine CL preparations.
Thus, suggesting that P^ may modify its own production via
substrate inhibition of HSD activity in the CL.
Testosterone and O0 u§) '^sre also found to be potent
inhibitors of HSD activity, in vitro (Caffrey et al., 1979b;


39
Gower and Cooke, 1933), but not in vivo (Caffrey er al.,
1979b). Similarly, microgram concentrations of testosterone
and E£ were found to inhibit LH-stimulatea P^ production but
not cyclic AMP accummulation in dispersed luteal cells
collected from 2 to 6 months of gestation in catle.
(Williams and Marsh, 1978). In vivo luteolytic interactions
were reported between E£ and PGF£a in hysterectomized ewes
(Gengenbach et al., 1977), and between E2 (0.5 ug) and
oxytocin (200 ralU) in dispersed luteal cells collected from
cyclic cattle (Tan and Biggs, 1984). However, Hixon and
Hansel (1979) were unable to demonstrate similar luteolytic
effects with subnanogram doses of E2 or E2 plus PGF2a in
dispersed bovine luteal cells from days 12 and 13 of the
cycle. Further support for a direct E2-CL interaction was
provided in a recent study (Glass et al., 1934) in which E2
receptors were characterized in large and small luteal cells
throughout the estrous cycle of the ewe. Concentrations of
E2 receptor rose gradually during the estrous cycle and were
localized primarily in large lutein cells. Additionally,
Sairam and Berman (1979) provided evidence that synthetic
estrogens prevented normal coupling of the gonadotropin-
receptor complex to its catalytic subunit, thus preventing
the initiation of intracellular gonadotropic-induced
responses.
Discrepancies with regard to the inhibitory role(s) of
E2 on CL P^ biosynthesis may be due to differences in dosage


40
of steroid employed, specie and status (cyclic or pregnant)
of the animal from which luteal tissue was collected, and in
vitro versus in vivo experimental conditions. Although
doses of E£ which inhibit CL biosynthesis seem high and
nonphysiological, they do compare favorably with
concentrations reported in follicular fluid of preovulatory
follicles (see: Fortune and Hansel, 1979)* Therefore, the
CL may be exposed to high intraovarian concentrations of
various steroids, viz., testosterone and E?, as would be
expected during preovulatory follicle development in the
late diestrum. The potential inhibitory nature on luteal P4
production exhibited by high concentrations of these
steroids supports a functional role in the initiation of CL
regression. In this regard, it is noteworthy that P4 plasma
concentrations begin to fall prior to any detectable rise in
peripheral plasma E2 concentrations or initiation of uterine
PG^2a Pulsatile activity (Chenault et al., 1975; Peterson et
al., 1975).
Beyond the luteotrophic effects initiated by LH, P4
production may be "protected" from steroid negative feedback
effects by several intraovarian processes.
For example, binding of steroids to proteins for short
term storage, viz., steroid carrier proteins (Goddard et
al., 1980; Willcox, 1983) or long-term storage in
cytoplasmic granules (Gemmell et al., 1974; Geramell and
Stacy, 1979; Sawyer et al., 1979) may reduce intracellular


41
concentrations of free steroid and reduce feedbacK
inhibition, in vivo. Furthermore, steroid sulfacion or
conjugation to fatty acids, viz., lipoidal steroids
(Hochberg et al., 1979; Albert et al., 1980; Schatz and
Hochberg, 1981; Albert et al., 1982), within the CL would
accomplish a similar function. Lastly, ovarian blood flow
parallels luteal production in cattle (Ford and Cnenault,
1931; Wise et al., 1982) thus providing an additional means
of reducing CL and ovarian steroid concentrations through
elevated clearance rates.
In recent years, several ovarian peptides, including
steroid-binding proteins (Willcox, 1983) LH-binding
inhibitors (Kumari et al., 1982; Ward et al., 1982),
gonadotropin releasing hormone (GnRH)-like peptides (Ying et
al., 1981; Fraser, 1982; Sharpe, 1982), angiogenic factor
(Gospodarowicz and Thakral, 1978), oxytocin (Fields et al.,
1983; Rodgers et al., 1983; Wathes et al., 1983),
vasopressin (Wathes et al., 1983) and relaxin-like peptides
(Fields et al., 1982) have been discovered. Although
biochemical evidence suggests that these peptides are
produced by the CL and/or follicle, little information is
available concerning their physiological roles in ovarian
processes.
Of the peptides mentioned above, oxytocin has been
evaluated most thoroughly. It is known that CL of the cow
and ewe contain high concentrations (ug/g) of oxytocin


42
(Fields at al., 1983; Flint and Sheldrick, 1983; Flint at
al., 1983; Wathes et al., 1983) which is synthesized and
stored in the large lutein cells (Rodgers et al., 1983).
The bovine CL contains neurophysin, lending further support
for the local synthesis of oxytocin within the CL (Wathes et
al., 1983). Additionally, circulating oxytocin
concentrations are rapidly reduced following ovariectomy
(Schams et al., 1982) and concentrations of immunoreactive
oxytocin were greater in ovarian venous plasma versus
uterine arterial or jugular venous plasma (Flint and
Sheldrick, 1982; Walters et al., 1984). Concentrations of
oxytocin and neurophysin in luteal tissue (Wathes et al.,
1984) and plasma (Sheldrick and Flint, 1981; Flint and
Sheldrick, 1983; Schams, 1983; Walters et al., 1984)
parallel CL production during the luteal phase, although
the decline in oxytocin concentrations occur prior to
during CL regression. Oxytocin and P^ are secreted from the
CL in a pulsatile fashion. Walters et al. (1984) reported
that only 29% of P^ episodes were associated with pulses of
oxytocin during the early luteal phase, due largely to the
occurrence of fewer oxytocin than P^ pulses at this time.
During the mid-luteal phase 86% of P^ and oxytocin episodes
occurred simultaneously. Remarkably, 97 to 100% of oxytocin
pulses, during all periods of the luteal phase, were
associated with P4 pulses. These observations suggest that


43
similar mechanisms may regulate the secretion of ?4 and
oxytocin from the CL.
Several lines of evidence support a systemic role for
oxytocin in the process of CL regression (reviewed in a
later section). Additional data suggests that oxytocin may
have an action within the ovary as well. Evidence reviewed
by Wathes (1984) indicates that oxytocin (and vasopressin)
may be involved in production during CL development since
oxytocin (and vasopressin) can promote P^ biosynthesis in
testicular cells through the inhibition of LH-stimulated
androgen biosynthesis. Oxytocin probably acts by selective
suppression of 17-hydroxylase and C17-20 desmolase
activities. Follicular androgen and estrogen biosynthesis
is suppressed in favor of P^ biosynthesis prior to and
following the preovulatory LH surge. Furthermore, oxytocin
has been measured in preovulatory follicles, ovarian tissue
(including all follicles) devoid of CL, and newly formed CL
(Wathes, 1984; Wathes et al., 1984). Therefore,
preovulatory follicle and luteal oxytocin biosynthesis,
possibly induced by LH, may prevent C-19 and C-18 steroid
biosynthesis and promote P^ formation in CL. High levels of
oxytocin in CL during the raid-luteal phase may reduce
follicular E£ production in a similar manner (Wathes,
1984). Results of Tan et al. (1982) also demonstrated
stimulatory effects of low levels of oxytocin (2 to 20 mill)
in CL collected from pregnant cattle. Likewise, exogenous


44
oxytocin, administered to cyclic cattle on days 12 and 1 ,
increased content of CL on day 14 (Mares and Casida,
1963). Conversely, high doses of oxytocin (200 co 4oD mill)
were inhibitory to in vitro P^ biosynthesis by CL of cyclic
(Tan and Biggs, 1984) and pregnant (Tan et al., 1982)
cattle.
Luteolytic prostaglandins appear to mediate oxytocin
secretion from the CL (Flint and Sheldrick, 1982, 1983;
Schallenberger et al., 1984). Similarly, PGF2a or analogues
of PGF2a stimulate rapid degranulation of large lutein calls
(Heath et al., 1983) which are known to be the synthetic and
storage site of oxytocin in the CL (Rodgers et al., 1983).
Consistent with the hypothesis of oxytocin storage in
granules are observations which demonstrate parallel
patterns of luteal oxytocin content (Wathes et al., 1984)
and secretion (Sheldrick and Flint, 1981; Flint and
Sheldrick, 1983; Schams, 1983; Walters et al., 1984) with
large luteal cell granule content (Gemmell et al., 1974).
Based on the nearly absolute synchrony with which tonic
oxytocin and P^ pulses occur, in vivo (Walters et al.,
1984), and the ability of PGF2a to induce oxytocin
secretion, it may be suggested that luteal prostaglandins
are involved in tonic oxytocin and P^ secretion during the
luteal phase of the estrous cycle. Evidence of luteal
prostaglandin biosynthesis (Shemesh and Hansel, 1975a;


45
Lukaszewska and Hansel, 1979; Milvae and Hansel, 1965a)
strengthens this supposition.
Bovine luteal tissue contains massive quantities (2 to
5 mg/g) of arachidonic acid, the bulk of which is esterified
to phospholipids (Lukaszewska and Hansel, 1979, 1980).
Thus, the potential for arachidonic acid metabolism in
either the cyclooxygenase or lipoxygenase pathway is
great. Metabolic pathways utilizing arachidonic acid as
precursor have been reviewed previously (Ramwell et al.,
1977). Characterization of local biosynthetic patterns and
functions of luteal prostaglandins and other arachidonic
acid metabolites have provided additional insight into
mechanisms controlling luteal production and regression.
Shemesh and Hansel (1975b) demonstrated an acute
increase in ovarian venous PGF2a concentrations following
injection of arachidonic acid into CL on days 12 or 13 in
cyclic cattle. Peripheral plasma P^ concentrations fall
rapidly from approximately 6 ng/ml to 2.5 ng/ml, and
remained constant until day 15 or 16 when the experiment
concluded. Additionally, CL weights on day 15 or 16 were
less in arachidonic acid-treated (4.3 1.4 g) than vehicle-
treated (6.3 0.5 g) cattle. Several studies have
demonstrated an inverse relationship between luteal PGF£a
and P^ content or production (Patek and Watson, 1976;
Lukaszewska and Hansel, 1979; Rothchild, 1981). However,
the means by which elevated luteal PGF2a contents reduce P^


46
production remain to be elucidated. Exogenous PGF?a
administration to cattle (Lukaszewska and Hansel, 1979),
sheep (Diekman et al., 1978) and rats (Hichens et al., 1974;
Grinwich et al., 1976) initiates a reduction in
production and the loss of LH receptors or luteal
sensitivity to LH. The authors suggested that loss of LH
receptor numbers or reduced sensitivity to LH were the
consequence rather than cause of luteolysis since reduced P^
production occurred considerably earlier tnan either of the
LH related phenomenon.
The PGF2a-induced decline in luteal P^ production
described above may be related more closely to changes in
ovarian vascular resistance. Blood flow to the CL-bearing
ovary parallels closely luteal P^ production (Niswender et
al., 1975; Ford and Chenault, 1981) and PGF^a is known to
reduce ovarian blood flow and luteal P^ production (Nett et
al., 1976; Niswender et al., 1976). Therefore, PGF2a
effects may be mediated through increasing ovarian vascular
resistance. However, metabolite requirements are generally
thought to dictate the degree of blood flow to a tissue. In
this light, reduced luteal metabolic activity may initiate
the accummulation of PGF2a and subsequent decline in luteal
blood flow.
Progesterone biosynthetic activity of bovine CL
collected during the estrous cycle is highly correlated
(r^=.93) with luteal prostacyclin (PGI2) biosynthesis


47
(Milvae and Hansel, 1983a). Sun at al. (1977) demonstraced
that PGI2 was the predominant prostaglandin product
following bovine CL membrane metabolism of PGH2-
Furthermore, PGI2 stimulated luteal P^ biosynthesis in vicro
and in vivo (Milvae and Hansel, 1980b). A recent report by
Milvae and Hansel (1985) demonstrated that indomethacin
inhibition of prostaglandin formation early in the estrous
cycle shortened luteal lifespan in cattle. Indomethacin was
administered into the uterine lumen twice daily from days 4
through 6. In conjunction with previous reports, these
authors proposed that luteotropic products of the
cyclooxygenase pathway, viz., PGI2 and PGE2, may be required
for normal CL development. The origin of these
prostaglandins may include both the CL and uterus.
Additionally, synthesis and content of PGI2 and PGF2a in
bovine CL are greatest early in the cycle (Milvae and
Hansel, 1983a). Luteal prostacyclin content was
approximately four times greater than PGF2a early in the
cycle although ratios of PGI2 and PGF2a gradually decline in
CL age increased (Milvae and Hansel, 1983a). Milvae et al.
(1985) provided several lines of evidence suggesting that
lipoxygenase products, viz., 5-hydroperoxy-eicosatetraenoic
acid (5-HPETE) and 5-hydroxyeicosatetraenoic acid (5-HETE),
regulate P^ production by the selective inhibition of
prostacyclin synthetase activity and PGI2 production in
bovine CL. Large quantities of 5-HETE are present in bovine


48
CL on days 10, 15 and 18 of the cycle and 5-HETE inhibited
in vitro PGI2 and biosynthesis in a dose dependent
fashion without influencing PGF2a production. Simultaneous
addition of LH was unable to prevent the 5-HETE-induced
reduction in luteal cell PGI2 and P^ production, in vitro.
In related experiments (Milvae et al., 1985), arachidonic
acid metabolism through the lipoxygenase pathway was
inhibited by nordihydro-guaiaretic acid (NDGA). When NDGA
was added to dispersed luteal cells, PGI2 production was
increased after a 2 hour incubation. Progesterone and PGF2a
synthesis had increased slightly, although not significantly
(P>.05). Arachidonic acid plus NDGA increased luteal
production of both PGI2 and PGF2a but not P^, suggesting an
antagonism between PGI2 and PGF2a in the regulation of
luteal P^ synthesis (Milvae et al., 1985). This possibility
is also suggested by the declining ratio of PGI2 and PGF2a
contents in bovine CL as luteolysis approaches (Milvae and
Hansel, 1985a). Collectively, these data suggest that HETE,
and perhaps other lipoxygenase products, are involved in
luteolysis by blocking synthesis of the luteotropin, PGI2,
and allowing additional arachidonic acid to be shunted
toward pathways involved in synthesis of luteolytic
prostaglandins.
Similar mechanisms controlling biosynthesis of
luteotropic and luteolytic prostaglandins may also occur


49
within the uterus (Milvae et al., 1985; Milvae and hansel,
1935).
Uterine Control of CL Function
There is little doubt that the uterus plays a key role
in regulation of CL lifespan during the estrous cycle. Loeb
(1923) first demonstrated that extirpation of the guinea pig
uterus resulted in extended CL function. Subsequent reports
in cattle (Wiltbank and Casida, 1956; Anderson et al., 1961;
Malven and Hansel, 1964; Anderson et al., 1965), sheep
(Wiltbank and Casida, 1956; Anderson et al., 1969), pigs
(Spies et al., 1958; Du Mesnil Du Buisson, 1966) and horses
(Ginther and First, 1971) provided similar results following
surgical removal of the uterus. Thus, a basis for uterine
involvement in the luteolytic process was developed. The
nature of this utero-ovarian relationship was further
defined following observations that cattle (Bland, 1970) and
sheep (McCracKin and Caldwell, 1960) with congenital absence
of the uterine horn (uterus unicornis) adjacent to the CL
bearing ovary exhibited prolonged luteal lifespans and cycle
lengths which suggested local regulation of CL function by
the ipsilateral uterine horn. Additionally, normal luteal
regression occurred following heraihysterectomy of the
contralateral, but not the ipsilateral uterine horn in


50
cyclic cattle and sheep (Inskeep and Butcher, i960; Ho or and
Rowson, 1966; Ginther, 1974, 1931).
As a consequence of these data, Wollm^rhaus (1964) and
Ginther (review, 1974) examined the anatomy and histology of
the utero-ovarian vascular architecture as a possible route
of exchange between the uterine horns and their adjacent
ovaries. In cattle and sheep, species which exhibit local
regulation of CL function by the uterus, exchange of
luteolytic substances from the uterine venous effluent
(ovarian vein, International Committee of Veterinary
Anatomical Nomenclature, 1968) into the ovarian arterial
supply is thought to occur just below the ovarian vascular
pedicle. At this point, the ovarian artery follows an
extremely tortuous and convoluted path over the surface of
the ovarian vein (containing both uterine and ovarian venous
blood) thus maximizing the area of contact between the two
vessels. Histologically, the adventitial layers of each
vessel were substantially thinner in the regions of vein-
artery apposition (Wollmerhaus, 1964; Del Campo and Ginther,
1974). From an anatomical and histological perspective,
these observations favored the existence of a functional
venoarterial exchange system between uterine horns and their
adjacent ovaries.
A series of physiological experiments, utilizing
surgical anastomoses of ovarian veins or ovarian arteries in
hemihysterectoraized cattle and sheep, provided conclusive


51
support for the functional ovarian vein and artery-
components of the local uteroovarian pathway (Gincher, 1974;
1981). In such experiments, luteal regression occurred when
either ovarian vein or ovarian artery blood from the
contralateral, uterine-intact side was diverted to the
appropriate vessel on the ipsilateral, hemihysterectomized
side. Conversely, CL were maintained if the surgical
anastomosis became occluded. Thus, it became clear that the
uterus was producing a blood borne product which could move
from the uterine venous drainage into the ovarian arterial
supply and initiate the process of luteolysis in a local
manner.
As early as 1940, Hechter and coworkers suggested that
some uterine-derived product may be responsible for
regulation of CL lifespan. However, it was not until the
resurgence of prostaglandin research in the 1950's that the
identity of the uterine luteolytic factor was elucidated.
The chemical structures of several prostaglandins, including
PGF2a, were confirmed in 1962 (Nelson et al., 1982) and by
1969, Pharris and Wyngarden provided evidence that the
vasoactive prostaglandin, PGF2a, may be the uterine
luteolytic substance in the rat. Subsequently, the
luteolytic activity of exogenous PGF2a in cattle has been
documented extensively (Hansel et al., 1973; Hafs et al.,
1974; Lauderdale, 1974; Thatcher and Chenault, 1976).
Several studies have provided evidence indicating that PGF2a


52
is an endogenous luteoiysin in cattle. For example,
elevated levels of PGF2a in uterine venous drainage
(Nancarrow et al., 1973; Shemesh and Hansel, 1975), uterine
tissue (Shemesh and Hansel, 1975) and uterine flushings
(Lamothe et al., 1977; Bartol et al., 1981a) coincide
closely with the period of expected luteal regression. In
addition, luteolysis is prevented in cows immunized
passively against PGF2a (Fairclough et al., 1981) or
following intrauterine administration of the prostaglandin
synthetase inhibitor, indomethacin, late in the estrous
cycle (Lewis and Warner, 1977).
The biological half-life of PGF2a is very short owing
to its rapid metabolism by the lungs (Piper et al., 1970)
and uterine endometrium (Thatcher et al., 1984; Chapter
3). Thus, measurements of its primary metabolite, 15-keto-
13,14-dihydro-PGF2a (PGFM; Granstrora and Kindahl, 1982),
provide an index of uterine PGF2a production. Peripheral
concentrations of PGFM are significantly correlated with the
uterine production of PGF2a (Thatcher et al., 1984; Chapter
3) and concentrations of PGF2a in uterine flushings (Bartol
et al., 1981a). Furthermore, three to five pulsatile
episodes of PGFM were always temporally associated with the
decline in plasma P^ concentrations during luteolysis in
cattle (Peterson et al., 1975; Kindahl et al., 1976;
Betteridge et al., 1984). Collectively, these data indicate


53
that the endogenous uterine luteolysis in cattle is likely
to be PGF2a.
Finally, in keeping with the concept of a local
venoarterial pathway, transfusion of PGF£a from the uterine
venous drainage into the adjacent ovarian arterial supply
was demonstrated. In an early study, Goding et al. (1971)
induced luteal regression, as demonstrated by the
precipitous decline in plasma P4 concentrations, following
the infusion of physiological concentrations of PGF2a into
the uterine venous drainage of diestrous cattle. Hixon and
Hansel (1974) reported elevated PGF2a concentrations in the
uterine venous drainage (ovarian vein) and ovarian artery,
ipsilateral to the CL bearing ovary, as a result of the
intrauterine deposition of PGF2a* Similarly, Thatcher et
al. (1984b) verified the functionality of the countercurrent
exchange system (PGF2a)' during the period of normal luteal
regression in cyclic cattle.
Regulation of Uterine PGF?^Production
Demise of the cyclic CL is uterine and PGF2a-dependent,
as reviewed above. However, interplay between ovarian
estrogen, progesterone and oxytocin with the uterine
endometrium regulate the timing and magnitude of luteolysin
production during the estrous cycle. Estrogens, of
follicular origin, appear to constitute an important
stimulatory component regulating uterine PGF2a production
and CL regression. Destruction of ovarian follicles in


54
cattle (Villa-Godoy et al., 1931) and sheep (Karsch et al.,
1970; Ginther, 1971) result in prolonged luteal lifespans
and heavier CL compared to follicle-intact controls.
Furthermore, administration of estrogens to late-luteal
phase cows and ewes resulted in production of PGF2a by the
uterus (Barcikowski et al., 1974; Thatcher et al., 1984b;
Chapter 3) and premature CL regression (Greenstein et al.,
1958; Wiltbank, 1966; Stormshak et al., 1960; Caldwell et
al., 1972; Eley et al., 1979). As anticipated, luteolytic
doses of E2 were ineffective in hysterectomized (Brunner et
al., 1969; Akbar et al., 1971; Caldwell et al., 1972) and
indomethacin-treated (Warren et al., 1979; Barcikowski et
al., 1974) cattle and sheep. In another study (Loy et al.,
1960), E2 administration to cattle on day 1 or 4 of the
estrous cycle had no effect on CL examined on day 14.
However, treatment with caused a reduction in CL weight,
P4 content, and number of functional luteal cells.
Administration of exogenous P^ for 1 to 5 days post-estrus
in cattle and sheep results consistently in shorter
interestrous intervals (Woody et al., 1967; Ginther, 1968,
1969; Thwaites, 1971; Lawson and Cahill, 1983), presumably
as a result of premature development and activation of the
uterine PGF2a secretory system (Baird et al., 1976; Ottobre
et al., 1980). These data suggest that the uterus requires
a period of P4 exposure in order to develop the capacity to
secrete luteolytic levels of PGF2C1 in response to


55
estrogens. For example, in the cow (Skjerven, 195*0;
Martinov and Lovell, 1968) and ewe (Brinsfield and Hawk,
1973) the degree of endometrial lipid droplet accumulation
varies cyclically, being more prevalent during P4 man E2
dominated phases of the estrous cycle. Additionally,
exogenous P4 stimulated accumulation of endometrial lipid
droplets while E2 administration decreased lipid stores
(Brinsfield and Hawk, 1973). Finally, Hansel et al. (1975)
extracted large quantities of arachidonic acid, the
principle substrate for PGF2a synthesis, from bovine
endometrium between days 10 and 14 of the luteal phase.
Collectively, these results support the concept that P4
directs endometrial accumulation of lipid and prostaglandin
precursor concentrations during the estrous cycle.
Estrogens induce the luteolytic response during late
diestrus.
Timing and magnitude of the uterine luteolytic response
are dependent on circulating patterns and concentrations of
P4 and E2 which, in turn, regulate the degree of uterine
sensitivity to ovarian steroids, viz., endometrial P4 and E2
receptor populations. Estrogen is known to stimulate
synthesis of endometrial estrogen and P4 cytoplasmic
receptors (Rc) (Clark et al., 1977; Koligian and Stormshak,
1977a). Conversely, P4 inhibits synthesis of E-Rc and
reduces nuclear retention of the estrogen-receptor complex
in endometrial tissue (Clark et al.,
1977; Koligian and


BtormshaK, 1977a; Katzenellenbogan et al., 1930).
Progesterone also reduces endometrial synthesis of its own
receptor (Schrader and O'Malley, 1973; Walters ana Ciar*:,
1930). With this in mind, direct measurements of steroid
receptors in bovine and ovine endometrium revealed elevated
concentrations of E-Rc during proestrus and metestrus, and
low E-Rc concentrations during diestrus (Senior, 1975;
Koligian and Stormshak, 1977b; Henricks and Harris, 1978).
Additionally, Zelinski et al. (1982) demonstrated a decline
in bovine endometrial P4~RC concentrations from the
proestrus to diestrus phases of the estrous cycle. Thus, as
the CL develops and plasma E2:P^ ratios decline,
concentrations of endometrial E-Rc fall precipitously.
Plasma P^ thereby inhibits the uterus from responding in an
estrogen-directed fashion. However, elevated plasma P^
concentrations progressively suppress endometrial production
of its own receptor such that estrogen-induced events (i.e.,
prostaglandin biosynthesis) are gradually removed from P^
inhibition.
Induction of uterine PGF2a production by E2 may be
attributed partially to E2-dependent increases in activities
of cyclooxygenase (Huslig et al., 1979) and phospholipase A2
(Dey et al., 1982). Calcium is probably involved in
activating phospholipase A2 since this enzyme is Ca+^
dependent (Brockerhoff and Jensen, 1974). Estrogen was also


57
shown to increase uterine Ca+^ availability in swine
(Geisert et al., 1932).
Estrogen-induced luteolysis is prevented by inhibition
of uterine DNA dependent RNA synthesis with actinomycin D
(French and Casida, 1973) suggesting that the process of
PGF2a production requires protein synthesis. Furthermore,
Roberts et al. (1976) and McCracken et al. (1931, 1984)
provided evidence for estrogen induction of endometrial
oxytocin receptors following a period of P^ priming in
sheep. Exogenous oxytocin stimulates uterine PGF2a
production in cyclic cattle (Newcomb et al., 1977; Milvae
and Hansel, 1980) and sheep (Roberts et al., 1975, 1976;
McCracken, 1981, 1984) and causes premature CL regression in
uterine-intact (Armstrong and Hansel, 1959; Hansel and
Wagner, 1960; Milne, 1963), but not hysterectomized (Ginther
et al., 1967) or indomethacin-treated (Cooke and Homeida,
1983) females. In addition, luteal function was extended in
ewes immunized against oxytocin (Sheldrick et al., 1980;
Scharas et al., 1983). Based on these results and reports
concerning secretion of oxytocin and P4 by the CL (reviewed
earlier), McCracken and coworkers (1981, 1934) proposed a
working hypothesis for CL regression in cyclic ewes.
Briefly, their proposed sequence of events are: 1)
declining endometrial P4 receptor population reduces P4
dominance on the uterus during late diestrus and permits
follicular E£ to stimulate endometrial estrogen and oxytocin


53
receptor formation and activate enzymes essential for
biosynthesis; 2) circulating luteal (or pituitary) oxytocin
binds to newly synthesized endometrial receptors and
initiates PGF2a secretion; 3) plasma P^ concentrations
decline slowly, further reducing its influence on the
uterus; 4) uterine-derived PGF^ induces a rapid dumping of
luteal oxytocin which reinforces uterine PGF2a production
and 5) PGF2a initiates a rapid decline in plasma P^
concentrations associated with CL demise.
Available data indicate that PGF2a is the primary
uterine luteolysin in numerous species (Horton and Poyser,
1976). However, recent data implicate additional
arachidonic acid metabolites in the luteolytic process. For
example, the 13,14-dihydro metabolite of PGF2a was as
effective as equivalent amounts of PGF2a in causing CL
regression in heifers (Milvae and Hansel, 1983).
Arachidonic acid metabolites of the lipoxygenase pathway may
also be physiological regulators of luteal lifespan (Milvae
et al., 1985). Lipoxygenase products, such as 5-hydroxy-
eicosatetraenoic acid (5-HETE), specifically inhibit luteal
synthesis of prostacyclin, a potent luteotropin (Milvae and
Hansel, 1980, 1983), thereby reducing luteal P4 production,
in vitro. When in vitro synthesis of 5-HETE and other
lipoxygenase products was blocked by nordihydroguaiaretic
acid NDGA, luteal cell prostacyclin and P4 production were
increased without influencing PGF2a production. Further,


59
twice daily intrauterine adrainistration of NDGA to cyclic
heifers on days 14 to 13 extended luteal function
approximately 5 days beyond control heifers. iaese data
suggest that lipoxygenase products, of uterine and/or
ovarian origin, are involved in the luteolytic process. in
a final study, Milvae and Hansel (1985) provided evidence
that uterine and/or ovarian prostaglandins are required for
normal CL development and function in cattle. Intrauterian
administration of indomethacin to cyclic heifers on days 4,
5 and 6 caused decreased production and early CL
regression. The authors implied that luteotropic
prostaglandins, such as PGE2 and prostacyclin, may be
involved in luteal development.
Conceptus-Associated Events During
Early Pregnancy
An opportunity for establishment of pregnancy is
provided with each estrous cycle. As a consequence of
luteal P^ production, glandular epithelium of the uterine
endometrium develops morphological/ultrastructural
characteristics, and functions of an active secretory tissue
(Skjerven, 1956b; Marinov and Lovell, 1968; Wathes and
Wooding, 1980). Several studies have demonstrated increased
endometrial tissue content or uterine secretion of amino
acids, proteins, fatty acids, lipids, carbohydrates, and
certain ions during the luteal phase of the estrous cycle or


O
in response to exogenous P4 (corf: Skjerven, 1956a; Fanning
et al., 1967; Carlson et al., 1970; Roberts and Parker,
1974; Hansel et al., 1975; Bartol et al., 1931; sneeo:
Brinsfield and Hawk, 1973; Roberts and Parker, 1976; pig:
Murray et al., 1972; Knight et al., 1974; Bazer, 1975; Bazer
et al., 1977; human: Shapiro et al., 1980). In addition,
elevated P4 concentrations reduce uterine tone and
myometrial contractility (Hays and Van Demark, 1953; Lehrar
and Schindler, 1974). Thus, during the first 15 to 16 days
post-estrus, a complex embryotrophic environment is
established within the uterine lumen.
During early pregnancy there is a clear requirement for
developmental synchrony between the conceptus and
endometrium. Embryo transfer studies in cattle and sheep
indicate that a small degree of asynchrony (1 or 2 days)
is tolerated with respect to stage of transferred conceptus
and recipient uterine development (Rowson and Moor, 1966;
Rowson et al., 1969; Sreeman, 1978; Seidel, 1981). However,
pregnancy success is highest when synchronizetion is precise
(Cook and Hunter, 1978).
As described earlier, P4 administered early in the
estrous cycle is thought to initiate premature development
and activation of the uterine PGF£a secretory system (Baird
et al., 1976; Ottobre et al., 1980). This system was
utilized recently to evaluate the role of P4 pretreatment,
early in the estrous cycle, on the embryonic state of the


61
ovine uterus (Lawson and Cahill, 1933). Progesterone
administered during the first 4 days of the ovine estrous
cycle accelerated development of the uterine luminal
environment such that the day 6 P^-treated uterus provided
acceptable support for normal development of transferred U
day old embryos. Luteolysis had not occurred in 8 of 12
ewes slaughtered on day 25* No day 10 conceptuses survived
following transfer to control (no P^ pretreatment) uteri on
day 6. Estrous cycles in these ewes were of normal
length. These data suggest that uterine development and
complexity of its luminal environment progress sequentially
under the influence of P^. Further, the conceptus must
develop in synchrony with this environment to survive.
The inability of embryos to develop beyond the
blastocyst stage, when cultured in vitro (Wright and
Bondioli, 1931) or confined within the oviduct
(Wintenberger-Torres, 1956; Murray et al., 1971; Pope and
Day, 1972; Heap et al., 1979), illustrate that unique
components of the uterine environment become essential for
further conceptus development. The essentiality of this
relationship may be due, in part, to increased complexity of
conceptus nutrient and hormonal requirements following
blastulation (Daniel, 1971; Biggers and Borland, 1976).
Additionally, Lawson et al. (1933) demonstrated an active
interaction between the uterine environment and rate of
conceptus development in sheep. In two experiments,


62
sixteen-cell embryos (day 4) were transferred to uteri of
ewes on days 4, 6 or 7 of the estrous cycle. The embryos
were then recovered and evaluated 4 or 6 days later.
Embryos transferred to older, more advanced uteri were
viable when recovered (days 8 to 12 of the estrous cycle)
and exhibited accelerated growth rates during the 4 to 6
days in tero as compared to synchronously transferred
controls. A third experiment examined conceptuses on days
12, 14 or 15 of the estrous cycle following synchronous
versus asynchronous embryo transfers, as described above.
Results indicated that viability and accelerated growth
rates of asynchronously transferred embryos were halted by
day 11 or 12 post-estrus. Further, these conceptuses were
unable to maintain CL function in recipient ewes.
Similarly, P^ has been implicated in regulating ovine
conceptus growth during normal pregnancy (Bindon, 1971) and
in superovulated recipients of embryo transfers
(Wintenberger-Torres, 1968; Wintenberger-Torres and
Rorabauts, 1968). Presumably, P4 effects on embryonic growth
are mediated by alterations in the uterine luminal milieu.
Collectively, these data demonstrate that the conceptus is
capable of responding to physiochemical cues in its uterine
environment or conversely, the uterus exerts a regulatory
influence on some aspects of conceptus development during
early pregnancy. Either of these viewpoints may explain how
small degrees of developmental asynchrony between the uterus


63
and conceptus are tolerated in large domestic species
(Rowson and Moor, 1966; Rowson et al., 1969; Sreenan, 197b;
Seidel, 1931). However, there appears to be a developmental
limit, beyond which the conceptus may not "catch up" to Its
uterine environment and prevent luteolysis (Lawson et al.,
1933).
Regardless of this physiological flexibility, a
considerable percentage of embryos do not survive beyond day
30 of pregnancy in large domestic species. In cattle, the
majority of embryonic mortality occurs between days 16 and
26 of pregnancy (Hawk et al., 1955; Boyd et al., 1969;
Ayalon, 1978; Ball, 1978; Hawk, 1979). Dramatic changes in
bovine conceptus growth, differentiation (Melton et al.,
1951; Chang, 1952; Greenstein and Foley, 1958a, b;
Greenstein et al., 1958; King et al., 1982) and endocrine
activity (Shemesh et al., 1979; Chenault, 1980; Lewis et
al., 1982; Eley et al., 1983; Bartol et al., 1985) occur
during this relatively short period. Pregnancy success
between days 16 to 30 of gestation depends upon 1)
appropriate physiochemical communication between conceptus
and endometrium such that CL maintenance is achieved, and
2) the initiation of placental (cotyledon and caruncle)
development and function. "Maternal recognition of
pregnancy" (Short, 1969), occurring by days 16 to 17 in
cattle (Betteridge et al., 1980; Northey and French, 1980;
Dalla Porta and Humblot, 1933), and day 12 in sheep (Moor


64
and Rowson, 1966; Rowson and Moor, 1967) and swine (Dhinasa
and Dziuk, 1968), represents the first period during early
gestation when production of conceptus signals becomes
essential for luteal maintenance and continued endometrial
secretory support. The conceptus does not appear to be
essential for CL maintenance or initiation of an embryonic
uterine environment prior to maternal recognition of
pregnancy. This was clearly demonstrated by Betteridge et
al. (1980) when pregnancies were established following
synchronous embryo transfers as late as day 16 in cyclic
cattle. Additionally, noticable histological alterations in
endometrial development between pregnant and cyclic cattle
do not occur prior to day 17 (King et al., 1981, 1982).
Thus, pregnancy associated events occurring prior to
maternal recognition of pregnancy may be thought of as being
mediated primarily by the CL and endometrium, whereas events
following this period are under direction of the conceptus.
Conceptus Development (Days 15 to 30)
As noted earlier, developmental phases including
maternal recognition of pregnancy througn definitive
placentorae formation occur between days 16 and 30 of
gestation in cattle. The large degree of embryonic
mortality during this period attests to the critical nature
of these developmental processes with regard to
establishment and maintenance of pregnancy.


65
On day 1b of gestation, the bovine conceptas is
characteristically bilaminar (trophectoderm and encoders),
with more advanced conceptuses exhibiting varying degrees of
mesodermal germ layer development (Chang, 1952; Greenstein
and Foley, 1958 a, b). Trophectodermal and endodermal germ
layers initiate a phase of rapid elongation by days 15 to 16
of pregnancy. Lengths of extraembryonic membranes vary
tremendously during this period (1.5 to 225 mm; Hawk et al.,
1955; Greenstein and Foley, 1958b; Betteridge et al., 1980)
with mean diameters calculated between 50 and 90 ram.
Histological and ultrastructural evaluation of rapid
and progressive transition of porcine conceptus forms, viz.
spherical, tubular and elongated filamentous, between days
10 and 12 of gestation (Geisert et al., 1982) provided
evidence that rapid conceptus elongation occurs as a result
of trophectodermal and endodermal reorganization and not
hyperplasia. Whether or not this phenomenon occurs during
conceptus elongation in the cow or sheep is not known.
By day 17, mean conceptus extraembryonic membrane
length reaches approximately 150 mm (Chang, 1952; Greenstein
and Foley, 1958b). Histochemical evaluation of
trophectodermal cell types (days 16 to 33; Greenstein et
al., 1958) based on staining characteristics with Sudan
Black B or Oil Red 0 (demonstration of lipids), Periodic-
Acid-Schiff (PAS; demonstration of glycogen, glycoproteins),
and phloxine-methylene blue (demonstration of cytoplasmic


66
basopnilia or acidophilia) delineated three distinct cyoes
of cells by day 16 and 17* These included: 1) PA3 ( + ),
trophoblastic giant cells (GC) which were occasionally
binucleated at this stage; 2) undifferentiated tropho'olast
"stem cells"; and 3) normal columnar trophoblast cells which
consistently contained basally located lipid inclusions.
The embryo proper, viz. germinal or embryonic disc, is
notably oval in shape by day 17 and demonstrates signs of
initiating organogenesis, as indicated by the appearance of
the primitive groove and node. Additionally,
ultrastructural appearance of day 17 endometrial epithelium
was indistinguishable in pregnant versus cyclic cattle (King
et al., 1981), suggesting that no physical interaction
between conceptus and endometrium had occurred at this stage
of pregnancy.
Conceptus adhesion to maternal endometrium was noted to
occur by days 18 to 20 (Wathes and Wooding, 1980; King et
al., 1980, 1981), although no microvillous interdigitation
was observed. This physical interaction between the
conceptus and endometrium is consistent with the observed
reduction in uterine epithelial cell height by day 18 of
pregnancy (Wathes and Wooding, 1980; King et al., 1981) and
the apparent migration of binucleated trophoblastic GC into
the uterine epithelial cell layers (Wathes and Wooding,
1980). Extraembryonic membranes continue to elongate,
extending throughout the gravid and nongravid uterine horns


67
by days 19 and 20. In addition, numbers of binucleated GC
continue to increase, constituting as much as 2CG of the
total trophectodermal cell population on day 20 (v/athes and
Wooding, 1980).
Between days 18 and 20, amnionic formation is completed
and the allantoic diverticulum develops via an evagination
of embryonic hindgut splanchnopleure (Greenstein and Foley,
1958a; Greenstein et al., 1958). Conversely, the highly
vascularized yolk sac reaches its developmental peak by day
20 and begins regressing thereafter as the allantois emerges
(Greenstein et al., 1958). This period of development marks
a coordinated transition from conceptus-derived (chorio-
vitelline placenta) to maternal-derived (chorioallantoic
placenta) nutritional support.
Mutual microvillous interdigitation between
trophoblastic and endometrial epithelium is initiated by day
24 (King et al., 1980, 1981; Wathes and Wooding, 1980) while
definitive attachment occurs by day 27 (King et al.,
1980). Additionally, immature fetal cotyledonary villi are
apparent adjacent to maternal caruncles from approximately
days 27 to 30 of gestation (Greenstein et al., 1958; King et
al., 1981). Considerably more lipid accumulation was noted
in trophectodermal epithelium from day 27 onward (King et
al., 1980) suggesting that functional exchange of nutrients
from maternal to conceptus tissues was accelerated following
definitive attachment. There is a continued increase in the


68
number of bi- and raultinucleated GC reported in
trophectoderm and endometrial epithelial cell layers
throughout this period (Greenstein et al., 1958; King et
al., 1980; Wathes and Wooding, 1980). Multinucleated GG
were reported to comprise 25% of total endometrial
epithelial cell population and 50% of cell area in the
gravid uterine horn on day 24 (Wathes and Wooding, 1980).
Regression of the yolk sac is complete by day 24.
However, allantois expansion and vascularization are not
completed until approximately days 30 to 33 (Greenstein et
al., 1958). Diameter of the allantois ranges from 0.8 to
55 cm on day 24 to over 35 cm on day 33. Dramatic
expansion and vascularization of the allantois is initiated
between days 28 to 30 (Greenstein et al., 1958). Continued
expansion of allantoic fluid volume and membrane diameter
during the first two months of gestation forces
extraembryonic membranes into apposition with the
endometrium, thus facilitating development of firm
cotyledon-caruncle attachment (Eley et al., 1978).
Establishment of a vascularized chorioallantoic placenta
preceeds and is essential for promotion of substantial fetal
weight gains during later gestation (Eley et al., 1978).
Conceptus Signals and Maternal Recognition
of Pregnancy
A functional CL is required if pregnancy in cattle is
to persist. Maintenance of the CL during pregnancy depends
on the presence of a viable conceptus within the uterine


69
luman and its ability to appropriately "signal" a receptive
maternal system, such that processes mediating cyclic
regression of the CL are circumvented. In cattle, maternal
recognition of pregnancy (Short, 1969) must be initiated by
days 16 to 17 post-estrus if pregnancy is to become
established. For example, Betteridge et al. (1980)
demonstrated that synchronous embryo transfer to recipients
on or prior to day 16, but not day 17 post-estrus, resulted
in normal pregnancies. Additionally, nonsurgical embryo
removal from pregnant cattle on days 16 through 19 resulted
in extended CL maintenance and interestrous intervals (range
4 to 8 days) compared to nonmated controls (Northey and
French, 1980; Dalla Porta and Humblot, 1983). Embryo
removal prior to day 16 of pregnancy, viz. days 9, 13, 14,
and 15, had no effect on cycle length or occurrence of
luteolysis. These data provided clear evidence that CL
maintenance required the presence of a viable conceptus
within the uterine lumen of cattle by days 1b to 17 post-
estrus. Further data indicated that twice daily
intrauterine injections of homogenized day 17 or 18
conceptuses (Northey and French, 1980) or freeze-killed day
16 conceptuses (Dalla Porta and Humblot, 1980) on days 14
through 18 or days 15 through 19, respectively, also
extended luteal function. Northey and French (1980) noted
that intrauterine injections of conceptus homogenates did
not stimulate luteal P^ production, but extended the


70
interval to CL regression. Incrauterine administration of
two freeze-killed, day 12 embryos/injection had no effect on
CL lifespan (Dalla Porta and Humblot, 1983). Thus,
potential for bovine conceptus production of potent,
biologically active substances by day 16 of pregnancy was
supported, as was their involvement in prolongation of CL
function.
Subsequent studies in cattle suggested that there are
probably several interdependent strategies by which
conceptus signals may initiate maintenance of the CL during
early pregnancy. Potential effectors, or biologically
active signals, which are synthesized and secreted by bovine
conceptuses (days 13 to 27) include steroids (Shemesh et
al., 1979; Chenault, 1980; Gadsby et al., 1980; Eley et al.,
1983; Chapter 2), prostaglandins (Shemesh et al., 1979;
Lewis et al., 1982; Lewis, 1984; Lewis and Waterman, 1984),
and proteins (Masters et al., 1982; Bartol et al., 1985;
Chapter 4). This subject has been reviewed recently by
Thatcher et al. (1984a, b; 1985) with respect to maternal
recognition of pregnancy in cattle.
In several studies, higher plasma P4 concentrations
were detected in pregnant versus bred-nonpregnant or cyclic
cattle from as early as days 9 to 10 post-estrus (Henricks
et al., 1971; Ford et al., 1979; Lukaszewska and Hansel,
1930), suggesting a luteotropic role of the conceptus during
early pregnancy. However, pregnancy-related elevations in


