Redacted for Privacy - COnnecting REpositories · Thanks also to Dr. Kenneth Rowe, Oregon State University, for his statistical guidance. ... extra special thank you to Brian, who

AN ABSTRACT OF THE THESIS OF

Patrice L. Prater for the degree of Master of Science in

Animal Science presented on November 14. 1990

Title: Physiological Factors Affecting Ovine Uterine

Estrogen and Progesterone Receptor Concentrations

Redacted for PrivacyAbstract Approved: w-A

Fredrick Stormshak

Two experiments were conducted to determine whether in

ewes uterine concentrations of estrogen and progesterone

receptors are affected by the presence of a conceptus or by

the hormonal milieu associated with extremes in photoperiod

to which ewes are exposed.

In Exp.1, nine mature ewes were unilaterally

ovariectomized by removing an ovary bearing the corpus luteum

(CL). The ipsilateral uterine horn was ligated at the

external bifurcation and a portion of the anterior ipsilateral

uterine horn was removed and assayed for endometrial nuclear

and cytosolic concentrations of estrogen receptor (ER) and

progesterone receptor (PR) by exchange assays. After a

recovery estrous cycle, ewes were bred to a fertile ram. On

day 18 of gestation a 10 ml jugular blood sample was collected

for measurement of serum concentrations of estradiol -178 (E2)

and progesterone by radioimmunoassay. Ewes were

relaparotomized on day 18 and the remaining uterine tissue was

removed. Endometrium from both the pregnant and nonpregnant

uterine horn was assayed for nuclear and cytosolic ER and PR

concentrations. Nuclear and cytosolic ER concentrations on

day 10 of the cycle were greater than in endometrium of gravid

and nongravid uterine horns on day 18 of gestation (p<.01).

Endometrial nuclear PR levels were also greater on day 10 of

the cycle than in the pregnant (p<.05) and nonpregnant horn

(p<.01) on day 18 of gestation. There were no differences in

nuclear and cytosolic ER and PR concentrations between the

pregnant and nonpregnant uterine horn on day 18. Serum levels

of E2 and progesterone on day 18 of gestation were 16.56 ±

2.43 pg/ml and 1.74 ± 0.57 ng/ml, respectively. These data

suggest that duration of exposure of the uterus to

progesterone and(or) the presence of the conceptus causes a

reduction in uterine concentrations of ER and PR. Further,

an effect of the conceptus, if any, is exerted via a systemic

route.

In Exp. 2, ten mature ewes were bilaterally

ovariectomized in early October. During the onset of the

winter solstice (late December), a 10 ml blood sample was

collected from five ewes for analysis of serum levels of E2

and progesterone. Ewes were then laparotomized and

approximately one-third to one-half of a uterine horn was

removed and assayed for endometrial nuclear and cytosolic ER.

The contralateral horn was ligated at the external bifurcation

and 10 Ag of E2 in 3 ml of physiological saline was injected

into the uterine lumen of the ligated horn. After 48 h, a

jugular blood sample was collected for steroid analysis and

a section of the E2_treated horn was removed and assayed for

endometrial cytosolic and nuclear ER. This procedure was

repeated on the remaining five ewes during the height of the

summer solstice (late June). Endometrial nuclear and

cytosolic concentrations of ER prior to and after exogenous

E2 stimulation were similar during the winter and summer

solstice (p>.05). However, treatment with E2 increased

endometrial nuclear and cytosolic concentrations of ER

compared with those of the nonstimulated uterine horn during

the winter and summer solstice (p<.05 for each). Serum levels

of E2 prior to luminal treatment of ewes with E2 during the

winter and summer solstice did not differ (16.55 ± 4.05 vs

16.00 ± 3.0 pg/ml, respectively, p>.05). Serum levels of E2

48 h after administration of E2 did not differ among ewes at

the winter and summer solstice (18.75 ± 2.4 vs 18.65 ± 1.65

pg/ml, respectively, p>.05). Serum levels of progesterone

were basal (<0.10 ng/ml) and did not differ in ewes prior to

and after E2 treatment at the winter and summer solstice

(p>.05). These data indicate that physiological factors

and(or) hormones such as prolactin and melatonin secreted in

response to extremes in photoperiod do not appear to influence

uterine concentrations of ER in ovariectomized ewes.