71
concentrations were not supported by others (.Batson at
al., 1972; Folraan et al., 1973; Hasler et al., 1930).
Progesterone production by dispersed luteal cells was
stimulated by homogenates and aqueous extracts of day 18
bovine conceptuses (Beal et al., 1981). Progesterone
stimulatory activity of conceptus extracts was lost
following heating or removal by dialysis of components less
than a relative molecular weight (Mr) of 12,000 Daltons.
Similarly, the elongating ovine conceptus contains products
which stimulate luteal P^ production, in vitro (Godkin et
al., 1978). The product(s) responsible for this luteotropic
activity in ewes is not proteinaceous since 100 ug of
conceptus protein had no effect on synthesis of P^ or cyclic
AMP by dispersed ovine luteal cells (Ellinwood et al.,
1979). Furthermore, no LH-like activity was detected in
bovine (day 1b to 20; Henricks and Poffenbarger, 1984) or
ovine (day 14 to 15; Ellinwood et al., 1979) conceptuses as
demonstrated under bioassay and RIA conditions,
respectively. Thus the presumptive luteotropin(s) may be a
steroid or prostaglandin. Marsh (1970) demonstrated that
PGE£ stimulated bovine luteal production of P4 in vitro.
However, evaluation of in vivo PGE2 effects on bovine luteal
function and maintenance are not clearly indicated.
Prostaglandin-E2 administered into the uterine lumen alone
(Gimenez and Henricks, 1983; Chenault, 1983) or in
combination with E2 (Reynolds et al., 1983) extends luteal


72
function only slightly beyond cessation of intrauterine PGt^
treatments. No stimulation of luteal P^ production was
noted in vivo. Systemic concentrations declined within
12 h following cessation of PGE£ treatment (Gimenez and
Henricks, 1983) or during the treatment period (Reynolds et
al., 1983) suggesting that PGE£ (plus E2) will not prevent
production and transfer of uterine luteolytic substances,
but affects luteal function directly (Marsh, 1970; Henderson
et al., 1977; Reynolds et al., 1931). Others have reported
no effect of intrauterine PGE2 administration on CL
maintenance in cattle (Dalla Porta and Humblot, 1983;
Chenault et al., 1984). As in cattle, reports in sheep
indicate that luteal function may be prolonged with PGE2 for
only short periods beyond the time of normal luteolysis
(Pratt et al., 1979; Huie et al., 1981; Magnuss et al.,
1981). In sheep, numerous studies have demonstrated that
the CL becomes refractory to exogenous PGF2a during early
pregnancy (Inskeep et al., 1975; Mapletoft et al., 197bb;
Pratt et al., 1977; Silvia and Niswender, 1984) or following
PGE2 administration (Pratt et al., 1977; Henderson et al.,
1977; Reynolds et al., 1981). Furthermore, PGE2 is
synthesized by bovine (Shemesh et al., 1979; Lewis et al.,
1982) and ovine (Hyland et al., 1982; Lacroix and Kann,
1982) conceptuses and endometrium. Luteal protective
substances, originating from the gravid uterine horn in
cattle, were demonstrated by Del Campo et al. (1980). In


73
this study, uterine horns were isolated surgically and
embryos transferred to uterine horns contralateral or
ipsilateral to the CL containing ovary. In three or four
cases, pregnancies in the ipsilateral horn maintained CL
viability, whereas all pregnancies on the contralateral side
failed to maintain CL function. However, when the vein
draining the uterus of the contralateral, pregnant horn was
anastomosed surgically with the ipsilateral, nonpregnant
uterine venous drainage, luteal regression was prevented in
three of three cows. Thus, it was apparent that some blood-
borne product(s) of pregnancy was able to circumvent the
luteolytic effects of the nonpregnant uterine horn. Similar
findings were reported in the ewe (Mapletoft et al.,
1976b). These results supported previous data demonstrating
existence of a unilateral uteroovarian relationship (veno
arterial pathway) during early pregnancy (Del Campo et al.,
1977) and luteolysis (Ginther, 1974; Ginther et al., 1974)
in cattle. Collectively, data generated in cattle and sheep
suggest that conceptus (and/or uterine)-derived luteal
protective substances provide the CL with some resiliency
against PGF2a-induced luteolysis during early pregnancy.
Evidence supports PGE2 as a possible luteal protective
product of conception.
Movement of substances to and from the pregnant uterus
and ipsilateral ovary may be regulated, to some degree, by
blood flow rates and intraovarian vascular hemodynamics


74
(Lamond and Drost, 1974; Ford and Chenault, 1930). During
early pregnancy (days 14 to 18) in cattle, blood flow to the
gravid uterine horn is transiently increased (Ford et al.,
1979; Ford and Chenault, 1980; Ford, 1982). Estrogens and
PGE£ are known to stimulate uterine blood flow in cattle
(Roman-Ponce et al., 1978; Thatcher et al., 1984b; Chapter
3). Elevated luminal concentrations of estrogen and PGE£ in
the gravid uterine horn (Bartol et al., 1981; Ford, 1982;
Lewis et al., 1982) and evidence supporting synthesis of
estrogens (Shemesh et al., 1979; Gadsby et al., 1980; Eley
et al., 1983; Chapter 2) and PGE2 (Lewis et al., 1982) by
bovine conceptuses during early pregnancy support a role for
these conceptus products in pregnancy-associated elevations
in uterine blood flow. Recent reports by Ford (1982) and
Ford and Reynolds (1983) suggested that hydroxylated
metabolites of estrogens viz., catecholestrogens, may
mediate uterine blood flow responses to follicular-(estrus)
and conceptus-derived (early pregnancy) E2. They propose
that an antagonistic interaction of catecholestrogens with
uterine vascular a-adrenergic receptors promote reduced
vascular contractility and facilitate elevated rates of
uterine blood flow. Additionally, increased endometrium
vascular permeability was suggested in pregnant ovine uteri
as evidenced by positive Pontamine Blue dye uptake (Boshier,
1970). Local induction of uterine blood flow and vascular
permeability by the conceptus may increase transfer


efficiency of luteotropic/luteal protective substances from
the pregnant uterus to the CL (Reynolds et al., 1935;
Thatcher et al., 1984b, 1985). However, evaluation of
tritiated PGF2a uptake in bovine endometrium and
uteroovarian vascular components suggested that PGF2a
permeability may be decreased during early pregnancy
(Thatcher et al., 1984b). Tissue samples, viz.,
endometrium, ovarian vein and ovarian artery, were collected
on day 17 of pregnancy or the estrous cycle from the side
ipsilateral to the CL-bearing ovary. Data demonstrated
reduced PGF2a permeability in pregnant versus cyclic tissues
on day 17. Furthermore, ovarian vein permeability to PGF2
was less than ovarian artery in pregnant cows, however, this
relationship was reversed in vascular samples collected on
day 17 of the estrous cycle. In pregnant tissues, responses
suggest that conceptus induced reductions in vessel PGF2a
permeability were greater with regard to vessel proximity to
the pregnant uterus. It may be hypothesized that conceptus
products responsible for reduced PGF2a permeability would be
more concentrated in the uterine venous drainage (ovarian
vein) versus ovarian artery, thus resulting in the responses
observed (Thatcher et al., 1984b). Further studies are
required to determine if this pregnancy-associated reduction
in tissue permeability is specific for PGF2a* For example,
elevated PGF£ a and contents of uterine flushings from
pregnant versus cyclic cattle (Bartol et al., 1981; Lewis et


76
al., 1982) may suggest sequestration within the pregnant
uterine lumen.
Another strategy by which the conceptus maintains CL
function involves attenuation of PGF2a production by the
uterine endometrium. In one of several studies, reviewed by
Thatcher et al. (1984a, b; 1985), in vitro production of
PGF2a by day 17 pregnant endometrial explants was reduced by
more than 50% of PGF2a produced by day 17 cyclic endometrial
explant cultures. Production of PGE2 was similar for
pregnant and cyclic endometrium. Endometrial prostaglandin
synthesis was stimulated following addition of exogenous
arachidonic acid to cultures, however, relative proportion
of prostaglandins (PGF2a> PGFM and PGE2) within and between
pregnancy status were unchanged. These data support an
antiluteolytic effect of the bovine conceptus on endometrial
PGF2C1 production. Levels of arachidonic acid cascade which
appear to be modified during early pregnancy involve phos
pholipase A2 (i.e., exogenous arachidonic acid stimulated
prostaglandin synthesis) and cyclooxygenase (i.e., PGF2ct
production was attenuated in pregnant endometrium with and
without the addition of arachidonic acid). Although PGE2
production did not differ between pregnancy status, it is
clear that the ratio of PGE2:PGF2a must become elevated with
suppression of PGF2a production during early pregnancy.
Such an increase in the PGE2:PGF2a ratio may be of
physiological importance. In vivo evaluation of temporal


77
PGF£a episodes in the ovarian arterial supply of cows from
days 17 to 20 of the estrous cycle or pregnancy (Thatcher et
al., 1984b) supported in vitro results demonstrating a
reduction in PGF2a during pregnancy. No differences ware
observed in frequency of PGF2a episodes between pregnancy
status. However, plasma concentrations of PGF2a during
episodic release were attenuated dramatically in pregnant
cattle.
Peripheral patterns of PGFM concentrations were used as
an index of uterine PGF2a episodic production during the
bovine estrous cycle (Peterson et al., 1975; Kindahl et al.,
1976), early pregnancy (Kindahl et al., 1976), and following
embryo transfer on day 16 post-estrus (Betteridge et al.,
1984). Luteal regression, during the estrous cycle and
following embryonic mortality, as determined by declining
plasma P^ concentrations, was always associated temporally
with three to five large episodes of PGFM. Conversely, PGFM
episodes are reduced or completely eliminated in early
pregnancy. Similar observations were also reported during
the estrous cycle and early pregnancy in buffaloes (Batra
and Pandey, 1983). The 11-ketotetranor metabolites of PGF2a
have been evaluated recently in 24 hour, pooled urine
samples of cyclic and pregnant cattle (Harvey et al., 1984;
Plante et al., 1984). Although detection of acute patterns
of uterine prostaglandin production are not possible using
this system, clear elevations of 11-ketotetranor PGF2a
are


73
temporally associated with a decline in plasma P^
concentrations during luteolysis. Peaks of tne urinary
metaoolite are absent during early pregnancy, however, oasal
concentrations begin a gradual increase as pregnancy
proceeds beyond 13 to 20 days. Similarly, day 17 pregnant
heifers had higher basal concentrations of PGFM than
nonpregnant heifers (Williams et al., 1933). It is probable
that conceptus prostaglandin biosynthesis during early
pregnancy contributes to elevated urinary and plasma
metabolites of PGF2a. Conceptus prostaglandin production
increases wirn gestational age in cattle (Lewis et al.,
1982). It should be noted, however, that elevations in
baseline prostaglandin concentrations during early pregnancy
are not characteristic of luteolytic pulses detected during
CL regression (Thatcher et al., 1984a). Additional support
for conceptus-derived anti-luteolytic effects in cattle was
reported following E2~induction of uterine PGF2a production
on day 18 of the estrous cycle, and day 18 and 20 of
pregnancy. The capacity of the uterus to produce PGF2a
(peripheral plasma PGFM) in pregnant cattle was reduced
significantly from day 18 cyclic cattle responses (Thatcher
et al., 1984b). Reduction in E2~induced PGFM response was
more dynamatic in day 20 versus day 18 pregnant cattle
suggesting that as conceptus elongation proceeds,
progressively larger regions of endometrium become exposed
to conceptus antiluteolytic products, resulting in a graded


79
decline in total uterine capacity to produce PGF2a-
Additionally, duration of endometrial exposure co conceptus
antiluteolytic signals may be important in tnis regard.
Inhibition or reduction of uterine PGF2ct production and
secretory patterns may be the primary mechanism by which the
developing bovine conceptus ensures CL maintenance during
early pregnancy. Luteotropic and luteal protective agents,
permeability and blood flow alterations may act as secondary
or support systems to further reduce, dilute, or otherwise
protect CL from luteolytic effects of the uterus. This
argument is based on the inability of putative luteotropic
and luteal protective signals to maintain CL function for
extended periods in cyclic cattle. In contrast, recent
reports in cattle and sheep provide evidence that
antiluteolytic proteins of conceptus origin significantly
extend CL lifespan and function.
Data evaluating in vitro protein synthesis and
secretion by elongating conceptuses are available for cattle
(Masters et al., 1982; Bartol et al., 1985), sheep (Godkin
et al., 1982b; 1984a) and swine (Godkin et al., 1982a). It
becomes evident from these studies that qualitative and
quantitative changes in array of proteins produced are
related to stage of conceptus development. In all three
species, total conceptus protein production increases with
gestational age (Godkin et al., 1982a, b; Bartol et al.,
1985). This trend is similar to data reported for bovine


80
conceptus steroid (Shemesh et al., 1979; Chanault, 1980;
Gadsby et al., 1980; Eley et al., 1983; Chaptar 2) and
prostaglandin (Lewis et al., 1982) biosynthetic activity.
Additionally, apparent total protein production par rag of
conceptus wet weight, in cattle, becomes elevated as rapid
trophectodermal elongation is initiated between days 1b and
17 of gestation (Knickerbocker et al., 1984; Chapter 4).
During this period of conceptus development, a distinct, but
similar family of low Mr, acidic polypeptides constituted a
major portion of proteins secreted into medium (cattle: Mr
22-26,000, pi 5.6-6.5; Bartol et al., 1985; sheep: Mr 17-
21,000, pi 55; Godkin et al., 1982b; Martal et al., 1984b;
swine: Mr 20-25,000, pi 5.6-6.2; Godkin et al., 1982a).
Transient conceptus production of low Mr polypeptide species
during respective periods of conceptus elongation and
maternal recognition of pregnancy in cattle, sheep and swine
suggest that these products may be involved in early
pregnancy maintenance. Experimental evidence in the cow and
ewe support this hypothesis.
Rowson and Moor (1967) demonstrated that intrauterine
administration of day 14 to 15 ovine conceptus homogenates
prolonged estrous cycles in ewes. However, no effect on
estrous cycle length was observed following intrauterine
administration of day 25 ovine conceptus homogenates or heat
treated, day 12 to 14 ovine conceptuses. Similarly,
introduction of homogenates or extracts of day 14 to 16


31
ovine conceptuses into the uterine lumen resulted in
prolonged CL function and estrous cycles in eight cf 12 ewes
(Mortal et al., 1979)* Only one of six ewes exhioited
prolonged luteal function and interestrous interval when day
21 to 23 conceptus homogenates were administered. Pronase
or heat pretreatment completely eliminate the ability of day
14 to 16 conceptus homogenates to extend CL function in the
cyclic ewe. Subsequent characterization of protein
production patterns by elongating bovine (days 16 to 24;
Bartol et al., 1935), ovine (days 13 to 21; Godkin et al.,
1982b), and porcine (days 10.5 to 12; Godkin et al., 1982a)
conceptuses verified transient biosynthesis and secretion of
major, low Mr polypeptides during early pregnancy.
Collectively, these data imply that biologically active
proteins of conceptus origin are involved in processes
leading to maternal recognition of pregnancy.
Recent data demonstrated that pooled conceptus
secretory proteins (CSP), collected from medium of cultured
bovine (days 16 to 18; Chapter 4) and ovine (days 15 and 16;
Godkin et al., 1984b) conceptuses, substantially extend CL
function and interestrous interval following intrauterine
administration to cyclic cattle (days 15 through 21) and
sheep (days 12 through 21), respectively. In addition,
intrauterine administration of the prominent, low Mr, acidic
polypeptide, termed ovine trophoblast protein-1 (oTP-1;
Godkin et al., 1984b), also extended luteal function in


82
cyclic ewes. Thus, oTP-1 may be a primary protein signal
responsible for luteal maintenance in sheep. Conceptus
protein signals probably act locally at the level of uterine
endometrium. For example, immunohistochemical data suggest
that oTP-1 is secreted by extraembryonic trophectoderm of
the ovine conceptus and binds specifically to receptors
localized on uterine endometrial epithelium (Godkin et al.,
1984a). In the same report, oTP-1 was shown to stimulate
secretion of specific endometrial proteins in culture. In
cattle (Chapters 4 and 5) and sheep (Fincher et al., 1984;
Fincher, 1984), pooled CSP dramatically reduced spontaneous
uterine PGF2a episodes (Chapter 4) and E2~induced production
of uterine PGF2a (peripheral plasma PGFM response; Fincher
et al., 1984; Fincher et al.; Chapter 5). Collectively,
these data demonstrate a role for conceptus-derived
secretory proteins as signals during early pregnancy in
cattle and sheep. It is proposed that proteinaceous
conceptus signals interact with the uterine endometrium such
that endometrial capacity to synthesize and secrete PGF2a is
dampened. At this time, it is not known whether CSP
antiluteolytic effects are exerted directly or mediated by
some induced endometrial product (Godkin et al., 1984a).
Direct antiluteolytic effects of CSP may include regulation
of steroid and/or oxytocin receptor populations on
endometrium. In the rat, both E^ and P^ nuclear receptors
are increased at the site of blastocyst implantation on day


93
5 and of gestation (Logeat et al., 1990). Findley et al.
(1982) reported a decline in caruncular E2 receptors
associated with side of conceptus on day 15 of pregnancy in
ewes. Additionally, caruncular and intercaruncular
endometrial production of protein in pregnant ewes was
greater than nonpregnant endometrium. A reduction in
endometrial E2 receptors may prevent E2~induction of
oxytocin receptors and subsequent activation of PGF2
synthesis (McCracken et al., 1984). Replenishment of
endometrial receptors could also result in decreased
nuclear accumulation of E2 receptor. In this light, Fincher
et al. (1984) demonstrated that oxytocin-induced uterine
PGF2a production in E2 primed ewes was decreased
significantly by intrauterine CSP administration.
Thatcher et al. (1984b) reported that day 17 pregnant
endometrial explants produced less PGF2a than cyclic
endometrium in the presence or absence of exogenous
arachidonic acid. They suggested that both phospholipase A2
and cyclooxygenase activity was reduced during early
pregnancy. Wlodawer et al. (1976) and Shemesh et al. (1981,
1984) reported the presence of prostaglandin inhibitors in
bovine endometrium and placental tissues, respectively. It
is possible that induction of such inhibitors during early
pregnancy may involve conceptus protein signals. In such a
scheme, endometrial PGF2ct biosynthesis may be selectively
inhibited (possibly at the level of phospholipase A2 and/or


84
cyclooxygenase) without influencing prostaglandin production
by the conceptus (Thatcher et al., 1985).
In cattle, low molecular weight conceptus polypeptides,
described by Bartol et al. (1985), may play key roles in
pregnancy maintenance as was demonstrated for oTP-1 in sheep
(Godkin et al., 1984a, b). Similarities in the nature and
function of conceptus protein signals in cattle and sheep
have been discussed. Relative to this discussion, Martal et
al. (1984a) provided evidence that conceptus signals
(presumably protein in nature) of cattle may be biologically
active in sheep and vice-versa. Two trophoblastic vesicles
(0.5 to 2 mm diameter), composed of extraembryonic
trophectoderm and endoderm (embryonic disc removal), from
day 13 bovine blastocysts were transferred to 11 ewes on day
12 of the estrous cycle. Two ewes exhibited prolonged
luteal maintenance until at least day 38 when ewes were
slaughtered. Interestingly, an elongated (>100 mm)
trophoblastic vesicle was recovered from the uterus in one
of these ewes, although conceptus membranes were undergoing
necrosis at this time. Similarly, when two trophoblastic
vesicles from day 11 to 13 ovine blastocysts were
transferred to ten cyclic cattle on day 12, two heifers
exhibited cycle lengths of 31 and 36 days. The authors
suggested that nonspecific conceptus signals in the cow and
ewe were sufficient for maintaining CL function and the


95
biologically active molecules responsible for CL maintenance
in these species may be very similar.
As discussed earlier in this review, conceptus
production of biologically active signals is closely
synchronized with conceptus elongation during maternal
recognition of pregnancy. Furthermore, precise
synchronization between development of the conceptus and
maternal uterine milieu are essential for successful
membrane elongation and signal emission by the conceptus.
Immunological rejection of transferred interspecie conceptus
membranes undoubtedly discourages this process (Allen, 1979;
Beer and Billingham, 1979; Rossant et al., 1982; Wielen and
King, 1984). In this light, observed extension of CL
function in 20% of cattle and sheep receiving interspecific
trophoblastic vesicle transfers (Martal et al., 1984a) seems
even more remarkable. Recent data (S.D. Helmer, W.W.
Thatcher, F.W. Bazer, P.J. Hansen and R.M. Roberts, 1984,
unpublished observations; see Thatcher et al., 1985) lend
additional support that conceptus antiluteolytic protein
signals may be similar in cattle and sheep. Antibodies
developed against oTP-1 (Godkin et al-, 1984a) were used in
an ouchterlony double immuno-diffusion system with
unfractionated bovine conceptus secretory proteins. Two
precipitin bands were formed against unfractionated bovine
coneptus secretory proteins, one of which showed partial
identity with either purified oTP-1 or unfractionated ovine


3
conceptus secretory proteins. Subsequently, an
imraunoprecipitate of tritiated bovine conceptus secretory
protein fraction which crossreacted with oTP-1 antiserum was
solubilized and analyzed by polyacrylamide gel
electrophoresis and fluorography. Immunoprecipitated oovine
conceptus secretory proteins were detected in the 20,000 and
22,000 Mr range. Collectively, these data suggest that
analogous mechanism of "Maternal Recognition of Pregnancy,
possibly involving the low Mr, acidic polypeptide species,
may exist in cattle and sheep.
The conceptus may also exert an indirect antiluteolytic
effect as a result of subtle suppression and acceleration in
folliculogenesis and atresia, respectively (Dufour et al.,
1984). Such a conceptus interaction may be important since
exogenous estrogens have been shown to stimulate uterine
PGFpct production in cattle (Chapter j>) resulting in luteal
regression (Greenstein et al., 1958; Wiltbank, 1966; Eley et
al., 1979). Estrogens may also act directly on the CL to
antagonize LH stimulated P^ production (Williams and Marsh,
1978). Conversely, CL function was extended following
destruction of ovarian follicles during the luteal phase of
the bovine estrous cycle (Villa-Godoy et al., 1981). Thus,
conceptus regulation of follicular development and hence,
estrogen production may complement the intrauterine
antiluteolytic efforts of the conceptus to suppress uterine
PGF2a synthesis during early gestation.


87
In conclusion, events associated with "Maternal
Recognition of Pregnancy" reflect dynamic as well as subtle
alterations in maternal and conceptus physiology. Numerous
putative conceptus signals have been identified, although
their precise roles in "Maternal Recognition of Pregnancy"
are just beginning to be understood. Maintenance of CL
function and an embryotrophic uterine milieu during early
pregnancy is accomplished by several interdependent,
conceptus-mediated strategies. Evaluation and clarification
of the complex interactions between conceptus and its
maternal host presents a considerable and exciting task for
the future.


CHAPTER 2
PATTERNS OF PROGESTERONE METABOLISM BY DAYS 19-23
BOVINE CONCEPTUS AND ENDOMETRIAL EXPLANTS
Introduction
Maternal recognition of pregnancy (Snort, 1909) in
cattle occurs by day 16 (Betteridge et al., 1930; Northey
and French, 1980; Dalla Porta and Humblot, 1983). During
this period the bovine conceptus initiates a phase of rapid
elongation (Hawk et al., 1955; Greenstein and Foley, 1958b;
Betteridge et al., 1980) and elevated endocrine activity
(Shemesh et al., 1979; Chenault, 1980; Lewis et al., 1982;
Eley et al., 1983; Bartol et al., 1985). Conceptus
endocrine products, in turn, direct events which are
essential for the establishment and maintenance of
pregnancy. Biosynthesis and possible mechanisms of action
for conceptus-derived prostaglandin and protein signals have
been discussed earlier (see: Literature Review). Recent
reports demonstrate the capacity for synthesis and
metabolism of various steroids by elongating bovine
conceptuses (Shemesh et al., 1979; Chenault, 1980; Gadsby et
al., 1980; Eley et al., 1983). Based on these studies it is
clear that small amounts of estrogen biosynthesis results
following conceptus incubation with androgen precursors
88


89
(androstenedione, A^; testosterone, T; dehydroepiandros-
terone, DHA). In addition, Shemesh et al (1979) reported
detectable amounts of immunoreactive P^, T and estradiol-176
(E2) in some bovine conceptus extracts on days 15 and 1b of
gestation. Total content of these steroids were elevated in
medium following 48 hour conceptus cultures in the absence
of exogenous precursors. Thus the bovine conceptus may
utilize C21 steroids, viz., and pregnenolone, as
precursors for androgen and estrogen production.
Conversely, Eley et al. (1983) were unable to demonstrate
tritiated-P^ (10 ng) conversion to estrogens when shorter
culture periods (3 to 6 hours) and less sensitive procedures
of identification (liquid column chromatography and
recrystallization) were utilized. Currently, no data are
available with regard to relative ratios of estrone (E-j), E2
and estriol (E^) production by the early bovine conceptus.
Indirect physiological data support conceptus estrogen
biosynthesis during maternal recognition of pregnancy.
Blood flow to the gravid uterine horn is transiently
elevated between days 14 and 18 of gestation in cattle (Ford
et al., 1979; Ford and Chenault, 1981). Uterine blood flow
during the bovine estrous cycle and early pregnancy are
highest during periods of low ?4:E2 ratios in plasma.
Furthermore, exogenous estrogens stimulate uterine blood
flow in cyclic cattle (Roman-Ponce et al.,
1978; Chapter 3).


Full Text
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PREGNANCY RECOGNITION IN CATTLE:
EFFECTS OF CONCEPTUS PRODUCTS ON
UTERINE PROSTAGLANDIN PRODUCTION
BY
JEFFREY JOHN KNICKERBOCKER
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
1985

ACKNOWLEDGMENTS
During the last several years, I have had the good
fortune of interacting with a host of generous and talented
individuals. The many contributions, insights and
friendships offered me by these individuals are acknowledged
greatfully.
I wish to extend my heartfelt thanks and appreciation
to Dr. William W. Thatcher, chairman of my supervisory
committee, for giving generously of his time, experience and
knowledge throughout my Ph.D. training. Dr. Thatcher's
insight and contributions with regard to my education and
research have made lasting impressions on my perception of
reproductive function and philosophy of basic reproductive
research. His friendship and interest in my professional
growth and personal well-being are deeply appreciated. Dr.
Thatcher will always be held in my highest esteem as a
scientist, teacher and friend.
Special thanks are given to Dr. Fuller W. Bazer,
cochairman, for his constant interest, availability and
input during the course of my research endeavors. His
substantial contributions to my research program and
understanding of reproductive physiology have been
invaluable. Dr. Bazer's friendship throughout the years and
ii

"support" during the final stages of this degree will always
be appreciated greatly.
Thanks are expressed to Dr. Michael J. Fields for our
numerous stimulating conversations, and for his guidance and
support during early aspects of ray research in his
laboratory. My association and friendship with Dr. Fields
over the years have been a pleasure. His contributions to
my research endeavors and education are sincerely
acknowledged.
Dr. R. Michael Roberts is acknowledged for his
important role in the development of technical and
conceptual insight relevant to conceptus protein
biochemistry and physiology. In this respect, Dr. Roberts
has been a tremendous asset in my research endeavors
relative to conceptus secretory protein function in
cattle. I thank Dr. Roberts for his interest and input
while serving on my supervisory committee.
Dr. Donald Catón is acknowledged greatfully for his
input, expert surgical training and assistance in numerous
surgeries at the 34th Street laboratory during our
characterization of uterine responses to estradiol. I am
indebted to Dr. Catón for his contributions while serving on
my supervisory committee and for his friendship.
Thanks are extended to Dr. Robert J. Collier for his
comments and critical evaluation of my dissertation. His
friendship and willingness to participate in my dissertation
iii

defense in the absence ox Dr
. Thatcher are appreciated
greatly.
I wish to extend my sincere appreciation and thanks to
Dr. Donald H. Barron and Dr. Maarten Drost for their
gracious and generous support, and valuable interactions and
insight throughout the course of my studies at the
University of Florida. It has been tremendously gratifying
for me to have worked with these gentlemen. Thank you.
Special thanks are given to fellow graduate students,
Dallas Foster, Dr. Louis Guilbault, Dr. Frank 'Skip' 3artol
and Kimberly Fincher for their valued friendships,
interactions and inestimable assistance over the years.
Friendships and support offered me by Dr. Charlie Wilcox,
Dr. Herb
Head, Dr.
Dave Beede, Dr
. Dan Sharp,
III,
Dr. Ray
Roberts,
Dr. David
Wolfenson, Dr.
Lee Fleeger
, Dr.
Wilfrid
Dubois, Dr. Rod Geisert, Dr. Randy Renegar, Dr. Jeff
Moffatt, Dr. Charlie Ducsay, Dr. Juaquim Moya, Harold
Fischer, John McDermott, Dr. Helton Saturino, Karen
McDowell, Marlin Dehoff, Lokenga Badinga, Deanne Morse, Fran
Romero, Sue Chiachimonsour, Jeff Valet, Kathy Hart, Dr.
George Baumbach and Leslie Smith are deeply appreciated.
Thank you.
I am greatful especially to Dr. Raul Schneider and son,
Jordan, for their generosity, patience, hospitality and
encouragement during these last 3 months.
iv

I thank Carol Underwood, Candy Stoner, Jesse Johnson
and Warren ClarK for their tolerance, technical assistance
and efficiency in the laboratory, and Cindy Zimmernan for
typing this manuscript so skillfully.
To special friends, Chuck and June Wallace, and Gail
and Bob Knight, most sincere thanks and love are extended
from my family and I. Keep in touch.
I wish to thank my parents, John and Jan, for their
continued love, support and confidence. This proud day
would not have been possible without the principles and
motivation instilled in me by them both.
Lastly, the major contributions and sacrifices made by
my wife, Holly, throughout the course of my studies are more
than greatfully acknowledged. Her constant love, support,
patience and understanding have given me the strength to
endure and reasons to succeed. My deepest love and
affections are hers always.
v

TABLE OF CONTENTS
P 3. 29
ACKNOWLEDGMENTS ii
A3STRACT viii
CHAPTERS
1 REVIEW OF LITERATURE 1
Introduction 1
The Bovine Estrous Cycle 2
The Corpus Luteum Life Cycle 17
Uterine Control of CL Function 49
Conceptus-Associated Events During Early
Pregnancy 59
2 PATTERNS OF PROGESTERONE METABOLISM BY
DAYS 19-23 BOVINE CONCEPTUS AND ENDOMETRIAL
EXPLANTS 88
Introduction 88
Materials and Methods 90
Results 102
Discussion 129
3 UTERINE PROSTAGLANDIN AND BLOOD FLOW
RESPONSES TO ESTRADIOL-170 IN CYCLIC CATTLE...139
Introduction 139
Materials and Methods 141
Results 146
Discussion 159
4 PROTEINS SECRETED BY DAY 16 TO 18 BOVINE
CONCEPTUS EXTEND CORPUS LUTEUM FUNCTION IN
CATTLE 166
Introduction 166
Materials and Methods 168
Results 176
Discussion 185
vi

5 INHIBITION OF ESTRADIOL-173 INDUCED UTERINE
PR0STAGLANDIN-F2a PRODUCTION BY BOVINE
CONCEPTUS SECRETORY PROTEINS 193
Introduction 193
Materials and Methods 195
Results 201
Discussion 209
6 GENERAL DISCUSSION 215
APPENDICES
A MANUFACTURE OF SEPHADEX LH20 COLUMNS 221
B STEROID ELUTION BY GAS/LIQUID
CHROMATOGRAPHY 222
C MASS CALCULATIONS FOR ESTROGENS 224
D ELUTION OF [3H]-CONCEPTUS METABOLITES AND
[14C]-MARKERS ON HPLC (ACETONITRILE:WATER,
54:46) 226
E ELUTION OF RADIOINERT STEROID STANDARDS
ON HPLC (ACETONITRILE : WATER, 54:46) 228
REFERENCES 229
BIOGRAPHICAL SKETCH 272
vii

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
PREGNANCY RECOGNITION IN CATTLE:
EFFECTS OF CONCEPTUS PRODUCTS ON
UTERINE PROSTAGLANDIN PRODUCTION
BY
JEFFREY JOHN KNICKERBOCKER
May, 1985
Chairman: William W. Thatcher
Cochairraan: Fuller W. Bazer
Major Department: Animal Science
In cattle, continued progesterone (P4) production by
the corpus luteum (CL) is required if pregnancy is to per¬
sist. Identification of putative conceptus-derived signals
and evaluation of their biological roles relative to CL
maintenance during early pregnancy were goals of this
research endeavor.
Using various chromatography systems, bovine con-
ceptuses (days 19 to 23) exhibited extensive metabolism of
tritiated P4 (90-98%), in vitro. A majority of conceptus
metabolites were 53-reduced pregnanes. A major conceptus
metabolite was 53-pregnan-3a-ol-20-one (5B-P). Conversely,
endometrial explant cultures metabolized 40 to 50% of P4
substrate to primarily 5a-reduced steroid products.
viii

An in vivo test system to evaluate uterine PGF2a pro¬
duction capacity was characterized in experiment two. Exo¬
genous estradiol-17B (5 3 mg I.V.) stimulated uterine
blood flow, and PGF£a production and metabolism. Concen¬
trations of the primary metabolite of PGF2a> 15-keto-13,14-
dihydro-PGF2a (PGFM) were significantly correlated (r=.66)
with the E2~induced uterine PGF2a production response. Use
of peripheral PGFM concentrations as an index of uterine
PGF2a production was supported.
In experiment three, CL function, interestrous inter¬
val, and spontaneous uterine PGF2» production were evaluated
in cyclic cows following intrauterine administration of
56-P, conceptus secretory proteins (CSP) or homologous serum
proteins (Control). Extensions in CL lifespan and
interestrous interval were detected in cows administered CSP
compared to 5&-P and Control group responses. Lifespan of
CL and estrous cycle lengths were not different (P>.25)
between 5B-P and Control groups. Spontaneous PGF2a episodes
were depressed in CSP-treated cows but not in cows admini¬
stered 5B-P or serum proteins. In experiment four, E2-
induced uterine PGF2a production (peripheral PGFM response)
was evaluated in cyclic cattle following interuterine
administration of bovine CSP or day 18 pregnant serum
proteins (Control). Mean concentrations of PGFM in CSP-
treated cows were depressed (PC.01) compared to Control
responses. Estradiol injection failed to elicit any PGFM
ix

response in three of five CSP-treated cows. In contrast,
all Control animals exhibited PGFM responses during the
period of E2~induced PGF2a production. Results support a
role for CSP in suppression of uterine PGF2a production
during early pregnancy in cattle.
x

CHAPTER 1
REVIEW OF LITERATURE
Introduction
The recurrent nature of estrous cycles in cattle
centers around development and regression of the ovarian
corpus luteum (CL). Progesterone (P4)* the primary product
of CL, directs uterine development and secretory activity
such that an embryotropic uterine environment is established
during each estrous cycle. Luteal maintenance and P4
production are essential if successful pregnancy is to be
established. The process of CL maintenance during early
pregnancy involves numerous and complex biochemical inter¬
actions between the conceptus and its maternal host. Our
evaluation of mechanisms by which the conceptus and maternal
units interact during early pregnancy depend upon identifi¬
cation of putative conceptus-derived 'signals' and develop¬
ment of biological test systems through which conceptus
product function may be assessed. It was in this light that
research described herein was conducted.
1

The Bovine Estrous Cycle
General Considerations
The nature of the estrous cycle has been characterized
extensively in cattle. Early observations and documentation
of the recurrence of sexual behavior in animals provided the
first clue as to the cyclical nature of reproductive
processes in the female (Heape, 1900; Marshall, 1922;
Hammond, 1927; Asdell, 1969). Wallace in 1876 and
Ellenberger in 1892 (Hammond, 1927; as cited in Marshall,
1922) were among the earliest to report accurately the
interval between "sexual periods" in domestic female cattle.
The cyclical recurrence of estrous behavior follows a
periodicity of approximately 21 days with a range of 17 to
25 days considered normal (Hammond, 1922; Asdell et al.,
1949; Olds and Seath, 1951; Cupps et al., 1969).
Nulliparous heifers tend to exhibit slightly shorter
(approximately 1 day) interestrous intervals than
multiparous cows (Hammond, 1927; Olds and Seath, 1951).
Unlike sheep (Goodman and Karsch, 1981) and horses
(Sharp, 1980), photoperiod does not influence consistently
annual reproductive patterns in mature cattle (Rzepkowski et
al., 1982). Thus, reproductive activities in domestic
cattle are not restricted to specific seasons of the year
(Heape, 1900; Hammond, 1927). However, attainment of

3
puberty in cattle is enhanced by increased daily exposure to
light (Hansen et al., 1933).
Loss of body condition due to decreased nutrient
availability, lactational stress and parasitism has
detrimental effects on cyclicity and reproductive efficiency
in cattle (Lamond and Bindon, 1969; Dunn et al., 1969; Rakha
and Igboeli, 1971). Thus, wild cattle are known to exhibit
limited breeding and calving seasons under natural
conditions (Marshall, 1922; Heape, 1900). When such
stresses are reduced via management schemes in the confines
of a zoo, wild cattle are capable of breeding at all times
of the year (Heape, 1900).
High ambient temperature and humidity are other
important environmental influences which reduce expression
of estrus, blood flow to the reproductive tract, and may
alter the normal endocrine milieu in cattle (Thatcher and
Collier, 1985). Consequently, heat stress results in
decreased reproductive efficiency.
Historically, observations regarding cyclical
alterations in behavior and ovarian morphology led Walter
Heape (1900) to propose terminology which established the
first classification of these cyclical changes into four
recurrent phases. Thus, the estrous cycle ("diestrus cycle"
by Heape's terminology) in cattle consists of four phases:
proestrus (day 19 to estrus), estrus (day 0), metestrus (day
1-3) and diestrus (day 4-18).

4
Physiology and Endocrinology
Sexual receptivity of the female, or estrus, is a
convenient behavioral landmark which is commonly employed to
mark the start of an estrous cycle (day 0). Estrus is often
characterized by increased restlessness (Kiddy, 1976),
vocalizations, mounting activity and, most notably, standing
to be mounted by other cattle (Howes et al., 1960). Other
variables related to estrus are discussed by Lewis and
Newman (1984). Average length of behavioral estrus
approaches 18 to 20 hours in mature cattle (Trimberger and
Hansel, 1955; Schams et al., 1977); however, cattle in
subtropical regions generally experience shorter (10 to 13
hour) phases of estrus (Branton et al., 1957; Chenault et
al., 1975).
The temporal sequence of events which culminate in
behavioral estrus, and later in the preovulatory gonado¬
tropin surge, involve a synchronized and interdependent
cascade of physiological changes in the central nervous
system, hypothalamus, pituitary, ovaries and uterus. Char¬
acterization of endocrine profiles and study of mechanisms
controlling their changes have, at least partially,
unravelled the dynamics of various reproductive processes.
Approximately 2 to 4 days before behavioral estrus and
the preovulatory surge of gonadotropins, regression of the
corpus luteum (CL) is initiated, as determined by a
precipitous decline in plasma progesterone (P4)

• 9
1 973; Peterson
• )
concentrations (Chenault et al., 1975; Peterson et al
1975). In association with this decrease in P_¡_, pulse
frequencies of LH (Rahe et al., 1980; Schallenberger et al.,
1984) and FSH (Schallenberger et al., 1984) increase, as do
basal concentrations of LH, but not FSH (Chenault et al.,
1975; Roche and Ireland, 1981; Milvae and Hansel, 19S3b;
Schallenberger et al., 1984).
Plasma concentrations of estradiol-17S (E2) begin to
rise during this period (Chenault et al., 1975; Walters and
Schallenberger, 1984; Schallenberger et al., 1984) as a
result of the initiation and accelerated growth of the
preovulatory follicle (Dufour et al., 1972; Merz et al.,
1981; Staigmiller et al., 1982; Ireland and Roche, 1982;
McNatty et al., 1984a,b). In fact, approximately ten times
more E2 was measured in ovarian vein plasma draining the
ovary containing the preovulatory follicle chan the contra¬
lateral ovary at estrus in cattle (McNatty et al., 1984a).
A significant correlation exists between LH and E2
pulse frequencies during all phases of the bovine estrous
cycle (Schallenberger et al., 1984; Walters and
Schallenberger, 1984; Walters et al., 1984). Prior to the
gonadotropin surge, LH pulsatile episodes occur every 30
minutes (high frequency) with E2 pulses being generated
responsively (Walters and Schallenberger, 1984). Addi¬
tionally, E2 pulse amplitudes gradually increase as estrus
approaches. However, these alterations in E2 pulse

6
amplitude are not reflected by similar amplitude elevations
in LH episodes (Walters and Schallenberger, 1984). Inis may
suggest that LH pulse frequency and follicle sensitivity to
LH are major regulatory factors for follicular E2 production
in the cow.
Before reviewing data in support of such a claim, a
brief description of follicular anatomy and the two-cell
mechanism of follicular estrogen biosynthesis is in order.
The ovarian follicle consists of two steroidogenica1ly
active cellular compartments: the vascularized theca
interna and the avascular granulosa, which is separated from
the theca interna by an acellular basal laminae (see:
McNatty et al., 1984b, for references). According to the
two-cell mechanism of follicular estrogen production
proposed by Falck (1959), the theca compartment metabolizes
C-21 steroids to androgens which are then utilized by the
granulosa compartment to synthesize estrogens. More recent
evaluation of steroidogenic pathways utilized by bovine
follicular compartments (Lacroix et al., 1974) support this
concept. The theca cell layer utilizes, almost exclusively,
the A^-pathway (pregnenolone and 17ct-hydroxy-pregnenolone)
to synthesize androgens, of which androstanedione (A^) is
the major product. In vivo evaluation of A^ concentrations
in ovarian vein plasma and A^ production by the ovary
throughout the estrous cycle in cattle (Wise et al., 1982)
agree with in vitro appraisals of thecal androgen

7
biosynthesis by Lacroix and coworkers (1974). Metabolism of
A4 to estrogens by the granulosa is very efficient.
Conversely, only small amounts of estrogen are synthesizeu
by the theca compartment. Lacroix and coworkers (1974)
observed greater yields of estrogen with combined
incubations of theca and granulosa cells than yields with
incubations of either cell type separately, indicative of a
synergism between the two follicle compartments (Falck,
1959).
The capacity of follicles to synthesize estrogens
depends upon their ability to respond to gonadotropins. In
the cow, 85% of large (_>_ 8 mm diameter) follicles on both
ovaries bound FSH to granulosa cells and hCG (LH) to theca
interna cells, while only 38% of these follicles also bound
hCG to granulosa. Follicles with granulosa hCG binding
sites were associated with enhanced E2 concentrations in
follicular fluid (England et al., 1981; Merz et ai.,
1981). Sheep also exhibit a high positive correlation
between hCG binding to granulosa and follicular E2 content
and production (Webb and England, 1979, 1982). McNatty et
al. (1984a) suggested that there was at least one follicle
greater than 5 mm (range 1 to 3 follicles per cow) with
granulosa cells possessing aromatase activity on day -5
through estrus. However, most, if not all, healthy bovine
follicles greater than 2 mm and many atretic follicles were
capable of secreting A4 in response to LH. In no cases were

8
follicles with granulosa aromatase activity found in the
absence of an LH-responsive thecal compartment. Likewise,
Bartol et al. (1931) demonstrated that large follicles
capable of significant E£ production were present in ovaries
of cattle throughout the estrous cycle.
In the rat (Nimrod et al., 1977), pig (Channing, 1975)
and sheep (Weiss et al., 1978) ovarian follicle, granulosa
binding sites for hCG/LH are induced by FSH, thereby
enhancing granulosa cell responsiveness- to LH.
Additionally, P^, dihydroxytestosterone (DHT) and E£ plus
FSH synergistically stimulated granulosa cell responsiveness
to LH above levels found with FSH alone (Rani et al.,
1931). Low concentrations of E2 enhance production by
isolated bovine theca cells (Fortune and Hansel, 1979),
suggesting that E£ may also regulate theca sensitivity to
LH.
Thus, follicular development of granulosa aromatase
activity appears to occur secondarily to the thecal capacity
to synthesize androgens. Androgen biosynthesis by the theca
interna compartment is enhanced by exposure to LH, while FSH
may be important in sensitization of granulosa cells to
LH. Steroids, such as P^, DHT and E£ may also potentiate
this FSH effect.
One apparent consequence of granulosa-binding of LH is
activation of the aromatase enzyme complex. Mechanisms
involved in the selection of follicles which become

9
LH-responsive probably depend upon factors other than FSH
alone, perhaps intraovarian factors (Alexander et al., 1973;
Darga and Reichert, 1978; Scnomberg, 1979; Hsueh et al.,
1933; Hansel and Convey, 1983), since granulosa cells in a
majority of bovine follicles appear to bind FSH, yet in only
one to three large follicles do granulosa cells develop the
ability to bind LH and acquire aromatase activity (Merz et
al., 1981; Bartol et al., 1981; McNatty et al., 1984).
As mentioned, steroidogenic capacity of ovarian folli¬
cular compartments depend upon their ability to respond to
gonadotropins. Additionally, periodicity and magnitude of
gonadotropin release from the anterior pituitary play
important roles in ovarian function. Gonadotropin patterns
are, in turn, regulated by gonadal steroids via long loop
feedback mechanisms. In ovariectomized sheep (Goodman and
Karsch, 1980), frequency of tonic LH episodes is decreased
by with no apparent effect on pulse amplitude, while £3
reduces LH pulse amplitude and does not influence frequency
of LH release. Similar observations were made by Rahe et
al. (1980) following characterization of LH episodes during
mid-luteal and follicular phases of the estrous cycle in
intact cows. High amplitude, low frequency LH episodes
which occur during P^-dominated phases of the cycle are
thought to result from P^ negative feedback on the central
nervous system's (CNS) oscillator regulating episodic
secretion of gonadotropin releasing hormone (GnRH) (Knobil,

10
1980) and pituitary sensitivity to GnRH (Padmanabnan et al.,
1982). During estrogen-dominated phases, prior to she
preovulatory surge of gonadotropins, initially decreases
pituitary sensitivity to GnRH resulting in less LH releasaa
per GnRH pulse (Kesner et al., 1981; Kesner and Convey,
1982). However, by approximately 8 hours before the
gonadotropin surge, when highest preovulatory plasma
concentrations of occur (Walters and Schallenberger,
1984), pituitary sensitivity to GnRH reaches its maximum
(Kesner et al., 1981; Kesner and Convey, 1982; Padmanabhan
et al., 1982). Yet during this period, amplitude (but not
frequency) of gonadotropin episodes remains low (Walters and
Schallenberger, 1984). Walters and Schallenberger (1984)
suggested that a negative feedback effect of the elevated E£
on the hypothalamus may explain these endocrine patterns
during the preovulatory period.
Regulation of FSH secretion by gonadal steroids appears
to be more subtle than observed for LH. Concentrations of
FSH are considerably higher than LH during all phases of tne
estrous cycle in cattle (Schallenberger et al., 1984;
Walters and Schallenberger, 1984; Walters et al., 1984).
Interpulse frequencies for FSH range from approximately 25
minutes during the preovulatory phase to 30-50 minutes
during the mid-luteal phase of the estrous cycle (Walters
and Schallenberger, 1984; Walters et al., 1984) with pulse
amplitudes declining during the preovulatory period. Thus,

11
P4 appears to exert only slight reductions in F3H pulse
frequency in cattle. Evidence suggests that E¿ exerts an
inhibitory effect on FSH release via the pituitary just
prior to the preovulatory gonadotropin surge (Kesner and
Convey, 1982; Walters and Schallenberger, 1984).
Additionally, exogenous E£ administered to ovariectomized
heifers reduced FSH concentrations to precastration levels
(Kesner and Convey, 1982). Follicular production of inhibin
may also regulate plasma FSH concentrations during periods
of follicular growth (De Jong, 1979; De Paolo et al., 1979;
Henderson and Franchimont, 1982; Padmanabhan et al., 1984),
possibly through decreasing pituitary release of FSH.
Low basal concentrations of P^ and peak follicular
production of E^ potentiate the onset of behavioral estrus
and trigger the preovulatory surge of LH and FSH from the
anterior pituitary (Scharas et al., 1977). Although estrous
behavior in cattle may be induced by estrogen treatment
alone (Melarapy et al., 1957), a period of P^ pre-exposure
enhances the sensitivity of brain centers responsible for
estrous behavior to E^ (Melampy et al., 1958). Therefore,
P4 priming, during the luteal phase of the cycle, may oe an
important aspect of estrous expression (see also: McEwen et
al., 1982; Pfaff and McEwen, 1983)- During physiological
states in which P^ priming is absent, as occurs in heifers
approaching puberty (Gonzalez-Padilla et al., 1975; Schams
et al., 1981) or in cows following postpartum anestrous

12
(Schams at al., 1978; Peters, 1934), behavioral estrus does
not accompany the first gonadotropin surge and ovulation.
Additionally, duration and magnitude of P4 exposure may
influence subsequent luteal function by modulating preovula¬
tory follicle development directly as was suggested Oy the
data of Snook et al. (1969) in heifers treated with bovine
LH antisera and McLeod and Haresign (1984) in the seasonally
anestrous ewe. Similarly, ovulations occurring witnout the
benefit of P4 priming in pubertal and postpartum cattle
often result in short-lived, less functional CL (Gonzalez-
Padilla et al., 1975; Schams et al., 1978; 1981; Peters,
1984).
Elevated P4 concentrations completely eliminate
induction of estrous behavior and the gonadotropin surge
(Hobson and Hansel, 1972; Short et al., 1979; Roche and
Ireland, 1981; Ireland and Roche, 1982; Padmanabhan et al.,
1982). With demise of the CL during the preovulatory
period, plasma P4 concentrations fall to below 1 ng/ml thus
removing the negative feedback on GnRH and gonadotropin
release rates. Follicular production of E£ provides
increasingly higher plasma E2 concentrations, the magnitude
of which is proportional to the size of the preovulatory
gonadotropin surge (Walters and Schallenberger, 1984).
Approximately 1 hour prior to the preovulatory surge of
gonadotropins, Walters and Schallenberger (1984) observed a
consistent decline in E^ concentrations and suggested that

13
this event removed E? negative feedback on the hypothalamus
and increased GnRH pulse amplitude which triggered one
gonadotropin surge from the maximally responsive pituitary
(Convey, 1973; Zolman et al., 1973; Kesner et al., 1931;
Kesner and Convey, 1982). The surge of LH and F3H occur
concomitantly near the onset of an 13 to 20 hour behavioral
estrus (Trimberger and Hansel, 1955; Schams et al., 1977)
and lasts 8 to 10 hours (Rahe et al., 1980; Kesner et al.,
1981; Kesner and Convey, 1982; Walters and Schallenberger,
1984). The gonadotropin surge is terminated due to the
refractoriness of the pituitary to GnRH (Kesner et al.,
1981; Kesner and Convey, 1982).
During and immediately following the preovulatory
gonadotropin surge, follicular (granulosa cell) production
of E£ and circulating levels of E£ decline rapidly as
follicular luteinization occurs (Chenault et al., 1975;
Ireland and Roche, 1982). Plasma concentrations of E£, P4
and LH remain low throughout a majority of the raetestrous
phase. Pulses of LH are absent for 6 to 12 hours post¬
gonadotropin surge. Similarly, FSH pulse amplitudes are
low; however, pulse frequencies remain at a rate comparable
to that observed during the preovulatory gonadotropin surge
(Walters and Schallenberger, 1984). Prior to ovulation and
4 to 12 hours following the gonadotropin surge, FSH
concentrations become elevated as a result of an increase in
FSH pulse amplitude (Kazmer et al., 1981; Hansel and Convey,

14
1983; Walters and Schallenberger, 1934). The second rise in
FSH is of lower magnitude than the FSH surge and may be a
result of reduced follicular inhibin production during the
ovulatory process (De Jong, 1979; De Paolo et al., 1979;
Henderson and Franchimont, 1981; Padmanabhan et al.,
1984). The function of this secondary rise in FSH is
currently unknown, however, FSH may play a role in oocyte
maturation (Plachot and Mandelbaum, 1978) or in the
recruitment of preantral follicles (Hansel and Convey,
1983).
Ovulation occurs on day 1 of the estrous cycle,
approximately 24 to 30 hours after the preovulatory surge of
LH and FSH (Chenault et al., 1975; Scharas et al., 1977;
Hansel and Convey, 1983). Following ovulation, development
of CL function is reflected by the gradual increase in CL
weight (Shemesh et al., 1976; Bartol et al., 1981) and P4
content (Shemesh et al., 1976), plasma P4 concentrations
(Chenault et al., 1975; Peterson et al., 1976; Schams et
al., 1977; Wise et al., 1981; Hansel and Convey, 1933) and
P4 production (Wise et al., 1981), and ovarian blood flow
(Ford and Chenault, 1981; Wise et al., 1981). Follicular
growth and atresia occur continuously throughout the estrous
cycle (Rajakoski, 1960; Marion et al., 1968; Ireland and
Roche, 1983) with moderate rises in plasma E2 concentrations
occurring responsively (Hansel et al., 1973; Hansel and
Convey, 1983; Ireland and Roche, 1983).

15
During the early luteal phase, both LH and FSH secre¬
tion patterns may be characterized as high frequency-low
amplitude (Rahe et al., 1980; Walters et al.f 1984). The
majority of LH and FSH pulses occur simultaneously during
this period. Similarly, pulsatile episodes of £2 anci are
high frequency. High amplitude pulses of E2 follow LH
pulses in 90 to 96$ of the cases observed, while occurrence
of low amplitude episodes were more similar to FSH
pulsatile patterns (Walters et al., 1984). These
gonadotropin-steroid pulsatile relationships become more
obvious during the mid-luteal phase. By day 10 to 12 of the
estrous cycle, frequency of LH episodes are significantly
decreased (Rahe et al., 1980; Walters et al., 1984). Rahe
and coworkers (1980) also described increased LH pulse
amplitude during the mid-luteal phase, however, elevations
in LH pulse amplitude were not evident in the data set of
Walters et al. (1984). Pulse amplitude of FSH was similar
during the early and mid-luteal phases, although FSH pulse
frequency decreased during the mid-luteal phase.
Nevertheless, 41 $ more FSH than LH episodes were observed
during the mid-luteal phase. Low amplitude E2 episodes were
consistently preceded by pulses of LH and 92 to 100$ of
concurrent FSH and LH pulses and 97$ of separate FSH pulses
were followed by high amplitude episodes of P^. In no case
were separate FSH pulses associated with episodes of E2
(Walters et al., 1984).

16
The dynamics of endocrine changes during the luteal
phase support previously described roles for FSH ana LH in
follicular steroid production. Additionally, it would
appear that FSH, in conjunction with LH, regulates
secretion patterns. The major site of P^ production is the
CL (Shemesh et al., 1976). The bovine CL possesses both FSH
(Mann and Niswender, 1983) and LH (Rao et al., 1979, 1983)
receptors and responds to these gonadotropins, in vitro,
with elevated P^ production (Romanoff, 1966; Hixon and
Hansel, 1979; Milvae et al., 1983).
In cyclic cattle, lifespan of the CL is regulated, to a
large extent, by interactions between the ovary and
uterus. During late diestrus-early proestrus phases of the
cycle, estrogens produced by the developing, large antral
follicle(s) are thought to initiate the process of luteal
regression. Exogenous estrogens are known to shorten the
lifespan of CL in cattle (Greenstein et al., 1958; tfiltbank,
1966; Eley et al., 1979). Luteolytic activity of estrogen
is thought to be mediated through stimulation of uterine
prostaglandin (PG)-F2a synthesis and release (Thatcher et
al., 1984b). The luteolytic activity of exogenous PGFpa in
cattle has been documented by several groups (Hansel et al.,
1973; Hafs et al., 1974; Lauderdale, 1974; Thatcher and
Chenault, 1976). Furthermore, episodic pulses of uterine
venous PGF2a (Nancarrow et al., 1973), and peripheral
measurements of its primary metabolite,

17
15-keto-13,14-dihydro-PGF2a (PGFM; Granstrom and Kindahl,
ly82), are always associated with spontaneous luteal
regression in cattle (Peterson et al., 1973; Kindahl et ai.,
1976; Betteridge et al., 1984). Uterine PGF2a production
requires a period of P^-priming which increases the tissue's
potential to synthesize PGF2a while suppressing copious
secretion of the luteolysin (Hansel et al., 1973; Horton and
Poyser, 197b; Rothchild, 1981).
An indepth discussion of CL characteristics, function
and mechanisms of regression will be reserved for a
subsequent section of this chapter.
The Corpus Luteum Life Cycle
Origin and Development of Corpus Luteum Cells
Cells which make up the corpus luteum (CL) are derived
from the preovulatory follicle. Two distinct luteal cell
types have been described in CL of cattle (Donaldson and
Hansel, 1985b; Ursely and Leymarie, 1979a,b; Koos and
Hansel, 1981; Weber et al., 1984; Alila and Hansel, 1984),
sheep (O'Shea et al., 1979» 1980; Fitz et al., 1982; Rogers
et al., 1983, 1984), swine (Lemon and Loir, 1977; Lemon and
Mauleon, 1982), rat (Kenny and Robinson, 1982), rabbit (Yuh
et al., 1982) and human (Gaukroger et al., 1979). These
cell types are described generally as small and large luteal
cells. Current dogma suggests that small luteal cells are

13
derived from the theca interna and large luteal cells from
the granulosa of the preovulatory follicle.
In the ewe, large luteal cells of the mature CL are
approximately six-times larger than small luteal cells and
occupy 25% or more of the CL volume (Niswender et al., 1976;
Rodgers et al., 1984) as compared to approximately 18% by
small luteal cells (Rodgers et al., 1984). Small luteal
cells outnumber large luteal cells by nearly five to one.
By far, the most numerous cell type within the mature ovine
CL is endothelium (Rodgers et al., 1984), which attests to
the high vascular density of this tissue.
Cellular mitotic activity in the CL is thought
generally to be at a minimum (Donaldson and Hansel, 1965;
Rodgers et al., 1984). Histological examination and DNA
determinations of the bovine preovulatory follicle (6 hours
following the onset of estrus) and CL throughout the estrous
cycle (Donaldson and Hansel, 1965b) indicated that
granulosa-luteal cell mitotic activity was reduced following
the preovulatory surge of LH and ceased altogether by
approximately day 4. The theca-derived luteal cells,
however, mi tosed freely until approximately day 7.
Neovascularization continues in CL until a maximum metabolic
potential is reached around days 10 to 12. In vivo exposure
of developing bovine CL to hCG or LH (Donaldson et al.,
1965a) stimulated mitotic activity in small luteal cells,
vascular endothelium and stromal cells, whereas mitotic

19
figures were rarely observed in large luteal cells
(Donaldson and Hansel, 1965). Thus, during CL development,
LH appears to limit granulosa cell division and enhance
theca-luteal cell mitosis (Donaldson and Hansel, 1965;
Friedrich et al., 1975; McNatty and Sawers, 1975).
In addition, estimates of granulosa cell numbers in
mature preovulatory follicles compare favorably with numbers
of granulosa-luteal cells in the CL (McNatty, 1979; McNatty
et al., 1984a; Rogers et al., 1984), suggesting that large
luteal cells are derived solely from follicular granulosa
cells. Furthermore, increases in follicular size are
correlated positively with follicular cell numbers (McNatty,
1979; McNatty et al., 1982; Ireland and Roche, 1983). Thus,
it may be assumed logically that events which regulate
viability, proliferation and biosynthetic potential of
preovulatory follicular cells markedly influence the
functionality of the CL subsequently formed post-ovulation
(see: McNatty, 1979; Richards, 1979, 1980; di Zerega and
Hodgen, 1981; Murdoch et al., 1983).
Observations in the cow (Donaldson and Hansel, 1965;
Alila and Hansel, 1984) and ewe (Fitz et al., 1981) support
the hypothesis that CL growth occurs initially via
hypertrophy and hyperplasia by both theca- and granulosa-
derived luteal cells. However, these authors suggest that
as the CL matures, theca-derived lutein cells enlarge to
form large luteal cells. It has further been suggested that

20
the large luteal cells (granulosa- or tneca-derived), which
possess receptors for PGF£a, play a key role in CL
regression (Fitz et al., 1932; Alila and Hansel, 1934; Hoyar
et al., 1984).
Factors Regulating CL Steroidogenesis
The formation of luteal tissue is begun prior to
ovulation as the preovulatory rise and surge release of LH
promote several biochemical (Savard, 1973) and cytological
(Donaldson and Hansel, 1965b; Blanchette, 1966a,b;
Priedkalns and Weber, 19Ó8; Enders, 1973; Van Blerkom and
Motta, 1978; 1979) changes in both theca and granulosa cells
of the preovulatory follicle.
Unlike the follicle, cells of the bovine CL lack the
endoplasmic reticulum-associated 17-hydroxylase and 19-
hydroxy lase-aroma tase enzyme systems (Savard and Telegdy,
1965; Savard, 1973). The obvious physiological
manifestations of these biochemical alterations are the
sudden loss of androgen and estrogen biosynthetic capacity
by luteal cells (Savard and Telegdy, 1965; Ireland and
Roche, 1982; Walters and Schallenberger, 1984). Thus,
bovine CL exhibit a very abbreviated steroidogenic pathway
from which P4 is the primary end-product, with lesser
amounts of 20B-hydroxy-4-pregnen-3-one (2O8-P4),
pregnenolone (P5), and 5a-reduced progestins (5a-P) being
generated (Wise and Fields, 1978; Albert et al., 1982).
Corpus luteum development and steroidogenic capacity may be

21
grossly assessed by monitoring plasma concentra cions.
Bovine CL mass increases rapidly from day 3 (_<_ d gram) to
day 7 (4 gram) and is maintained at a weight of 5 to ó grams
from days 10 through 17. Likewise, plasma concentrations of
P4 are low (_<_ 1 ng/ml) on day 3, but rise rapidly to levels
of approximately 5 ng/ml by day 7 and are maintained between
6 and 10 ng/ml from days 10 through 17 (Donaldson and
Hansel, 1965b; Erb et al., 1971; Rao et al., 1979; Milvae
and Hansel, 1983a). During regression of the CL, a loss in
function (reduced plasma P^ concentrations) consistently
precedes any decline in CL weight.
Luteal steroidogenic potential is regulated by several
factors. Luteinizing hormone is considered the primary
luteotropin in cattle. During the past 20 years, a
tremendous number of studies have been performed which
support this concept. Administration of exogenous LH
(Donaldson and Hansel, 1965a), hCG (i/iiltbank et al., 1961),
and GnRH-agonists, to induce elevated endogenous LH
concentrations (Milvae et al., 1984), increased plasma P4
concentrations and prolonged CL function in cattle. Several
in vitro studies demonstrated increased P4 production by
dispersed luteal cells or luteal slices when incubated with
LH or hCG (Hansel and Seifart, 1967; Hansel et al., 1973;
Hixson and Hansel, 1979; Milvae and Hansel, 1983a; Tan and
Biggs, 1984; Milvae et al., 1985). Antiserum to LH
prevented this luteotropic effect of LH on luteal slices in

22
vitro (Hansel and Seifart, 1967) and induced a decline in CL
weight and content of CL in intact heifers (Snook at al.,
1969). In hysterectomized heifers, LH treatment increased
P4 content of CL (Brunner et al., 1969) while hysterectomy
plus hypophysectomy or hypophysectomy alone caused a decline
in content of CL, lower plasma P4 concentrations, and a
reduction in CL weight in cattle (Henricks et al., 1989).
Lastly, oxytocin and PGF2a induced CL regression was
circumvented by administration of LH or GnRH to cyclic
cattle (Hansel and Seifart, 1967; Thatcher and Chenault,
1976). Therefore, maximal function of the bovine CL is
contingent upon LH stimulation.
Direct binding studies, using radiolabelled ligands,
revealed the presence of specific, high affinity-low
capacity receptors for LH/hCG in bovine luteal cell
membranes (Rao et al., 1979, 1983) and intracellular
organelles (Rao et al., 1983). Furthermore, luteal
concentrations of LH receptor sites are correlated with P^
secretion during the estrous cycle. Other ligands (to be
discussed later) which bind specifically to luteal tissue
include FSH (Mann and Niswender, 1983), PGF2a (Kimball and
Lauderdale, 1975; Rao et al.,1979; Bartol et al., 1981; Fitz
et al., 1982), PGE^ and PGE2 (Kimball and Lauderdale, 1975;
Fitz et al., 1982), and E2 (Glass et al., 1984).
Recent application of cell separation techniques have
enabled researchers to better characterize and evaluate the

23
function of specific luteal ceil populations with regard to
their roles in production and luteolysis. As described
previously, small luteal cell numbers exceed large luteal
cells by approximately five-fold although large luteal cells
comprise more of the CL volume. In cattle (Ursely and
Leymarie, 1979a,b; Koos and Hansel, 1981; Weber et al.,
1984), sheep (Fitz et al., 1982; Rodgers and O'Shea, 1982;
Rodgers et al., 1983), and swine (Lemon and Loir, 1977;
Lemon and Mauleon, 1982) incubations enriched in large
luteal cells produce consistently more tnan equivalent
numbers of small luteal cells. However, only small luteal
cells were capable of responding to LH/hCG stimulation, as
determined by a significant increase in P^ production over
nonstimulated cells. This observation suggested, as was
demonstrated by Fitz and coworkers (1982), that the
luteotropic effect of LH/hCG was due to the interaction of
LH/hCG with specific receptor sites on small luteal cells.
Small cell steroidogenesis was also stimulated by cyclic AMP
and PGEp. The deficiency of LH/hCG receptors in the large
luteal cell population and a constant secretion of P¿¡_ in the
presence or absence of secretagogues (Fitz et al., 1982)
indicate that peak steroidogenic activity in large luteal
cells does not require receptor-mediated cyclic AMP
generation as it does in small luteal cells (Hoyer et al.,
1984). Instead, maximum potential for P4 production by

24
large lutein cells appears to be dependent upon precursor
(primarily cholesterol) availability.
Lemon and Mauleon (1962) demonstrated an interaction
between small and large luteal cell types in the porcine CL
with regard to production. These authors utilized a
superfusion system in which medium was pumped from one
chamber, containing small luteal cells, through a second
chamber, containing large luteal cells, and vise-versa.
Progesterone production by the small cell to large cell 'in
series' superfusion exceeded (20%) the sum total of P^
produced by each cell type alone. No such increase in P^
production was detected when the superfusion was conducted
with cell populations in a reversed order. Thus, products
derived from small luteal cells stimulated P^ production by
large luteal cells. When steroid precursors were added to
superfusions of either small or large luteal cells, P^ was
metabolized to P^ equally well by both cell types. In
contrast, only large luteal cells responded to exogenous
cholesterol with increased P^_ production compared to control
cells superfused without cholesterol. Metabolism of
cholesterol by large cells was not stimulated by exogenous
LH. Pulses of exogenous LH to small luteal cell
superfusions stimulated P^ production responsively.
However, P^ production in response to LH was not affected by
the addition of cholesterol to small luteal cells. Lemon
and Mauleon (1982) suggested that local transfer of

25
cholesterol from small to large luteal cells was responsible
for enhanced P^ production by large luteal cells.
Furthermore, LH-stiraulated P^ production and cnolesoerol
mobilization in small luteal cells would provide additional
substrate to large luteal cells. The end result would be a
coordinated increase in P^ production by both cell types.
The following discussion will summarize some of the
important processes involved in cellular cholesterol
regulation. Pertinent references may be found in reviews by
Savard, 1973; Brown et al., 1981; Kaplan, 1981; Savion et
al., 1982; Strauss et al., 1982.
Steroidogenic cells, and extrahepatic cells in general,
synthesize very little cholesterol. Instead, the bulk of
cholesterol required for steroidogenesis and membrane
synthesis is derived from plasma and stored as cholesteryl
esters within the cell.
Approximately 70% of the total plasma cholesterol pool
resides within low density lipoprotein (LDL) particles in an
esterified form. These LDL particles are approximately 220
o
A in diameter and contain a core of nearly pure cholesteryl
ester surrounded by two copies of the 250,000 Dalton
glycoprotein, apoprotein B. Cellular uptake of cholesterol
involves binding of LDL to specific, high affinity receptors
which become localized on specialized regions of the plasma
membrane called coated pits (Pastan and Willingham,
1981a,b). Receptor recognition of specific regions of

26
apoprotein B ensures the selective uptake of LDL. Receptor
binding affinity to LDL is Ca+2-dependent, the importance of
which will become obvious. Receptor-LDL complexes are
rapidly internalized enmass via receptor mediated
endocytosis (Pastan and Dillingham, 1981b). Coated pits,
containing numerous receptor-LDL complexes, invaginate and
pinch off to form endocytotic coated vesicles which
ultimately fuse with lysosomal membranes in the cytoplasm.
Receptor-ligand complexes dissociate allowing the LDL
receptors to be recycled for further use. Dissociation is
thought to occur as a result of low Ca+2 concentrations
within the endocytotic vesicle and lysosome. Hydrolysis of
LDL within the lysosome frees cholesterol for use or storage
within the cell.
Intracellular cholesterol concentrations are highly
regulated by the cell. As intracellular cholesterol
concentrations rise following hydrolysis of LDL in the
lysosomes, cellular cholesterol biosynthesis is suppressed
via a decrease in the activity of a pivitol enzyme in the
cholesterol biosynthetic pathway, HMG-CoA reductase.
Secondly, acyl-CoA:cholesterol acyltransferase activity is
enhanced, thus catalyzing the re-esterification of
cholesterol for storage. Most importantly, LDL receptor
biosynthesis is suppressed, preventing further uptake of
cholesteryl ester-rich LDL from the plasma.

These data suggest that small and large luteal ceils
also adjust to intracellular cholesterol concentrations in
accordance with changing cellular requirements. Thus, large
lutein cells, which synthesize approximately 80% of the
total P4 in unstimulated CL (Hoyer et al., 1984), would be
expected to maintain a higher cholesterol requirement than
small luteal cells. Furthermore, it may be suggested that
large luteal cells in the pig (Lemon and Mauleon, 1932) were
in a cholesterol-deficient state, with regard to their
maximum steroidogenic capacity, since P^ production was
elevated following exposure to exogenous cholesterol.
Conversely, small luteal cells, which utilize cholesterol
less rapidly, may possess larger stores of intracellular
cholesterol and would not be expected to exhibit elevations
in P4 production in response to exogenous cholesterol (Lemon
and Mauleon, 1932). Demand for cholesterol by LH-sensitive
small luteal cells increases following stimulation with LH
or cyclic AMP. However, intracellular stores are apparently
sufficient to accomodate this demand (Lemon and Mauleon,
1982).
Luteinizing hormone and cyclic AMP increase cholesterol
esterase and cholesterol side chain cleavage (SCC) enzyme
activities, lipoprotein uptake and P^ production (Savard,
1973; Bisgaier et al., 1979; Caffrey et al., 1979a; Strauss
et al., 1982). However, specific mechanisms of LH action on
luteal cells are not defined clearly.

23
Intracellular Ca+^ and cyclic AMP concentrations, and
cyclic AMP-dependent protein kinase activation are known to
play essential roles in transduction of receptor-mediated
signals (see: Rasmussen, 1981). General concepts regarding
the mechanism of action of peptide hormones on target cells
have centered around intracellular accumulation of Ca+^ and
cyclic AMP, and activation of cyclic AMP-dependent protein
kinase(s) (Catt and Dufau, 1976; Marsh, 1976). This enzyme
mediates the effects of cyclic AMP via phosphorylation of
specific substrates which in turn regulate cell function.
Cyclic AMP-dependent protein kinase(s) has been localized in
the cytoplasm and numerous cellular oganelles (plasma
membrane, mitochondria, ribosomes, nucleus and endoplasmic
reticulum) suggesting that diversity of cellular responses
to cyclic AMP may depend on availability of specific
substrates within compartmentalized regions of the cell
(Langan, 1973).
Recent evaluation of membrane structure and function
has expanded our understanding of mechanisms involved in
receptor-mediated information flow into the cell. Over the
past decade, it has become apparent that the organization of
phospholipids and proteins within membranes is more complex
than described by the "fluid mosaic" membrane model of
Singer and Nicolson (1972). Several components of
biological membranes (i.e., receptors, enzymes,
phospholipids) are asymmetrically distributed between the

29
inner and outer bilayer (Rothman and Lenard, 1977; Lodish
and Rothman, 1979)* Furthermore, membrane components may
aggregate preferentially in specific domains of the Dilayar
structure (Chapman et al., 1979; Karnovsky et al., 1932).
Assembly of phospholipid and protein domains facilitate
events such as intercellular communications (Loewenstein,
1970; Garfield et al., 1979; Hertzberg et al., 1931),
endocytosis (Pearse, 1980; Schlessinger, 1980; Pastan and
Willingham, 1981a,b), ion movement and enzymatic activity
(Savard, 1973; Strauss et al., 1982; Lentz et al., 1933).
Membrane components are in a state of constant
reorganization, synthesis and breakdown in response to
external stimuli. Current data suggest that, in addition to
Ca+2 and cyclic AMP messenger systems, rapid and transient
alterations in membrane phospholipid composition,
biosynthesis and metabolism are essential in stimulus-
response coupling in cells (Hirata and Axelrod, 1980; Davis
et al., 1931; Crews, 1982; Farese, 1983; Milvae et al.,
1983). Phospholipid methylation (Hirata and Axelrod, 1980;
Crews, 1982; Milvae et al., 1983) and phosphotidylinositol
(PI) metabolism (Davis et al., 1981; Crews, 1982; Farese,
1983; Nishizuka, 1934b) are two mechanisms by which membrane
phospholipids are transiently altered in response to
receptor-ligand binding.
Methylation of phospholipids is dependent upon two
phospholipid methy1transferase (PMT) enzymes which are

30
distributed on the inner (PMT-1) and outer (PiVlT-2) aspects
of the cell membrane bilayer. Likewise, the phospholipid
substrates for PMT-1 (phosphatidylethanoiamine, P¿) ana PMT-
2 (phosphatidyl-N-monomethylethanolamine, PME) are
asymmetrically distributed within the membrane. Formation
of PME requires the PMT-1 catalyzed transfer of a single
methyl group to PE. Conversion of PME to PC requires two
successive methyl transfers by PMT-2. Successive
methylations of PE by this system result in translocation of
methylated phospholipids (PME and phosphatidylcholine, PC)
toward the outer aspects of the membrane bilayer.
As a result of these chemical alterations in
phospholipid composition, the membrane becomes less
viscous. Enhanced membrane fluidity is thought to result
from the accumulation of deeply embedded PME within the
phospholipid bilayer. With the increase in membrane
fluidity, lateral mobility of membrane proteins is
potentiated thereby enhancing the coupling of surface
receptors with adenylate cyclase and cyclic AMP formation
(Rimon et al., 1978; Axelrod, 1983). Additionally,
formation and deposition of PC in the outer phospholipid
layer of the membrane promotes the unmasking of cryptic
membrane receptors. Thus, receptor availability is enhanced
(Hirata and Axelrod, 1930; Crews, 1982).
Milvae et al. (1983) demonstrated the importance of
phospholipid methylation in expression of LH-stimulated P^

31
production by dispersed bovine luteal cells. Incorpora cion
of [5H] -methyl-groups into PME, PC and PI was stimulated by
LH, in vitro. An endogenous methyl-donor, 3-aaenosy1-L-
methionine (SAM), enhanced LH-induced P^ production while
methylation inhibitors, 3-deazaadenosine (DZA) and S-
adenosyl-L-homocysteine (SAH), prevented the luteotropic
action of LH on dispersed luteal cells. Cyclic AMP effects
on P^ production were not influenced by DZA or SAH,
supporting the view that inhibition of phospholipid
methylation prevents coupling of ligand-receptor complexes
to adenylate cyclase, cyclase activation and cyclic AMP
generation in the bovine CL.
The degree of membrane phospholipid methylation also
appears to influence cellular Ca+2 fluxes, possibly via
regulation of calcium ion channels and Ca+^-dependent
adenosinetriphosphatase (ATPase) activity (Hirata and
Axelrod, 1980; Crews, 1982). Regulation of cell function by
calcium ion concentrations and intracellular Ca+^ binding
proteins, such as calmodulin, have been reviewed by several
authors (Cheung, 1979; Means, 1981; Rasmussen, 1981).
Receptor mediated Ca+^ influx occurs secondarily to
methylation of phospholipids and, like cyclic AMP
generation, is prevented by methylation inhibitors.
Activation of Ca+^-dependent phospholipase A£ parallels the
influx of Ca+2, as demonstrated by elevated free arachidonic
acid and lysolecitnin (lysophosphatidylcholine)

32
concentrations following receptor activation (Crews,
1932). These observations suggest that hydrolysis of newly
synthesized methylated phospholipids by Ca+^-dependent
phospholipases serve to regulate methylated phospholipid-
induced membrane effects, viz., increased fluidity, Ca+~
influx, receptor availability. It is interesting to note
that Ca+^ is not required for membrane phospholipid
methylation or cyclic AMP generation in several cell
systems, although many of the physiological responses to
cyclic AMP are Ca+^-dependent (Rasmussen, 1970).
In addition to phospholipid methylation, considerable
interest has been directed toward receptor-mediated
alterations of the phosphatidate-inositide cycle (Crews,
1982; Farese, 1983; Nishizuka, 1984b). There are two known
mechanisms by which the phosphatidate-inositide cycle may be
affected by receptor activation. The first involves the
hydrolysis of phosphatidylinositides to diacylglyceride (DG)
and inositol phosphates via phospholipase C catalyzed
removal of the phosphorylated inositide head group. This
mechanism is thought to regulate plasma membrane Ca+^ fluxes
and intracellular Ca+2 distribution. The phosphatidylino-
sitides are localized primarily in the inner phospholipid
layer of the plasma membrane bilayer, and membranes of
endoplasmic reticulum and mitochondria as mono-, di- and
triphosphorylated inositol phospholipids (PI, PIP and PIP2>
respectively). These phospholipids bind Ca+“
avidly and

33
provide a releasable Ca+- pool upon hydrolysis. Recent
evidence, reviewed by Nishizuka (1984b), suggests that PIP^,
rather than PI or PIP, is rapidly degraded following
receptor activation. Once hydrolysis of PIP2 is initiated
in the plasma membrane the process is thought to self-
propagate as a result of elevated free Ca+^ concentrations,
increased Ca+^-dependent phospholipase activity, and
extended hydrolysis of phosphatidylinositides within the
plasma membrane and intracellular organelles. One
metabolite of PIP2 hydrolysis, inositol triphosphate, may
serve as a mediator for Ca+^ release from intracellular
stores (Nishizuka, 1984b). A second metabolite, DG, arises
following phospholipase C catalyzed hydrolysis of PIP2> PIP
or PI. Diacylglycerol may serve as a precursor for the
regeneration of phosphatidylinositides as well as PE and
PC. The phosphatidylinositides and DG contain primarily
arachidonate at fatty acid position -2. Thus, hydrolysis by
Ca+^-dependent phospholipase A2 provides free arachidonic
acid for use in prostaglandin and hydroxyperoxylipid
biosynthetic pathways (Ramwell et al., 1977), and various
lysolecithins, many of which are required for membrane
fusion reactions during uptake and secretion of products
(Crews, 1982). Additionally, DG activates the Ca+^- and
phospholipid-dependent protein kinase C (Nishizuka,
1984a,b). Protein kinase C activation has been implicated
in the elicitation of cell proliferation (Nishizuka, 1984a)

34
and inhibition of gonadal steroidogenesis (Welsh et al.,
1984). It is interesting to note that protein Kinase C
serves as a receptor for tumor promoting phorbol esters
(Nishizuka, 1984afb) which are thought to irreversibly
activate the enzyme. Phorbol esters possess a similar
chemical structure to DG, the endogenous activator of
protein kinase C. Phorbol esters have been shown to innibit
gonadotropin-induced steroidogenesis, in vitro (Welsh et
al., 1984), supporting a similar role for endogenous DG in
the regulation of steroid production. Therefore,
steroidogenesis may be antagonized as a consequence of
phosphatidylinositide hydrolysis.
Conversely, de novo phosphatidate-inositide
biosynthesis may be important in the mediation of
gonadotropin effects on steroidogenesis (Davis et al., 1981;
Crews, 1982; Farese, 1984). Several studies have
demonstrated that LH and ACTH stimulate biosynthesis of
phosphatidylinositides and phosphatidic acid (PA) in gonadal
and adrenal cortex cells, respectively (Davis et al., 1981;
Strauss et al., 1982; Farese, 1984). Data reviewed by
Farese (1984) indicated that identical responses were
triggered by cyclic AMP, suggesting that phospholipid
biosynthesis occurred after cyclic AMP generation. Both
hormone- and cyclic AMP-induced phosphatidate-inositide
biosynthesis, and steroidogenesis are blocked by protein
synthesis inhibitors, puromycin and cyclohexiraide.

35
Apparently, the two enzymes responsible for PA biosynthesis,
viz., glycerol-3-phosphate acyltransferase and diglyceriie
kinase, are substrate activated and require protein
synthesis. Hormone activation of these enzyme systems is
indirect and thought to occur by increasing glycerol
phosphate, glucose, fatty acid and diglyceride availability
through stimulation of glycogenolysis, glycolysis, glucose
uptake and lipolysis. Phosphatidylinositol kinase catalyzes
the conversion of PA plus ATP to PI, and subsequent
phosphorylations to PIP and PIP2.
Phosphatidate-inositol biosynthesis parallels
steroidogenic activation in adrenal and luteal tissues
following exposure to ACTH and LH, respectively.
Furthermore, addition of certain polyphosphorylated
phospholipids, viz., the phosphatidylinositides and
cardiolipin, to adrenal cortex and luteal cell cultures
stimulated steroid biosynthesis in a dose dependent manner
(Strauss et al., 1982; Farese, 1984). Effects of these
"steroidogenic" phospholipids are not prevented by
cycloheximide or puromycin.
The site of phospholipid action in both adrenal cortex
and luteal cells appears to be the mitochondria. Specifi¬
cally, steroidogenic phospholipids stimulate cholesterol
side chain cleavage (SCC) by intact cells and acetone
powders of luteal mitochondria (Strauss et al., 1982;

3b
Farese, 1984), although the mechanism through whicn this is
accomplished remains unknown.
At present, it is known that trophic hormones, such as
LH and ACTH, stimulate steroidogenesis by influencing
several biochemical steps. Free intracellular cholesterol
concentrations are elevated via enhanced cellular uptake of
cholesterol from plasma and mobilization of cholesterol from
intracellular stores following cholesterol esterase
activation. Apparently, hormone induced elevations in free
intracellular cholesterol are not affected by cycloheximide,
but are blocked by cytochalasin B suggesting that microfila¬
ments, but not protein synthetic events, are crucial in the
acquisition of substrata for steroid biosynthesis (Gemmel
and Stacy, 1977; Sawyer et al., 1979; Silavin et al., 1980;
Rasmussen, 1981; Gwynne and Condon, 1982). Microtubule
involvement in steroidogenesis is, however, less clear with
evidence reported both pro (Gemmel and Stacy, 1977; Sawyer
et al., 1979) and con (Gwynne and Condon, 1982).
In addition to elevating intracellular substrate
concentrations, LH and ACTH increase mitochondrial content
of cholesterol, presumably as a result of enhanced
cholesterol transfer from cytosol to mitochondria (see:
Rasmussen, 1981, for references). This event requires
protein synthesis and microfilament action. Hormone- and
cyclic AMP-induced biosynthesis of the "steroidogenic"
phospholipids are also dependent on protein synthetic

37
events, although their stimulatory effects on mitochondrial
SCC activity are not (Strauss et al., 1932; Farese, 1934).
Furthermore, mitochondrial SCC activity is substrate- and
Ca+2-dependent. Thus, it appears as though hormone-induced
steroidogenic activity involves primarily processes which
increase intracellular cholesterol content and transfer of
cholesterol from the cytosol to the SCC enzyme complex,
viz., sterol carrier proteins (see: Savard, 1973, for
references). Translocation of cholesterol across the
mitochondrial membranes may be regulated by the phospha-
tidylinositides and other "steroidogenic" phospholipids
(Strauss et al., 1982; Farese, 1984), the synthesis of which
is dependent on some, as yet undetermined, protein synthetic
event. Increased SCC activity may also involve structural
changes in the enzyme-phospholipid domain (Savard, 1973;
Strauss et al., 1982), and altered mitochondrial Ca+^
concentrations (Crews, 1982; Farese, 1984).
It is becoming more apparent that membrane phospholipid
dynamics, in addition to cyclic AMP and Ca+2 second
messenger effects, are of paramount importance in cellular
information flow and stimulus-response coupling. The reader
is referred to Rasmussen (1981) for further discussions on
this broad and rapidly developing area of cell physiology.
Following side chain cleavage of cholesterol, newly
synthesized P5 is transferred out of the mitochondria to the
endoplasmic reticulum. Here, the membrane bound

33
3p-hydroxy-A^-steroid dehydrogenase/a^-a^ isomerase enzyme
complex (H3D) catalyzes the rapid conversion of zo P,
the primary steroid end product of the CL. These latter
steps in the biosynthesis of do not appear to be
regulated by trophic hormones. However, luteal P^
production may be modified by local and systemic mechanisms
involving steroids, arachidonic acid metabolites, and
ovarian peptide hormones.
Intraovarian control of CL function
The dependency of CL production on LH has been
extensively documented in the literature, as has the
importance ox uterine-derived PGF2a in luteolytic events.
However, the level and duration of luteal P^ production may
also be determined by the local ovarian environment in which
the CL resides.
A large body of evidence supports a role for steroids
in the regulation of steroidogenic enzyme activities in the
ovary, testes, adrenal and placenta (see: Rothchild, 1981;
Gower and Cooke, 1983). Caffrey et al. (1979b) demonstrated
that the affinity of HSD for its product, P^, was
substantially greater than for P^ substrate and that P^ (IQ-
50 ug) inhibited HSD activity in ovine CL preparations.
Thus, suggesting that P^ may modify its own production via
substrate inhibition of HSD activity in the CL.
Testosterone and OO uS) were also found to be potent
inhibitors of HSD activity, in vitro (Caffrey et al., 1979b;

39
Gower and Cooke, 1983), but not in vivo (Caffrey at al.,
1979b). Similarly, microgram concentrations of testosterone
and E£ were found to inhibit LH-stimulatea P^ production but
not cyclic AMP accummulation in dispersed luteal cells
collected from 2 to 6 months of gestation in cattle.
(Williams and Marsh, 1978). In vivo luteolytic interactions
were reported between E£ and PGF£a in hysterectomized ewes
(Gengenbach et al., 1977), and between E2 (0.5 ug) and
oxytocin (200 ralU) in dispersed luteal cells collected from
cyclic cattle (Tan and Biggs, 1984). However, Hixon and
Hansel (1979) were unable to demonstrate similar luteolytic
effects with subnanogram doses of E2 or E2 plus PGF2a in
dispersed bovine luteal cells from days 12 and 13 of the
cycle. Further support for a direct E2-CL interaction was
provided in a recent study (Glass et al., 1934) in which E2
receptors were characterized in large and small luteal cells
throughout the estrous cycle of the ewe. Concentrations of
E2 receptor rose gradually during the estrous cycle and were
localized primarily in large lutein cells. Additionally,
Sairam and Berman (1979) provided evidence that synthetic
estrogens prevented normal coupling of the gonadotropin-
receptor complex to its catalytic subunit, thus preventing
the initiation of intracellular gonadotropic-induced
responses.
Discrepancies with regard to the inhibitory role(s) of
E2 on CL P^ biosynthesis may be due to differences in dosage