Physiological Factors Affecting Ovine Uterine

Estrogen and Progesterone Receptor Concentrations

by

Patrice L. Prater

A THESISsubmitted to

Oregon State University

in partial fulfillment ofthe requirements for the

degree of

Master of Science

Completed November 14, 1990

Commencement June, 1991

APPROVED:

Redacted for PrivacyProfessor of Animal Science and Biochemistry and Biophysicsin charge of the major

Redacted for PrivacyHead of Department of Animal Science

Dean of C

Redacted for Privacy

Date thesis presented: November 14, 1990

Typed for Researcher by: Patrice L. Prater

ACKNOWLEDGEMENTS

I would like to take the opportunity to express my thanks

to all of the individuals who helped make this thesis a

reality. Sincere thanks to Dr. "Stormy" Stormshak for his

guidance and friendship, and most importantly, for teaching

me perseverance to "take the bull by the tail on a uphill

pull" as there will always be worthwhile and attainable goals

to pursue. I would also like to thank Ligaya Tumbelaka, Anna

Cortell-Brown, Ov Slayden, and Sue Leers-Sucheta for their

help and friendship during surgeries and laboratory

tribulations. Appreciation also to the U.S.F.S, LaGrande

research station and Dr. John Stellflug of the U.S. Sheep

Experimental Station, Dubois, Idaho for their contributions

toward this research. Thanks also to Dr. Kenneth Rowe, Oregon

State University, for his statistical guidance.

In hindsight, I realize that without the support and

encouragement of those most dear to me, this degree would not

have been realized. Therefore, I would like to extend a very

special thank you to my family (Moores and Praters) and my

husband Brian for believing in me and for giving me the

foundation of love and encouragement on which to build. An

extra special thank you to Brian, who as a husband and best

friend has shown me that prosperity in life is found through

love and laughter.

TABLE OF CONTENTS

Page

REVIEW OF LITERATURE 1

Introduction 1

ESTROGEN AND PROGESTERONE 4

Origin of Estrogen and Progesterone 4

Physiological Responses to Estrogens 5

Physiological Responses to Progesterone 7

Biosynthesis of Estrogen and Progesterone 7

Steroid Hormone Transport 11

Steroid Hormone Receptors 12

THE ESTROUS CYCLE 17

Follicular Phase 17

Luteal Phase 18

Luteolysis 20

EARLY GESTATION 23

Maternal Recognition of Pregnancy 23

Implantation or Attachment 26

SEASONAL REPRODUCTION 30

Melatonin 31

Prolactin 34

EXPERIMENT 1 38

Introduction 38

Materials and Methods 39

Animals 39

Nuclear Estrogen Receptor Assay 40

Cytosolic Estrogen Receptor Assay 42

Nuclear Progesterone Receptor Assay 43

Cytosolic Progesterone Receptor Assay 44

DNA Determination 45

Serum Progesterone Radioimmunoassay 46

Serum Estradiol Radioimmunoassay 47

Statistical Analysis 49

Results 49

Discussion 52

EXPERIMENT 2 55

Introduction 55

Materials and Methods 56

Animals 56Nuclear and Cytosolic Estrogen Receptor Assay 57

TABLE OF CONTENTS (CONT.)

Page

DNA Determination 57

Serum Progesterone and EstradiolRadioimmunoassay 58

Statistical Analysis 58

Results 58

Discussion 61

BIBLIOGRAPHY 64

LIST OF FIGURES

Figure Page

1. Specific binding of Clflestradio1-178 tonuclear and cytosolic estrogen receptors (mean ±SE) in endometria of ewes on day 10 of the estrouscycle and in endometria of gravid and non-graviduterine horns of ewes on day 18 of gestation. Meannuclear and cytosolic concentrations of estrogenreceptors, day 10 vs day 18 gravid and nongravidhorns differ (p<.05).