40
of steroid employed, specie and status (cyclic or pregnant)
of the animal from which luteal tissue was collected, and in
vitro versus in vivo experimental conditions. Although
doses of E£ which inhibit CL biosynthesis seem high and
nonphysiological, they do compare favorably with
concentrations reported in follicular fluid of preovulatory
follicles (see: Fortune and Hansel, 1979)* Therefore, the
CL may be exposed to high intraovarian concentrations of
various steroids, viz., testosterone and £9, as would be
expected during preovulatory follicle development in the
late diestrum. The potential inhibitory nature on luteal P4
production exhibited by high concentrations of these
steroids supports a functional role in the initiation of CL
regression. In this regard, it is noteworthy that P4 plasma
concentrations begin to fall prior to any detectable rise in
peripheral plasma E2 concentrations or initiation of uterine
PG^2a Pulsatile activity (Chenault et al., 1975; Peterson et
al., 1975).
Beyond the luteotrophic effects initiated by LH, P4
production may be "protected" from steroid negative feedback
effects by several intraovarian processes.
For example, binding of steroids to proteins for short¬
term storage, viz., steroid carrier proteins (Goddard et
al., 1980; Willcox, 1983) or long-term storage in
cytoplasmic granules (Gemmell et al., 1974; Geramell and
Stacy, 1979; Sawyer et al., 1979) may reduce intracellular

41
concentrations of free steroid and reduce feedbacK.
inhibition, in vivo. Furthermore, steroid sulfacion or
conjugation to fatty acids, viz., lipoidal steroids
(Hochberg et al., 1979; Albert et al., 1980; Schatz and
Hochberg, 1981; Albert et al., 1982), within the CL would
accomplish a similar function. Lastly, ovarian blood flow
parallels luteal production in cattle (Ford and Cnenault,
1931; Wise et al., 1982) thus providing an additional means
of reducing CL and ovarian steroid concentrations through
elevated clearance rates.
In recent years, several ovarian peptides, including
steroid-binding proteins (Willcox, 1983)» LH-binding
inhibitors (Kumari et al., 1982; Ward et al., 1982),
gonadotropin releasing hormone (GnRH)-like peptides (Ying et
al., 1981; Fraser, 1982; Sharpe, 1982), angiogenic factor
(Gospodarowicz and Thakral, 1978), oxytocin (Fields et al.,
1983; Rodgers et al., 1983; Wathes et al., 1983),
vasopressin (Wathes et al., 1983) and relaxin-like peptides
(Fields et al., 1982) have been discovered. Although
biochemical evidence suggests that these peptides are
produced by the CL and/or follicle, little information is
available concerning their physiological roles in ovarian
processes.
Of the peptides mentioned above, oxytocin has been
evaluated most thoroughly. It is known that CL of the cow
and ewe contain high concentrations (ug/g) of oxytocin

42
(Fields at al., 1983; Flint and Sheldrick, 1983; Flint at
al., 1983; Wathes et al., 1933) which is synthesized and
stored in the large lutein cells (Rodgers et al., 1933).
The bovine CL contains neurophysin, lending further support
for the local synthesis of oxytocin within the CL (Wathes et
al., 1983). Additionally, circulating oxytocin
concentrations are rapidly reduced following ovariectomy
(Scharas et al., 1982) and concentrations of imraunoreactivs
oxytocin were greater in ovarian venous plasma versus
uterine arterial or jugular venous plasma (Flint and
Sheldrick, 1982; Walters et al., 1984). Concentrations of
oxytocin and neurophysin in luteal tissue (Wathes et al.,
1984) and plasma (Sheldrick and Flint, 1981; Flint and
Sheldrick, 1983; Schams, 1983; Walters et al., 1984)
parallel CL production during the luteal phase, although
the decline in oxytocin concentrations occur prior to
during CL regression. Oxytocin and P^ are secreted from the
CL in a pulsatile fashion. Walters et al. (1984) reported
that only 29% of P^ episodes were associated with pulses of
oxytocin during the early luteal phase, due largely to the
occurrence of fewer oxytocin than P^ pulses at this time.
During the mid-luteal phase 86% of P^ and oxytocin episodes
occurred simultaneously. Remarkably, 97 to 100% of oxytocin
pulses, during all periods of the luteal phase, were
associated with P4 pulses. These observations suggest that

43
similar mechanisms may regulate the secretion of ?4 and
oxytocin from the CL.
Several lines of evidence support a systemic role for
oxytocin in the process of CL regression (reviewed in a
later section). Additional data suggests that oxytocin may
have an action within the ovary as well. Evidence reviewed
by Wathes (1984) indicates that oxytocin (and vasopressin)
may be involved in production during CL development since
oxytocin (and vasopressin) can promote P^ biosynthesis in
testicular cells through the inhibition of LH-stimulated
androgen biosynthesis. Oxytocin probably acts by selective
suppression of 17“-hydroxylase and C17-20 desmolase
activities. Follicular androgen and estrogen biosynthesis
is suppressed in favor of P^ biosynthesis prior to and
following the preovulatory LH surge. Furthermore, oxytocin
has been measured in preovulatory follicles, ovarian tissue
(including all follicles) devoid of CL, and newly formed CL
(Wathes, 1984; Wathes et al., 1984). Therefore,
preovulatory follicle and luteal oxytocin biosynthesis,
possibly induced by LH, may prevent C-19 and C-18 steroid
biosynthesis and promote P^ formation in CL. High levels of
oxytocin in CL during the raid-luteal phase may reduce
follicular E£ production in a similar manner (Wathes,
1984). Results of Tan et al. (1982) also demonstrated
stimulatory effects of low levels of oxytocin (2 to 20 mlU)
in CL collected from pregnant cattle. Likewise, exogenous

44
oxytocin, administered to cyclic cattle on days 12 and 1 ,
increased content of CL on day 14 (Mares and Casida,
1963). Conversely, high doses of oxytocin (200 co 4oD mill)
were inhibitory to in vitro P^ biosynthesis by CL of cyclic
(Tan and Biggs, 1934) and pregnant (Tan et al., 1932)
cattle.
Luteolytic prostaglandins appear to mediate oxytocin
secretion from the CL (Flint and Sheldrick, 1932, 1983;
Schallenberger et al., 1934). Similarly, PGF2a or analogues
of PGF2a stimulate rapid degranulation of large lutein calls
(Heath et al., 1983) which are known to be the synthetic and
storage site of oxytocin in the CL (Rodgers et al., 1983).
Consistent with the hypothesis of oxytocin storage in
granules are observations which demonstrate parallel
patterns of luteal oxytocin content (Wathes et al., 1984)
and secretion (Sheldrick and Flint, 1981; Flint and
Sheldrick, 1983; Schams, 1983; Walters et al., 1984) with
large luteal cell granule content (Gemmell et al., 1974).
Based on the nearly absolute synchrony with which tonic
oxytocin and P^ pulses occur, in vivo (Walters et al.,
1984), and the ability of PGF2a to induce oxytocin
secretion, it may be suggested that luteal prostaglandins
are involved in tonic oxytocin and P^ secretion during the
luteal phase of the estrous cycle. Evidence of luteal
prostaglandin biosynthesis (Shemesh and Hansel, 1975a;

45
Lukaszewska and Hansel, 1979; Milvae and Hansel, 1965a)
strengthens this supposition.
Bovine luteal tissue contains massive quantities (2 to
3 mg/g) of arachidonic acid, the bulk of which is esterified
to phospholipids (Lukaszewska and Hansel, 1979, 1980).
Thus, the potential for arachidonic acid metabolism in
either the cyclooxygenase or lipoxygenase pathway is
great. Metabolic pathways utilizing arachidonic acid as
precursor have been reviewed previously (Ramwell et al.,
1977). Characterization of local biosynthetic patterns and
functions of luteal prostaglandins and other arachidonic
acid metabolites have provided additional insight into
mechanisms controlling luteal production and regression.
Shemesh and Hansel (1975b) demonstrated an acute
increase in ovarian venous PGF2a concentrations following
injection of arachidonic acid into CL on days 12 or 13 in
cyclic cattle. Peripheral plasma P^ concentrations fell
rapidly from approximately 6 ng/ml to 2.5 ng/ml, and
remained constant until day 15 or 16 when the experiment
concluded. Additionally, CL weights on day 15 or 16 were
less in arachidonic acid-treated (4.3 ± 1.4 g) than vehicle-
treated (6.3 ± 0.5 g) cattle. Several studies have
demonstrated an inverse relationship between luteal PGF2a
and P^ content or production (Patek and Watson, 1976;
Lukaszewska and Hansel, 1979; Rothchild, 1981). However,
the means by which elevated luteal PGF2a contents reduce P^

46
production remain to be elucidated. Exogenous PGF?a
administration to cattle (Lukaszewska and Hansel, 1979),
sheep (Diekman et al., 1978) and rats (Hichens et al., 1^74;
Grinwich et al., 1976) initiates a reduction in
production and the loss of LH receptors or luteal
sensitivity to LH. The authors suggested that loss of LH
receptor numbers or reduced sensitivity to LH were the
consequence rather than cause of luteolysis since reduced P^
production occurred considerably earlier tnan either of the
LH related phenomenon.
The PGF2a-induced decline in luteal P^ production
described above may be related more closely to changes in
ovarian vascular resistance. Blood flow to the CL-bearing
ovary parallels closely luteal P^ production (Niswender et
al., 1975; Ford and Chenault, 1981) and PGF2a is known to
reduce ovarian blood flow and luteal P^ production (Nett et
al., 1976; Niswender et al., 1976). Therefore, PGF2a
effects may be mediated through increasing ovarian vascular
resistance. However, metabolite requirements are generally
thought to dictate the degree of blood flow to a tissue. In
this light, reduced luteal metabolic activity may initiate
the accummulation of PGF2a and subsequent decline in luteal
blood flow.
Progesterone biosynthetic activity of bovine CL
collected during the estrous cycle is highly correlated
(r^=.93) with luteal prostacyclin (PGI2) biosynthesis

47
(Milvae and Hansel, 1983a). Sun at al. (1977) demonstrated
that PGI2 was the predominant prostaglandin product
following bovine CL membrane metabolism of PGH2-
Furthermore, PGI2 stimulated luteal P^ biosynthesis in vitro
and in vivo (Milvae and Hansel, 1980b). A recent report by
Milvae and Hansel (1985) demonstrated that indomethacin
inhibition of prostaglandin formation early in the estrous
cycle shortened luteal lifespan in cattle. Indomethacin was
administered into the uterine lumen twice daily from days 4
through 6. In conjunction with previous reports, these
authors proposed that luteotropic products of the
cyclooxygenase pathway, viz., PGI2 and PGE2, may be required
for normal CL development. The origin of these
prostaglandins may include both the CL and uterus.
Additionally, synthesis and content of PGI2 and PGF2a in
bovine CL are greatest early in the cycle (Milvae and
Hansel, 1983a). Luteal prostacyclin content was
approximately four times greater than PGF2a early in the
cycle although ratios of PGI2 and PGF2a gradually decline in
CL age increased (Milvae and Hansel, 1983a). Milvae et al.
(1985) provided several lines of evidence suggesting that
lipoxygenase products, viz., 5-hydroperoxy-eicosatetraenoic
acid (5-HPETE) and 5-hydroxyeicosatetraenoic acid (5-HETE),
regulate P^ production by the selective inhibition of
prostacyclin synthetase activity and PGI2 production in
bovine CL. Large quantities of 5-HETE are present in bovine

4a
CL on days 10, 15 and 13 of the cycle and 5-HETE inhibited
in vitro PGI2 and biosynthesis in a dose dependent
fashion without influencing PGF2a production. Simultaneous
addition of LH was unable to prevent the 5-HETE-induced
reduction in luteal cell PGI2 and P^ production, in vitro.
In related experiments (Milvae et al., 1985), arachidonic
acid metabolism through the lipoxygenase pathway was
inhibited by nordihydro-guaiaretic acid (NDGA). When NDGA
was added to dispersed luteal cells, PGI2 production was
increased after a 2 hour incubation. Progesterone and PGF2a
synthesis had increased slightly, although not significantly
(P>.05). Arachidonic acid plus NDGA increased luteal
production of both PGI2 and PGF2a but not P^, suggesting an
antagonism between PGI2 and PGF2a in the regulation of
luteal P^ synthesis (Milvae et al., 1985). This possibility
is also suggested by the declining ratio of PGI2 and PGF2a
contents in bovine CL as luteolysis approaches (Milvae and
Hansel, 1985a). Collectively, these data suggest that HETE,
and perhaps other lipoxygenase products, are involved in
luteolysis by blocking synthesis of the luteotropin, PGI2»
and allowing additional arachidonic acid to be shunted
toward pathways involved in synthesis of luteolytic
prostaglandins.
Similar mechanisms controlling biosynthesis of
luteotropic and luteolytic prostaglandins may also occur

49
within the uterus (Milvae et al., 1985; Milvae and nansel,
1935).
Uterine Control of CL Function
There is little doubt that the uterus plays a key role
in regulation of CL lifespan during the estrous cycle. Loeb
(1923) first demonstrated that extirpation of the guinea pig
uterus resulted in extended CL function. Subsequent reports
in cattle (Wiltbank and Casida, 195*5; Anderson et al., 19*51;
Malven and Hansel, 1964; Anderson et al., 1965), sheep
(Wiltbank and Casida, 1956; Anderson et al., 1969), pigs
(Spies et al., 1958; Du Mesnil Du Buisson, 1966) and horses
(Ginther and First, 1971) provided similar results following
surgical removal of the uterus. Thus, a basis for uterine
involvement in the luteolytic process was developed. The
nature of this utero-ovarian relationship was further
defined following observations that cattle (Bland, 1970) and
sheep (McCracKin and Caldwell, 1960) with congenital absence
of the uterine horn (uterus unicornis) adjacent to the CL
bearing ovary exhibited prolonged luteal lifespans and cycle
lengths which suggested local regulation of CL function by
the ipsilateral uterine horn. Additionally, normal luteal
regression occurred following heraihysterectomy of the
contralateral, but not the ipsilateral uterine horn in

50
cyclic cattle and sheep (Inskeep and Butcher, i960; Ho or and
Rowson, 1966; Ginther, 1974, 1931).
As a consequence of these data, Wollm^rhaus (1964) and
Ginther (review, 1974) examined the anatomy and histology of
the utero-ovarian vascular architecture as a possible route
of exchange between the uterine horns and their adjacent
ovaries. In cattle and sheep, species which exnibit local
regulation of CL function by the uterus, exchange of
luteolytic substances from the uterine venous effluent
(ovarian vein, International Committee of Veterinary
Anatomical Nomenclature, 1968) into the ovarian arterial
supply is thought to occur just below the ovarian vascular
pedicle. At this point, the ovarian artery follows an
extremely tortuous and convoluted path over the surface of
the ovarian vein (containing both uterine and ovarian venous
blood) thus maximizing the area of contact between the two
vessels. Histologically, the adventitial layers of each
vessel were substantially thinner in the regions of vein-
artery apposition (Wollmerhaus, 1964; Del Campo and Ginther,
1974). From an anatomical and histological perspective,
these observations favored the existence of a functional
venoarterial exchange system between uterine horns and their
adjacent ovaries.
A series of physiological experiments, utilizing
surgical anastomoses of ovarian veins or ovarian arteries in
hemihysterectoraized cattle and sheep, provided conclusive

51
support for the functional ovarian vein and artery-
components of the local uteroovarian pathway (Gincher, 1974;
1981). In such experiments, luteal regression occurred when
either ovarian vein or ovarian artery blood from the
contralateral, uterine-intact side was diverted to the
appropriate vessel on the ipsilateral, hemihysterectomized
side. Conversely, CL were maintained if the surgical
anastomosis became occluded. Thus, it became clear that the
uterus was producing a blood borne product which could move
from the uterine venous drainage into the ovarian arterial
supply and initiate the process of luteolysis in a local
manner.
As early as 1940, Hechter and coworkers suggested that
some uterine-derived product may be responsible for
regulation of CL lifespan. However, it was not until the
resurgence of prostaglandin research in the 1950's that the
identity of the uterine luteolytic factor was elucidated.
The chemical structures of several prostaglandins, including
PGF2a, were confirmed in 1962 (Nelson et al., 1982) and by
1969, Pharris and Wyngarden provided evidence that the
vasoactive prostaglandin, PGF2a, may be the uterine
luteolytic substance in the rat. Subsequently, the
luteolytic activity of exogenous PGF2a in cattle has been
documented extensively (Hansel et al., 1973; Hafs et al.,
1974; Lauderdale, 1974; Thatcher and Chenault, 1976).
Several studies have provided evidence indicating that PGF2a

52
is an endogenous luteoiysin in cattle. For example,
elevated levels of PGF2a in uterine venous drainage
(Nancarrow et al., 1973; Shemesh and Hansel, 1975), uterine
tissue (Shemesh and Hansel, 1975) and uterine flushings
(Lamothe et al., 1977; Bartol et al., 1981a) coincide
closely with the period of expected luteal regression. In
addition, luteolysis is prevented in cows immunized
passively against PGF2a (Fairclough et al., 1981) or
following intrauterine administration of the prostaglandin
synthetase inhibitor, indomethacin, late in the estrous
cycle (Lewis and Warner, 1977).
The biological half-life of PGF2a is very short owing
to its rapid metabolism by the lungs (Piper et al., 1970)
and uterine endometrium (Thatcher et al., 1984; Chapter
3). Thus, measurements of its primary metabolite, 15-keto-
13,14-dihydro-PGF2a (PGFM; Granstrora and Kindahl, 1982),
provide an index of uterine PGF2a production. Peripheral
concentrations of PGFM are significantly correlated with the
uterine production of PGF2a (Thatcher et al., 1984; Chapter
3) and concentrations of PGF2a in uterine flushings (Bartol
et al., 1981a). Furthermore, three to five pulsatile
episodes of PGFM were always temporally associated with the
decline in plasma P^ concentrations during luteolysis in
cattle (Peterson et al., 1975; Kindahl et al., 1976;
Betteridge et al., 1984). Collectively, these data indicate

53
that the endogenous uterine luteolysis in cattle is likely
to be PGF2a.
Finally, in keeping with the concept of a local
venoarterial pathway, transfusion of PGF£a from the uterine
venous drainage into the adjacent ovarian arterial supply
was demonstrated. In an early study, Goding et al. (1971)
induced luteal regression, as demonstrated by the
precipitous decline in plasma P4 concentrations, following
the infusion of physiological concentrations of PGF2a into
the uterine venous drainage of diestrous cattle. Hixon and
Hansel (1974) reported elevated PGF2a concentrations in the
uterine venous drainage (ovarian vein) and ovarian artery,
ipsilateral to the CL bearing ovary, as a result of the
intrauterine deposition of PGF2a* Similarly, Thatcher et
al. (1984b) verified the functionality of the countercurrent
exchange system (PGF2a)' during the period of normal luteal
regression in cyclic cattle.
Regulation of Uterine PGF?^Production
Demise of the cyclic CL is uterine and PGF2a-dependent,
as reviewed above. However, interplay between ovarian
estrogen, progesterone and oxytocin with the uterine
endometrium regulate the timing and magnitude of luteolysin
production during the estrous cycle. Estrogens, of
follicular origin, appear to constitute an important
stimulatory component regulating uterine PGF2a production
and CL regression. Destruction of ovarian follicles in

54
cattle (Villa-Godoy et al., 1931) and sheep (Karsch et al.,
1970; Ginther, 1971) result in prolonged luteal lifespans
and heavier CL compared to follicle-intact controls.
Furthermore, administration of estrogens to late-luteal
phase cows and ewes resulted in production of PGF2a by the
uterus (Barcikowski et al., 1974; Thatcher et al., 1934b;
Chapter 3) and premature CL regression (Greenstein et al.,
1958; Wiltbank, 1966; Stormshak et al., 1960; Caldwell et
al., 1972; Eley et al., 1979). As anticipated, luteolytic
doses of E2 were ineffective in hysterectomized (Brunner et
al., 1969; Akbar et al., 1971; Caldwell et al., 1972) and
indomethacin-treated (Warren et al., 1979; Barcikowski et
al., 1974) cattle and sheep. In another study (Loy et al.,
1960), E2 administration to cattle on day 1 or 4 of the
estrous cycle had no effect on CL examined on day 14.
However, treatment with caused a reduction in CL weight,
P4 content, and number of functional luteal cells.
Administration of exogenous P^ for 1 to 5 days post-estrus
in cattle and sheep results consistently in shorter
interestrous intervals (Woody et al., 1967; Ginther, 1968,
1969; Thwaites, 1971; Lawson and Cahill, 1983), presumably
as a result of premature development and activation of the
uterine PGF2a secretory system (Baird et al., 1976; Ottobre
et al., 1980). These data suggest that the uterus requires
a period of P4 exposure in order to develop the capacity to
secrete luteolytic levels of PGF2a in response to

55
estrogens. For example, in the cow (Skjerven, 195b;
Martinov and Lovell, 1968) and ewe (Brinsfield and Hawk,
1973) the degree of endometrial lipid droplet accumulation
varies cyclically, being more prevalent during man E2
dominated phases of the estrous cycle. Additionally,
exogenous stimulated accumulation of endometrial lipid
droplets while E£ administration decreased lipid stores
(Brinsfield and Hawk, 1973). Finally, Hansel et al. (1975)
extracted large quantities of arachidonic acid, the
principle substrate for PGF2a synthesis, from bovine
endometrium between days 10 and 14 of the luteal phase.
Collectively, these results support the concept that P^
directs endometrial accumulation of lipid and prostaglandin
precursor concentrations during the estrous cycle.
Estrogens induce the luteolytic response during late
diestrus.
Timing and magnitude of the uterine luteolytic response
are dependent on circulating patterns and concentrations of
P4 and E2 which, in turn, regulate the degree of uterine
sensitivity to ovarian steroids, viz., endometrial P4 and E2
receptor populations. Estrogen is known to stimulate
synthesis of endometrial estrogen and P4 cytoplasmic
receptors (Rc) (Clark et al., 1977; Koligian and Stormshak,
1977a). Conversely, P4 inhibits synthesis of E-Rc and
reduces nuclear retention of the estrogen-receptor complex
in endometrial tissue (Clark et al.,
1977; Koligian and

BtormshaK, 1977a; Katzenellenbogan et al., 1930).
Progesterone also reduces endometrial synthesis of its own
receptor (Schrader and O'Malley, 1973; Walters ana Ciar*:,
1930). With this in mind, direct measurements of steroid
receptors in bovine and ovine endometrium revealed elevated
concentrations of E-Rc during proestrus and metestrus, and
low E-Rc concentrations during diestrus (Senior, 1975;
Koligian and Stormshak, 1977b; Henricks and Harris, 1978).
Additionally, Zelinski et al. (1982) demonstrated a decline
in bovine endometrial P4~RC concentrations from the
proestrus to diestrus phases of the estrous cycle. Thus, as
the CL develops and plasma E2:P^ ra’*:^os decline,
concentrations of endometrial E-Rc fall precipitously.
Plasma thereby inhibits the uterus from responding in an
estrogen-directed fashion. However, elevated plasma P^
concentrations progressively suppress endometrial production
of its own receptor such that estrogen-induced events (i.e.,
prostaglandin biosynthesis) are gradually removed from P^
inhibition.
Induction of uterine PGF2a production by E2 may be
attributed partially to E2-dependent increases in activities
of cyclooxygenase (Huslig et al., 1979) and phospholipase A2
(Dey et al., 1982). Calcium is probably involved in
activating phospholipase A2 since this enzyme is Ca+^
dependent (Brockerhoff and Jensen, 1974). Estrogen was also

57
shown to increase uterine Ca+^ availability in swine
(Geisert et al., 1932).
Estrogen-induced luteolysis is prevented by inhibition
of uterine DNA dependent RNA synthesis with actinomycin 0
(French and Casida, 1973) suggesting that the process of
PGF2a production requires protein synthesis. Furthermore,
Roberts et al. (1976) and McCracken et al. (1931, 1984)
provided evidence for estrogen induction of endometrial
oxytocin receptors following a period of P^ priming in
sheep. Exogenous oxytocin stimulates uterine PGF2a
production in cyclic cattle (Newcomb et al., 1977; Milvae
and Hansel, 1980) and sheep (Roberts et al., 1975, 1976;
McCracken, 1981, 1984) and causes premature CL regression in
uterine-intact (Armstrong and Hansel, 1959; Hansel and
Wagner, 1960; Milne, 1963), but not hysterectomized (Ginther
et al., 1967) or indomethacin-treated (Cooke and Homeida,
1983) females. In addition, luteal function was extended in
ewes immunized against oxytocin (Sheldrick et al., 1980;
Scharas et al., 1983). Based on these results and reports
concerning secretion of oxytocin and P4 by the CL (reviewed
earlier), McCracken and coworkers (1981, 1934) proposed a
working hypothesis for CL regression in cyclic ewes.
Briefly, their proposed sequence of events are: 1)
declining endometrial P4 receptor population reduces P4
dominance on the uterus during late diestrus and permits
follicular E2 to stimulate endometrial estrogen and oxytocin

53
receptor formation and activate enzymes essential for
biosynthesis; 2) circulating luteal (or pituitary) oxytocin
binds to newly synthesized endometrial receptors and
initiates PGF2a secretion; 3) plasma P^ concentrations
decline slowly, further reducing its influence on the
uterus; 4) uterine-derived PGF^ induces a rapid dumping of
luteal oxytocin which reinforces uterine PGF2a production
and 5) PGF2a initiates a rapid decline in plasma P^
concentrations associated with CL demise.
Available data indicate that PGF2a is the primary
uterine luteolysin in numerous species (Horton and Poyser,
1976). However, recent data implicate additional
arachidonic acid metabolites in the luteolytic process. For
example, the 13,14-dihydro metabolite of PGF2a was as
effective as equivalent amounts of PGF2a in causing CL
regression in heifers (Milvae and Hansel, 1983).
Arachidonic acid metabolites of the lipoxygenase pathway may
also be physiological regulators of luteal lifespan (Milvae
et al., 1985). Lipoxygenase products, such as 5-hydroxy-
eicosatetraenoic acid (5-HETE), specifically inhibit luteal
synthesis of prostacyclin, a potent luteotropin (Milvae and
Hansel, 1980, 1983), thereby reducing luteal P4 production,
in vitro. When in vitro synthesis of 5-HETE and other
lipoxygenase products was blocked by nordihydroguaiaretic
acid NDGA, luteal cell prostacyclin and P4 production were
increased without influencing PGF2a production. Further,

59
twice daily intrauterine adrainistration of NDGA to cyclic
heifers on days 14 to 13 extended luteal function
approximately 5 days beyond control heifers. Ihese data
suggest that lipoxygenase products, of uterine and/or
ovarian origin, are involved in the luteolytic process. in
a final study, Milvae and Hansel (1985) provided evidence
that uterine and/or ovarian prostaglandins are required for
normal CL development and function in cattle. Intrauterian
administration of indomethacin to cyclic heifers on days 4,
5 and 6 caused decreased production and early CL
regression. The authors implied that luteotropic
prostaglandins, such as PGE2 and prostacyclin, may be
involved in luteal development.
Conceptus-Associated Events During
Early Pregnancy
An opportunity for establishment of pregnancy is
provided with each estrous cycle. As a consequence of
luteal P^ production, glandular epithelium of the uterine
endometrium develops morphological/ultrastructural
characteristics, and functions of an active secretory tissue
(Skjerven, 1956b; Marinov and Lovell, 1968; Wathes and
Wooding, 1980). Several studies have demonstrated increased
endometrial tissue content or uterine secretion of amino
acids, proteins, fatty acids, lipids, carbohydrates, and
certain ions during the luteal phase of the estrous cycle or

ÓO
in response to exogenous P4 (cow: Skjerven, 195ba; Fanning
et al., 1967; Carlson et al., 1970; Roberts and Parker,
1974; Hansel et al., 1975; Bartol et al., 1931; sheep:
Brinsfield and Hawk, 1973; Roberts and Parker, 1976; pig:
Murray et al., 1972; Knight et al., 1974; Bazer, 1975; Bazer
et al., 1977; human: Shapiro et al., 1980). In addition,
elevated P4 concentrations reduce uterine tone and
myometrial contractility (Hays and Van Demark, 1953; Lehrar
and Schindler, 1974). Thus, during the first 15 to 16 days
post-estrus, a complex embryotrophic environment is
established within the uterine lumen.
During early pregnancy there is a clear requirement for
developmental synchrony between the conceptus and
endometrium. Embryo transfer studies in cattle and sheep
indicate that a small degree of asynchrony (±1 or 2 days)
is tolerated with respect to stage of transferred conceptus
and recipient uterine development (Rowson and Moor, 1966;
Rowson et al., 1969; Sreeman, 1978; Seidel, 1981). However,
pregnancy success is highest when synchronizetion is precise
(Cook and Hunter, 1978).
As described earlier, P4 administered early in the
estrous cycle is thought to initiate premature development
and activation of the uterine PGF£a secretory system (Baird
et al., 1976; Ottobre et al., 1980). This system was
utilized recently to evaluate the role of P4 pretreatment,
early in the estrous cycle, on the embryonic state of the

61
ovine uterus (Lawson and Cahill, 1933). Progesterone
administered during the first 4 days of the ovine estrous
cycle accelerated development of the uterine luminal
environment such that the day 6 P^-treated uterus provided
acceptable support for normal development of transferred ID
day old embryos. Luteolysis had not occurred in 8 of 12
ewes slaughtered on day 25* No day 10 conceptuses survived
following transfer to control (no P^ pretreatment) uteri on
day 6. Estrous cycles in these ewes were of normal
length. These data suggest that uterine development and
complexity of its luminal environment progress sequentially
under the influence of P^. Further, the conceptus must
develop in synchrony with this environment to survive.
The inability of embryos to develop beyond the
blastocyst stage, when cultured in vitro (Wright and
Bondioli, 1931) or confined within the oviduct
(Wintenberger-Torres, 1956; Murray et al., 1971; Pope and
Day, 1972; Heap et al., 1979), illustrate that unique
components of the uterine environment become essential for
further conceptus development. The essentiality of this
relationship may be due, in part, to increased complexity of
conceptus nutrient and hormonal requirements following
blastulation (Daniel, 1971; Biggers and Borland, 1976).
Additionally, Lawson et al. (1933) demonstrated an active
interaction between the uterine environment and rate of
conceptus development in sheep. In two experiments,

62
sixteen-cell embryos (day 4) were transferred to uteri of
ewes on days 4, 6 or 7 of the estrous cycle. The embryos
were then recovered and evaluated 4 or 6 days later.
Embryos transferred to older, more advanced uteri were
viable when recovered (days 8 to 12 of the estrous cycle)
and exhibited accelerated growth rates during the 4 to 6
days in útero as compared to synchronously transferred
controls. A third experiment examined conceptuses on days
12, 14 or 15 of the estrous cycle following synchronous
versus asynchronous embryo transfers, as described above.
Results indicated that viability and accelerated growth
rates of asynchronously transferred embryos were halted by
day 11 or 12 post-estrus. Further, these conceptuses were
unable to maintain CL function in recipient ewes.
Similarly, P^ has been implicated in regulating ovine
conceptus growth during normal pregnancy (Bindon, 1971) and
in superovulated recipients of embryo transfers
(Wintenberger-Torres, 1968; Wintenberger-Torres and
Rorabauts, 1968). Presumably, P4 effects on embryonic growth
are mediated by alterations in the uterine luminal milieu.
Collectively, these data demonstrate that the conceptus is
capable of responding to physiochemical cues in its uterine
environment or conversely, the uterus exerts a regulatory
influence on some aspects of conceptus development during
early pregnancy. Either of these viewpoints may explain how
small degrees of developmental asynchrony between the uterus

63
and conceptus are tolerated in large domestic species
(ñowson and Moor, 1966; Rowson et al., 1969; Sreenan, 1978;
Seidel, 1931). However, there appears to be a developmental
limit, beyond which the conceptus may not "catch up" to Its
uterine environment and prevent luteolysis (Lawson et al.,
1933).
Regardless of this physiological flexibility, a
considerable percentage of embryos do not survive beyond day
30 of pregnancy in large domestic species. In cattle, the
majority of embryonic mortality occurs between days 16 and
26 of pregnancy (Hawk et al., 1955; Boyd et al., 1969;
Ayalon, 1978; Ball, 1978; Hawk, 1979). Dramatic changes in
bovine conceptus growth, differentiation (Melton et al.,
1951; Chang, 1952; Greenstein and Foley, 1958a, b;
Greenstein et al., 1958; King et al., 1982) and endocrine
activity (Shemesh et al., 1979; Chenault, 1980; Lewis et
al., 1982; Eley et al., 1983; Bartol et al., 1985) occur
during this relatively short period. Pregnancy success
between days 16 to 30 of gestation depends upon 1)
appropriate physiochemical communication between conceptus
and endometrium such that CL maintenance is achieved, and
2) the initiation of placental (cotyledon and caruncle)
development and function. "Maternal recognition of
pregnancy" (Short, 1969), occurring by days 16 to 17 in
cattle (Betteridge et al., 1980; Northey and French, 1980;
Dalla Porta and Humblot, 1933), and day 12 in sheep (Moor

64
and Rowson, 1966; Rowson and Moor, 1967) and swine (Dhinasa
and Dziuk, 1968), represents the first period during early
gestation when production of conceptus signals becomes
essential for luteal maintenance and continued endometrial
secretory support. The conceptus does not appear to be
essential for CL maintenance or initiation of an embryonic
uterine environment prior to maternal recognition of
pregnancy. This was clearly demonstrated by Betteridge et
al. (1980) when pregnancies were established following
synchronous embryo transfers as late as day 16 in cyclic
cattle. Additionally, noticable histological alterations in
endometrial development between pregnant and cyclic cattle
do not occur prior to day 17 (King et al., 1981, 1982).
Thus, pregnancy associated events occurring prior to
maternal recognition of pregnancy may be thought of as being
mediated primarily by the CL and endometrium, whereas events
following this period are under direction of the conceptus.
Conceptus Development (Days 15 to 30)
As noted earlier, developmental phases including
maternal recognition of pregnancy througn definitive
placentorae formation occur between days 16 and 30 of
gestation in cattle. The large degree of embryonic
mortality during this period attests to the critical nature
of these developmental processes with regard to
establishment and maintenance of pregnancy.

65
On day 1b of gestation, the bovine conceptas is
characteristically bilaminar (trophectoderm and encoders),
with more advanced conceptuses exhibiting varying degrees of
mesodermal germ layer development (Chang, 1952; Greenstein
and Foley, 1958 a, b). Trophectodermal and endodermal germ
layers initiate a phase of rapid elongation by days 15 to 16
of pregnancy. Lengths of extraembryonic membranes vary
tremendously during this period (1.5 to 225 mm; Hawk et al.,
1955; Greenstein and Foley, 1958b; Betteridge et al., 1980)
with mean diameters calculated between 50 and 90 ram.
Histological and ultrastructural evaluation of rapid
and progressive transition of porcine conceptus forms, viz.
spherical, tubular and elongated filamentous, between days
10 and 12 of gestation (Geisert et al., 1982) provided
evidence that rapid conceptus elongation occurs as a result
of trophectodermal and'endodermal reorganization and not
hyperplasia. Whether or not this phenomenon occurs during
conceptus elongation in the cow or sheep is not known.
By day 17, mean conceptus extraembryonic membrane
length reaches approximately 150 mm (Chang, 1952; Greenstein
and Foley, 1958b). Histochemical evaluation of
trophectodermal cell types (days 16 to 33; Greenstein et
al., 1958) based on staining characteristics with Sudan
Black B or Oil Red 0 (demonstration of lipids), Periodic-
Acid-Schiff (PAS; demonstration of glycogen, glycoproteins),
and phloxine-methylene blue (demonstration of cytoplasmic

66
basopnilia or acidophilia) delineated three distinct rypes
of cells by day 16 and 17* These included: 1) PA3 (+),
trophoblastic giant cells (GC) which were occasionally
binucleated at this stage; 2) undifferentiated tropho'olast
"stem cells"; and 3) normal columnar trophoblast cells which
consistently contained basally located lipid inclusions.
The embryo proper, viz. germinal or embryonic disc, is
notably oval in shape by day 17 and demonstrates signs of
initiating organogenesis, as indicated by the appearance of
the primitive groove and node. Additionally,
ultrastructural appearance of day 17 endometrial epithelium
was indistinguishable in pregnant versus cyclic cattle (King
et al., 1981), suggesting that no physical interaction
between conceptus and endometrium had occurred at this stage
of pregnancy.
Conceptus adhesion to maternal endometrium was noted to
occur by days 18 to 20 (Wathes and Wooding, 1980; King et
al., 1980, 1981), although no microvillous interdigitation
was observed. This physical interaction between the
conceptus and endometrium is consistent with the observed
reduction in uterine epithelial cell height by day 18 of
pregnancy (Wathes and Wooding, 1980; King et al., 1981) and
the apparent migration of binucleated trophoblastic GC into
the uterine epithelial cell layers (Wathes and Wooding,
1980). Extraembryonic membranes continue to elongate,
extending throughout the gravid and nongravid uterine horns

67
by days 19 and 20. In addition, numbers of binucleated GC
continue to increase, constituting as much as 2CG of the
total trophectodermal cell population on day 20 (v/athes and
Wooding, 1980).
Between days 18 and 20, amnionic formation is completed
and the allantoic diverticulum develops via an evagination
of embryonic hindgut splanchnopleure (Greenstein and Foley,
1958a; Greenstein et al., 1958). Conversely, the highly
vascularized yolk sac reaches its developmental peak by day
20 and begins regressing thereafter as the allantois emerges
(Greenstein et al., 1958). This period of development marks
a coordinated transition from conceptus-derived (chorio-
vitelline placenta) to maternal-derived (chorioallantoic
placenta) nutritional support.
Mutual microvillous interdigitation between
trophoblastic and endometrial epithelium is initiated by day
24 (King et al., 1980, 1981; Wathes and Wooding, 1980) while
definitive attachment occurs by day 27 (King et al.,
1980). Additionally, immature fetal cotyledonary villi are
apparent adjacent to maternal caruncles from approximately
days 27 to 30 of gestation (Greenstein et al., 1958; King et
al., 1981). Considerably more lipid accumulation was noted
in trophectodermal epithelium from day 27 onward (King et
al., 1980) suggesting that functional exchange of nutrients
from maternal to conceptus tissues was accelerated following
definitive attachment. There is a continued increase in the

68
number of bi- and raultinucleated GC reported in
trophectoderm and endometrial epithelial cell layers
throughout this period (Greenstein et al., 1958; King et
al., 1980; Wathes and Wooding, 1980). Multinucleated GG
were reported to comprise 25% of total endometrial
epithelial cell population and 50% of cell area in the
gravid uterine horn on day 24 (Wathes and Wooding, 1980).
Regression of the yolk sac is complete by day 24.
However, allantois expansion and vascularization are not
completed until approximately days 30 to 33 (Greenstein et
al., 1958). Diameter of the allantois ranges from 0.8 to
5«5 cm on day 24 to over 35 cm on day 33. Dramatic
expansion and vascularization of the allantois is initiated
between days 28 to 30 (Greenstein et al., 1958). Continued
expansion of allantoic fluid volume and membrane diameter
during the first two months of gestation forces
extraembryonic membranes into apposition with the
endometrium, thus facilitating development of firm
cotyledon-caruncle attachment (Eley et al., 1978).
Establishment of a vascularized chorioallantoic placenta
preceeds and is essential for promotion of substantial fetal
weight gains during later gestation (Eley et al., 1978).
Conceptus Signals and Maternal Recognition
of Pregnancy
A functional CL is required if pregnancy in cattle is
to persist. Maintenance of the CL during pregnancy depends
on the presence of a viable conceptus within the uterine

69
luman and its ability to appropriately "signal" a receptive
maternal system, such that processes mediating cyclic
regression of the CL are circumvented. In cattle, maternal
recognition of pregnancy (Short, 1969) must be initiated by
days 16 to 17 post-estrus if pregnancy is to become
established. For example, Betteridge et al. (1980)
demonstrated that synchronous embryo transfer to recipients
on or prior to day 16, but not day 17 post-estrus, resulted
in normal pregnancies. Additionally, nonsurgical embryo
removal from pregnant cattle on days 16 through 19 resulted
in extended CL maintenance and interestrous intervals (range
4 to 8 days) compared to nonmated controls (Northey and
French, 1980; Dalla Porta and Humblot, 1983). Embryo
removal prior to day 16 of pregnancy, viz. days 9, 13, 14,
and 15, had no effect on cycle length or occurrence of
luteolysis. These data provided clear evidence that CL
maintenance required the presence of a viable conceptus
within the uterine lumen of cattle by days 1b to 17 post-
estrus. Further data indicated that twice daily
intrauterine injections of homogenized day 17 or 18
conceptuses (Northey and French, 1980) or freeze-killed day
16 conceptuses (Dalla Porta and Humblot, 1980) on days 14
through 18 or days 15 through 19, respectively, also
extended luteal function. Northey and French (1980) noted
that intrauterine injections of conceptus homogenates did
not stimulate luteal P^ production, but extended the

70
interval to CL regression. Incrauterine administration of
two freeze-killed, day 12 embryos/injection had no effect on
CL lifespan (Dalla Porta and Humblot, 1983). Thus,
potential for bovine conceptus production of potent,
biologically active substances by day 16 of pregnancy was
supported, as was their involvement in prolongation of CL
function.
Subsequent studies in cattle suggested that there are
probably several interdependent strategies by which
conceptus signals may initiate maintenance of the CL during
early pregnancy. Potential effectors, or biologically
active signals, which are synthesized and secreted by bovine
conceptuses (days 13 to 27) include steroids (Shemesh et
al., 1979; Chenault, 1980; Gadsby et al., 1980; Eley et al.,
1983; Chapter 2), prostaglandins (Shemesh et al., 1979;
Lewis et al., 1982; Lewis, 1984; Lewis and Waterman, 1984),
and proteins (Masters et al., 1982; Bartol et al., 1985;
Chapter 4). This subject has been reviewed recently by
Thatcher et al. (1984a, b; 1985) with respect to maternal
recognition of pregnancy in cattle.
In several studies, higher plasma P4 concentrations
were detected in pregnant versus bred-nonpregnant or cyclic
cattle from as early as days 9 to 10 post-estrus (Henricks
et al., 1971; Ford et al., 1979; Lukaszewska and Hansel,
1930), suggesting a luteotropic role of the conceptus during
early pregnancy. However, pregnancy-related elevations in

71
concentrations were not supported by others (.Batson et
al., 1972; Folraan et al., 1973; Hasler et al., 1930).
Progesterone production by dispersed luteal cells was
stimulated by homogenates and aqueous extracts of day 18
bovine conceptuses (Beal et al., 1981). Progesterone
stimulatory activity of conceptus extracts was lost
following heating or removal by dialysis of components less
than a relative molecular weight (Mr) of 12,000 Daltons.
Similarly, the elongating ovine conceptus contains products
which stimulate luteal P^ production, in vitro (Godkin et
al., 1978). The product(s) responsible for this luteotropic
activity in ewes is not proteinaceous since 100 ug of
conceptus protein had no effect on synthesis of P^ or cyclic
AMP by dispersed ovine luteal cells (Ellinwood et al.,
1979). Furthermore, no LH-like activity was detected in
bovine (day 1b to 20; Henricks and Poffenbarger, 1984) or
ovine (day 14 to 15; Ellinwood et al., 1979) conceptuses as
demonstrated under bioassay and RIA conditions,
respectively. Thus the presumptive luteotropin(s) may be a
steroid or prostaglandin. Marsh (1970) demonstrated that
PGE£ stimulated bovine luteal production of P4 in vitro.
However, evaluation of in vivo PGE2 effects on bovine luteal
function and maintenance are not clearly indicated.
Prostaglandin-E2 administered into the uterine lumen alone
(Gimenez and Henricks, 1983; Chenault, 1983) or in
combination with E2 (Reynolds et al., 1983) extends luteal