2. Specifically bound (3H)R5020 to nuclearprogesterone receptors (mean ± SE) in endometria ofewes on day 10 of the estrous cycle and in endometriaof the gravid and nongravid uterine horns of ewes onday 18 of gestation. Day 10 vs day 18 gravid andnongravid horns differ in concentrations ofprogesterone receptors (p<.05).

3. Specifically bound [3H]estradio1-178 tonuclear and cytosolic estrogen receptors (mean ± SE)in nonstimulated and exogenous estradiol-stimulatedendometria of ewes during the winter and summersolstice. Stimulated vs nonstimulated endometrium;nuclear (p<.05), cytosolic (p<.01).

51

51

60

PHYSIOLOGICAL FACTORS AFFECTING OVINE UTERINEESTROGEN AND PROGESTERONE RECEPTOR CONCENTRATIONS

REVIEW OF LITERATURE

INTRODUCTION

The mechanisms of steroid hormone action have long been

an area of fascination and scientific inquiry. In the female,

estrogen and progesterone play a primary role in promoting the

development of an intrauterine environment necessary for

successful procreation.

Prior to 1960 most research on estrogens and progesterone

entailed studies with various mammalian species to elucidate

the physiological effects of these steroids on the development

of the uterus and mammary glands. During the 1960's interest

in the effects of these hormones shifted from physiological

to biochemical. Indeed, it was during the late 1960's that

experimental evidence was first presented to suggest that the

biological responses evoked by estrogens were the consequence

of this steroid being bound within the target cell by a

macromolecular receptor. Subsequently, research demonstrated

the existence of target tissue receptors for other steroids,

including progesterone.

Because the availability of estrogen and progesterone

receptors directly affects the biological activity of these

steroids, much research has been conducted to quantify

2

estrogen and progesterone receptors in uteri of laboratory

animals. However, comparatively little is known about the

changes in uterine concentrations of estrogen and progesterone

receptors that occur during various reproductive states in

domestic animals.

One of the leading causes of reduced reproductive

efficiency in many species is embryonic mortality. Most

embryos are lost before or soon after implantation. Many

factors influence the ability of an embryo to implant

including adequate production of ovarian hormones and

appropriate uterine responses to these hormones. However,

whether embryo wastage is, in part, attributable to impaired

action of estrogen and progesterone is unknown. Basic

information on estrogen and progesterone receptor

concentrations in the uterus during early gestation could

provide insight about factors that maintain or interfere with

successful pregnancy.

In the northern hemisphere, sheep are seasonal breeders

that exhibit behavioral estrus during the fall and winter and

then become anestrus during the late spring and early summer.

The underlying cause for the annual reproductive cycle of ewes

has been attributed to changes in duration of melatonin

secretion that occurs in response to seasonal changes in

photoperiod. Secretion of melatonin from the pineal gland of

ewes is maximal as hours of daylight decrease and minimal as

hours of daylight increase. In a number of species, daily

3

profiles of secretion of melatonin and prolactin are inversely

related. Indeed, experimental evidence suggests that

melatonin can inhibit the secretion of prolactin from the

adenohypophysis. It is not known whether systemic

concentrations of prolactin or melatonin can alter the

responsiveness of the uterus of ewes to steroids. These

hormones have been implicated as having a direct effect on the

uterus in the mink, pig, hamster and rabbit. Therefore,

research is needed to determine whether uterine concentrations

of steroid receptors in the ewe are influenced by seasonal

fluctuations in the secretion of melatonin and prolactin.

The following review of literature will encompass the

physiological and biochemical responses of the uterus to

estrogen and progesterone. Much of the review will rely on

data acquired from the study of laboratory animals but when

available, information on domestic animals and primates will

be included.

4

ESTROGEN AND PROGESTERONE

ORIGIN OF ESTROGEN AND PROGESTERONE

The female mammal is born with all of the oocytes she

will need throughout her reproductive life. In order for an

oocyte to be ovulated it must first go through several stages

of maturation. In the initial stages of folliculogenesis,

each oocyte is surrounded by a single layer of granulosa

cells, together forming a primordial follicle (Ireland, 1987).

Just prior to or following birth the primordial follicle

enters and remains in a resting state until the appropriate

hormonal signals cue it to resume maturation in preparation

for ovulation or atresia (Ireland, 1987). Follicular growth

and maturation occur in a series of events whereby the

primordial follicle first develops into a primary, then

secondary follicle, followed by formation of the fluid filled

tertiary follicle and finally the ovulatory or Graafian

follicle (Bjersing, 1967; Turnbull et al., 1977). Only

follicles that undergo "recruitment" actually ovulate. Most

follicles instead become atretic and are subsequently replaced

by new follicles (Smeaton and Robertson, 1971). In the

bovine, this maturation process occurs in sequential waves of

development until the ensuing ovulation occurs (Sirois and

Fortune, 1988). Soon after ovulation, the ruptured follicle

is transformed into a corpus luteum (CL).

5

During the estrous or menstrual cycle, ovarian follicles

and the CL are the primary sources of steroid synthesis and

secretion (Falck, 1959; Hafs and Armstrong, 1968; Moor, 1977).

Follicle steroidogenesis begins as the specialized cells that

comprise the follicle wall (granulosa and theca cells) grow

and proliferate. It has been well established that follicles

are the primary source of estrogens in the nonpregnant animal.

Similarly, once the CL has formed it also becomes a major

source of steroid synthesis with progesterone being the major

hormone secreted. During pregnancy, depending on the species,

the developing conceptus and later the placenta may become a

rich source of estrogens and progestins. Estrogens and

progestins are also synthesized and secreted by the adrenal

cortex, however, this source accounts for only a minor

fraction of the total estrogen and progesterone in the

systemic circulation. Without steroid synthesis and secretion

the complex and highly synchronized events that permit

reproduction could not occur.

PHYSIOLOGICAL RESPONSES TO ESTROGENS

Depending upon the mammalian species, there are several

forms of estrogens that are synthesized and circulate

throughout the body. However, the most important and

prevalent of the estrogens are estradio1-17B and estrone.

Estradio1-17B is approximately ten times more potent than

estrone and is the most biologically active of all steroids

6

produced by the ovary (Hisaw, 1959). A third estrogen known

as estriol is found in high concentrations in primates during

gestation. In general, estriol is considered the least potent

of these three estrogenic forms (Hisaw, 1959). During the

estrous or menstrual cycle, estrogens are responsible for

stimulating cellular proliferation and growth of the female

reproductive tract, regulation of utero-ovarian function and

intrauterine environment, and development and maintenance of

secondary sex characteristics. The following discussion will

focus on the role of estrogens in regulating utero-ovarian

function and the intrauterine environment.

Estrogens act on the uterine myometrium to increase both

the frequency and amplitude of contractions, which are

requisite for sperm and accelerated ovum transport, and in

some species, migration and spacing of embryos (Pope et al.,

1982). Estrogens initiate endometrial stromal edema (Astwood,

1938) as a result of increased uterine capillary and venule

permeability (Halkerston et al., 1960; Martin et al., 1973;

Espey, 1980). In response to estrogens, the glands of the

endometrium become increasingly tortuous as the nuclear RNA

polymerases and mRNA activate and facilitate protein

synthesis, cell hypertrophy and cell hyperplasia. Estrogens

are also responsible for increasing uterine blood flow

(Williams, 1948; Greiss and Anderson,1970), which has been

suggested to be a result of hormone-induced vasodilation of

the uterine vasculature (Greiss and Anderson, 1970). The

7

resultant increased mass of both the uterine endometrium and

myometrium as well as increased uterine vascularization is

essential for implantation and development of the embryo.