72
function only slightly beyond cessation of intrauterine PGE2
treatments. No stimulation of luteal P^ production was
noted in vivo. Systemic concentrations declined within
12 h following cessation of PGE2 treatment (Gimenez and
Henricks, 1983) or during the treatment period (Reynolds et
al., 1983) suggesting that PGE2 (plus E2) will not prevent
production and transfer of uterine luteolytic substances,
but affects luteal function directly (Marsh, 1970; Henderson
et al., 1977; Reynolds et al., 1931). Others have reported
no effect of intrauterine PGE2 administration on CL
maintenance in cattle (Dalla Porta and Humblot, 1983;
Chenault et al., 1984). As in cattle, reports in sheep
indicate that luteal function may be prolonged with PGE2 for
only short periods beyond the time of normal luteolysis
(Pratt et al., 1979; Huie et al., 1981; Magnuss et al.,
1981). In sheep, numerous studies have demonstrated that
the CL becomes refractory to exogenous PGF2a during early
pregnancy (Inskeep et al., 1975; Mapletoft et al., 197bb;
Pratt et al., 1977; Silvia and Niswender, 1984) or following
PGE2 administration (Pratt et al., 1977; Henderson et al.,
1977; Reynolds et al., 1981). Furthermore, PGE2 is
synthesized by bovine (Shemesh et al., 1979; Lewis et al.,
1982) and ovine (Hyland et al., 1982; Lacroix and Kann,
1982) conceptuses and endometrium. Luteal protective
substances, originating from the gravid uterine horn in
cattle, were demonstrated by Del Campo et al. (1980). In

73
this study, uterine horns were isolated surgically and
embryos transferred to uterine horns contralateral or
ipsilateral to the CL containing ovary. In three or four
cases, pregnancies in the ipsilateral horn maintained CL
viability, whereas all pregnancies on the contralateral side
failed to maintain CL function. However, when the vein
draining the uterus of the contralateral, pregnant horn was
anastomosed surgically with the ipsilateral, nonpregnant
uterine venous drainage, luteal regression was prevented in
three of three cows. Thus, it was apparent that some blood-
borne product(s) of pregnancy was able to circumvent the
luteolytic effects of the nonpregnant uterine horn. Similar
findings were reported in the ewe (Mapletoft et al.,
1976b). These results supported previous data demonstrating
existence of a unilateral uteroovarian relationship (veno¬
arterial pathway) during early pregnancy (Del Campo et al.,
1977) and luteolysis (Ginther, 1974; Ginther et al., 1974)
in cattle. Collectively, data generated in cattle and sheep
suggest that conceptus (and/or uterine)-derived luteal
protective substances provide the CL with some resiliency
against PGF2a-induced luteolysis during early pregnancy.
Evidence supports PGE2 as a possible luteal protective
product of conception.
Movement of substances to and from the pregnant uterus
and ipsilateral ovary may be regulated, to some degree, by
blood flow rates and intraovarian vascular hemodynamics

74
(Lamond and Drost, 1974; Ford and Chenault, 1930). During
early pregnancy (days 14 to 18) in cattle, blood flow to the
gravid uterine horn is transiently increased (Ford et al.,
1979; Ford and Chenault, 1980; Ford, 1982). Estrogens and
PGE2 are known to stimulate uterine blood flow in cattle
(Roman-Ponce et al., 1978; Thatcher et al., 1984b; Chapter
3). Elevated luminal concentrations of estrogen and PGE2 in
the gravid uterine horn (Bartol et al., 1981; Ford, 1982;
Lewis et al., 1982) and evidence supporting synthesis of
estrogens (Shemesh et al., 1979; Gadsby et al., 1980; Eley
et al., 1983; Chapter 2) and PGE2 (Lewis et al., 1982) by
bovine conceptuses during early pregnancy support a role for
these conceptus products in pregnancy-associated elevations
in uterine blood flow. Recent reports by Ford (1982) and
Ford and Reynolds (1983) suggested that hydroxylated
metabolites of estrogens viz., catecholestrogens, may
mediate uterine blood flow responses to follicular-(estrus)
and conceptus-derived (early pregnancy) E2. They propose
that an antagonistic interaction of catecholestrogens with
uterine vascular a-adrenergic receptors promote reduced
vascular contractility and facilitate elevated rates of
uterine blood flow. Additionally, increased endometrium
vascular permeability was suggested in pregnant ovine uteri
as evidenced by positive Pontamine Blue dye uptake (Boshier,
1970). Local induction of uterine blood flow and vascular
permeability by the conceptus may increase transfer

efficiency of luteotropic/luteal protective substances from
the pregnant uterus to the CL (Reynolds et al. , 1935;
Thatcher et al., 1984b, 1985). However, evaluation of
tritiated PGF2a uptake in bovine endometrium and
uteroovarian vascular components suggested that PGF2a
permeability may be decreased during early pregnancy
(Thatcher et al., 1984b). Tissue samples, viz.,
endometrium, ovarian vein and ovarian artery, were collected
on day 17 of pregnancy or the estrous cycle from the side
ipsilateral to the CL-bearing ovary. Data demonstrated
reduced PGF2a permeability in pregnant versus cyclic tissues
on day 17. Furthermore, ovarian vein permeability to PGF2»
was less than ovarian artery in pregnant cows, however, this
relationship was reversed in vascular samples collected on
day 17 of the estrous cycle. In pregnant tissues, responses
suggest that conceptus induced reductions in vessel PGF2 permeability were greater with regard to vessel proximity to
the pregnant uterus. It may be hypothesized that conceptus
products responsible for reduced PGF2a permeability would be
more concentrated in the uterine venous drainage (ovarian
vein) versus ovarian artery, thus resulting in the responses
observed (Thatcher et al., 1984b). Further studies are
required to determine if this pregnancy-associated reduction
in tissue permeability is specific for PGF2«• For example,
elevated PGF£ a and contents of uterine flushings from
pregnant versus cyclic cattle (Bartol et al., 1981; Lewis et

76
al., 1982) may suggest sequestration within the pregnant
uterine lumen.
Another strategy by which the conceptus maintains CL
function involves attenuation of PGF2ct production by the
uterine endometrium. In one of several studies, reviewed by
Thatcher et al. (1984a, b; 1985), in vitro production of
PGF2a by day 17 pregnant endometrial explants was reduced by
more than 50% of PGF2a produced by day 17 cyclic endometrial
explant cultures. Production of PGE2 was similar for
pregnant and cyclic endometrium. Endometrial prostaglandin
synthesis was stimulated following addition of exogenous
arachidonic acid to cultures, however, relative proportion
of prostaglandins (PGF2 PGFM and PGE2) within and between
pregnancy status were unchanged. These data support an
antiluteolytic effect of the bovine conceptus on endometrial
PGF2C1 production. Levels of arachidonic acid cascade which
appear to be modified during early pregnancy involve phos¬
pholipase A2 (i.e., exogenous arachidonic acid stimulated
prostaglandin synthesis) and cyclooxygenase (i.e., PGF2ct
production was attenuated in pregnant endometrium with and
without the addition of arachidonic acid). Although PGE2
production did not differ between pregnancy status, it is
clear that the ratio of PGE2:PGF2a must become elevated with
suppression of PGF2a production during early pregnancy.
Such an increase in the PGE2:PGF2a ratio may be of
physiological importance. In vivo evaluation of temporal

77
PGF£a episodes in the ovarian arterial supply of cows from
days 17 to 20 of the estrous cycle or pregnancy (Thatcher et
al., 1984b) supported in vitro results demonstrating a
reduction in PGF2a during pregnancy. No differences were
observed in frequency of PGF2a episodes between pregnancy
status. However, plasma concentrations of PGF2a during
episodic release were attenuated dramatically in pregnane
cattle.
Peripheral patterns of PGFM concentrations were used as
an index of uterine PGF2a episodic production during the
bovine estrous cycle (Peterson et al., 1975; Kindahl et al.,
1976), early pregnancy (Kindahl et al., 1976), and following
embryo transfer on day 16 post-estrus (Betteridge et al.,
1984). Luteal regression, during the estrous cycle and
following embryonic mortality, as determined by declining
plasma P^ concentrations, was always associated temporally
with three to five large episodes of PGFM. Conversely, PGFM
episodes are reduced or completely eliminated in early
pregnancy. Similar observations were also reported during
the estrous cycle and early pregnancy in buffaloes (Batra
and Pandey, 1983). The 11-ketotetranor metabolites of PGF2a
have been evaluated recently in 24 hour, pooled urine
samples of cyclic and pregnant cattle (Harvey et al., 1984;
Plante et al., 1984). Although detection of acute patterns
of uterine prostaglandin production are not possible using
this system, clear elevations of 11-ketotetranor PGF2a
are

73
temporally associated with a decline in plasma P^
concentrations during luteolysis. Peaks of tne urinary
metaoolite are absent during early pregnancy, however, oasal
concentrations begin a gradual increase as pregnancy
proceeds beyond 13 to 20 days. Similarly, day 17 pregnant
heifers had higher basal concentrations of PGFM than
nonpregnant heifers (Williams et al., 1933). It is probable
that conceptus prostaglandin biosynthesis during early
pregnancy contributes to elevated urinary and plasma
metabolites of PGF2a. Conceptus prostaglandin production
increases wirn gestational age in cattle (Lewis et al.,
1982). It should be noted, however, that elevations in
baseline prostaglandin concentrations during early pregnancy
are not characteristic of luteolytic pulses detected during
CL regression (Thatcher et al., 1984a). Additional support
for conceptus-derived anti-luteolytic effects in cattle was
reported following E2~induction of uterine PGF2a production
on day 18 of the estrous cycle, and day 18 and 20 of
pregnancy. The capacity of the uterus to produce PGF2a
(peripheral plasma PGFM) in pregnant cattle was reduced
significantly from day 18 cyclic cattle responses (Thatcher
et al., 1984b). Reduction in E2~induced PGFM response was
more dynamatic in day 20 versus day 18 pregnant cattle
suggesting that as conceptus elongation proceeds,
progressively larger regions of endometrium become exposed
to conceptus antiluteolytic products, resulting in a graded

79
decline in total uterine capacity to produce PGF2a-
Additionally, duration of endometrial exposure co conceptus
antiluteolytic signals may be important in tnis regard.
Inhibition or reduction of uterine PGF2ct production and
secretory patterns may be the primary mechanism by which the
developing bovine conceptus ensures CL maintenance during
early pregnancy. Luteotropic and luteal protective agents,
permeability and blood flow alterations may act as secondary
or support systems to further reduce, dilute, or otherwise
protect CL from luteolytic effects of the uterus. This
argument is based on the inability of putative luteotropic
and luteal protective signals to maintain CL function for
extended periods in cyclic cattle. In contrast, recent
reports in cattle and sheep provide evidence that
antiluteolytic proteins of conceptus origin significantly
extend CL lifespan and function.
Data evaluating in vitro protein synthesis and
secretion by elongating conceptuses are available for cattle
(Masters et al., 1982; Bartol et al., 1985), sheep (Godkin
et al., 1982b; 1984a) and swine (Godkin et al., 1982a). It
becomes evident from these studies that qualitative and
quantitative changes in array of proteins produced are
related to stage of conceptus development. In all three
species, total conceptus protein production increases with
gestational age (Godkin et al., 1982a, b; Bartol et al.,
1985). This trend is similar to data reported for bovine

80
conceptus steroid (Shemesh et al., 1979; Chanault, 1980;
Gadsby et al., 1980; Eley et al., 1983; Chaptar 2) and
prostaglandin (Lewis et al., 1982) biosynthetic activity.
Additionally, apparent total protein production par rag of
conceptus wet weight, in cattle, becomes elevated as rapid
trophectodermal elongation is initiated between days 1b and
17 of gestation (Knickerbocker et al., 1984; Chapter 4).
During this period of conceptus development, a distinct, but
similar family of low Mr, acidic polypeptides constituted a
major portion of proteins secreted into medium (cattle: Mr
22-26,000, pi 5.6-6.5; Bartol et al., 1985; sheep: Mr 17-
21,000, pi 5«5; Godkin et al., 1982b; Martal et al., 1984b;
swine: Mr 20-25,000, pi 5.6-6.2; Godkin et al., 1982a).
Transient conceptus production of low Mr polypeptide species
during respective periods of conceptus elongation and
maternal recognition of pregnancy in cattle, sheep and swine
suggest that these products may be involved in early
pregnancy maintenance. Experimental evidence in the cow and
ewe support this hypothesis.
Rowson and Moor (1967) demonstrated that intrauterine
administration of day 14 to 15 ovine conceptus homogenates
prolonged estrous cycles in ewes. However, no effect on
estrous cycle length was observed following intrauterine
administration of day 25 ovine conceptus homogenates or heat
treated, day 12 to 14 ovine conceptuses. Similarly,
introduction of homogenates or extracts of day 14 to 16

31
ovine conceptuses into the uterine lumen resulted in
prolonged CL function and estrous cycles in eight cf 12 ewes
(Mortal et al., 1979)* Only one of six ewes exhioited
prolonged luteal function and interestrous interval when day
21 to 23 conceptus homogenates were administered. Pronase
or heat pretreatment completely eliminate the ability of day
14 to 16 conceptus homogenates to extend CL function in the
cyclic ewe. Subsequent characterization of protein
production patterns by elongating bovine (days 16 to 24;
Bartol et al., 1935), ovine (days 13 to 21; Godkin et al.,
1982b), and porcine (days 10.5 to 12; Godkin et al., 1982a)
conceptuses verified transient biosynthesis and secretion of
major, low Mr polypeptides during early pregnancy.
Collectively, these data imply that biologically active
proteins of conceptus origin are involved in processes
leading to maternal recognition of pregnancy.
Recent data demonstrated that pooled conceptus
secretory proteins (CSP), collected from medium of cultured
bovine (days 16 to 18; Chapter 4) and ovine (days 15 and 16;
Godkin et al., 1984b) conceptuses, substantially extend CL
function and interestrous interval following intrauterine
administration to cyclic cattle (days 15 through 21) and
sheep (days 12 through 21), respectively. In addition,
intrauterine administration of the prominent, low Mr, acidic
polypeptide, termed ovine trophoblast protein-1 (oTP-1;
Godkin et al., 1984b), also extended luteal function in

82
cyclic ewes. Thus, oTP-1 may be a primary protein signal
responsible for luteal maintenance in sheep. Conceptus
protein signals probably act locally at the level of uterine
endometrium. For example, immunohistochemical data suggest
that oTP-1 is secreted by extraembryonic trophectoderm of
the ovine conceptus and binds specifically to receptors
localized on uterine endometrial epithelium (Godkin et al.,
1984a). In the same report, oTP-1 was shown to stimulate
secretion of specific endometrial proteins in culture. In
cattle (Chapters 4 and 5) and sheep (Fincher et al., 1984;
Fincher, 1984), pooled CSP dramatically reduced spontaneous
uterine PGF2a episodes (Chapter 4) and E2~induced production
of uterine PGF2a (peripheral plasma PGFM response; Fincher
et al., 1984; Fincher et al.; Chapter 5). Collectively,
these data demonstrate a role for conceptus-derived
secretory proteins as signals during early pregnancy in
cattle and sheep. It is proposed that proteinaceous
conceptus signals interact with the uterine endometrium such
that endometrial capacity to synthesize and secrete PGF2a is
dampened. At this time, it is not known whether CSP
antiluteolytic effects are exerted directly or mediated by
some induced endometrial product (Godkin et al., 1984a).
Direct antiluteolytic effects of CSP may include regulation
of steroid and/or oxytocin receptor populations on
endometrium. In the rat, both E^ and P^ nuclear receptors
are increased at the site of blastocyst implantation on day

93
5 and ó of gestation (Logeat et al., 1990). Findley et al.
(1982) reported a decline in caruncular E£ receptors
associated with side of conceptus on day 15 of pregnancy in
ewes. Additionally, caruncular and intercaruncular
endometrial production of protein in pregnant ewes was
greater than nonpregnant endometrium. A reduction in
endometrial E£ receptors may prevent E2~induction of
oxytocin receptors and subsequent activation of PGF2
synthesis (McCracken et al., 1984). Replenishment of
endometrial receptors could also result in decreased
nuclear accumulation of E2 receptor. In this light, Fincher
et al. (1984) demonstrated that oxytocin-induced uterine
PGF2c( production in E2 primed ewes was decreased
significantly by intrauterine CSP administration.
Thatcher et al. (1984b) reported that day 17 pregnant
endometrial explants produced less PG^a than cyclic
endometrium in the presence or absence of exogenous
arachidonic acid. They suggested that both phospholipase A2
and cyclooxygenase activity was reduced during early
pregnancy. Wlodawer et al. (1976) and Shemesh et al. (1981,
1984) reported the presence of prostaglandin inhibitors in
bovine endometrium and placental tissues, respectively. It
is possible that induction of such inhibitors during early
pregnancy may involve conceptus protein signals. In such a
scheme, endometrial PGF2a biosynthesis may be selectively
inhibited (possibly at the level of phospholipase A2 and/or

84
cyclooxygenase) without influencing prostaglandin production
by the conceptus (Thatcher et al., 1985).
In cattle, low molecular weight conceptus polypeptides,
described by Bartol et al. (1985), may play key roles in
pregnancy maintenance as was demonstrated for oTP-1 in sheep
(Godkin et al., 1984a, b). Similarities in the nature and
function of conceptus protein signals in cattle and sheep
have been discussed. Relative to this discussion, Martal et
al. (1984a) provided evidence that conceptus signals
(presumably protein in nature) of cattle may be biologically
active in sheep and vice-versa. Two trophoblastic vesicles
(0.5 to 2 mm diameter), composed of extraembryonic
trophectoderm and endoderm (embryonic disc removal), from
day 13 bovine blastocysts were transferred to 11 ewes on day
12 of the estrous cycle. Two ewes exhibited prolonged
luteal maintenance until at least day 38 when ewes were
slaughtered. Interestingly, an elongated (>100 mm)
trophoblastic vesicle was recovered from the uterus in one
of these ewes, although conceptus membranes were undergoing
necrosis at this time. Similarly, when two trophoblastic
vesicles from day 11 to 13 ovine blastocysts were
transferred to ten cyclic cattle on day 12, two heifers
exhibited cycle lengths of 31 and 36 days. The authors
suggested that nonspecific conceptus signals in the cow and
ewe were sufficient for maintaining CL function and the

35
biologically active molecules responsible for CL maintenance
in these species may be very similar.
As discussed earlier in this review, conceptus
production of biologically active signals is closely
synchronized with conceptus elongation during maternal
recognition of pregnancy. Furthermore, precise
synchronization between development of the conceptus and
maternal uterine milieu are essential for successful
membrane elongation and signal emission by the conceptus.
Immunological rejection of transferred interspecie conceptus
membranes undoubtedly discourages this process (Allen, 1979;
Beer and Billingham, 1979; Rossant et al., 1982; Wielen and
King, 1984). In this light, observed extension of CL
function in 20% of cattle and sheep receiving interspecific
trophoblastic vesicle transfers (Martal et al., 1984a) seems
even more remarkable. Recent data (S.D. Helmer, W.W.
Thatcher, F.W. Bazer, P.J. Hansen and R.M. Roberts, 1984,
unpublished observations; see Thatcher et al., 1985) lend
additional support that conceptus antiluteolytic protein
signals may be similar in cattle and sheep. Antibodies
developed against oTP-1 (Godkin et al-, 1984a) were used in
an ouchterlony double immuno-diffusion system with
unfractionated bovine conceptus secretory proteins. Two
precipitin bands were formed against unfractionated bovine
coneptus secretory proteins, one of which showed partial
identity with either purified oTP-1 or unfractionated ovine

3ó
conceptus secretory proteins. Subsequently, an
imraunoprecipitate of tritiated bovine conceptus secretory
protein fraction which crossreacted with oTP-1 antiserum was
solubilized and analyzed by polyacrylamide gel
electrophoresis and fluorography. Immunoprecipitated oovine
conceptus secretory proteins were detected in the 20,000 and
22,000 Mr range. Collectively, these data suggest that
analogous mechanism of "Maternal Recognition of Pregnancy,
possibly involving the low Mr, acidic polypeptide species,
may exist in cattle and sheep.
The conceptus may also exert an indirect antiluteolytic
effect as a result of subtle suppression and acceleration in
folliculogenesis and atresia, respectively (Dufour et al.,
1984). Such a conceptus interaction may be important since
exogenous estrogens have been shown to stimulate uterine
PGFpct production in cattle (Chapter j>) resulting in luteal
regression (Greenstein et al., 1958; Wiltbank, 1966; Eley et
al., 1979). Estrogens may also act directly on the CL to
antagonize LH stimulated P^ production (Williams and Marsh,
1978). Conversely, CL function was extended following
destruction of ovarian follicles during the luteal phase of
the bovine estrous cycle (Villa-Godoy et al., 1981). Thus,
conceptus regulation of follicular development and hence,
estrogen production may complement the intrauterine
antiluteolytic efforts of the conceptus to suppress uterine
PGF2a synthesis during early gestation.

87
In conclusion, events associated with "Maternal
Recognition of Pregnancy" reflect dynamic as well as subtle
alterations in maternal and conceptus physiology. Numerous
putative conceptus signals have been identified, although
their precise roles in "Maternal Recognition of Pregnancy"
are just beginning to be understood. Maintenance of CL
function and an embryotrophic uterine milieu during early
pregnancy is accomplished by several interdependent,
conceptus-mediated strategies. Evaluation and clarification
of the complex interactions between conceptus and its
maternal host presents a considerable and exciting task for
the future.

CHAPTER 2
PATTERNS OF PROGESTERONE METABOLISM BY DAYS 19-23
BOVINE CONCEPTUS AND ENDOMETRIAL EXPLANTS
Introduction
Maternal recognition of pregnancy (Snort, 1909) in
cattle occurs by day 16 (Betteridge et al., 1930; Northey
and French, 1980; Dalla Porta and Humblot, 1983). During
this period the bovine conceptus initiates a phase of rapid
elongation (Hawk et al., 1955; Greenstein and Foley, 1958b;
Betteridge et al., 1980) and elevated endocrine activity
(Shemesh et al., 1979; Chenault, 1980; Lewis et al., 1982;
Eley et al., 1983; Bartol et al., 1985). Conceptus
endocrine products, in turn, direct events which are
essential for the establishment and maintenance of
pregnancy. Biosynthesis and possible mechanisms of action
for conceptus-derived prostaglandin and protein signals have
been discussed earlier (see: Literature Review). Recent
reports demonstrate the capacity for synthesis and
metabolism of various steroids by elongating bovine
conceptuses (Shemesh et al., 1979; Chenault, 1980; Gadsby et
al., 1980; Eley et al., 1983). Based on these studies it is
clear that small amounts of estrogen biosynthesis results
following conceptus incubation with androgen precursors
88

89
(androstenedione, A^; testosterone, T; dehydroepiandros-
terone, DHA). In addition, Shemesh et al (1979) reported
detectable amounts of immunoreactive P^, T and estradiol-176
(E2) in some bovine conceptus extracts on days 15 and 1b of
gestation. Total content of these steroids were elevated in
medium following 48 hour conceptus cultures in the absence
of exogenous precursors. Thus the bovine conceptus may
utilize C21 steroids, viz., and pregnenolone, as
precursors for androgen and estrogen production.
Conversely, Eley et al. (1983) were unable to demonstrate
tritiated-P^ (10 ng) conversion to estrogens when shorter
culture periods (3 to 6 hours) and less sensitive procedures
of identification (liquid column chromatography and
recrystallization) were utilized. Currently, no data are
available with regard to relative ratios of estrone (E-j), E2
and estriol (E^) production by the early bovine conceptus.
Indirect physiological data support conceptus estrogen
biosynthesis during maternal recognition of pregnancy.
Blood flow to the gravid uterine horn is transiently
elevated between days 14 and 18 of gestation in cattle (Ford
et al., 1979; Ford and Chenault, 1981). Uterine blood flow
during the bovine estrous cycle and early pregnancy are
highest during periods of low ?4:E2 ratios in plasma.
Furthermore, exogenous estrogens stimulate uterine blood
flow in cyclic cattle (Roman-Ponce et al.,
1978; Chapter 3).

90
The bovine conceptus possesses an extremely active 93-
reductase enzyme system (Chenault, 1980; Eley et al., 1989)
whereas the endometrium produces primarily 5«-reduced
steroids (Eley et al., 1983). Metabolites identified were
hydroxylated in an a-configuration at the 3 and/or 17 (A^
substrate) and 20 (P^ substrate) positions. Roles of 58-
reduced steroids during early pregnancy in cattle have not
been studied.
It was the objective of this study to characterize
patterns of metabolism to 58/«-reduced products and
estrogens by bovine conceptus and endometrial tissues during
early pregnancy and to identify major conceptus metabolites
for future evaluation of physiological roles during early
pregnancy (Chapter 4).
Materials and Methods
Materials
Minimum essential medium (MEM; GIBCO, Grand Island, NY)
supplemented with non-essential amino acids (GIBCO, Grand
Island, NY), antibiotic/antimycotic (GIBCO, Grand Island,
NY), 200 units of insulin/L (Sigma Chemical Company, St.
Louis, MO) and 1 g glucose/L (Fisher Scientific, Orlando,
FL) was used for rinsing and incubating conceptus and
endometrial tissues. All radiolabelied steroids were
purchased from New England Nuclear, Boston, MA. Tritiated

91
progesterone (P^; [1,2,b,7-^H(N)J, 97 Ci/mmola) was used as
substrate for tissue metabolism. Tritiated 53-pregnan-
3a,20a-diol (L1,2-^H(N)], 55 Ci/mmole) was utilized as a
radiolabelled marker to examine elution of this steroid with
respect to conceptus steroid metabolites in high pressure
liquid chromatography (HPLC) systems. Carbon-14-labe1led
marker steroids used were androstenedione (A^; [4-^C], 57-4
mCi/mmole), testosterone (T; [4-^C], 57 mCi/mmole),
estradiol-17 6 (; [4-^C], 57 mCi/mmole) and estrone (E-j ;
[4-^C], 57 mCi/mmole). Radiolabelled steroids were judged
to be greater than 95% pure based on liquid chromatography
or HPLC trials.
Several radioinert steroids (P^, A^, T, E>| , E£>
estriol, 5a/56-androstane-5,17-dione, 5a/5B-pregnane-3,20-
dione, 3a-hydroxy-56-pregnan-20-one) were used as markers
for unknown, tritiated P^ metabolite identification in HPLC
and gas/liquid chromatography (GLC) systems. All radioinert
steroids were purchased from Steraloids, Wilton, NH, with
the exception of 3a-hydroxy-50-pregnan-2O-one, which was a
generous gift from Dr. John R. Chenault of the Upjohn
Company, Kalamazoo, MI. Purity of radioinert steroids was
verified by HPLC and GLC analyses. All reagent grade
solvents (Fisher Scientific, Fair Lawn, NJ; Eastman Kodak,
Rochester, NY) were distilled before use. Acetonitrile
(HPLC grade; Fisher Scientific, Fair Lawn, NJ) and various

92
laboratory grade chemicals were used as received. Douola
distilled-deionized water was prepared in the laboratory.
Animals and Tissue Collection
Angus cows (n=11) were serviced naturally at observed
estrus (day 0) by an intact Brown Swiss bull and slaughtered
on days 19 (n=5), 21 (n=1), 22 (n=1) or 23 (n=4) post¬
mating. Reproductive tracts were excised following
exsanguination, sealed in a plastic bag, and placed on ice
while being transported to the laboratory. Uterine horns
were trimmed of excess tissue, and ovaries and oviducts
removed. Serosal and myometrial tissue layers of both horns
were gently peeled back along the mesometrial border until
the outside of the endometrial layer was visable. Care was
taken not to crush the uterine-contained conceptus or expose
the uterine lumen during this procedure. When the uterine
lumen was opened, conceptus tissue was removed with sterile
forceps. Conceptuses (n=11) were washed in 15 ml of culture
medium (MEM) before being transferred to culture flasks.
Endometrial tissue was collected from eight of these cattle
on days 19 (n=3), 21 (n=1) and 23 (n=4) of gestation.
Endometrium was dissected from both uterine horns with
Metzenbaum (curved) scissors and minced into 1 to 3 mnP
pieces. Minced endometrial tissue was rinsed in 15 ml of
MEM and approximately 500 mg (497 ±8.1 mg) transferred to
culture flasks.

93
Uteri from an additional seven Angus cows were
nonsurgica1ly flushed with sterile Dulbeccos' pnosphate
buffered saline (pH 7.4; Dulbecco and Vogt, 1954) on day 19
post-mating. Conceptuses (n=7) were recovered and
transferred to culture flasks within 15 minutes of
collection. It should be noted that these seven cows had
been injected with estradiol-178 (» 5 rag I.V.) on day 18
of pregnancy as part of another experiment (Thatcher et al.,
1984b). Conceptus metabolic results relevant to this point
are discussed later.
Tissue Incubations and Extraction of Steroids
Approximately 20 ng of [^Hj-P^ in organic solvent were
aliquoted into 25 ml flasks. Solvents were evaporated under
N2 gas, 3 ml of warm (37 C) MEM were added to each flask and
contents vortexed for several minutes. Whole conceptuses
and minced endometrial tissue (Table 2-1) were weighed and
placed individually into incubation flasks, gased with
Ü2:C02 (95:5) and gently rocked in a Dubnoff Metabolic
Shaking Incubator (Precision Scientific Group, Cnicago, IL)
at 37 C. Incubations were stopped at the end of 3 hours
with addition of 5 to 10 ml acetone to each flask.
Incubation flasks and contents were stored at -20 C until
steroid extraction. Approximately 5000 counts per minute
(cpm) of [^Cj-P^, [14c]_t and [^Cj-E^ markers were added
to flasks to account for procedural losses during extraction
and chromatography. Incubation flask contents were

94
•Table 2-1 . Tissue incubations
Day
Endometrium
Incubations
(no. cows)
Mean ± S.E.M.
Wet weight (mg)
19
5
(3)
499.5 ± 16.7
21-23
13
(5)
496.0 ± 9.2
TOTAL
18
(8)
497.0 ± 8.1
Day
Conceptus
Incubations
(no. cows)
Mean ± S.E.M.
Wet weight (mg)*
19
12
(12)
265.0 ± 22.5
21-23
6
(6)
358.0 ± 5.4
TOTAL
18
(18)
296.0 ± 18.9
* Pieces of conceptus tissue recovered from days 19 (n=2)
and 21 (n=1) pregnant cattle were not included in the wet
weight calculations; wet weights for these tissues were:
46.5 ± 2.5 and 53 mg, respectively.

95
transferred to individual, 50 ml, stainless steel centrifuge
tubes. Flasks were rinsed thrice with 10 ml of acetone,
each rinse being deposited into the appropriate sample
tubes. Tissues were then homogenized with a Brinkman
polytron (Westbury, NY). The polytron shaft was cleaned of
tissue fragments and rinsed (10 ml acetone) into sample
tubes. Samples were centrifuged at 18,000 x g for 20
minutes. Supernatant (5 ml MEM plus 40 ml acetone) was
poured into 125 ml separatory funnels. Tissue pellets were
reextracted from one to three times with 10 ml of acetone
each and centrifuged. Supernatants following reextraction
were combined with previous acetone extracts. Acetone was
evaporated under N£ gas leaving an aqueous MEM volume of 3
ml in each separatory funnel. Thirty milliliters of
methylene dichloride was added to each MEM fraction and
vigorously agitated for several minutes. Following complete
separation of the MEM (top layer) and methylene dichloride
(bottom layer) phases, the methylene dichloride fraction was
drained into glass conical tubes, dried under gas, and
stored at -20 C until liquid-column chromatography. Aqueous
MEM was collected into glass vials and 100 yl counted for
radioactivity.
Liquid-Column Chromatography
Sephadex LH-20 (Sigma Chemical Company, St. Louis, MO)
column chromatography was employed for initial separation of
conceptus and endometrial steroid extracts. The solvent

96
system consisted of a cyclohexane (CyH), benzene (Bz) and
methanol (MeOH) mixture (90:25:5)* Column dimensions were
12 x 1.5 cm. Procedures for preparation of Sephadex LH20
and pouring of columns are discussed in Appendix A.
Steroid extracts were reconstituted in 0.5 ml of
CyH:Bz:MeOH (90:25:5) and loaded carefully on the top of the
Sephadex LH20 bed. Collection of 1 ml fractions was begun
at this point. Sample containers were rinsed with an
additional 0.5 ml of the solvent mixture and applied to the
column immediately after the first extract had moved into
the column bed. A 1 ml solvent 'cushion' was then loaded
after which the glass column was filled with solvent mixture
and a 300 ml solvent reservoir placed in line with the
column to maintain a constant head pressure. Column flow
rates were adjusted and maintained between 6 and 8 drops per
minute (1 drop per 7 to 9 seconds). Eighty 1 ml fractions
were collected using an automated fraction collector (Model
328, ISCO, Lincoln, NE) followed by a single 40 ml fraction
collected at the end of each 80 ml run. One hundred
raicroliters of each 1 ml fraction was counted to evaluate
radioactivity elution profiles. Columns were washed with 50
to 100 ml of CyH:Bz:MeOH (90:25:5) between each sample run.
Steroid metabolites were divided into eight areas based
on peaks of tritiated radioactivity (Eley et al., 1983) and
relative elution to [^C]-markers (P^, T, E^).

97
Gas-Liquid Chromatography (GLC)
A Varian, model 2740, gas-liquid chromatograph (Palo
Alto, CA) was utilized to further separate and identify
radiolabelled metabolites. The column used was a 168 cm
Supelcoport, coiled glass column packed with
trifluoropropyl silicone on a Supelcoport 100/120 mesh (SP-
2401; Bellefonte, PA). Elution of radioinert steroids were
registered on a strip chart recorder following 10:1 sample
splitting and flame ionization detection. Nitrogen gas
pressure was maintained at 26.5 p.s.i., and hydrogen and air
flow rates were 33 and 300 ml/minute, respectively.
Temperatures for the column, injector and detector were 240,
262 and 265 C, respectively.
Prior to running samples on GLC, elution patterns of 42
radioinert steroids (Steroid Reference Collection, National
Research Council, London, England) were determined (Figure
2-1) to aid in the identification of unknown conceptus
metabolites. Steroid elution times and mobilities relative
to P4 (Rf/P^) are listed in Appendix B.
Approximately 5000 cpm of tritiated radioactivity in
single conceptus (n=3) areas 2, 3 and 5 (Sephadex LH20) were
solubilized in carbon disulfide and injected separately on
GLC with radioinert standards. One minute fractions were
collected by condensing radiolabelled compounds in cold,
'U'-shaped, glass collection tubes inserted over the column
exit port. Collection tubes (I.D., 2 mm) consisted of a 10

Figure 2-1. Retention times for radioinert steroids using gas liquid
chromatography. Steroids were donated from the Steroid Reference
Collection, International Research Council, London, England.

5/3- pregnone-
3/3, 20a-diol
5/3- pregnane -
3/9, 20/3-diol
5a - Androstane-
3/9, 17/3-diol
5a - Androslone-
3/9, l7a-diol
5a -Androstane^.
3a, 17/3-diol
5/3- Androstane-
3a, 17/9- diol
4- Androslene-.
3/9,17/3 -diol
5/3-Androstane-
3a, 17a-diol
5a-Androstane-
3a, l7a-diol —
5/3-Androstane-
3/3, 17/3-diol ~
3/3-hydroxy-4-
pregnen- —
20-one
5/3-Androstone
3/9,17a-diol
5/3- pregnane-
30, 20/9-diol
- 3/3-hydroxy-
5/9-Androstan
17-one
- 5a - pregnane-
30, 20a -diol
- 5a - pregnane-
3/3, 20/3 - diol
5/3- pregnane-
30, 20a-diol
. 5a- pregnane-
30 , 20/3-diol
3a - hydroxy-
’ 5a- Androstan-
17-one
5a -pregnane
3/3, 20a-diol
3a - hydroxy-
5/9-Androstan
17- one
3/3-hydroxy -
5a -Androstan
17-one
7a-hydroxy -
5/3-Androslan-
3-one
3/3-hydroxy-
5/3-pregnan-
20-one
17/3-hydroxy-
5/3 -Androstan -
3- one
17a -hydroxy-
5a - Androstan-
3-one
3a-hydroxy-
5a -pregnan-
20-one
17/3-hydroxy-
5a -Androstan-
3-one(DHT)
3a -hydroxy -
5/3-pregnan-
20-one
3/3-hydroxy-
5a - pregnan -
20- one
17a-hydroxy-
„ 4- Androstene
3-one(Epitestosterone)
¡Ti 7/3-hydroxy-4-Androstene
3-one (Testosterone)
5/3-Androstone-
3, 17-dione
5a-Andros!ane-
3, 17-dione
^5/3-pregnane-
3, 20- dione
20/3-hydroxy-
4-pregnen- 3-one
Flame Ionization 10:1 Splitter
3% SP-2401 (tri(luropropyl)
100/120 Mesh Supelcoport
Col Temp 240° C 5 '/z Col
4-pregnene-
3/3, 20/9-diol
5a - pregnane-
3, 20-dione
25 30
20 a-hydroxy -
pregnen-3-one
4-pregnene-
3, 20-dione
4 Androslene-
3, 17 dione
VO
VO
MINUTES

100
cm straight section followed by a 15 cm loop. Collection
tubes were rinsed with 5 ml acetone into glass scintillation
vials. Acetone was evaporated under N2 gas, 5 ml
scintillation fluid added to each vial, and vials counted
for radioactivity. Radioactivity elution patterns were then
collated with radioinert steroid chromatograms. Identical
procedures were used to identify conceptus (n=7) and
endometrial (n=4) steroid products following chromic
trioxide (CrO^) oxidation (Bush, 1961) of steroids in pooled
areas 1 through 6 (Sephadex LH20).
High Performance Liquid Chromatography (HPLC)
Further identification and verification of conceptus
and endometrial steroid metabolites were accomplished on
HPLC. Two HPLC systems were used during estrogen
determinations: 1) Waters Autosampler (WISP-710B; Waters
Associates Inc., Milford, PA) and variable UV wavelength
detector (275-280 model 450; Waters Associates Inc.,
Milford, PA); 2) Perkin Elmer Series 4 Liquid Chromatograph
and Spectrophotometrie Detector (275-280 \; model LC-85;
Perkin Elmer Inc., Norwalk, CT). Identical octadecylsilica
(ODS) columns (Radial Pack, C-18, 10 u; Waters Associates
Inc., Milford, PA) were used in both systems. Solvent
mixtures used in systems 1 and 2 were acetonitrile:H20
(45:55) and acetonitrile:H20 (55:45), respectively (Table
2-8). Estrogen fractions from Sephadex LH20 column

101
A A
chromatography ware pooled (viz., area of [ ^CJ-E-j elution
to end of column run) from single conceptos (n=9) and
endometrial (n=10) samples. Approximately 10,000 cpra of
tritium and 200 to 500 cpm of and E£ markers were
dissolved in 100 yl of acetonitrile containing radioinerc
E-|, E2 and E^ standards and injected for HPLC analysis.
Injection volumes were 20 to 50 yl and solvent flow rates
were between 1.5 and 2.0 ml/minute. Fractions were
collected in scintillation vials on an ISCO fraction
collector (model 528; Lincoln, NE) every 0.1 or 0.2 minutes;
5.5 ral of 25% Triton-X (Research Products International, Elk
Grove Village, IL) scintillation fluid were added to each
vial, and vials counted for radioactivity. Radioactivity
([^H] and [^C]) was then collated with chromatograms of
radioinert estrogen standards.
Production of neutral steroid metabolites were
evaluated on the Perkin-Elmer HPLC system, described
earlier. A 5 y guard column (RP-18 OD-GU; Brownlee Labs
Inc., Santa Clara, CA) and Perkin Elmer 3 y octadecylsilica
column (H3-3 C18; Perkin Elmer Inc., Norwalk, CT) were used
for separation of steroids. An acetonitrile and water
(54:46) solvent system was employed at a flow rate of 1.5
ml/minute. Fractions were collected every 0.2 minutes and
evaluated as described earlier. Radioactivity in single
conceptus (n=15) and endometrial (n=10) samples from
Sephadex LH20 areas 1 through 6 were pooled, oxidized and

102
injected on HPLC with [^Cj-A^ and markers, and
radioinert standard steroids, viz. T, A^, 5a/5s-
androstanedione, P^, 58-pregnan-3a-ol-20-one and 5a/5B-
pregnanedione. Similarly, radioactive metabolites were
evaluated in individual and pooled conceptus samples from
Sephadex LH20 areas 2 (n=15), 3 (n=15) and 5 (n=13).
Results
LH2Q Column Chromatography of Conceptus and
Endometrial Metabolites
Recoveries of [1^C] markers were calculated in 10
randomly selected samples of conceptus and endometrial
incubations to evaluate procedural losses of radioactivity
during extraction and LH20 column chromatography. Based on
[^C] radioactivity counted following LH20 chromatography,
recoveries (mean ± S.E.) for P^, T and E^ were 91.0 ± 5.2,
76.0 ± 3.2 and 83.5 ± 3*7 percent, respectively. Mean ±
S.E. elution (ml) of [1^C] markers was 9.7 ± 0.2 (P^; n=37),
31.5 ± 0.8 (T; n=10) and 47.5 ± 1.3 (E1; n=37).
Eight areas of [*Hj metabolite elution were defined
following column chromatography (Table 2-2). No differences
were observed between the metabolic patterns of endometrial
incubations from day 19 versus days 21 to 23 of pregnancy
(Table 2-3). Progesterone metabolism was incomplete with
35.4 ± 2.1 (n=17) percent of radioactivity remaining in the
substrate peak (area 2) after 3 hours of incubation. Major

103
Table 2-2. Elution of progesterone metabolites with
reference to ['4C]-estrone after sephadex LH20 column
chromatography (CyH:Bz:MeOH [90:25:5])
Areaa
N
Peak elution (ml)
Range (ml)
RfSlb
1
10
4.21
3-5
.088
2
10
9.42
8-11
.202
3
10
14.33
12-16
.305
4
10
19.80
17-24
.413
5
10
29.43
26-35
.639
6
10
38.32
36-44
.829
7
10
54.20
51-61
1.183
3
9
67.60
62-73
1.460
a Metabolite areas from bovine endometrium and conceptus
incubations
b Peak elution of [*H] metabolites relative to ["^C]-E^;
for estrone (E-j) = 1.000

Table 2-3. Percent distribution of bovine endometrial [^Hj-progesterone metabolites
following LH20 column chromatography (CyH:Bz:MeOH [90:25:5])
ENDOMETRIAL INCUBATIONS (DAY)
Area
1(19)
2(19)
3(19)
4(19)
5(19)
Day 19
X ± S.E.
6(21)
7(21)
8(21)
9(23)
1
0
5.2
0
2.2
0
1.5 ± 1.0
21.7
14.6
5.0
5.4
2
44.2
43.8
50.3
32.9
32.6
40.8 ± 3.5
20.8
35.2
49.4
35.9
3
21 .0
22.9
32.9
33.2
25.8
27.2 ± 2.5
21 .8
16.4
17.5
18.2
4
14.5
13.9
14.9
16.5
21.0
16.2 ± 1.3
11.2
12.2
18.2
21 .6
5
11.7
8.7
18.7
6.1
9.1
10.9 ± 2.1
12.3
9.3
7.7
9.6
6
1.4
2.1
3.3
3.5
3.5
2.8 ± 0.4
3.9
4.7
1.5
3.8
7
1 .2
0.7
3.3
1.3
1.6
1.6 ± 0.4
1.0
4.3
0.2
2.4
8
5.9
3.3
9.5
4.3
5.8
5.8 ± 1.0
7.4
3.3
0.4
3.0
Days 21-23
Area
10(23)
11(23)
12(23)
13(23)
14(23)
15(23)
16(23)
17(23)
X ±
S.E.
1
1.6
3.2
1.1
6.7
14.8
1.6
1.4
4.9
6.8
± 1 .8
2
37.9
32.9
38.9
17.9
26.7
35.2
39.8
26.7
33.1
± 2.4
3
22.3
20.7
19.8
24.5
19.7
21.1
17.4
21.1
20.0
±0.7
4
21.4
13.8
22.0
19.4
-
23.5
25.1
18.5
18.8
± 1 .3
5
12.4
16.9
13.1
-
-
9.7
15.3
12.8
11.9
± 0.9
6
1.6
5.5
1.9
-
-
2.4
7.6
5.4
3.8
± 0.6
7
0.7
1 .8
0.8
-
-
0.9
5.6
5.0
2.3
± 0.6
8
1.9
5.1
2.2
-
-
4.9
3.2
5.6
3.7
± 0.6
104

105
percentage of P4 metabolites elated in areas 5 (22.1 ± 1.5;
n = 17), 4 (18.0 ± 1.0; n=l6) and 5 (11.6 ± 0.9; n=15), with
less than 10 percent residing in areas ó through 8 (Table
2-3, Figure 2-2). In contrast, day 19 to 23 conceptus
tissues actively metabolized substrate (Table 2-4,
Figure 2-3) during the 3 hour incubation period. Extent of
P4 metabolism tended to be greater by day 23 (97.4 ± 0.2)
versus days 21 to 22 (91.2 ± 1.3) or day 19 (88.1 ± 1.7)
conceptus tissues (Table 2-4). Increased tissue mass of day
23 conceptuses (Table 2-1) may contribute to the apparent
increased P4 metabolic capacity at this stage. However,
tissue metabolic activity may be increased as well since
tissue fragments from two day 19 conceptuses (43 and 50 mg
wet weight) and a day 21 conceptus (53 mg wet weight)
metabolized 81.8 ±1.0 versus 93 percent of available P4
substrate, respectively (see Table 2-4, conceptus numbers 1-
19, 5-19 and 10-21). Progesterone metabolic profiles were
qualitatively different by day 19 versus days 21 to 23
conceptuses (Table 2-4; Figures 2-3 and 2-4). Most notable
was the shift in P4 metabolism to more polar steroids in
area 6, eluting just prior to [^CJ-E^. Chromatograms from
day 19 conceptus incubations exhibited a double peak of
radioactivity in area 6 containing 9.5 ±1.0 percent of
total eluted radioactivity. By day 23, area 6 contained
34.5 ± 4.7 percent of total eluted radioactivity with over
50% eluting in the earlier peak (Figure 2-4). In addition,

Figure 2-2.
Sample elution pattern of tritiated endometrial (day 19) metabolites
following a 3 hour incubation with tritiated progesterone. Sephadex LH2
column chromatography (CyH:Bz:MeOH [90:25:5])*

DPM X 10“
ro
END0METRIALC3HD-P4 metabolite elution profile
AFTER LH-20 COLUMN CHROMATOGRAPHY (CyH:Bz:MeOHll90:25:5D)

Figure 2-3. Sample elution pattern of tritiated conceptus (day 19) metabolites
following a 3 hour incubation with tritiated progesterone. Sephadex LH20
column chromatography (CyH:Bz:MeOH [90:25:5]).

CONCEPTUS C3HU-P4 METABOLITE ELUTION PROFILE
AFTER LH-20 COLUMN CHROMATOGRAPHY (CyH^Bz'MeOH L90:25:5U)

Table 2-4. Percent distribution of bovine conceptus [^^-progesterone metabolites
following LH20 column chromatography (CyH:Bz:MeOH [90:25:5])
CONCEPTUS INCUBATIONS (DAY)
Area
1(19)
2(19)
3(19)
4(19)
5(19)
6(19)
7(19)
8(19)
9(19)
Day
X ±
19
S.E.
1
5.2
11.0
18.3
12.7
6.2
12.7
12.2
8.2
15.1
11.3 ±
1.3
2
19.6
7.0
13.2
12.7
16.8
15.9
9.4
9.9
2.3
11.9 ±
1.7
3
40.5
50.8
38.1
36.5
43.2
38.4
52.8
57.4
47.5
45.0 ±
2.3
4
7.8
1.5
5.4
4.4
5.5
5.4
1.4
1.6
4.1
4.1 ±
.7
5
10.3
6.6
8.6
16.1
12.1
14.1
12.5
8.5
12.4
11.2 ±
.9
6
9.8
12.7
9.3
13.3
6.1
8.8
5.5
6.4
13.5
9.5 ±
1.0
7
4.9
3.7
3.8
2.1
6.1
2.2
4.1
2.1
2.5
3.5 ±
.4
8
1.9
1.2
3.4
2.2
4.0
2.5
1.4
1.8
2.6
2.3 ±
.3
Da^s 21-23
Area
10(21)
11(22)
12(23)
13(23)
14(23)
15(23)
X ± S.E.
1
5.8
3.5
9.6
3.7
8.0
9.6
7.7 ± 1 .9
2
7.0
10.6
3.3
2.7
2.3
2.3
5.1 ± 1.4
3
57.9
18.6
36.5
30.9
39.0
28.8
39.6 ± 6.5
4
-
11.6
4.2
9.3
6.8
5.1
7.8 ± 0.8
5
6.1
5.8
10.5
7.0
10.1
6.7
8.7 ± 1.5
6
14.2
31.1
23.9
46.4
26.8
41 .0
30.6 ± 4.4
7
4.9
11.0
3.7
1.0
0.7
0.8
3.0 ± 1.3
8
4.0
2.5
2.1
5.8
6.4
5.5
4.5 ± 0.7

Figure 2-4. Sample elution pattern of tritiated conceptúa (day 23) metabolites
following a 3 hour incubation with tritiated progesterone. Sephadex LH20
column chromatography (CyH:Bz:MeOH [90:25:5]).

100
90
80
? 24
o
x 20
2
£ '6
12
0
4
0
C0NCEPTUSC3h:]-P4 metabolite elution profile
AFTER LH-20 COLUMN CHROMATOGRAPHY(CyH:Bz:MeOH C90:25:5l])
hH-2-4-3 h-4—I 5—I 6 1 7 1 8 1

113
definitive peaks of radioactivity in area 4 were not
observed in day 19 conceptus incubations but were
consistently present following conceptus incubations on days
22 and 23.
GLC of Conceptus and Endometrial Metabolites
Base structures of conceptus (n=7) and endometrial
(n=4) steroid metabolites were determined following CrO^
oxidation and GLC analysis of pooled areas 1 through 6
(Tables 2-5 and 2-6). The oxidation procedure converted a
and 8 hydroxyl isomers at positions 3, 17 and 20 to keto-
groups. It should be noted that 17 hydroxylated progestins
would be converted to either or 5a/56-androstanedione
following CrO-j oxidation (Bush, 1961). Loss of the C21
side-chain was verified in our system, as resulted from
oxidation of 17a-hydroxy-P^ (data not shown). Separation of
radioinert standards and their respective coeluting
radioactive metabolites are depicted in figures 2-5 and
2-6. Endometrial tissues metabolized P^ to predominantly
5a-reduced pregnane steroids which were converted to 5a-
pregnanedione following CrO-^ oxidation. Small amounts of
56-pregnanedione and A^ were also observed after oxidation
of endometrial metabolites. In agreement with results from
LH20 column chromatography, P^ was the major steroid present
in oxidized endometrial samples suggesting that metabolic
activity of endometrial tissue was less than that of the
conceptus. In contrast to endometrial metabolism, reduction

114
Table 2-5. Percent distribution of radioactivity eluted
with standard steroid peaks after CrO^ oxidation and gas
chromatography of endometrial metabolites
Endometrium (Day)
Metabolite3
1 (19)
2(19)b
5(21)
4(25)
X ±
S
. E.c
56 -A
0.17
0.50
0.15
0.50
0.27
±
0.11
5a-A
0.45
1 .00
0.50
0.80
0.51
±
0.15
5B-P
5.00
o
o
•
ro
11 .10
9.10
7.75
±
2.44
5a -P
27.10
52.70
27.20
50.80
28.57
±
1.22
a4
5.80
2.80
2.55
1 .90
5.55
±
1 .25
P4
41 .00
18.40
49.60
55.40
42.00
±
4.15
Total
77.50
88.60
90.70
78.50
82.25
±
4.24
a A = androstanedione; P = pregnanedione; = andro-
stenedione; P^ = progesterone
b 21 hour incubation
c x±S.E. does not include endometrium 2(19)

Table 2-6.
after CrO^
Percent
oxidation
distribution of radioactivity eluted
and gas chromatography of conceptus
with standard steroid ]
metabolites
peaks
Metabolite3
Conceptus (Day)
S
. E.c
1(19)
2(19)
3(19)
4(19)b
5(21)
6(22)
7(23)
X ±
5 3-A
3.5
2.8
3.5
8.3
3.1
3.0
1.3
2.83
±
0.32
5«-A
5.9
3.8
6.6
12.5
2.3
6.0
2.5
4.52
±
0.77
58-P
59.9
67.4
42.1
32.4
27.6
15.2
14.9
37.85
±
9.17
5 a-P
9.9
5.5
3.8
2.6
7.7
16.1
12.3
9.22
±
1.85
A4
1.3
1 .6
3.1
1 .8
5.1
7.4
12.5
5.17
±
1 .74
P4
4.1
5.7
3.7
12.6
13.0
12.2
15.5
9.03
±
2.09
Total
84.4
86.8
65.9
70.2
58.8
59.9
59.0
69.13
±
5.32
3 A = androstanedione; P = pregnanedione; = androstenedione; = progesterone
3 21 hour incubation
c Mean±S.E. does not include Conceptus 4(19)
115

Figure 2-5* Elution of endometrial steroid metabolites on gas liquid chromatography
following CrO^ oxidation.

PERCENT RADIOACTIVITY ELUTED WITH STANDARD
STEROIDS AFTER GAS/LIQUID CHROMATOGRAPHY OF ENDOMETRIAL METABOLITES

Figure 2-6. Elution of conceptúa steroid metabolites on gas liquid chromatography
following Cr05 oxidation.

DPM
PERCENT RADIOACTIVITY ELUTED WITH STANDARD
STEROIDS AFTER GAS/LIQUID CHROMATOGRAPHY OF CONCEPTUS METABOLITES
70
51 48 39 36 33 30 27 24
MINUTES

120
of P4 by conceptus tissues resulted in formation of
predominantly 50-pregnane derivatives. Lesser amounts of
radioactivity eluted with 5a-oregnanedione, A4 and 5a/53-
androstanedione. Although sample numbers are too small to
make any firm conclusions, it was interesting to note that
percent of 50-pregnane derivatives appeared to decline in
conceptus incubations from day 19 to 23 (Table 2-6; 56.5 ±
6.1 versus 19.2 ± 3.4). This trend was associated with a
slight increase in 5a-pregnane derivatives (6.4 ±1.5 versus
12.0 ± 2.0).
Limited data were generated from GLC analysis of
radiolabelled steroid metabolites in areas 2, 3 and 5 of
three day 19 conceptus incubations (Table 2-7). The major
percent of radioactivity (38.3) in area 2 (n=1) eluted at 4
minutes, prior to the earliest eluting GLC standard (Figure
2-1; Appendix B). Progesterone and 50-pregnanedione were
also major steroids in this area. Small amounts of A4 were
also detected. Area 2 contained 45% of total eluted
radioactivity after steroid separation on Sephadex LH20
columns (Table 2-4). Over 60% of the radioactivity in area
3 (n=3) coeluted with 50-pregnan-3a-ol-2O-one on GLC.
Nearly a third of radioactivity in area 5 (n=2) coeluted
with the GLC standard, 50-pregnanediol. Although L^Cj-T
markers eluted in area 5 on LH20 column chromatography, no
[3HJ -T was observed in these samples. These data, although
based on limited observations, provide preliminary

Table 2-7. Gas liquid chromatography of major conceptus steroid metabolites from LH20
column chromatographic areas
Area
(N)a
GLC Standard (minutes)
% [3h]
X ± S.E.
2
(1)
Unknown
(4)
38.3
38.3
5 a-Androstañedione
(22)
2.2
2.2
5 g-Pregnanedione
(26)
16.8
16.8
5 a-Pregnanedione
(28)
4.5
4.5
Androstenedione
(32)
4.0
4.0
Progesterone
(42)
17.3
17.3
3
(3)
5 g-pregnan-3 a-ol-20-one
(14)
55.5,68.2,60
61.2 ± 3.0
5 g-pregnanedione
(26)
3.9,2.3,3.6
3.3 ± 0.4
5
(2)
5 g-pregnane-3 a, 20 a-diol
(10)
32.3,26.9
29.6±1.9
a
Individual day 19 bovine conceptuses

122
indications as to the identity of major steroids synthesized
by the bovine conceptus relative to their elution and
sephadex LH20 columns (CyH:Bz:MeOH [90:25:5]).
HPLC of Conceptus and Endometrial Metabolites
Procedures followed during identification of conceptus
(n=15) and endometrial (n=10) steroid metabolites on HPLC
were similar to those used for GLC analyses. Radioinert
steroid standards were used as markers to evaluate base
structures of oxidized radioactive metabolites. Retention
times for radioinert and ["*^C]-steroid markers used in HPLC
analyses are listed in Table 2-8. Nearly 40 percent of
total eluted radioactivity from oxidized endometrial
incubations were collated with P^ standards (Table 2-9).
The major oxidized metabolite was 5a-pregnanedione (28.8 ±
2.3%). No major differences were observed between days 19
(n=4) and 21 to 23 (n=6) endometrial metabolic patterns,
although percent radioactivity eluted with 5“-pregnanedione
declined slightly, but increased in 5B-pregnandione
fractions as gestational age increased. Little radio¬
activity coeluted with P^ standards in oxidized conceptus
samples (table 2-10), attesting to the high metabolic
activity of conceptus tissues. Approximately 45 percent of
radioactivity eluted with 58-pregnanedione standards. Days
19 (n=9) and 21 to 23 (n=6) oxidized conceptus metabolite
patterns differed in percent of radioactivity accumulated in
56-pregnanedione and 5ct-androstanedione fractions. As

123
Table 2-8. Retention times of steroid markers on HPLC
SYSTEM Aa
Mean ± S.E.
Radioinert Steroids
N
Retention (min)
Testosterone
45
2.01±.003
Androstenedione
44
2.39±.005
5 8-Androstanedione
43
2.88±.005
5a-Androstanedione
41
3•46±.009
Progesterone
46
4.83+.010
5 S-Pregnan-3a-ol-20-one
45
5.89±.013
5 8-Pregnanedione
45
7.08±.016
5a-Pregnanedione
45
7.90±.022
Radioactive Steroids
[^C]-Androstenedione
37
2.55±.015
['C]-58-Pregnane-3a,20a-
[ ' 4c]-Progesterone
diol 10
4.70±.015
37
5.05±.017
SYSTEM Bb
SYSTEM Cc
Mean ± S.E.
Mean ± S.E.
Radioinert Steroids N
Retention (min)
N
Retention (min)
Estriol 38
2.43±.006
18
2.37±.004
Estradiol-178 38
4.87±.017
18
8.35±.010
Estrone 38
5.85±.023
18
12.09±.018
Radioactive Steroids
Estriol
18
2.56±.016
Estradiol-178 10
5.00±.035
18
8.56±.014
Estrone 39
6.03±.020
18
12.24±.019
a Columns: 5v guard column (RP-18 OD-GU; Brownlee Labs.
Inc., Santa Clara, CA) and 3v octadecylsilica column (HS-3
C18; Perkin Elmer Inc., Norwalk, CT); solvents: aceto¬
nitrile : water (54:46); isocratic, 1.5 ml/minute flow rate.
Columns: 10u octacecylsilica column (Radial Pack; Waters
Associates Inc., Milford, PA); solvents: acetonitrile:
water (55:45); isocratic, 1.5 ral/minute flow rate.
c Columns: (same as b); solvents: acetonitrile:water
(45:55); isocratic, 2 ml/minute flow rate.

Table 2-9. Percent distribution of radioactivity eluted with standard steroids after
CrO^ oxidation and HPLC of endometrial metabolites
Day 19
a4
5e-A
5a-A
UNK
P4
53-P
5 a-P
88-1
0
1.0
2.8
8.71
42.73
5.28
31.33
88-2
3.07
1.82
5.0
7.5
31 .1
2.56
23.54
6/55-1
1.05
1 .02
2.0
4.3
42.55
0
43.47
6/55-2
1.14
1.17
2.68
4.3
38.09
1.76
44.04
Mean
1 .32
1.25
3.12
6.2
38.62
2.4
35.6
±S.E.
0.55
0.17
0.56
0.97
2.36
0.95
4.3
Days 21-23
A4
5g-A
5 a-A
UNK
P4
5B-P
5a-P
8/131-1
1.47
0.47
2.66
3.3
48.02
9.32
33.6
96-2
3.4
2.4
6.11
7.43
15.68
8.32
28.2
8/24-1
0
0
2.6
5.15
61 .08
6.01
15.04
8/24-2
0
0
5.1
4.8
46.16
11.99
27.05
75-4
2.72
1.79
4.21
9.8
33.31
7.13
20.1
75-5
2.22
2.22
5.78
8.0
28.7
6.34
21 .3
Mean
1 .64
1.15
4.41
6.41
38.83
8.19
24.22
i: S • E •
0.53
0.42
0.57
0.90
6.01
0.83
2.48
Overall Mean
1.5
1 .2
3.9
6.3
38.7
5.9
28.8
±S. E.
±.4
±.3
±.5
±.7
±3.7
±1.1
±2.9
Abbreviations: (androstenedione), 5 3-A (5 3-androstanedione) , 5“-A(5“-
androstanedione), UNK (unknown), P^ (progesterone), 5 B-P (5 6-pregnanedione) , 5 a-P (5 a-
pregnanedione).
124

Table 2-10. Percent distribution of radioactivity eluted with standard steroids after
CrO^ oxidation and HPLC of conceptus metabolites
Day 19
a4
5B-A
5a-A
P4
3“-0H
50-P
5a-P
88
4.51
2.8
11.07
1.59
9.67
40.91
4.17
6/55
2.75
2.59
13.2
1.0
12.95
47.31
1 .9
71
4.13
2.98
10.29
2.07
8.03
39.2
2.19
8/24
1.74
2.37
3.07
1.91
5.04
65.26
4.35
8/34
2.33
2.36
10.31
2.55
3.87
55.95
5.18
6/95
4.65
2.91
6.99
3.11
6.11
48.54
5.03
5/195
3.43
5.14
5.61
4.6
7.8
52.46
7.07
1/80
3.41
3.42
5.13
2.97
7.92
51 .05
4.26
9/96
2.91
2.49
7.96
1.81
11 .13
45.32
6.28
Mean
3.32
3.01
8.18
2.40
8.09
49.56
4.49
±S. E.
0.31
0.27
1.03
0.33
0.91
2.49
0.53
Days 21-23
A4
5 0-A
5 a-A
P4
3 «-0H
5 0-P
5 a-P
8/173 "
5.1
6.69
30.49
2.77
5.38
25.6
2.07
8/131
1.19
3.69
21 .14
2.12
9.23
41 .96
1.0
8/24
1 .61
2.81
13.65
2.55
8.46
43.0
2.05
96
3.75
8.06
14.29
2.98
9.13
40.46
3.71
75
3.94
5.94
11 .56
2.07
10.38
40.84
4.01
5/195
1.0
5.1
27.25
1.43
8.22
35.97
1 .9
Mean
2.77
5.38
19.73
2.32
8.47
38.0
2.4o
±S.E.
0.64
0.72
2.92
0.21
0.63
2.43
0.43
Overall Mean
3.10
3.96
12.80
2.37
8.22
44.92
3.68
±S.E.
±0.32
±0.45
±1 .97
±0.22
±0.60
±2.31
±0.44
Abbreviations:
A. (androstenedione) , 5 0-A
(5 0-androstanedione) ,
5 a-A(5 a-
androstanedione), P.
(progesterone),
3 a-OH
(5 0-pregnan-
3 a-ol-20-
one), 5 0-P
(5 0-
pregnanedione)
, 5 Ctr-P
(5 a-pregnanedione) .
125

126
observed in GLC analyses, 50-pregnanedione associated radio¬
activity decreased as gestational age of the con-
ceptus increased (Table 2-10). Conversely, percent
radioactivity eluting with 5a-androstanedione on HPLC
increased by approximately 10 percent in days 21 to 23
versus day 19 conceptus incubations. Incomplete oxidation
of 58-pregnane metabolites was suggested since 8 percent of
radioactivity eluted with 5B-pregnan-3a-ol-20-one
standards. This result may suggest that a major percent of
the 50-pregnane metabolite pool consisted of 5S-pregnan-3a-
ol-20-one (Tables 2-7 and 2-11). High performance liquid
chromatographic analyses of metabolites in separate LH20
column chromatographic areas 2, 3 and 5 were performed on
pooled days 19 (n=9) and 21 to 23 (n=6) conceptus
incubations (Table 2-11). Results are similar to data
generated on GLC (Table 2-7). Conceptus area 2 was composed
largely of P4, 50-pregnanedione and 5B-pregnan-3a-ol-20-
one. Radioactivity eluted with 5 B-pregnan-3oc-ol-20-one
probably represented residual spillover from conceptus area
3 from LH20 column chromatography. Two regions of radio¬
activity were not associated with radioinert HPLC standards
(unknown 1 and 2). The earlier unknown eluted with the void
volume. Elution times of [-%]-5S-pregnane-3a,20a-diol
standards, evaluated in separate HPLC runs, were very
similar to elution time of the latter unknown. However, it
is doubtful that unknown 2 radioactivity in area 2 samples

Table 2-11. HPLC of major conceptus steroid metabolites from LH20 column
chromatographic areas; % of total eluted radioactivity
UNK1
A4
POOLED
56-A
DAY 19
5a-A
CONCEPTUSES
UNK2 P4
3«-0H
50-P
5a-P
AREA 2 (n=9)
11 .8
3.0
2.5
6.0
15.8
18.7
17.1
20.0
5.2
AREA 3 (n=9)
11.0
1 .2
1 .7
5.2
4.0
7.9
61.5
5.8
0
AREA 5 (n=7)
34.5
0
5.3
5.9
8.21
0
34.3
3.7
0
OXIDIZED
AREA 5 (n=7)
4.2
0
6.1
7.6
0
0
11 .4
64.3
0
POOLED DAYS 21-23 CONCEPTUSES
AREA 2 (n=6)
13.3
3.6
2.8
10.7
13.3
22.0
14.6
15.1
4.5
AREA 3 (n=6)
12.0
0
2.8
6.1
7.6
6.7
55.6
6.1
0
AREA 5 (n=6)
19.1
0
3.1
5.3
11.3
0
50.7
5.4
0
OXIDIZED
AREA 5 (n=6)
0
0
2.5
4.9
0
0
15.5
69.9
0
Abbreviations: UNK-1 (early eluting unknown), A. (androstenedione), 5^-A (5^-
androstanedione), 5a-A(5a-androstanedione), UNK-2 (late eluting unknown), Pa
(progesterone), 3 a-OH (5 B-pregnan-3 o-ol-20-one) , 5 &-P (5 8-pregnanedione) , 5^-P (5 a-
pregnanedione).
127

128
represent 58-pregnane-3a,20a-diol since this standard elutes
in area 6 and 7 when run on LH20 columns (CyH:3z:MeOH
[90:25:5]). Small percentages of and 5s/5a-reduced
androgens were also present in pooled area 2 samples. The
predominant (approximately 60%) radioactive conceptus meta¬
bolite in area 3 coeluted with the 5S-pregnan-3a-ol-20-one
standard on HPLC. This is in agreement with GLC data
described earlier (Table 2-7). Pooled area 5 radioactivity
eluted largely with 5S-pregnan-3a-ol-20-one standards and in
unknown regions 1 and 2. Radioactivity in unknown 1 eluted
in a broad region from 0.6 to 2.2 minutes on HPLC. A por¬
tion of unknown 1 radioactivity eluted in the region of
radioinert T standards. Oxidation of the 176-hydroxyl group
on T would yield (Bush, 1961). Therefore, area 5 was
oxidized to evaluate possible T production by conceptus
tissues. No radioactivity eluted with A^ standards
following oxidation suggesting that T was not a component of
radioactivity in area 5 (unknown 1). Additionally,
radioactivity in unknown regions 1 and 2 appeared to be
converted to 5S-pregnanedione following CrO^ oxidation. No
changes in percent radioactivity eluting with 5a/5g-reduced
androgens was observed. Thus, radioactive metabolites in
unknown regions 1 and 2 appeared to be composed of
hydroxylated 56-pregnane metabolites.

129
Conceptus Estrogen Production (HPLC Analyses)
Conceptus (n=15) and endometrial (n=10) estrogen
production was evaluated in pooled estrogen fractions, viz.,
from [^Cj-E-j elution to approximately 120 ml, collected
following LH20 column chromatography. Retention times for
radioinert and [^4C]-estrogen markers used in HPLC analyses
are listed in table 2-8). No estrogens were identified from
endometrial incubations, however a major peak of
radioactivity (48.7 ± 5.2%) eluted between E^ and E£
standards. A similar, although smaller, peak of
radioactivity eluted between E^ and E£ standards in
conceptus incubations (Figure 2-7).
Definitive peaks of [^Hj-metabolites coeluted with
[14C]- and radioinert E^, E£ and E-^ markers in all conceptus
incubations (Table 2-12; Figure 2-7). Estradiol-178 appears
to be the primary estrogen synthesized from substrate
with lesser amounts of E^ and E^ being produced. Mean ±
S.E. of total estrogen production was 44.12 ± 5*47
pg/conceptus incubation (Appendix C). No differences in
conceptus estrogen production were noted between days 19 and
21 to 23 of gestation.
Discussion
Results of the present study support previous data
which indicated a differential metabolism of neutral
steroids (P4 and A4) by bovine conceptus and endometrial

Table 2-12. Bovine conceptúa estrogen production; HPLC determinations
Conceptus
No.
Wet wt. (mg)
Day
E3
(pg total)3
e2
(pg total)
El
(pg total)
Total
Estrogens
(Pg)
88
312
19
1 .54
5.83
1 .02
8.39
9/96
207.6
19
12.0
9.83
9.13
30.96
71
284
19
N.D.
12.14
N.D.
12.14
6/55
291.3
19
1.53
23.30
2.27
27.1
5/195
140
19
1.28
44.52
1.94
47.74
8/24
43
19
2.61
54.32
3.46
60.39
8/34
50
19
1.97
73.20
2.85
78.02
6/95
159
19
2.06
65 • 65
2.31
70.02
1/80
255
19
3.11
88.80
2.29
94.2
Mean
193.54
2.9
41 .95
2.81
47.66
±S.E.
32.02
1.1
9.6
0.81
9.5
75
53
21
3.72
28.89
1 .88
34.49
8/173
364.7
22
1 .07
34.83
2.19
38.09
96
336
23
5.83
13.00
22.25
41 .08
8/24
356.2
23
3.39
31.74
4.83
39.96
5/195
370.6
23
1.15
32.11
4.73
37.99
8/131
360.5
23
1.10
35.84
4.35
41.29
Mean
306.8
2.71
29.40
6.70
38.81
±S. E.
46.56
.73
3.13
2.88
0.95
Overall Mean
238.83
2.82
36.93
4.37
44.12
±S. E.
30.35
.73
6.10
1 .34
5.47
a
See Appendix C for mass calculations
130

Figure 2-7. Sample elution pattern of conceptus (day 23) estrogens analyzed by high
performance liquid chromatography (acetonitrile:water, 45:55).

ESTRIOL
UNKNOWN
estradiol-170
HPLC/CONCEPTUS
[l^Cl MARKER
I ^111 SAMPLE
MINUTES

133
tissues, viz., concsptus 56-reouctase activity versus
endometrial 5a-reductase activity (Chenault, 1930; Sley et
al., 1983). In addition, GLC and HPLC analyses of conceptus
and endometrial P^ metabolism demonstrated production of
small quantities of and 5ct/58 reduced androgens.
Conceptus and endometrial incubations also contained small
quantities of 5a and 56-reduced progestins, respectively.
This apparent 5a/58 reduction of P^ by conceptus and
endometrial tissues, respectively, may represent endogenous
reductase enzyme activities or result from cross¬
contamination of conceptus and endometrial tissues or
cells. Endometrial 56-reductase activity, as determined by
GLC and HPLC analyses of oxidized samples, appeared to
increase slightly after day 19 of gestation. During this
period, conceptus-derived binucleated giant cells are known
to migrate and fuse with endometrial epithelial cells such
that by day 24 of gestation multinucleated giant cells were
reported to comprise 25% of the total endometrium epithelial
cell population and 50% of cell area in the gravid uterine
horn (Wathes and Wooding, 1980). Based on this evidence, it
may be suggested that endometrial 58-reductase activity is a
result of conceptus cell migration into this tissue.
Reduction of P4 to 5a-androstane and pregnane derivatives
appeared greatest in later stage conceptuses in the present
study. This was most evident when total 5a based steroids
were evaluated in oxidized samples. Our results conflict in

134
this respect with observations of Chenault (1980) and Eley
et al. (1983), however, these authors evaluated 5u/5s
reduction in specific chromatographic fractions as opposed
to total oxidized conceptus pools in the present study.
Increased 5a-reductase activity may reflect conceptus
developmental changes, viz., embryo organogenesis, allantois
expansion, yolk sac regression, vascularization of the
chorioallantois (Greenstein and Foley, 1958a,b; Greenstein
et al., 1958). Endometrial cell contamination of conceptus
incubations seems doubtful since firm attachment between
these tissues does not occur until day 27 (King et al.,
1980). However, this possibility cannot be excluded.
Identification of 58-pregnan-3a-ol-20-one as a major P^
metabolite in conceptus incubations agrees with findings of
Eley et al. (1983). Over 40 percent of radioactivity
following conceptus incubations with eluted in a
single sharp peak comprising area 3 on LH20 column
chromatography. Sixty percent of radioactivity in this peak
coeluted with 5S-pregnan-3a-ol-20-one on GLC and HPLC.
Thus, conceptus tissues produced nearly 5 ng of this steroid
during 3 hour incubations with [-^Hj-P^ (20 ng). In
addition, day 21 to 23 conceptus incubations metabolized P^
to more polar compounds eluting as a double peak in area 6
(30 percent of total radioactivity). Unfortunately,
metabolites in this area were not evaluated in the present
study. However, Eley et al. (1983) identified

135
5B-pregnane-3a,20a-diol as a major concaptus metabolite of
P4. It is possible that area 6 radioactivity represents
dihydroxylated pregnane and androstane derivatives in the
present study. Production of more polar compounds as
conceptus age increases may be due to increased tissue mass
and tissue activity, as suggested by data in the present
study and Eley et al. (1933). Based on proportions of 5g-
pregnanedione, 5B-pregnan-3a-ol-20-one and 5g-pregnane-
3a,20a-diol identified in the present study and by Eley et
al. (1983), it may be suggested that sequential P^
metabolism occurs by 58-reduction (5B-reductase) and rapid
3a-hydroxylation (3a-hydroxysteroid dehydrogenase; HSD),
followed by 20a hydroxylation (20a-HSD) in later stage
conceptuses.
The present report consistently demonstrated production
of E^| , Eg and Ej following bovine conceptus tissue
incubations with P^. The ability of bovine conceptus
tissues to utilize C21 steroids as precursors for estrogen
biosynthesis was suggested by Shemesh et al. (1979).
However, Eley et al. (1983) was unable to demonstrate
estrogen production using [-^Hj-P as substrate. Sensitivity
of HPLC techniques employed to isolate estrogens in the
present study may be superior to methods used by Eley et al.
(1983), viz., column chromatography, phenolic extraction,
acetylation and recrystallization, thus explaining the
apparent discrepancies in our two reports. Extradiol-17b

136
was the primary estrogen identified from days 19 to 23
conceptus incubations in the present study. This is
interesting in light of observations during later gestation
in cattle when production predominates (Eley et al.,
1979a; Robertson and King, 1979)*
Functions of conceptus 53-reduced steroids and
estrogens during early pregnancy in cattle are not known.
However, Chenault (1980) and Eley et al. (1983) have cited
evidence suggesting that these steroids may be involved in
conceptus development processes and alterations in maternal
uterine physiology. For example, 53-reduced steroids have
been shown to stimulate erythropoiesis (Gordon et al., 1970;
Grass and Goldwasser, 1972; Singer and Adamson, 1976),
hemoglobin biosynthesis (Necheles and Rai, 1969; Levere et
al., 1967), and activity of ¿-aminolevulinic acid
synthetase, the rate limiting enzyme of porphyrin-heme
biosynthesis (Edwards and Elliott, 1975). Thus, 53-reduced
steroids may play a role in development of the conceptus
circulatory system during early pregnancy (see: Greenstein
and Foley, 1958a,b; Greenstein et al., 1958; Grimes et al.,
1958). Kubli-Garfias et al. (1979) demonstrated that 53-
reduced progestins were several times more potent than in
reducing uterine myoraetrial contractions, whereas 5“-reduced
progestins were less effective than in this regard.
These 53-steroids may decrease myometrial contractility via
regulation of myometrial Ca+^ availability during early

137
pregnancy (Crankshaw et al., 1979)* Human mammary tumor 56-
reductase activity was negatively correlated with estrogen
receptor concentrations in that tissue (Abul Hajj, 1979)-
In addition, Findlay et al. (1982) reported that decreased
caruncular E2 receptor concentrations in ovine umeri were
associated with location of conceptus tissues within the
uterus. Estrogens stimulate uterine PGF2a production
(Chapter 3) and initiate luteolysis (Eley et al., 1979b) in
cattle. This process may be prevented during early
pregnancy by conceptus 58-steroid effects on endometrial E2
receptor populations. Conversely, 58-reductase activity by
conceptus tissue may be a means of eliminating excessive
aromatizable substrate or otherwise reducing quantities of
active steroids within the uterine lumen.
Initiation of bovine conceptus estrogen biosynthesis
(Shemesh et al., 1979; Chenault, 1980; Gadsby et al., 1980;
Eley et al., 1983) coincides with transient elevations in
uterine blood flow during early pregnancy. Furthermore,
exogenous estrogens are known to stimulate uterine blood
flow in cyclic cattle (Roman-Ponce et al., 1979; Chapter
3). Catecholestrogens, hydroxylated metabolites of
estrogen, were suggested to mediate the E2-induced uterine
blood flow response (Ford, 1978, 1982; Ford and Reynolds,
1983)* These estrogen metabolites also induce production of
receptors in the rat uterus (Kirchhoff et al., 1983)-
Thus, conceptus derived estrogens and estrogen metabolites


CHAPTER 3
UTERINE PROSTAGLANDIN AND BLOOD FLOW RESPONSES
TO ESTRADIOL-176 IN CYCLIC CATTLE
Introduction
Free and conjugated estrogens shorten lifespan of the
corpus luteum (CL) in cattle if administered during the
luteal phase of the estrous cycle (Greenstein et al., 1958;
Wiltbank, 1966; Eley et al., 1979). Luteolytic activity of
estrogen is thought to be mediated through stimulation of
uterine prostaglandin ?2a (PGF2a) synthesis and release.
The luteolytic activity of exogenous PGF2a in cattle has
been documented extensively (Hansel et al., 1973; Hafs et
al., 1974; Lauderdale, 1974; Thatcher and Chenault, 1976).
Elevated levels of PGFpa in uterine venous drainage
(Nancarrow et al., 1973; Sheraesh and Hansel, 1974), uterine
tissue (Shemesh and Hansel, 1975) and uterine flushings
(Lamothe et al., 1977; Bartol et al., 1981a) coincide with
the transient rise in follicular estradiol prior to
ovulation in cyclic cattle (Nancarrow et al., 1973; Chenault
et al., 1975). Collectively, these observations support the
concept that follicular estrogen secretion regulates onset
of luteolysis.
139

140
Peterson et al. (1975) and Kindahl et al. (197b)
reported elevations in peripheral plasma 13,14-dihydro-15-
keto-PGF2a (PGFM), the inactive metabolite of PGF£a» which
were temporally associated with CL regression in cyclic
heifers. Thatcher and coworkers (1979) also utilized mea¬
surements of PGFM in the peripheral circulation to charact¬
erize indirectly the uterine response to exogenous estradiol
in cyclic heifers. Concentrations of PGFM began to rise by
3 h, reached peak levels by 6 h, and returned to basal
values by 9 h post-estradiol administration. In addition,
estrous cycle length was reduced in estradiol-treated
heifers as compared with heifers treated with vehicle
alone. An additional study indicated that this increase in
peripheral plasma PGFM concentration was associated with an
elevation of PGF2a in uterine flushings collected at 6 h
after estradiol injection (Bartol et al., 1981b). Such a
response further supports the use of PGFM measurement in
peripheral circulation as an index of uterine PGF2a produc¬
tion.
In cyclic cattle, luteolysis is thought to occur via a
local, countercurrent exchange of PGF2a from the ovarian
vein, draining the uterine horn ipsilateral to the ovary
bearing the CL, into the adjacent ovarian artery (see
reviews: Baird, 1978; Ginther, 1981). Hixon and Hansel
(1974) presented evidence supporting such an exchange of
PGF2a from the uterine venous drainage into the ovarian

141
arterial supply following intrauterine deposition of PGF2a
in cyclic cattle. In a subsequent experiment (Shemesh and
Hansel, 1975), a countercurrent exchange of PGF£a ^as not
detected. However, only single samples were collected at
surgery and a more chronically sustained sampling regime is
warranted to test the countercurrent exchange concept.
Objectives of this experiment were 1) to examine
directly the bovine uterine response to exogenous estradiol-
178 (E2) by characterizing uterine venous concentrations of
PGF2a and PGFM, uterine blood flow (UBF) and uterine produc¬
tion of PGF2a and PGFM; 2) to describe the temporal rela¬
tionship between peripheral plasma PGFM concentrations and
uterine production of PGF2a and PGFM following ¿2 admini¬
stration; and 3) to examine the possible countercurrent
exchange of PGF2 ovarian arterial supply during the period of E2~induced
PGF2a production by the uterus.
Materials and Methods
Normal cyclic dairy cows (n=7) were used to charac¬
terize uterine responses to exogenous E2 injection. Animals
were prepared for surgery on day 17 of the estrous cycle
(day of estrus = day 0). Five grams of sodium thiamylal
(BIO-TAL, Bio-Ceutic Laboratories, Inc., St. Joseph, MO)
were dissolved in 40 ml of sterile saline and injected via

142
the jugular vein. An endotracheal tube was fitted and cows
maintained under general anesthesia with a flucthane
(Vescor, Orlando, FL) and oxygen gas mixture. The surgical
field was shaved, disinfected and covered with a sterile
drape. The reproductive tract was exposed following a mid-
ventral laparotomy and specific vessels (Ginther, 1976)
ipsilateral to the CL were catheterized: uterine branch of
the ovarian vein (with the catheter tip located 15 to 20 cm
into the main ovarian vein (0V, n=7) and uterine branch of
the ovarian artery (UBOA, n=5). Ideally, blood from the
uterine branch of the ovarian vein should be used for mea¬
suring uterine venous prostaglandin concentrations. How¬
ever, maintaining patency of the catheter in this vessel was
difficult. Since little ovarian prostaglandin contribution
was expected, the larger ovarian vein was utilized. Cathe¬
ters were secured within the vessels by ligation at the
point of catheter entry into the vessel. This prevented
blood flow past the point of catheter entry. An electro¬
magnetic blood flow transducer was placed and secured around
the main uterine artery (UA, n=3) or its primary branch
(n=2) both ipsilateral to the CL, and a catheter placed in
the facial artery (FA, catheter tip inserted into the
internal carotid artery, n=7). Blood flow transducers were
calibrated prior to surgery within a flow range of 0 to 300
ml/min using excised uterine arteries and heparinized bovine
blood (Roman-Ponce et al., 1978). A Narcomatic, Model

143
RT-500, blood flow meter was used with a Narco Desk Model
DMP-4B physiograph (Narco Biosystems, Inc., Houston, TX) to
monitor UBF continuously during the experiment. Ampicillin
(POLYFLEX, Bristol Laboratory, Syracus, NY) was administered
intra-abdominally (375 mg) and intramuscularly (875 mg)
prior to closure of the incision.
On day 18, blood samples were drawn concurrently from
the three catheters every 30 min, UBF monitored continuously
from 1 h pre- to 12 h post-S2 administration (3 mg in 6 ml
of an ET0H:0.9% NaCl [50:50] solution via jugular vein).
Blood samples were drawn into heparinized syringes and
placed immediately on ice until centrifugation at 12,000 x g
(within 15 min of collection). Plasma was stored frozen at
-20 C until assayed for PGF2a and PGFM. Blood flow trans¬
ducers were recovered following conclusion of the experiment
via flank laparotomy. Both placement and calibration of the
transducers were reconfirmed. One month following the
experiment, animals were slaughtered and reproductive tracts
dissected to verify placement of uterine catheters.
Unextracted plasma samples (.05, .1 and/or .2 ml) were
assayed for PGFM and PG^a* A polyethylene glycol radio¬
immunoassay system for PGFM was performed in accordance to
procedures described by Guilbault et al. (1984). Inter- and
intraassay coefficients of variation for a reference sample
(100 pg/.2 ml) assayed in duplicate for 10 assays (103-4 ±
2.9 pg/.2 ml) were 17.0% and 12.5%, respectively.

144
Prostaglandin F2a was measured using an antibody (gift
from Dr. K. Kirton of the Upjohn Company, Kalamazoo, MI)
generated in goats against PGF? conjugated to bovine serum
albumin at the C^-position. Tritiated PGF2a
([5,6,8,9,11,12,14,15-^H(N)], specific activity 150 to 180
Ci/mmole), was purchased from New England Nuclear (Boston,
MA). Cross-reactivities of the PGF2a antiserum with other
prostaglandins were 53.6% for PGF>| a; .22% for 15-keto-PGF2a;
<.04% for PGFM, PGE^, PGE2 and arachidonic acid. Standard
curves were prepared by adding known amounts of radioinert
PGF2ct (0, 10, 25, 50, 100, 250, 500, 1000 and 2500 pg) to
bovine plasma (.2 ml) obtained from cattle treated with
flunixon meglamine (BANAMINE, Schering Corp., Kenilworth,
NJ), an inhibitor of cyclo-oxygenase, to suppress prosta¬
glandin synthesis. Cattle were injected twice with Banamine
(20 ml each; 50 mg/ml) at 1700 h and 0800 h, and blood
collected at 1100 h. Immunoreactive PGF2a in this plasma
was below detectable levels (10 pg). Using an antibody
dilution of 1:2000, sensitivity of the assay was 10 pg.
Unextracted plasma samples were assayed for PGF2a in
duplicate using aliquots of either 50, 100 or 200 ul. No
significant differences were found between like concen¬
trations of PGF2a measured using these aliquot volumes
(P>.10). A plasma sample (from uterine branch of the
ovarian vein of a day 2 postpartum cow) that contained
approximately 8000 pg/ml of immunoreactive PGF2a
was assayed

145
serially in sample volumes of 10, 25, 50, and 100 ul.
Samples were brought to a 200 ul assay volume with addition
of plasma from Banamine treated cattle. A quantitative
linear displacement curve was achieved (Y = 2.43 - 2.052X;
= logit or natural log of bound/free, X = log10 of sample
volumes). Test for homogeneity of regression between this
curve and the standard curve (Y = 4.9590 - 2.2967X) did not
show them to differ (P>.10; i.e., there was no evidence for
lack of parallelism between the standard curve and the
displacement curve of bovine plasma). Accuracy of the assay
procedure was further characterized by measuring known
quantities of exogenous PGF2a added to 200 ul of plasma
(from cattle treated with Banamine) at concentrations of 25,
50, .100, 125, 200, 250 and 500 pg. Differences among
concentrations were described by a linear regression of
added vs. measured PGF2a [Y = -8.7 + 1.01X; Y = amount PGF2a
measured (pg/200 uO> X = amount PGF2a added (pg/200 ul), R2
= .97]. The intraassay coefficient of variation for the
validation procedure was 14.8%. Inter- and intraassay
coefficients of variation for 12 assays were 8.1% and 12.9%,
respectively, when duplicate estimates were run in the assay
for a 500 pg/.2 ml (582.1 ± 13«9) reference sample.
Uterine production of PGF2a or PGFM (yg/min) from the
uterine horn ipsilateral to the CL was calculated by sub¬
tracting peripheral (FA) prostaglandin concentrations from
OV concentrations and multiplying this difference (OV - FA;
< >-<

146
pg/ral) by the UBF (ml/min x 10“~). Least squares mean
values of blood flow for a 30 min period were calculated
from means of 15 consecutive 1 min intervals prior to each
30 min blood sample. Data were analyzed by method of least
squares analysis of variance according to General Linear
Models Procedures of the Statistical Analysis System (Barr
et al., 1979). Sources of variation included in the
mathematical models were cow and time to describe the time
trends post-injection of estradiol.
Results
Mean concentrations (pg/ml) of PGF2ct and PGFM in the OV
and PGFM in the FA were elevated (PC.01) following £2
injection as compared to pre-treatment concentrations.
However, mean concentrations of PGF2a in FA for both pre-
and post-E2 periods were basal (Table 3-1)* Least squares
regression curves (Figure 3-1; Table 3-2) for net
concentrations of PGF2a, PGFM and total prostaglandin in OV
(i.e., OV - FA pg/ml) following E2 injection indicated an
initial rise above baseline concentrations between 2 to 3 h,
a peak at 6 h and a decline to near baseline concentrations
by 10 h. A secondary rise in net ovarian vein PGF2a and
total prostaglandin concentrations occurred between 10.5 and
12 h. In contrast, net PGFM concentrations in the OV
continued to decline until 12 h.

Table 3-1. Least squares means ± S.E. for concentrations of PGFpa, PGFM (pg/ml) and
uterine blood flow (ml/min) during the pre- and post-estradiol-170 injection periods
Response
UBF*
Ovarian Vein
Facial
Artery
UB0A +
(ml/min)
PGF2a
PGFM
PGF2a
PGFM
PGF2a
PGFM
Pre-E2
77.0
95.7
121.5
86.8
75.8
76.5
132.3
±1 .9
±9.3
±10.2
±5.6
±7.7
±15.8
±2.1
Post-E2**
179.4
1382.8
955.5
91 .0
206.9
599.9
685.2
±4.1
±128.4
±63.6
±5.5*
±12.7
±115.0
±110.3
* Uterine Blood Flow
+ Uterine Branch of the Ovarian Artery
**Least squares means listed in the post-E2 injection period are greater than the
pre-E2 injection period (P<.01)
t No significant difference between pre- and post-E2 concentration (P>.10)
147

Figure 3-1 •
Least squares means (symbols) and regression
lines for ovarian vein (i.e., OV-FA) PGFpa
(A, ), PGFM (â–¡ , ) and total
prostaglandin (PGFpa+PGFM; +, )
concentrations following ¿2 injection.

UTERINE VENOUS PROSTAGLANDINS (pg/ml)
149

Table 3-2. Least squares polynomial regression equations for dependent variables
Dependent
variables3
B0
B1
b2
B3
b4
B5
N
R2
0V PGFM
6201.3
-3037.60Xb
514.287X2
-36.6753X3
1.17411X4
-0.013998X3
148
.39
net pg/ml
0V PGF
7960.9
-3605.78X
502.720X2
-25.6031X3
0.43174X4
148
.43
net pg/ml
0V TOTAL
9127.6
-4131.35X
598.002X2
-30.8017X3
0.51962X4
148
.42
net pg/ml
PGFM PRD
pg/min
934882
-465343.OX
80231.44X2
-5895.256X3
196.5855X4
-2.45785X5
85
.35
66.9110X4
PGF PRD
pg/min
1226671
-554791.2X
79092.40X2
-4025.961X3
85
.47
TOTAL PRD
1284674
-597977.6X
88012.32X2
-4501.612X3
74.5083X4
85
.43
pg/min
FA PGFM
2197.9
-1001.41X
158.494X2
-10.484X3
0.3035X4
-0.00316X5
167
.47
pg/ml
FA PGF
83.2
-1.24X
167
.52
pg/ml
UBF
ml/min
118.8
-65.14X0
23.456X2
-2.3930X3
0.09570X4
-0.001331X3
116
.69
a OV=ovarian vein; FA=facial artery; PGFM=15-keto-13,14-dihydro-PGF; PGF=prostaglandin
Fpai TOTAL=PGF+PGFM; UBF=uterine blood flow; PRD=net production; net=OV concentration-
FA concentration.
b X=coded sampling sequence 3 12 h post-E2 injection.
c X=coded sampling sequence 1 pre- to 12 h post-E2 injection.
150

151
During the 1 h pre-treatment period, U3F was stable and
concentrations of PGF? and PGFM remained low in all vessels
(Table 5-1). Fifteen to 30 min following administration,
UBF increased to peak values between 2.5 and 3.5 h (Figure
3-2; Table 3-2) and declined over the period from 4 to 8.5
n. A small secondary rise and fall in UBF occurred between
9 and 12 h.
Profiles of uterine PC^a’ PGFM and total prostaglandin
production (yg/min; Figure 3-3; Table 3-2) were similar to
profiles of net 0V concentrations depicted in figure 3-1 .
Maximal production of uterine PGF2a (.93 ± .25 yg/rain), PGFM
(.35 ± .08 yg/min), and total prostaglandin (1.26 ± .32
yg/min) were observed at 6 h post-E2* Facial artery PGFM
concentrations were positively correlated (within cow
partial correlation) with uterine PGF2a production (r=.6b,
P<.001; Figure 3-4) total uterine prostaglandin production
(r=.59, P<.001) and other uterine prostaglandin responses
(Table 3-3). Peripheral (FA) PGF2a concentrations were not
correlated (r=.05, P>.10) with any measured uterine
prostaglandin response (Figure 3-4).
To detect the potential presence of a countercurrent
exchange system for PGF2a between the uterine venous and
ovarian arterial vasculature, concentrations of PGF2a were
compared between the UBOA and FA. A positive difference in
PGF2a concentration between the UBOA and FA would be
indicative of an exchange between the 0V and UBOA. As

Figure 3-2. Least squares means ( â–¡ ) and regression line
depicting the uterine blood flow response to
injection.

153
e2 hours

Figure 3-3« Least squares means (symbols) and regression
lines for Ep-induced production of PGFn
(a» ), PGFM (□ , ) and total
prostaglandin (PGFp +PGFM; +, ) by the
uterus.

UTERINE PROSTAGLANDIN PRODUCTION (¿Jg/min)
155

Figure 3-4. Least squares means (symbols) and regression
lines for uterine PGF^c, production (+, ),
and peripheral (FA) PGF2a (â–¡) and PGFM
(A, ) concentrations.

157
PERIPHLRAL PCIM (pg/ml;- *) AND PGI/J (|)R/ml; a)

158
Table 3-3* Correlations of PGFM in facial artery witn
uterine PGF2a and PGFM responses
Gross
Correlation*
Within' Cow
Partial Correlation**
OV-FA PGFo
¿a
.52
.64
OV-FA PGFM
•
IV)
.22* '
OV-FA PGFo + PGFM
¿a
.53
.55
PGFp Production
¿a
.64
. 66
PGFM Production
.42
o
•
Total (PGF2 + PGFM)
Production
CM
vO
•
.59
**P<.01
* P<.10

159
illustrated in table 5-4, concentrations of PGF£a in the
UBOA were greater than those in the FA in three of five
cows. An utero-ovarian arterial anastomosis (UA to UBOA)
was detected below the point of catheter entry in one cow
following dissection of the reproductive tract at
slaughter. Such a link between the uterine and ovarian
arteries may have diluted PGF2a concentrations in the
ovarian artery with peripheral blood, low in PGF2a> and
could explain our inability to demonstrate a countercurrent
response in this cow. However, evidence for a countercur¬
rent exchange system was detected in three cows and a signi¬
ficant cow x vessel interaction was detected (PC.01). Such
a system would require PGF2a to pass down a concentration
gradient from plasma of the OV (1382.8 pg/ral; Table 3-1)
into the ovarian artery (UBOA-FA, 508 pg/ml; Table 3-1)*
Discussion
Results demonstrated that E2 stimulated an increase in
UBF and induced prostaglandin production by the diestrus
bovine uterus (day 18). Uterine blood flow increased within
15 to 30 min following E2 administration reaching maximal
values by 2.5 h. Similar UBF responses have been reported
for cattle (Roman-Ponce et al., 1978) and sheep (Huckabee et
al., 1970; Kiliara et al., 1973; Barcikowski et al., 1974)
following estrogen administration. The mechanisms by which

160
Table 3-4. Evaluation of differences in PGF? (pg/ml)
between uterine branch of ovarian artery (U3ÓÁ) and facial
artery (FA) post-E2 injection
Cow
UBOA
FA
1
2653.7 ±
680.4
50.0 ±
0.0
4
30.0 ±
0.0
50.0 ±
0.0
5a
78.9 ±
11.6
124.8 ±
21 .6
7
94.0 ±
15.1
54.6 ±
3.7
8
192.3 ±
71.4
50.0 ±
0.0
Cow x Vessel, P<.001
a An utero-ovarian arterial anastomoses was detected below
the point of UBOA catheter entry in this cow (see text).

161
estrogen induces UBF are not known, however, processes
dependent upon protein synthesis may be involved. Induction
of UBF by estrogen was completely inhibited during uterine
arterial infusion of cyclohexamide (an inhibitor of RNA
dependent protein synthesis) in ovariectomized sheep.
Inhibition was overcome 25 min following cessation of
cyclohexamide treatment (Kiliara et al., 1973)» In contrast,
local administration of actinomycin D to the uterus had no
effect on the ability of estrogen to increase UBF even
though 44 to 74# of DNA dependent RNA synthesis had been
inhibited (Resnik et al., 1975). Thus post-translational
control of protein synthesis appears to play an essential
role in regulating the estrogen induced UBF response.
Ford (1982) and Ford and Reynolds (1983) indicated that
vasodilatory effects of estrogen on the uterine vasculature
may be mediated through an antagonistic interaction of
catechol-estrogens (2 or 4 hydroxylated metabolites of
estrogen) on a-adrenergic receptors. Such a hypothesis is
supported by 1) the similarity in structure of the
catechol-estrogens and the catecholamine, norepinephrine,
and 2) initiation of immediate UBF elevations following
administration of phentolamine, an a-adrenergic
antagonist. A delay of 15 to 30 min for E^ to induce an
increase in UBF, in the present study, may represent the
time required for tissue hydroxylation of E2 and subsequent
action of catechol-estrogen on the uterine vasculature.

162
In contrast to the rapid UBF response, elevations in
uterine prostaglandin production were not evident until 2 to
3 h following administration of E£* A decline in UBF after
3 h occurred simultaneously with the increase in uterine
PGF2a production (Figures 3-2 and 3-3). One contributing
factor to the reduction in UBF may be an increase in vascu¬
lature resistance due to the vasoconstrictive effect of
PGF2a on the uterine arterial vasculature (Clark et al.,
1973; Ford et al., 1976; Ford, 1982). Time trends, post-E2
injection, for uterine venous prostaglandin concentrations
in the present study (Fig. 3-1) were essentially parallel to
uterine prostaglandin production profiles (Fig. 3-3). This
demonstrated that prostaglandin concentrations in uterine
venous drainage were a function of uterine tissue synthesis
and release and not simply alterations due to changing UBF.
Protein synthesis appears to be involved in estrogen
stimulated prostaglandin synthesis and release. The 2 to 3
h latency period until uterine prostaglandin production
increased may have reflected an estrogen induced protein
synthetic process through which uterine PGF2a synthesis
depends. French and Casida (1973) demonstrated that the
luteolytic effect of estrogen was prevented when uterine
protein synthesis was inhibited with actinomycin D.
Infusion of E^ into the arterial supply of autotransplanted
ovine uteri induced an increase of PGF~ in tha uterine
2a
venous drainage (Barcikowski et al., 1974). Prostaglandin

163
F2a was markedly reduced when a concurrent Infusion of ¿2
and indomethacin was evaluated. Indoraethacin specifically
inhibits cyclo-oxygenase, a rate limiting enzyme for synthe¬
sis of prostaglandin (Ramwell et al., 1977). Huslig et al.
(1979) reported cyclical changes in concentrations of
uterine cyclo-oxygenase in ewes which occurred simultaneous¬
ly with changes in uterine PGF2a secretion. They suggested
that exposure of the progesterone primed uterus to estrogen
was responsible for synthesis of this rate limiting enzyme.
In addition to stimulation of uterine PGF2a synthesis,
E2 also increased uterine metabolism of PGF2« as demon¬
strated by the PGFM production profile in the present study
(Fig. 3-3). Data provide indirect evidence that estrogen
may regulate enzymatic activity responsible for uterine
metabolism of PGF2C1 (i.e., 15-hydroxyprostaglandin dehydro¬
genase and A^-reductase) as well as those for PGF201 syn¬
thesis (phospholipase A2 and cyclo-oxygenase). Rapid
metabolism of PGF2 tissues (Piper et al., 1970; Ramwell et al., 1977; Granstrom
and Kindahl, 1982) precludes the parent molecule from being
used as a peripheral marker for uterine prostaglandin pro¬
duction. As demonstrated in this study, peripheral concen¬
trations of PGF2ci were basal during the pre- and post-E2
phases (Table 3-1; Fig. 3-4) and were not correlated with
any uterine prostaglandin response (Fig. 3-4). In contrast,
measurement of the primary metabolite of PGF2a>
PGFM, in

164
peripheral plasma was related to uterine PGF.pa prouuction
(r=.66; Fig. 3-4). Measurement of peripheral concentrations
of PGFM have been used to monitor uterine PGF2a during the
estrous cycle (Peterson et al., 1975; Kindahl et al., 1976),
early pregnancy (Kindahl et al., 1976; Betteridge at al.,
1984), postpartum (Guilbault et al., 1984a,b) and following
estrogen injection (Thatcher et al., 1979; Bartol et al.,
1981b; Rico et al., 1981) in cattle. Direct evidence in
support of the assumption that peripheral PGFM concentra¬
tions reflected endogenous uterine PGF2a production was
shown by Bartol et al. (1981b) and is demonstrated clearly
in the present study. These results support the use of
peripheral PGFM as an index of uterine prostaglandin
production in cattle.
The countercurrent exchange of PGF2a from the uterine
venous drainage to the ovarian arterial supply (Hixon and
Hansel, 1974; Baird, 1978; Ginther, 1981) was examined
during a period of E2~induced PGF2a production. Three of
five cows exhibited higher (PC.01) concentrations of PGF2a
in the UBOA versus FA supporting the existence of such an
exchange (Table 3-4). Variations of PGF2a concentrations
were greater (PC.01) in plasma from the UBOA than FA. This
was true regardless of whether cow number one (Table 3-4)
was included or deleted from the analyses. Increased varia¬
tion in PGF2a concentrations in the UBOA suggests that
concentrations rose interraittently above those of the FA

165
during the 12 h sampling period following injection.
•These data, in addition to the significant cow x vessel
interaction, provide support for the existence of a
countercurrent exchange system in cattle.-
Shemesh and Hansel (1975) were unable to substantiate
the existence of a PGF2a countercurrent exchange system in
luteal phase, cyclic cattle following the collection of
single blood samples at surgery. Data in the present study
were based upon numerous blood samples collected from
conscious cattle and provides additional support for a
countercurrent exchange system. The vasoactive effects of
E2 and PGF2d on the uterine and ovarian vasculature and
their potential influence on efficiency of the
countercurrent exchange warrant additional investigation.
In summary, exogenous E2 induced an increase in UBF and
uterine prostaglandin production and metabolism. Measure¬
ment of peripheral PGFM concentrations was a good index of
uterine prostaglandin production. Three of five cows
demonstrated a countercurrent exchange of PGF2a during a
period of E2~induced PGF2a production.
The E2 injection scheme may provide a good experimental
model with which to investigate uterine prostaglandin syn¬
thetic and metabolic capacity during various physiological
states in cattle and a means to study conceptus-uterine
interactions during the period of maternal recognition of
pregnancy.

CHAPTER 4
PROTE I IMS SECRETED BY DAY 16 TO 18 BOVINE
CONCEPTUS EXTEND CORPUS LUTEUM FUNCTION IN CATTLE
Introduction
An essential manifestation of pregnancy in large
domestic species is extended corpus luteum (CL) function.
During the first 15-16 days of pregnancy in cattle,
progesterone (P4) production by the CL establishes a complex
uterine environment essential for conceptúa (embryo plus
extraembryonic membranes) growth and development. Embryos
may be transferred, and pregnancies established as late as
day 16 or 17 post-estrus (Betteridge et al., 1980).
Consequently, presence of a conceptas within the uterine
lumen prior to day 16 is not a requirement for initiation of
an embryotrophic uterine environment. Beyond this point,
however, a viable conceptus within the uterine lumen must
play an active role in the perpetuation of its embryotrophic
environment by maintenance of the CL (Betteridge et al.,
1980; Northey and French, 1980; Dalla Porta and Hurablot,
1983).
The bovine conceptus produces an array of potential
"signals" during early pregnancy including steroids (Shemesh
et al., 1979; Chenault, 1980; Gadsby et al., 1980; Eley et
166

167
al., 1983), prostaglandins (Shemash at al., 1979; Lewis at
al., 1982) and proteins (Bartol et al., 1984). However,
conceptus factors responsible for luteal maintenance during
early pregnancy and their mechanisms of action have not been
demonstrated clearly in cattle. Numerous studies have
evaluated the effect of prostaglandin (PG)-E2 on luteal
maintenance in cattle with mixed results. Prostaglandin-Ep
administered into the uterine lumen alone (Chenault, 1983;
Giraenez and Henricks, 1933) or in combination with
estradiol-17g (Reynolds et al., 1983) extends luteal
function only slightly beyond the cessation of intrauterine
PGE2 treatments. In addition, systemic P4 concentrations
declined within 12 h following PGE2 treatment (Gimenez and
Henricks, 1983) or during the treatment period (Reynolds et
al., 1983) suggesting that PGE2 (plus estradiol-17e) will
not prevent production and transfer of uterine luteolytic
substances, but affects luteal function directly (.Marsh,
1970; Henderson et al., 1977; Reynolds et al., 1981).
Others have reported no affect of intrauterine PGE?
administration on CL maintenance in cattle (Dalla Porta and
Humblot, 1983; Chenault et al., 1984).
Extension of CL maintenance and cycle length were
demonstrated following intrauterine administration of
conceptus homogenates to cyclic cattle (Northey and French,
1980). Likewise, ovine conceptus extracts and homogenates
(Rowson and Moor, 1967; Ellinwood et al.,
1979; Martal et

168
al., 1979) or conceptus secretory proteins (Godkin et al.,
1984) have been shown to extend CL function and cycle length
when administered into the uterine lumen of cyclic ewes.
However, luteotropic and/or antiluteolytic actions of major
conceptus-produced steroids and proteins have not been
evaluated in cattle. Objectives of the present experiment
were to examine the effects of a major bovine conceptus-
produced steroid (Eley et al., 1983; Chapter 2), 5B-pregnan-
3a-ol-20-one, and bovine conceptus secretory proteins on
luteal function, cycle length and spontaneous uterine PGF2a
production in cyclic cattle.
Materials and Methods
Conceptus Collection and Culture
Dairy and beef cows (n=49) served as conceptus donors
after being mated during estrus (day 0). Between days 16 to
18 post-mating (17.2 ± 0.6 days), the uterine horn
ipsilateral to the corpus luteum (CL) bearing ovary was
nonsurgically flushed. A French Foley catheter (size no.
16, 18 or 20; American Hospital Supply, Jacksonville, FL)
was inserted through the cervical os and positioned at the
base of the uterine horn just anterior to the uterine
body. Sterile Dulbeccos phosphate buffered saline (pH 7.4;
PBS; Dulbecco & Vogt, 1954) was warmed to 37 C and injected
into the uterine lumen (I.V.) at a volume of 60 ml per

169
flush. Medium containing conceptuses (n=33) was collected
into sterile glass containers, maintained at 37 C, and
transported to the laboratory within 30 min of recovery.
Following the last uterine flush, 20 ml of an antibiotic
solution (aqueous Procaine Penicillin G; 300,000 units/ral;
Pfizer Incorporated; New York, NY) were infused into the
uterine lumen, and Foley catheter removed. Donor cows were
bred and flushed up to three times during successive estrous
cycles.
An additional four cows were superovulated, bred and
slaughtered at 17 days post-mating. The uterus was removed
following exsanguination, sealed in a plastic bag, and
placed on ice while being transported to the laboratory
(within 60 min of slaughter). Uterine horns were trimmed of
excess tissue, ovaries and oviducts removed, and a large,
curved, Rochester-Ochsner forcep applied to the anterior
cervix. The anterior tip of the uterine horn ipsilateral to
the CL containing ovary was cut to provide an enlarged
opening. A plastic, 50 ml syringe fitted with an 18 gauge
needle was used to administer two 30 ml flushes (sterile
PBS, warmed to 37 C) into the uterine lumen through the tip
of the uncut uterine horn. Medium containing conceptuses
was collected into sterile plastic culture dishes.
Approximately 15 to 20 conceptuses (n=15 incubations) were
obtained from superovulated animals. All conceptuses,
collected either nonsurgically or at post-mortem flush, were

170
washed in and transferred to sterile culture dishes
containing 15 ail of warm Minimum Essential Medium (MEM;
GIBCO, Grand Island, NY) supplemented with non-essential
amino acids (GIBCO, Grand Island, NY), antibiotic/anti¬
mycotic (GIBCO, Grand Island, NY), 200 units of insulin/L
(Sigma Chemical Co., St. Louis, MO) and 1 g glucose/L
(Fischer Scientific; Orlando, FL). Conceptuses were
cultured for 24 h on a rocker platform (Bélico Glass
Company, Vineland, NJ) and maintained at 57 C in a gaseous
environment of nitrogen:oxygen:carbon dioxide (50:45:5).
Following a 24 h incubation, tissues and medium were
separated by centrifugation (10,000 x g; 20 min) at 4 C.
Medium (supernatant) from each culture was collected and
frozen individually. Conceptus wet weights were recorded.
Preparation of Material for Intrauterine Injections
Medium from individual incubations (15 ml) were dia¬
lyzed (Spectrapore 6, 1000 Mr cutoff, Spectrum Medical
Industries, Los Angeles, CA) extensively (4 L changed thrice
daily for 5 days) against 10 raM Tris-HCl buffer, pH 7.2
(TRIS). Following dialysis, an aliquot of medium from each
culture (n=47) was used for determination of protein
concentration (Lowry et al., 1951). All culture medium was
then pooled and concentrated by ultrafiltration (1000 Mr
cutoff; Amicon Corporation, Danvers, MA) to a volume of
approximately 75 ml. The concentrated filtrate was
processed through a sterilization filter unit (0.45 ym pore

171
size; Sybron/Nalgene, Rochester, NY) and dispensed into 2 mi
aliquots designated as pooled conceptus secretory proteins
(CSP) at a protein concentration of 740 ug/2 ml.
A serum sample from each experimental animal (n=9) was
collected on day 10 of the estrous cycle. Serum samples
were dialyzed individually, diluted in TRIS and sterilized
as described for CSP. Equal masses (740 ug) of homologous
serum proteins were added to each 2 ml injection of CSP (n=3
cows; 12 injections/cow; Group 1).
Two milligrams of 5B-pregnan-3a-ol-20-one (5b-P) were
dissolved in 2 ml of ethanol and mixed with 80 ml of TRIS.
Ethanol was evaporated from the solution using N£ gas and
gentle heating. The steroid solution was sterilized as
described previously and dispensed into 2 ml aliquots (50 yg
each). Homologous serum proteins (740 yg) were added to
each 2 ml injection of 5b~P (n=3 cows; 12 injections/cow;
Group 2). Group 3 cows (n=3) received homologous serum
proteins alone (1480 ug/2 ml TRIS; 12 injections/cow). All
treatment mixtures were frozen (-20 C) until time of
intrauterine injection.
Animal Preparations
Nine cyclic Holstein cows were assigned randomly to the
three previously described treatment groups. Animals were
prepared for surgery (Chapter 3) on day 10 of the estrous
cycle. Utilizing a midventral laparatomy, uterus and
ovaries were exposed and location of the CL recorded. A

172
sterile polyvinyl catheter (V—6; Bolab Incorporated, Lane
Havasu City, AZ) was inserted via an incision in the isthmus
of the oviduct and secured 30 to 50 mm into the anterior
portion of the uterine lumen, ipsilateral to the CL. The
catheter was exteriorized via a small flank incision. An
additional catheter (Sialastic tubing, I.D. 1.57 mm x O.D.
3.18 mm; Dow Corning Corporation, Midland, MI) was advanced
approximately 1.07 m into the saphenous vain to a position
in the dorsal vena cava slightly anterior to the point of
uteroovarian venous drainage. Jugular catheterizations
(V—9; Bolab Incorporated, Lake Havasu City, AZ) were
performed, if necessary, at the time of vena cava catheter
failure during the experiment. Antibiotics (Polyflex, 167
mg/ml; Bristol Laboratories, Syracuse, NY) were administered
on the day of surgery (10 ml I.M., 10 ral I.P. and 1 ml I.U.)
and one day post-surgery (10 ml I.M. and 1 ml I.U.).
Function of the CL was monitored by measuring plasma P4
concentrations from blood samples collected twice daily
(0800 and 2000 h) beginning on day 12 and continuing until
detection of oestrus. Three acute bleedings were conducted
to monitor spontaneous uterine prostaglandin (PG)
production. Plasma concentrations of PGF2a were measured in
blood samples drawn every 15 min from 0800 to 1400 h on days
18 through 20. Intrauterine injections of experimental
materials (2 ml; see above) were initiated at 2000 h on day
15 and continued every 12 h until 0800 h of day 21. All

173
animals were fitted with oestrous-mount detection devices
(KaMar Incorporated, Steamboat Springs, CO), maintained on
pasture in the presence of an intact bull, and observed two
to four times daily for oestrous behavior. Corpus luteura
regression was verified by rectal palpation post-oestrus.
When the experiment had concluded, all animals were
slaughtered and reproductive tracts dissected to assess
uteroovarian condition and catheter placement.
Progesterone and Prostaglandin Radioimmunoassay
Progesterone (P4) was measured in heparinized plasma
samples utilizing a specific antiserum generated in sheep
against P4 conjugated to bovine serum albumin (BSA) at the
C11-position. Tritiated P4 ([1,2,6,7-^H(N)], specific
activity 90-115 Ci/mM) was purchased from New England
Nuclear (Boston, MA). Cross-reactivity of the P4 antiserum
was < 1% with 17a-hydroxy-P4, 20a-hydroxy-P4, 206-hydroxy-
P4, cortisol, testosterone, androstenedione and estradiol-
176. Standard curves were prepared by adding known amounts
of radioinert P4 to phosphate buffered saline (pH 7.4)
containing 1 g/L of gelatin (PBSg). Final range of P4
concentrations were: 0, 15.6, 31.2, 62.4, 125, 250, 500 and
1000 pg/100 ul of PBSg. Utilizing an antiserum dilution of
1:35,000, sensitivity of the assay was 15.6 pg. A plasma
sample containing approximately 4.5 ng/ml of immunoreactive
P4 was aliquoted in triplicate into sample volumes of 50,

174
100, 200 and 300 yl. Progesterone from plasma samples were
extracted by vortexing for 2 min with 2 ml of freshly
distilled benzene and hexane (1:2). Solvent containing the
extracted P4 was dried under N2 and brought to a 500 yl
assay volume with the addition of PBSg. A quantitative
linear recovery was achieved [Y = 20.965 + 4.111X; Y =
concentration of P4 (pg/100 y1) and X = plasma volumes
extracted (y1)]. No significant differences were found
between concentrations of P4 (pg/100 yl) measured using
either 50, 100, 200 or 300 yl aliquot volumes (P>0.25).
Exogenous P4 was added to a plasma sample (X = 1.8 ng/ml) at
doses of 0, 0.5, 1.0, 5-0 and 10 ng/ml. All doses were
replicated four times. A linear regression equation of
added vs measured P4 [Y = 1626.1 + 1.0855X; Y = amount of P4
measured (pg/ml) and X = amount of P4 added (pg/ml); R =
0.975] described differences among concentrations. The
intra-assay coefficient of variation (CV) for the validation
was 13*4$. Intra- and interassay CV for eight assays
were: 10.4 and 7.5%, respectively, when duplicate estimates
were run in the assays for a 100 pg/500 yl (94.2 ±4.3
pg/500 yl) reference plasma sample, and 6.1 and 4.3%,
respectively, when duplicate estimates were run for a 250
pg/500 yl (269.5 ± 7.0 pg/500 yl) references plasma sample.
Prostaglandin-F2a was assayed in unextracted plasma
samples (50, 100 and 200 ul). Concentrations of PGF2a were
determined in a dextran coated charcoal radioimmunoassay

175
system previously validated for use in our laboratory
(Knickerbocker et al., 1982; Chapter 3). Antiserum used in
the assay was generated in goats against PCFpa conjugated to
A
BSA at the C -position. This antiserum crossreacts 53-b%
with PGF-] a and < 1% with 15-keto-PGF2a, 15-keto-13,14-
dihydro-PGF2a (PGFM), PGE^, PGE2 and arachidonic acid.
Sensitivity of the assay was 10 pg. The intra- and inter¬
assay CV for 13 assays were: 20.0 and 12.7%, respectively,
when duplicate estimates were run for a 50 pg/200 yl (60.9 ±
2.5 pg/200 ul) plasma reference, and 10.6 and 5.6%,
respectively, when duplicate estimates were run for a 500
pg/200 U1 (489.9 ± 10.1 pg/200 yl) plasma reference.
Statistical Analyses
Data for P^ and PGF2a concentrations in plasma, and
accumulated PGF2a were analyzed using the General Linear
Models procedure of the Statistical Analysis System (SAS
Institute Incorporated, 1982) for a split-plot analysis of
variance with repeated measurements over time. Analysis of
variance considered variability due to treatment (CSP, 5g-P
and Control), cow nested within treatment, time (for P^:
days 12-38.5; for PGF2a: samples 1-75 which consisted of 25
samples on each of days 18-20), treatment by time, and cow
nested within treatment by time. Estimates for PGF2a
produced over the 6 h sampling periods on days 18 through 20
were generated by sequential summation of PGF2a
concentrations measured in plasma on days 18 through 20.

176
Using this method, a series of PGF2a values were generated
over days for each sample within cow. Each PGFp value
corresponded to the PGFp a concentration in the respective
sample plus a summation of PGF2a concentrations in all prior
samples (accumulated PGF2a). To provide estimates of time
trends, P^ concentrations and accumulated PGF2a values were
analyzed by least squares regression analyses, and
differences in treatment means evaluated by orthogonal
comparisons (CSP versus 5S-P and Control; 58-P versus
Control). Differences in polynomial regressions were tested
by examining for homogeneity of regression between treatment
response curves. These data were analyzed with time as a
continuous, independent variable. Data pertaining to cycle
length, days with P^ concentrations > 1 ng/ml, and mean
accumulated PGF2a (Table 4-1), as well as age effects for
conceptus wet weight, and yg protein produced/rag conceptus
wet weight (Table 4-2) were analyzed by a one-way analysis
of variance. Treatment differences were evaluated by
orthogonal comparisons (refer to appropriate taoles for
information on the specific orthogonal comparisons).
Results
Evaluation of Reproductive Tracts
Cows were palpated per rectum following detected
oestrus. In all cases, a single regressing CL was detected

177
Table 4-1. Effects of intrauterine treatments on cycle
length, luteal function and production30
Treatment
Cycle length
Days with P¿
>1 ng/ml
Accumulated PGF2a
pg/cow/day (n)
CSP
55•42±2.49**
30.35±1.85**
39.17± 35.75(6)**
5 6-P
24.67±0.83
22.67±1.01
1322.44±544.42(7)**
Control
23.50+0.50
22.35±0.60
496.41±210.75(3)
3 Mean ± S.E.M.
0 Orthogonal comparisons: CSP vs. 5^-P and Control; 5^-P
vs. Control.
** P<0.01 .
Table 4-2. Bovine conceptus wet weight and protein
production30
Age (days)
N
Wet Weight (mg)c
ug Protein/mg Wet Weight^
16.5
9
51-74±7.75
4•95±2.59*
17.0
15
55.08±5.63
15•76±5.17
17.5
4
27.58±7.99
12.04±4.07
18.0-18.5
6
55.78+8.55*
15•88±5•25
3 Data from conceptuses collected following superovulation
are not included in analysis (n=15 cultures; 22.30±1.97 ug
protein/mg wet weight).
0 Mean ± S.E.M.
0 Orthogonal comparison: 18.0-18.5 vs. 1b.5, 17.0, 17.5.
Orthogonal comparison: 16.5 vs. 17.0, 17-5, 18.0-18.5.
* P<0.05.

178
on the same ovary bearing a functional CL at surgery. Upon
dissection of reproductive tracts post-slaughter, all
catheters were intact and patency was verified. General
appearance of endometrium and uteroovarian tissues were
normal for all experimental animals.
Effects of Intrauterine Injections on
Interestrous Interval and Corpus Luteum Function
Estrous cycle lengths immediately preceding the
experimental estrous cycle were not different (P>0.25) among
cows assigned to CSP, 58-P and Control groups (20.57 ± 0.89
days). Intrauterine administration of CSP to cyclic cows
resulted in extended estrous cycle lengths (P<0.01) of 30,
31.75 and 38.5 days as compared to cows which received 5g-P
(23, 25*5 and 25.5 days) and Control (22.5, 24 and 24 days)
injections. No differences were detected (P>0.25) in
interestrous intervals between cows of the 5 8-P and Control
groups (Table 4-1).
Analysis of plasma P^ concentrations verified observa¬
tions associated with rectal palpations and interoestrous
intervals (Fig. 4-1, Table 4-1). Function of the CL (P^
concentrations > 1 ng/ml) was maintained for 28, 29 and 34
days following CSP treatments, whereas CL lifespan of cows
in the 5B-P (21, 22.5 and 24.5 days) and Control (21.5, 22
and 23.5 days) groups were shorter (P<0.01). A major
conceptus-produced steroid, 5s-pregnan-3a-ol-20-one, did not
influence CL lifespan as compared to Control cows treated
with homologous serum proteins alone. Vena cava catheters

Figure 4-1. Least squares means and regressions of plasma
progesterone concentrations for groups of cows
(n=3 per group) receiving intrauterine
injections (days 15.5 to 21) of homologous
serum proteins (control), 56-pregnan-3“-ol-20-
one, or conceptus secretory proteins.
Horizontal lines above each graph represents
duration of vena cava catheter patency in each
cow.

180
DAYS OP TREATMENT CYCLE

181
were maintained to approximately day 21 of the experimental
cycle (21.17 ± 1.05; Fig. 4-1) at which time jugular
catheters were installed (eight of nine cows). Following
loss of vena cava catheter patency, seven of eight
experimental cows exhibited jugular concentrations of (X
± S.E.) in excess of 1 ng/ml (6.1 ±1.8 ng/ml), which
indicated presence of a functional CL. Concentrations of P^
were two to ten times higher in vena cava plasma immediately
prior to failure of the vena cava catheter (17.5 ± 6.3
ng/ml) versus concentrations in jugular plasma.
Effects of Intrauterine Injections on Plasma
Prostaglandin Concentrations
Analysis of prostaglandin responses suggested that
proteins secreted by bovine conceptuses reduced uterine
production of PGF2a (Fig. 4-2). Circulating PGFpa exhibits
a relatively short half-life (7-3 min; Kindahl et al., 1976)
due to its rapid metabolism by the lung and peripheral
tissues (Granstrom and Kindahl, 1982). Therefore, plasma
samples from the vena cava were utilized for PGF2a
determinations. Higher plasma concentrations of P^ in vena
cava versus jugular vein samples were used as verification
of catheter placement prior to PGF2a data analysis. The
number of cows contributing to PGF2a responses in vena cava
plasma for each group on days 18, 19 and 20 were CSP, 2,2,2;
5B-P, 3,3,1; and Control, 3,3,3, respectively. A
significant treatment by time interaction (P<0.05) was
detected for plasma PGF2a concentrations in the vena cava

Figure 4-2. Least squares means of vena cava PGF2a
concentrations. Blood samples were collected
every 15 minutes for 6 hours on days 18, 19 and
20. Cyclic cattle received intrauterine
injections of homologous serum proteins
(control), 58**pregnan-5a-ol-20-one or conceptus
secretory proteins from days 15.5 to 21.

PROSTAGLANDIN-F9rt (pg/ml)
183

184
and supported a role for CSP in the reduction of PGF2a
production. Pulsatile episodes of PGF2a concentrations in
the vena cava were apparent in plasma samples collected from
all cows of the 5^-P treatment group during days 18 tnrough
20. Similarly, spontaneous elevations of PGF2a in vena cava
plasma were detected in two of three, three of three, and
one of two Control cows on days 18, 19 and 20, respec¬
tively. In contrast, no measurable PGF2a was detected in
vena cava plasma from two of two cows in the CSP treatment
group on days 18 and 19. One plasma sample did contain
measurable amounts of PGF2« in one of two cows on day 20.
Profiles of PGF2a concentrations (least squares means) for
each group are depicted in Fig. 4-2. Concentrations of
PGF2a also were accumulated over the 3 days sample and
analyzed statistically. Orthogonal comparisons of treatment
means indicated that intrauterine administration of CSP
resulted in a mean accumulation per cow of less PGF2a than
cows in 58-P and Control treatments (Table 4-1; P<0.01).
Additionally, accumulation of PGF2a was greater in cows
administered 5 6-P compared to cows which received homologous
serum proteins alone (p<0.01).
Bovine Conceptus Observations
Thirty-two conceptuses were classified according to
their age (days 16.5, 17, 17.5 and 18-18.5) at the time of
nonsurgical flushing (Table 4-2). Although conceptus wet
weights did not increase significantly until day 18.0 to

135
18.5 post-insemination, tissue secretory activity (ug
protein produced/mg conceptus wet weight) increased
approximately three-fold in conceptuses 17 days and older
(PC0.05).
Discussion
Maternal recognition of pregnancy occurs by day 1b to
17 in cattle (Betteridge et al., 1980; Northey and French,
1980; Dalla Porta and Humblot, 1983). This represents a
critical period during early gestation when production of
conceptus signals becomes essential to luteal maintenance
and continued endometrial secretory activity (histotroph).
A phase of rapid conceptus elongation and differentiation is
intimately associated with this period of signal
transmission by the bovine conceptus (Chang, 1952;
Greenstein et al., 1958; Greenstein and Foley, 1958a,b).
Developmental stage of the conceptus may therefore determine
the timing of conceptus signal production and secretion
during early pregnancy (Bartol et al., 1984).
In the present study, total protein production per mg
of conceptus tissue increased after the initiation of
conceptus elongation on day 16. This increase in protein
secretory activity was observed prior to any detectable
increase in conceptus mass (Table 4-2). Conceptus mass may
not be an appropriate index of conceptus expansion since

186
Geisert et al. (1982) concluded that the initial rapid
elongation of the porcine conceptus was due to cellular
remodelling and not hyperplasia, whereas subsequent con¬
ceptus elongation was associated with increasing DNA and RMA
content. In bovine conceptus incubations, [^Hj-leucine
incorporation into nondialyzable secretory products
(presumably proteins) supported an increase in production of
labelled proteins from days 16 to 19 and 22 (Bartol et al.,
1984). Thus, an increase in conceptus tissue secretory
activity occurs concomitantly with bovine conceptus
elongation.
Maintenance of CL function in cattle is thought to
involve both luteotrophic and antiluteolytic processes which
are initiated by the conceptus during early pregnancy. Del
Campo et al. (1980) provided convincing evidence for the
presence of a blood-borne luteotrophic and/or luteal
protective factor(s) originating from the pregnant uterine
horn during early gestation in cattle. Furthermore, their
results support a local, countercurrent transfer of this
substance(s) from the venous drainage of the pregnant
uterine horn into the adjacent ovarian arterial supply,
ipsilateral to the CL.
Uterine-dependent luteolysis in nonpregnant cattle also
involves an ipsilateral, venoarterial pathway (Mapletoft et
al., 1976). Prostaglandin-F^a is the presumed uterine
luteolysin in cattle and numerous other species (Horton and

187
Poyser, 1976). However, participation of other active FGFoo
metabolites (Milvae and Hansel, 1983) and products of the
lipoxygenase pathway (Milvae and Hansel, 1934) in bovine
luteolysis is possible. In cattle, elevated concentrations
(Shemesh and Hansel, 1975) and high amplitude pulses
(Nancarrow et al., 1973) of PGF2a in uterine venous blood,
and PGFM in peripheral blood (Peterson et al., 1975; Kindahl
et al., 1976) are temporally associated with the decline in
plasma P4 concentrations during luteal regression. During
early pregnancy, the bovine conceptus may exert an
antiluteolytic effect within the uterus since peripheral
PGFM pulses are reduced or abolished in pregnant heifers
(Kindahl et al., 1976; Betteridge et al., 1934). Similarly,
acute elevations in PGF2 with luteal regression were absent during early pregnancy in
heifers (Harvey et al., 1984; Plante et al., 1934).
However, basal levels of plasma PGFM (Williams et al., 1933)
and PGF2a metabolites in urine (Harvey et al., 1934; Plante
et al., 1984) are elevated by days 17 to 20 of gestation.
Evidence that blood flow is increased to the gravid uterine
horn (Ford et al., 1979) and that bovine conceptuses are
capable of significant prostaglandin production during early
gestation (Shemesh et al., 1979; Lewis et al., 1982),
suggest that elevated basal prostaglandin metabolite
concentrations observed in pregnant cattle may result from

183
conceptus production of PGF2a which is transported out of
the gravid uterus during early pregnancy. However, this
increase in basal prostaglandin concentration is not of
sufficient magnitude to initiate luteolysis. Additional
support for conceptus-derived antiluteolytic effects in
cattle was demonstrated recently. Intravenous injection of
estradiol-176 stimulates uterine production of PGF2a which
may be accurately indexed by measurements of PGFM in
peripheral plasma (Knickerbocker et al., 1982; Chapter 3).
This experimental model was utilized to evaluate uterine
capacity to produce PGF2a on day "1® of the oestrous cycle
and days 18 and 20 of pregnancy (Thatcher et al., 1984b).
Estradiol-induced uterine prostaglandin production was
reduced significantly in pregnant cows on days 13 and 20.
Furthermore, bovine endometrium at day 17 of pregnancy
produced substantially less PGF2a, in vitro, than
endometrium collected on day 17 of the oestrous cycle
(Thatcher et al., 1984b). However, no differences in PGE2
production, in vitro, were apparent between pregnant and
cyclic endometrium.
Based on the P^ profiles (Fig. 4-1, Table 4-1) and
PGF2 data (Fig. 4-2, Table 4-1), conceptus secretory
proteins appeared not to stimulate luteal synthesis of P^
(luteotrophic), but allowed for extended luteal function via
local suppression of spontaneous uterine PGF2a production
(antiluteolytic). Results of a more recent study

189
demonstrated a significant reduction in estradiol-induced
uterine PGF£a production following intrauterine admini¬
stration of bovine CSP to cyclic cows (Chapter 5).
Collectively, these data indicate that proteins secreted by
the bovine conceptus are involved in mediation of anti-
luteolytic effects during early pregnancy. Reduction in
uterine luteolytic activity by conceptus secretory proteins
may involve regulation at several levels of the arachidonic
acid metabolic cascade (Milvae and Hansel, 1984; Thatcher et
al., 1984b), control of endometrial oxytocin and steroid
receptor populations (McCracken et al., 1981; McCracken,
1984) or stimulation of endometrial prostaglandin inhibitors
(Wlodawer et al., 1976; Shemesh et al., 1984).
In this study, protein signals secreted in culture by
elongating bovine conceptuses (day 16.5 through 18.5)
extended luteal function and interestrous interval to
approximately 30 and 33 days, respectively, when admini¬
stered into the uterine lumen of cyclic cattle on days 15
through 21 post-estrus. Similar data were reported recently
(Godkin et al., 1984) following intrauterine injection of
conceptus secretory proteins to cyclic ewes. Intrauterine
administration of a prominent, low molecular weight (Mr),
acidic polypeptide, termed ovine trophoblast protein-1
(oTP-1), also extended luteal function in cyclic ewes.
Thus, oTP-1 may be the primary protein responsible for
luteal maintenance in sheep. Roles of specific proteins

190
secreted by the bovine conceptus have not been examined.
However, several recent reports suggest that there may be
similarities in the nature and function of conceptas protein
signals in cattle and sheep during early pregnancy.
Major components of the protein synthesized and
released in culture by day 16 to 24 bovine conceptuses are
low Mr, acidic polypeptides (Bartol et al., 1984) similar in
nature to those produced by the elongating ovine conceptus
(day 13-21); Martal et al., 1984d; Godkin et al., 1y82b).
When bovine (day 14) and ovine (day 11-13) trophoblastic
vesicles (TV), composed of extraembryonic trophectoderra and
endoderm, were transferred and allowed to develop in cyclic
cattle and sheep, respectively, a majority of the recipients
exhibited prolonged CL maintenance (Heyman et al., 1984;
Martal et al., 1984b). These data support previous reports
(Godkin et al., 1932b, 1984) that the extraembryonic
trophectodermal layer of the conceptus secretes
proteinaceous signals responsible for CL maintenance in
early pregnancy. In a related study (Martal et al., 1984a),
cross-species transfers of bovine and ovine TVs led to
extended CL function in approximately 20$ of ovine and
bovine recipients. The authors suggested that nonspecific
conceptus signals in the cow and ewe were sufficient for
maintaining CL function, and the biologically active
molecules responsible for CL maintenance in these species
may be very similar. Additional support for this argument

191
was supplied recently when antibodies produced against oTP-1
(Godkin et al., 1984) were found to cross-react with low Mr
polypeptides secreted by the bovine conceptus (S.D. Helmer,
W.W. Thatcher, F.W. Bazer, P.J. Hansen and R.M. Roberts,
1984 unpublished data). These observations indicate that
luteal maintenance during early gestation may be achieved by
analogous mechanisms in cattle and sheep.
It is interesting to note that duration of CL extension
in recipient cattle (range 25-37 days) following species-
specific TV transfers (Heyman et al., 1984), and ovine TV
transfers (31 and 35 days; Martal et al., 1984a) is similar
to luteal lifespans achieved in this study (Fig. 4-1, Table
4-1) following intrauterine treatments with pooled bovine
conceptus secretory proteins in cyclic cattle (range 28-34
days).
In contrast to the observed antiluteolytic activity of
conceptus secretory proteins, 5B-pregnan-3a-ol-20-one, a
major, conceptus-produced steroid, did not influence CL
lifespan, cycle length or decrease uterine PGF2a production.
In conclusion, intrauterine administration of bovine
conceptus secretory proteins to cyclic cattle was shown to
extend CL lifespan and interestrous interval, and attenuate
spontaneous uterine PGF2a production (PGF2a in vena cava
plasma). Data presented provide strong evidence that
protein signals secreted by the bovine conceptus during the

192
first 2 to 3 weeks of gestation are required for the
establishment of pregnancy in cattle.

CHAPTER 5
INHIBITION OF ESTRADIOL-17B INDUCED UTERINE
PROSTAGLANDIN-F?a PRODUCTION BY
BOVINE CONCEPTUS SECRETORY PROTEINS
Introduction
Pregnancy maintenance in cattle requires continued
secretion of progesterone (P4) by the corpus luteum (CL).
To circumvent CL regression, the bovine conceptus must ini¬
tiate the process of pregnancy recognition by days 15 to 17
post-estrus (Betteridge et al., 1980; Northey and French,
1980; Dalla Porta and Humblot, 1983). Previous studies in
cattle suggest that there are probably several interdepen¬
dent strategies by which the conceptus may initiate main¬
tenance of the CL during early gestation. Luteotrophic
and/or luteal protective substances, originating from the
gravid uterine horn in cattle, were demonstrated by Del
Campo and coworkers (1980). Conceptus stimulated uterine
blood flow during early gestation (Ford et al., 1979; Ford
and Chenault, 1931) has been proposed to increase transfer
efficiency of potential luteotrophic substances from the
pregnant uterus to the CL (Reynolds et al., 1933; Thatcher
et al., 1984b). Conversely, endometrial and uteroovarian
vascular permeability to prostaglandin (PG) F2a is reduced
during early pregnancy and may thus insure a reduction in
193

194
the delivery of luteolytic agents to the CL (Thatcher at
al., 1984a,b).
Another process by which the conceptus may provide for
an extension of luteal function involves attenuation of
PGF2a production by the uterine endometrium. Thatcher and
coworkers (1984b) demonstrated that in vitro production of
PGF2a by day 17 pregnant endometrial explants was reduced
significantly compared to production by cyclic day 17 endo¬
metrium. These data support an antiluteolytic effect of the
bovine conceptus on endometrial PGF2a synthesis.
Patterns of uterine PGF2a production, as determined by
peripheral measurements of the primary metabolite, 15-keto-
13,14-dihydro-PGF2a (PGFM; Granstrom and Kindahl, 1982),
have been characterized during the estrous cycle (Peterson
et al., 1975; Kindahl et al., 1976; Betteridge et al., 1984)
and early pregnancy (Kindahl et al., 1976; Betteridge et
al., 1984) in cattle. Luteal regression, as determined by
declining P^ concentrations, was always associated with
three or more episodic pulses of PGFM. In contrast, pre¬
sence of a viable conceptus within the uterine lumen reduced
or completely eliminated the PGFM episodes.
Injection of estradiol-17S (S2) initiates an acute and
reproducible increase in uterine PGF2a production (Thatcher
et al., 1984b; Chapter 3). Furthermore, uterine PGF2a pro¬
duction was correlated significantly with concentrations of
PGFM measured in peripheral plasma. Using the E2~injection

195
model, Rico and coworkers (1981; see also: Thatcher at al.,
1984b) evaluated the capacity of the uterus to synthesize
and secrete PGF2a (PGFM response) at day 18 of the estrous
cycle versus days 18 and 20 of pregnancy in cows. Mean
peripheral concentrations of plasma PGFM, following E2
injection, were significantly less in pregnant versus cyclic
cows. Thus the bovine conceptus appears to exert an
antiluteolytic effect via modification of uterine PGF2a
production. The present study has focused on the possible
antiluteolytic role of bovine conceptus secretory proteins
(CSP), since intrauterine treatment with CSP to cyclic
cattle (Chapter 4) and sheep (Godkin et al., 1984) resulted
in extensions of CL lifespan and interestrous intervals.
The objective of the present experiment was to determine if
bovine conceptus secretory proteins attenuate the E2-induced
increase in uterine PGF2a production (Thatcher et al.,
1984b; Chapter 3).
Materials and Methods
Conceptus Collection and Culture
Thirty-eight Angus and crossbred beef cows were bred
during estrus (day 0) and artificially inseminated approxi¬
mately 12 h later. All animals were slaughtered on day 17
or 18 post-mating and reproductive tracts recovered
following exsanguination. Reproductive tracts were sealed

196
in a plastic bag and placed on ice while being transported
to the laboratory. Uterine horns were trimmed of excess
tissue, ovaries and oviducts removed, and a large, curved,
Rochester-Ochsner forcep applied to the anterior cervix.
The anterior tip of the uterine horn ipsilateral to the CL
containing ovary was cut to provide an enlarged opening. A
plastic, 50 ml syringe fitted with an 18 gauge needle was
used to administer two 30 ml flushes (sterile saline, warmed
to 37 C) into the uterine lumen through the tip of the uncut
uterine horn. Saline containing conceptuses (n=29) was
collected into sterile plastic culture dishes. All con¬
ceptuses were washed immediately in 15 ml of 37 C, sterile
modified (15) minimum essential medium (MEM) and transferred
to sterile culture dishes containing 15 ml of MEM. Con¬
ceptuses were cultured for 30 h on a rocker platform (Bélico
Glass Company, Vineland, NJ) and maintained at 37 C in a
gaseous atmosphere of nitrogen:oxygen:carbon dioxide
(50:45:5). Following a 30 h incubation, tissues and medium
were separated by centrifugation (10,000 x g; 20 min) at 4
C. Medium (supernatant) from each incubation was collected,
pooled and frozen (-20 C). Conceptus wet weights were
recorded.
Preparation of Protein Solutions
Pooled medium from conceptus incubations (435 ml) was
dialyzed (Spectrapore 6, 1000 Mr cutoff; Spectrum Medical
Industries, Los Angeles, CA) extensively (4 L changed thrice

197
daily for 5 days) against 10 mi-i Tris-HCl buffer, pH 7.2
(TRIS). Following dialysis, culture medium was concentrated
by ultrafiltration (1000 Mr cutoff filter; Araicon
Corporation, Danvers, MA) to a volume of approximately 120
ml. An aliquot of medium was then used for determination of
protein concentration (Lowry et al., 1951). Concentrated
medium was dispensed into 2 ml aliquots (n=S0) containing
0.6 mg of CSP (Treatment group). Serum proteins from a day
18 pregnant cow were dialyzed and protein concentrations
determined as described for conceptus culture medium. The
serum protein dialysate was appropriately diluted in TRIS
and dispensed into 2 ml aliquots (n=60) containing 0.6 mg of
day 18 pregnant serum proteins (Control group). Antibiotics
(Polyflex, 167 mg/ml; Bristol Laboratories, Syracuse, NY)
were added (200 pi) to each aliquot and frozen (-20 C) until
time of intrauterine injection.
Animal Preparations
Cyclic Jersey cows (n=10) were assigned randomly to
either Control or Treatment groups and prepared for surgery
(Chapter 3) on day 10 of the estrous cycle. Utilizing a
midventral laparotomy, the uterus and ovaries were exposed
and location of the CL recorded. A sterile polyvinyl cathe¬
ter (V-6; Bolab Incorporated, Lake Havasu City, AZ) was
inserted and secured 45 mm into the anterior lumen of each
uterine horn and exteriorized via a small flank incision. A
6 cm length of tape was folded around each catheter at the

193
point of exit from the body cavity and anchored to the skin
with suture. Exteriorized uterine catheters wera placed in
a pack, consisting of an 8 x 16 cm plastic bag reinforced
with tape (Ethikon; Johnson and Johnson Products
Incorporated, New Brunswick, NJ), and secured to the flank
with suture. Antibiotics (Polyflex, 167 mg/ml; Bristol
Laboratories, Syracuse, NY) were administered on the day of
surgery (10 ml I.M., 10 ml I.P. and 1 ral/each uterine horn)
and one day'post-surgery (10 ml I.M. and 1 ml/each uterine
horn).
Intrauterine injections (n=12/cow; see above) were
administered into each uterine horn at 12 h intervals (2.4
mg protein/ uterus/day) from 2000 h on day 15 to 0800 h on
day 18. Blood was collected daily via jugular venipuncture
from days 12 through 17 post-estrus. On day 17, all experi¬
mental cows were fitted with a jugular catheter. A jugular
vein was punctured via a stainless steel, 10 cm, 12 gauge
needle and a sterile polyvinyl catheter (V-9; Bolab
Incorporated, Lake Havasu City, AZ) threaded through the
needle barrel into the jugular vein. The needle was then
withdrawn leaving approximately 40 cm of catheter in the
jugular vein. The catheter was flushed with sterile,
heparinized saline (Sodium Heparin, 100 units/ml; Sigma
Chemical Company, St. Louis, MO), occluded with a hemostat
and knotted to prevent blood flow through the catheter.
Catheter remaining outside the vein was coiled and secured

199
to the neck with tape (Ethikon; Johnson and Johnson Products
Incorporated, New Brunswick, NJ) and glue (KaMar
Incorporated, Steamboat Springs, CO). Beginning at 0700 h
on day 18, jugular blood samples were collected at 30 min
intervals from 1 h prior to E£ injection (3 mg in 6 ml of
ethanol:0.9 % saline [1:1] via jugular vein) to 12 h
post-Ep. Jugular catheters were removed immediately
following the 13 h acute bleeding period. Experimental
animals were fitted with estrous detection patches (Kamar
Incorporated, Steamboat Springs, CO) and monitored twice
daily for estrous behavior. Twice daily blood samples were
drawn via jugular venipuncture from day 19 until detected
estrus. Catheter placement and verification of CL regres¬
sion were determined by rectal palpation at behavioral
estrus in all cows.
Radioimmunoassay of Plasma Hormones
Validation of extraction and assay procedures for the
P4 radioimmunoassay used in the present study were identical
to those described by Knickerbocker and coworkers (Chapter
3). Intra- and interassay coefficients of variation (CV)
for 5 assays were 20.1 and 6.1%, respectively, when dupli¬
cate estimates were run in the assay for a 100 pg/500 ul
(100.2 ± 6.8 pg/500 u) plasma reference, and 12.4 and 17.9%,
respectively when duplicate estimates were run for a 250
pg/500 ul (261.2 ± 17.3 pg/500 ^1) plasma references.
Sensitivity of the P4 assay was 30 pg/ml*

200
Unextracted plasma samples (50, 100 and 200 yl) were
assayed for PGFM using a polyethylene glycol radioimmuno¬
assay system previously validated in our laboratory by
Guilbault et al. (1984). The intra- and interassay CV were
13-1 and 6.1%, respectively, for a reference sample of 1000
pg/ml (1066.6 ± 35*9 pg/ml) measured in duplicate for each
of 6 assays. Sensitivity of the PGFM assay was 50 pg/ml.
Statistical Analysis
Data for P^ and PGFM concentrations in plasma, and
plasma PGFM concentrations accumulated sequentially over the
12 h post-E2 injection period (accumulated PGFM) were ana¬
lyzed using the General Linear Models procedure of the
Statistical Analysis System (1982) for a split-plot analysis
of variance with repeated measurements over time. Analysis
of variance considered variability due to treatment (CSP and
Control), cow nested within treatment, time (for P^: days
12-17; for PGFM: 30 min sequential periods for 12 h post-
E2), treatment by time, and residual. To provide estimates
of time trends, plasma PGFM concentrations and accumulated
PGFM values were analyzed by least squares regression ana¬
lysis of variance. Differences in polynomial regressions
for PGFM concentrations and accumulated PGFM time trends
were tested by examining for homogeneity of regression
between treatment response curves. These data were analyzed
with time as a continuous, independent variable. Data

201
pertaining to cycle lengths and days with P4 concentrations
> 1 ng/ml were analyzed by one-way analysis of variance.
Results
Thirty-six milligrams of pooled CSP were harvested from
medium following 30 h incubations of day 17 to 18 bovine
conceptuses (n=29). Mean (± S.E.M.) wet weight for con-
ceptuses was 105 ± 13 mg. A mean of 1.24 mg of CSP were
produced per conceptus per 30 h and mean CSP production per
mg of conceptus wet weight was 11.9 ug for a 30 h incuba¬
tion.
Bovine conceptus secretory proteins administered into
the uterine lumen of cyclic cattle (days 15 through 18)
resulted in an attenuation (P<0.01) of E2~induced uterine
PGF2a production (PGFM response) on day 18 compared to con¬
trol cows which received intrauterine injections of serum
proteins (Table 5-1, Fig. 5-1). Overall means for PGFM con¬
centrations following the E£ challenge were 74.4 ± 5.2 pg/ml
for cows in the CSP treatment group and 170.6 ± 14.3 pg/ml
for cows in the Control group. A treatment by time inter¬
action also was detected (P<0.01; Fig. 5-1). Mean responses
for the Control group exhibited two distinct phases during
which PGFM concentrations were transiently elevated above
baeline (Fig. 5-1). The initial rise occurred between 2 to
3.5 h with peak PGFM concentrations at approximately

202
Table 5-1. Progesterone (P^) concentrations prior to Ep
injection (3 mg) and prostaglandin, luteal lifespan and
interestrous interval following E£ in cyclic cows treated with
serum proteins (CONTROL) or conceptus secretory proteins (CSP)
CONTROL
Cow
P4 (ng/ml)a
Accumulated13 Luteal
PGFM (pg) Lifespan (days)
Interestrous
Interval (days)
J164
9.6±1.3
5063
20.0
22.0
J85
10.2±1.5
4720
19.0
23.0
J190
11.7±1.0
2402
20.0
21 .0
J240
7.3±1.3
2184
21 .0
24.0
J160
10.0±0.6
708
21.0
21.5
Overall
Mean
9.7
3016
20.2
22.3
± S.E.
±1.3
± 821
± 0.4
± 0.5
CSP
Cow ^4
(ng/ml)
Accumulated
PGFM (pg)
Luteal
Lifespan (days)
Interestrous
Interval (days)
J97
3.5±0.2
1505
20.0
21 .0
J193
4.9±0.7
1408
20.0
22.0
J216
7.9±1.5
102
22.5
'25.5
J181
10.6±0.5
33
25.0
26.0
J155
9.9±0.5
0
22.5
24.5
Overall
Mean
7 • 4ns
610**
22.0ns
23•8ns
± S.E.
±1.4
± 347
± 0.9
± 1 .0
a Mean plasma progesterone calculated from days 12-17, prior
to Ep.
b Total 15-keto-13,14-dihydro-PGF2a accumulated over the 12 h
period post-Ep.
ns nonsignificant difference; P>0.1.
** P<0.01; (CSP
Figure 5-1. Least squares means of plasma 15-keto-13,1 4-
dihydro-PGFpa (PGFM) concentrations in response
to an estraaiol-17B (Ep) challenge (3 mg; I.V.)
in cyclic Jersey cows receiving intrauterine
infusions (days 15*5-18) of serum proteins from
a day 18 pregnant cow (control; n=5) or
conceptus secretory proteins (n=5).

204

205
2.5 h. A second and more substantial peak was initiated by
approximately 6 to 6.5 h, peaked at 7.5 h, and returned to
baseline concentrations by 8.5 to 9.5 h post-E2- Mean PGFM
response in CSP-treated cows was approximately 20% of that
observed for the control group as determined by areas under
the two PGFM peaks and mean accumulated PGFM concentrations
following E£ injection (Table 5-1). Individual PGFM
responses for each cow are depicted in figure 5-2. All cows
in the Control group responded to exogenous E2 by having
elevations in plasma PGFM concentrations. In one animal
(J164), a spontaneous pulse of PGFM (2725 pg/ml) was
detected prior to the E2 challenge which suggested that
luteal regression had already been initiated. Concentra¬
tions of plasma P^ had declined from 6.4 ng/ml on day 17 to
2.1 ng/ml on the day of E2 injection (day 18). Uterine
PGF2ct Production was most dramatic in this cow as
demonstrated by the induced PGFM peak at 6.5 h (1225 pg/ml).
In contrast, three of five cows treated with CSP failed
to respond to injection of Ep. The remaining two cows exhi¬
bited PGFM responses similar to low responders (n=3) in the
control group. Interestingly, mean P^ concentrations from
days 12 to 17 of the estrous cycle (Table 5-1) were consid¬
erably lower in the two cows which did not exhibit a clear
antiluteolytic effect of CSP post-E2 injection (3.5 ± 0.2,
4.9 ± 0.7 ng/ml versus 7.9 ± 1*5* 9.9 ± 0.5, 10.6 ± 0.5
ng/ml). Cows in the Control group had P^ concentrations

Figure 5-2. Individual cow plasma concentrations of 15-
keto-13»14,-dihydro-PGF^a (PGFM) in response to
an estradiol-17B (E2 challenge (3 mg; I.V.) in
cyclic Jersey cows receiving intrauterine
infusions (days 15.5-18) of serum proteins from
a day 18 pregnant cow (control) or conceptus
secretory proteins.

PGFM (PG/ML) P6FM (PG/ML)
207

208
which fluctuated between 5 and 14 ng/ml (X ± S.E., 9.7 ±1.3
ng/ral) during the same period. The PGFM responses were
evaluated further by accumulating PGFM concentrations over
the 12 h period following E£ injection (Table 5-1). On the
average, cows in the Control group responded to an E£
challenge by accumulating approximately five times more PGFM
(P<0.01) than cows which received intrauterine treatments of
CSP (Table 5-1). This point is illustrated (Figure 5-2) by
comparing profiles for PGFM over the 12 h post-E2 period in
Control and CSP groups.
No differences (P>0.25) were detected in CL lifespan
(days with P^ > 1 ng/ml) or estrous cycle length responses
between the two experimental groups (Table 5-1). However,
the three cows in the CSP group which accumulated the least
PGFM tended to have extended CL maintenance and longer
interestrous intervals.
Rectal palpation of the reproductive tracts at estrus
confirmed catheter placements within the tip of each uterine
horn. No uterine or ovarian abnormalities were apparent.
Ovarian palpation per rectum and analysis of plasma P^ con¬
centrations verified regression of the CL which was observed
at surgery.

209
Discussion
Bovine conceptus secretory proteins significantly
attenuated uterine production of as determined by
plasma PGFM concentrations, following injection of a luteo-
lytic dose of E2 to cyclic cows. These data are consistent
with responses described previously for cyclic cattle
treated with bovine CSP (Chapter 4) in which a significant
reduction in vena cava plasma concentrations of PGF2a was
associated with extended CL function and interestrous inter¬
val. Collectively, these reports indicate that the local
antiluteolytic influence of the bovine conceptus, in útero
(Kindahl et al., 1976; Betteridge et al., 1984) results from
an interaction between biologically active conceptus-derived
proteins and the uterine endometrium (Thatcher et al.,
1984a).
However, mechanisms by which bovine CSP reduce endo¬
metrial production of PGF2a are not known. Data in sheep,
as in cattle, demonstrated that intrauterine administration
of conceptus derived secretory proteins to cyclic ewes re¬
sulted in prolonged CL maintenance and interestrous interval
(Godkin et al., 1984), and attenuated E2~stimulated rise in
plasma PGFM concentrations (Fincher et al., 1984). Further¬
more, ovine trophoblast protein-1 (oTP-1), the major con¬
ceptus produced protein during maternal recognition of
pregnancy in sheep, extended luteal function when introduced

210
into the uterine lumen (Godkin et al., 1984a), bound
specifically to endometrial receptors and stimulated
secretion of specific endometrial proteins in vitro (Godkin
et al., 1984b). Although no specific functions have been
attributed to these endometrial proteins in sheep, there are
reports which demonstrate the presence of inhibitors to
prostaglandin synthesis in bovine endometrium (Wlodawer et
al., 1976) and caruncular (Shemesh et al., 1984) tissues.
Additionally, Thatcher and coworkers (1984b) demonstrated a
reduction in de novo PGF2a synthesis by bovine endometrial
explants at day 17 of pregnancy compared to day 17 of the
estrous cycle. Arachidonic acid supplementation to
endometrial incubations increased PGF2a production in both
pregnant and cyclic tissues. However, PGF2a production was
less for pregnant endometrium. It is possible that bovine
conceptus secretory proteins activate endometrial
biosynthesis of inhibitors to prostaglandin production
resulting in selective inhibition of uterine PGF2a
production (Thatcher et al., 1984b; Chapter 4) while
permitting biosynthesis of prostaglandins by the developing
conceptus (Shemesh et al., 1979; Lewis et al., 1982).
Additionally, conceptus-induced uterine prostaglandin
inhibitors may modify the amounts and/or classes of
prostaglandins synthesized by the developing conceptus and
endometrium during early gestation (Lewis and Waterman,

211
1983; Lewis, 1984), as well as placentome tissues during
later gestation (Shemesh et al., 1981; 1984).
In the present study, the maternal endocrine milieu may
influence uterine responsiveness to conceptus signals during
the critical period of pregnancy recognition. Intrauterine
treatments with CSP, in two of five cows, did not prevent
the E2~stimulated rise in plasma PGFM concentrations. These
animals had 50 to 60% lower P^ concentrations (4.2 ± 0.3
ng/ml) during the luteal phase of the estrous cycle (days 12
through 17) compared to P^ concentrations exhibited by three
cows (9.5 ± 0.7 ng/ml) in which CSP completely inhibited the
E2-induced PGFM response. Progesterone is known to regulate
uterine physiological development in numerous species. The
rate and extent of uterine and conceptus development (syn¬
chrony) may be altered by variations in length of P4
exposure and P4 concentrations during early gestation
(Lawson and Cahill, 1983; Lawson et al., 1983). Thus,
inferior CL quality, as reflected by low plasma P^
concentrations, may preclude appropriate uterine development
and result in an inadequate maternal response to conceptus
signals.
Estrogen stimulation of uterine PGF2a production
(Chapter 3) is thought to involve uterine protein synthesis,
since the luteolytic effect of estrogen may be prevented by
inhibiting uterine DNA dependent RNA synthesis with actino-
mycin D (French and Casida, 1973). Elevated estrogen

212
production by ovarian follicles may initiate luteolysis in
cattle, as destruction of follicles during the luteal phase
of the estrous cycle prolongs CL function (Villa-Godoy et
al., 1981). Additionally, Huslig and coworkers (1979)
reported cyclic changes in concentrations of uterine cyclo¬
oxygenase in ewes which occurred simultaneously with changes
in uterine PGF2a secretion. They suggested that exposure of
the primed uterus to estrogen was responsible for synthe¬
sis of this rate limiting enzyme. Estrogen is also believed
to induce the formation of endometrial oxytocin receptors
during the estrous cycle (Roberts et al., 1976; McCracken et
al., 1981). Exogenous oxytocin stimulates uterine PGF2a
production in cyclic cattle (Newcomb et al., 1977; Milvae
and Hansel, 1980) and causes premature CL regression
(Armstrong and Hansel, 1959). Oxytocin is believed to
augment uterine PGF2a production as the estrogen:P^ ratio
increases following initiation of luteolysis and thus
ensures rapid and complete CL regression (Wathes, 1984). In
sheep, endometrial oxytocin receptor concentrations are
significantly reduced at day 16 of pregnancy versus day 16
of the estrous cycle (McCracken et al., 1981) and the
oxytocin induced rise in plasma PGFM concentrations are
attenuated in E2~primed cyclic ewes following intrauterine
CSP administration (Fincher et al., 1984). Therefore,
bovine and ovine CSP may regulate synthesis or availability
of steroid receptors responsible for oxytocin receptor

213
induction, or CSP may modify the formation or availability
of endometrial oxytocin receptors directly to prevent
luteolysis.
A biphasic PGFM response (at 2.5 and 7.5 h) to E£ was
observed in all control cows and two of five CSP treatment
cows. Timing of the first PGFM peak coincided with maximal
uterine blood flow response following stimulation by exo¬
genous E£ in cattle (Roman-Ponce et al., 1978; Chapter 3),
and probably represents a washing out of residual prosta¬
glandins from an active uterus. The second and more sub¬
stantial peak of PGFM corresponded to the period, post-E2*
when uterine PGF2a production is activated (Chapter 3).
Lastly, bovine CSP have been shown previously to extend
CL function and interestrous interval in cyclic cows when
administered into the uterine lumen at 12 h intervals from
days 15 to 21 post-estrus (Chapter 4). However, mean CL
lifespan and interestrous intervals in the present study did
not differ significantly between experimental groups. The
difference in response to bovine CSP in the first (Chapter
4) and present study are probably due to the shorter period
of CSP treatments (4 versus 7 days), and the post-CSP chal¬
lenge with a luteolytic dose of E2 (3 mg) in the present
study. There was a tendency, however, for extended CL func¬
tion and cycle length in CSP treatment cows which accumu¬
lated the least PGFM (Table 5-1).

214
In conclusion, these data support a role for conceptus
secretory proteins in suppression of uterine PGF2a produc¬
tion during the period of pregnancy recognition in cattle.

CHAPTER 6
GENERAL DISCUSSION
Advances in our understanding of mechanisms controlling
luteal maintenance during early pregnancy have been
considerable during the last decade. Characterization of
conceptus products and their biosynthetic patterns have
provided an important foundation from which many of the
exciting findings reported herein stem. The bovine
conceptus produces an array of biologically active steroids,
prostaglandins and proteins during early pregnancy which are
involved in conceptus developmental processes and
alterations in maternal physiological events directed toward
pregnancy maintenance (see: Chapter 1). Elevated endocrine
activity by bovine conceptus tissues occurs concomitantly
with rapid conceptus elongation and maternal recognition of
pregnancy. Maternal recognition of pregnancy represents the
first critical period during early gestation when conceptus
signals must be synthesized and received subsequently by the
maternal system such that cyclical events regulating corpus
luteum (CL) regression are circumvented. Currently,
evidence suggests that several conceptus-mediated strategies
are involved to this end. These strategies may be
classified broadly as 1) luteotropic (stimulation of luteal
215

216
production); 2) luteal protective (block luteolytic
effects of PGF2d at the level of the CL); and 3)
antiluteolytic (attenuation of uterine PGF2a production).
Antiluteolytic effects of conceptus products were
evaluated in two experiments (Chapters 4 and 5). Data from
these studies demonstrated that bovine conceptus-derived
protein signals extended CL function and interestrous
intervals, and attenuated spontaneous and E2~induced uterine
PGF2a production. Durations of CL and interestrous interval
extension following intrauterine administration (days 15.5
to 21) of bovine CSP were 30.3 and 33-4 days, respectively,
compared to 22.3 and 23.5 days, respectively for control
cows (Chapter 4). Bovine CSP influence on the uterus
remained effective for one to two weeks (range: 7 to 13
days) following the last intrauterine injection. This
response differs from responses reported previously to
intrauterine administration of PGE2 (Chenault, 1983; Giraenez
and Henricks, 1983; Reynolds et al., 1983). Prostaglandin-
E2 effects on CL maintenance are short-lived in cyclic
cattle suggesting that this substance will not prevent
production and transfer of uterine luteolytic substances to
the CL. Available data support luteotropic (Marsh, 1970)
and/or luteal protective (Henderson et al., 1977; Reynolds
et al., 1981) roles for PGE2 during early pregnancy. At
present, precise mechanism(s) by which bovine CSP attenuate
uterine PGF2a production is unknown. However, in ovine

217
endometrium, estrogen (Findlay et al., 1982) and oxytocin
(Roberts et al., 1976; McCracken et al., 1984) receptor
concentrations are reduced during early pregnancy.
Likewise, exogenous E2- (cow: Knickerbocker et al., 1984;
Chapter 5; sheep: Fincher et al., 1984) and oxytocin-
(sheep: Fincher et al., 1984) induced uterine PGF2a
production are attenuated following intrauterine CSP
administration. Conversely, decline in endometrial estrogen
and oxytocin receptors may result from maintenance of
endometrial sensitivity to P^ (viz., P^ receptors).
Progesterone inhibits replenishment of E2 receptors and
estrogen dependent responses (see: Chapter 1) such as
induction of endometrial oxytocin receptor production
(McCracken et al., 1984). Maintenance or increased
endometrial P4 receptor concentrations during early
pregnancy may be influenced by CSP, however, modifications
in steroid receptor populations are generally thought to be
steroid regulated (see: Chapter 1). Other conceptus
products, such as estrogens (Chapter 2) or catecholestrogens
(hydroxylated estrogen metabolites) may mediate endometrial
P4 sensitivity. Alternatively, effects of antiluteolytic
bovine CSP may be directed at reducing synthesis or activity
of arachidonic acid metabolic enzymes within the uterine
endometrium. This effect may occur directly or indirectly
via stimulation of uterine inhibitors of prostaglandin
synthesis. The extended antiluteolytic effect of bovine CSP

218
detected after cessation of intrauterine treatments (Chapter
4) support the hypothesis that CSP induce endometrial
production of inhibitors to prostaglandin synthesis. An
extended antiluteolytic effect of the conceptus was also
observed in endometrial explant cultures of day 17 pregnant
cattle (Thatcher et al., 1984b). Synthesis of specific
endometrial proteins, in vitro, were induced by an ovine
conceptus protein, oTP-1 (Godkin et al., 1984a). Lastly,
Wlodawer et al. (1976) reported that bovine endometrium
contains an inhibitor of prostaglandin biosynthesis.
Mediation of conceptus antiluteolytic effects through
induced endometrial production of prostaglandin synthesis
inhibitors is an attractive concept for the following
reasons: 1) uterine PGF2a biosynthesis may be prevented
without inhibiting prostaglandin production by the
developing conceptus; 2) ratio of PGE2:PGF2a within the
uterine lumen may be elevated as a result of continued
conceptus prostaglandin production and decreased uterine
PGF2a production; and 3) conceptus estrogen production
(Chapter 2) may stimulate uterine blood flow and facilitate
movement of luteotropic (PGE2, PGl^/luteal protective
(PGE2) prostaglandins to the CL without stimulating uterine
PGF2a biosynthesis. Similarly, conceptus induced
endometrial inhibitors may regulate biosynthesis of products
from the lipoxygenase pathway during early pregnancy.
Milvae and coworkers (Milvae and Hansel, 1980, 1983; Milvae

219
et al., 1985) have demonstrated (in vivo and in vitro) that
PGI2 is luteotropic in cattle and may be essential for
normal luteal development and continued biosynthesis.
Prostacyclin biosynthesis is inhibited by products of the
lipoxygenase pathway, e.g., 5-HETE. Furthermore,
intrauterine administration (days 14 to 18) of a
lipoxygenase pathway blocker, NDGA, to cyclic cattle
extended luteal function to day 25. Thus, 5-HETE and other
lipoxygenase products may be involved in normal luteolytic
events. Inhibition of lipoxygenase products during early
pregnancy may therefore facilitate PGl£ biosynthesis
(uterus, conceptus, CL) and luteotropic actions on the CL.
In contrast to the clear antiluteolytic effect of
conceptus secretory proteins, a major conceptus steroid
product, 5B-pregnan-3ct-ol-20-one (56—P; Chapter 2), had no
effect on luteal function or cycle length when administered
into the uterine lumen of cyclic cattle (Chapter 4). In
fact, total spontaneous PGF2a measured in vena cava samples
were greater (P<.01) in cows treated with 5B-P than
homologous serum proteins (Control). No adequate
explanation for this result can be offered at this time.
However, recent data (Thatcher et al., 1984b) have
demonstrated decreased endometrial and uteroovarian vascular
permeability to PGF2a in day 17 pregnant cows. If
intrauterine administration of 5B-P reduced tissue
permeability to PGF2a without preventing or attenuating

220
uterine PGF2a production, an accumulation of PGF2a might be
expected to occur within the uterine lumen. Such elevated
uterine luminal concentrations of PGF2a may translate
ultimately into greater uterine venous concentrations of
PGF2a as maximum tissue capacity is reached. Alternatively,
progestational activity of 5&-P may be sufficient to
stimulate endometrial accumulation of lipid stores and
prostaglandin precursors, thereby resulting in greater
uterine capacity to synthesize PGF2a. Other putative roles
for the 50-reduced steroids are discussed in Chapter 2.
Further research directed toward understanding conceptus and
endometrial steroid function is warranted.
Collectively, research described herein support the use
of peripheral PGFM concentrations as an index of uterine
PGF2a synthetic activity. The use of the E2~challenge
scheme (characterized in Chapter 3) proved an effective tool
for evaluating uterine capacity to synthesize PGF2a
following intrauterine treatments. Spontaneous PGF2a
episodes and E2~induced uterine PGF2a Production were
dampened significantly by intrauterine administration of
bovine CSP, but not 5g-pregnan-3a-ol-20-one, a major
conceptus steroid product. Bovine CSP did not influence CL
production of P^ suggesting that CSP act locally at the
uterine level.

APPENDIX A
MANUFACTURE OF SEPHADEX LH20 COLUMNS
1. Prepare a slurry of Sephadex LH20 beads in freshly
distilled solvents. Solvent mixture should be identical
to that which is employed during steroid separation.
2. Allow beads to swell for 12 to 24 hours at room
temperature in a sealed container.
3. Degas slurry under a vacuum of approximately 25 p.s.i.
for 15 to 20 minutes before pouring column.
4. Glass columns (35 x 1.5 cm) are filled with the solvent
mixture and stopcock moved to the open position.
5. Sephadex slurry is pipetted into the column until the
desired column height is reached. At this point,
approximately 50 ml of the solvent mixture is run over
the column to aid in packing. Do not let column run
dry.
6. Turn column stopcock off, add solvent mixture to
approximately 25 cm on the glass column, and seal column
top.
7. Columns are allowed to settle and pack for 12 to 24
hours before use.
221

APPENDIX B
STEROID ELUTION BY GAS/LIQUID CHROMATOGRAPHY
Steroid3
minutes
Rf/P4b
5B-androstane-38,17a-diol
5.85
.140
4-pregnen-38-ol-20-one
6.0
.144
5 8-androstane-38,17 8-diol
6.25
.150
5«-androstane-3a,17«-diol
6.5
.156
5 8-androstane-3«,17°-diol
6.6
.158
5a-androstane-3«,17 3-dio1
6.75
.162
5 6-androstane-3a,176-diol
6.75
.162
4-androstene-38,178-diol
6.75
.162
5«-androstane-3B,17a-diol
7.1
.170
5a-androstane-3S,178-diol
7.25
.174
58-pregnane-3 6,206-dio1
8.5
.204
58-pregnane-3 8,20a-diol
9.1
.218
5 8-pregnane-3«,206-dio1
9.4
.225
58-androstan-38-o1-17-one
9.6
.230
5«-pregnane-3a,20a-diol
9.8
.235
5«-pregnane-38,208-diol
10.1
.242
5 a-pregnane-3 a,20 8-diol
10.25
.246
5 6-pregnane-3«,20a-diol
10.25
.246
5 a-androstan-3 a-ol-17-one
10.25
.246
5«-pregnane-38,20a-diol
10.9
.261
5 8-androstan-3 a-ol-17-one
11.0
.263
5 a-androstan-3 S-ol-17-one
11.75
.281
5 8-androstan-17 a-ol-3-one
12.1
.290
5 6-pregnan-3 8-ol-20-one
12.5
.299
5 8-androstan-17 8-ol-3-one
12.75
.305
5a-androstan-17 a-ol-3-one
12.9
.309
5 a-pregnan-3 a-ol-20-one
13.5
.323
5 a-androstan-17 8-ol-3-one
13.6
.326
5 8-pregnan-3a-ol-20-one
13.75
.329
5a-pregnan-3 B-ol-20-one
15.0
.359
4-androsten-17 a-ol-3-one
19.25
.461
4-androsten-17 6-ol-3-one
20.1
.481
5 8-androstane-3,17-dione
20.75
.497
5 «-androstane-3,17-dione
22.25
.533
222

223
Steroid3
minutes
Rf/P4b
5 8-pregnane-3,20-dione
26.1
.625
4-pregnen-20 8-ol-3-one
27.4
. 656
4-pregnen-3 8,20 8-diol
27.75
. 665
5 B-pregnane-3,20-dione
28.1
.673
4-pregnen-3,17-dione
31.0
.743
4-androstene-3,17-dione
32.4
.776
4-pregnene-3,20-dione
41 .75
1.000
4-pregnene-17 a-ol-3,20-dione
61.0
1.461
Note: Column specifications: 3% trifluropropyl-silicone
(SP-2401) on Supelcoport 100/120 mesh (Supelcoport
Inc., Bellefonte, PA)
Detector: flame ionization
Column temperature: 240 C
Injector temperature: 265 C
Detector temperature: 260 C
N£ pressure: 26.5 p.s.i.
Gas flow rates (ml/min): H2 (20); air (300)
a Gift from National Research Council, Steroid Reference
Collection, London, England.
D Retention time relative to 4-pregnene-3,20-dione.

cr (u
APPENDIX C
MASS CALCULATIONS FOR ESTROGENS
STEP 1. Convert sample cpm to dpm.
Sample cpma = Sample dpm
Counting Efficiency
during liquid scintillation
STEP 2.
Calculate dpm/pg
estrogen.
A.
Specific Activity
substrate
(Ci/mmole)
of 1,2,6,7-[5H](N)-P4
B.
2.22 x 1012 dpm =
1 Ci
C.
Molecular weights:
Estrone
270.3
Estradiol-176 272.3
Estriol 288.3
Calculations:^ AxB = dpm/mg estrogen
C
then divided by 10^ = dpm/pg estrogen
STEP 3. Calculate pg of estrogen in sample.
Calculation: Sample dpm = pg estrogen
dpm/pg estrogen
Corrected for background cpm and [^C] spillover.
These calculations assume that the specific activity of
estrogen products equal the specific activity of tritiated
P4 substrate. This assumption is justifed by two
points. First, placement of tritiated hydrogens on the
1,2,6, and 7 positions of P4 are in an a-configuration
(Dr. Mayo Cabell, New England Nuclear, Boston, MA;
personal communication). Second, enzymatic steps in
aromatization involve the loss of A-ring hydrogens at the
13 or 26 sites (Fishman, 1982). Thus, loss of 1a, 2a, 6a
or 7a tritiums during aromatization are not warranted.
224

APPENDIX D
ELUTION OF RADIOINERT STEROID STANDARDS ON HPLC
(ACETONITRILE:WATER, 54:46)

226
„ 0 /0 _ 2.0 , 4 J 4.0 MIN. , 6.0 8.0
Testosterone (2.02 min.), Androstenedione (2.41 min.), 5B-Androstanedione (2.91 min.),
5a-Androstanedione (3.50 min.), Progesterone (4.96 min.), 5B-pregnan-3a-ol-20-one
(6.00 min.), 5 0-pregnanedione (7.18 min.), 5 ct-pregnanedione (8.06 min.).

APPENDIX E
ELUTION OF [3H]-CONCEPTUS METABOLITES AND
[14C]-MARKERS ON HPLC (ACETONITRILErWATER, 54:46)

Abbreviations: UNK (unknown), A. (androstenedione), 5“-A
(5a-androstanedione, P. (progesterone), 3a-0H (56-pregnan-
3a-ol-20-one), 5^-P (50-pregnanedione), 5a-P (5a-
pregnanedione).
228

REFERENCES
Albert, D.H., Ponticorvo, L., Welch, M., Lieberraan, S.
(1980). Identification of Fatty Acid Esters of
Pregnenolone and Allopregnanolone From Bovine Corpora
Lútea. J. Biol. Chem. 255:10618-10623.
Albert, D.H., Prasad, V.V.K., Lieberman, S. (1982). The
Conversion of Progesterone Into 5a-pregnane-3,20-dione,
36-hydroxy-5a-pregnan-20-one, and Its Fatty Acid Esters
by Preparations of Bovine Corpora Lútea. Endocrinology
111:17-23.
Alexander, J.S., Rigby, B.M., Jedwitz-Rigby, F. (1978).
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BIOGRAPHICAL SKETCH
Jeffrey John Knickerbocker, eldest of four children
born to John Vearon Knickerbocker and Janice Danforth, was
born in Eatontown, New Jersey, on April 17, 1955. During
his first 15 years, he acquired a taste for travel as his
parents made several work-related moves, including a
memorable two years in Frankfurt, West Germany. In 1973, he
graduated from Neptune Senior High School, Neptune, New
Jersey, and attended the University of Delaware, home of the
"Fighting Blue Hens," until 1977 when he graduated with a
Bachelor of Science in animal science. He began his
graduate study in the fall of 1977 at Clemson University,
South Carolina, under the guidance of Dr. Joseph F. Dickey
in the Department of Dairy Science. On August 5, 1978, he
was married to Margaret 'Holly* Hollywood Dougherty of
Claymont, Delaware. In 1982, he completed his degree
requirements for the Master of Science in food and animal
industries, Clemson University, South Carolina.
During the fall of 1979, he and his wife arrived in
Gainesville, Florida, where he began a program of research
under the direction of Dr. William W. Thatcher in the
Department of Dairy Science, University of Florida,
Gainesville. His training in reproductive biology at the
272

273
University of Florida may be said to have been a truly
fertile experience. During this period, two children,
Lauren (June 23, 1980) and Daniel John (December 11, 1983)
were born to him and Holly.
Upon completion of the Doctor of Philosophy degree, the
author will further his training in the laboratory of Dr.
Gordon D. Niswender, Colorado State University, Fort
Collins, as a post-doctoral fellow.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
/¿4U~ a/
William W. Thatcher, Chairman
Professor of Animal Science and
Dairy Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Michael Z/. Fields
Associate Professor
Science
7
of Animal
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
R. Michael Roberts
Professor of Biochemistry and
Molecular Biology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Professor of Anesthesiology and
Obstetrics and Gynecology
This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate School, and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
May, 1985
its/ C
, V
of.
L
Dean, College of Agripülture
Dean for Graduate Studies and
Research

UNIVERSITY OF FLORIDA
3 1262 08556 7583



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