PHYSIOLOGICAL RESPONSES TO PROGESTERONE

Progesterone is commonly referred to as the hormone of

pregnancy and is the principal secretory product of the CL

(reviewed by Rothchild, 1981). It is absolutely crucial for

normal fetal survival and development in all mammalian species

known to date. Progesterone stimulates secretory changes in

the intrauterine environment that prepares the endometrium for

implantation of the blastocyst and promotes development of

decidual cells that supply nutrients to the early embryo

(Auletta and Flint, 1988; Clark and Markaverich, 1988). In

addition, it decreases the frequency and intensity of uterine

contractions thereby preventing expulsion of the implanted

embryo (reviewed by Clark and Markaverich, 1988). Capacity

of the CL to secrete progesterone and thus to sustain a normal

pregnancy is regulated by hormones synthesized and secreted

by the hypothalamus, pituitary, placenta and by the CL itself.

BIOSYNTHESIS OF ESTROGEN AND PROGESTERONE

As stated previously, the primary source of estrogen and

progesterone synthesis and secretion in the nonpregnant female

mammal is the ovarian follicle and CL, respectively.

Synthesis of these steroids encompasses several factors,

8

namely, follicle maturation, selection and luteinization.

Changes in steroidogenesis associated with folliculogenesis

and luteal development are dependent initially, on the amount

of follicle stimulating hormone (FSH) available to the

maturing follicle, and later the ratio of FSH and luteinizing

hormone (LH) during the preovulatory and luteal phase of the

cycle. Steroid hormones are derivatives of cholesterol of

either dietary or de novo origin. Cholesterol in systemic

blood is generally transported in the form of low density or

high density lipoprotein (LDL or HDL, respectively). Low

density lipoprotein or HDL binds to specific receptors on the

surface of the theca and granulosa cell plasma membranes. The

lipoprotein-receptor complex then enters the cell by way of

endocytosis, becoming enclosed in a coated vesicle. Lysosomes

then enzymatically degrade the coated vesicle containing

lipoprotein-receptor complexes thereby liberating free

cholesterol. This free cholesterol is then available for

conversion into steroids. Excess intracellular cholesterol

is esterified and stored in lipid droplets for future use.

Cholesterol can also be synthesized de novo from small

molecule precursors such as acetate. This pathway of

synthesis is activated when cells are deprived of LDL or HDL.

Cholesterol is transported to the mitochondria where it is

converted to pregnenolone by the cytochrome P - 45O complex

(Ichii et al., 1963; Roberts et al., 1967). Pregnenolone is

then converted to progesterone by 38-hydroxysteroid

9

dehydrogenase in the smooth endoplasmic reticulum and

progesterone or subsequent metabolites of this steroid are

secreted into the extracellular space to ultimately enter the

blood stream (Miller and Turner, 1963; Niswender and Nett,

1988) .

Ovarian steroid synthesis and secretion is dynamically

regulated by the anterior pituitary gonadotropins FSH and LH.

(Kaltenbach et al., 1968b; Hixon and Clegg, 1969; Karsch et

al., 1971; Denamur et al., 1973; Niswender et al., 1976).

These protein hormones regulate the biosynthesis and secretion

of steroids in the ovary by binding to high-affinity, low

capacity plasma membrane receptors. The formation of the

hormone-receptor complex activates the enzyme adenylate

cyclase which in turn catalyzes the formation of cyclic

adenosine-3'5'-monophosphate (cAMP) from adenosine

triphosphate (ATP) (Marsh, 1975). Cyclic AMP then mediates

the activation of specific protein kinases (Ling and Marsh,

1977), which phosphorylate enzymes or other proteins necessary

for the conversion of cholesterol into steroids via the

cholesterol/steroid biosynthetic pathway (Caron et al., 1975;

Caffrey et al., 1979).

Ovarian follicles consist of two predominant and

distinctly different cell types. Theca cells, which are

arranged along the outer surface of the basement membrane of

the follicle, and granulosa cells that lie along the inner

surface of the basement membrane. The interaction of

10

pituitary gonadotropins with the theca and granulosa cells of

the follicle have formed the basis of the two cell-two

gonadotropin model of ovarian steroidogenesis (Falck, 1959;

Short, 1962; Liu and Hsueh, 1986). During the early

follicular stage of development ovarian thecal cells contain

LH but no FSH receptors (Zeleznik et al., 1974; Carson et al.,

1979). Luteinizing hormone is responsible for stimulating the

conversion of cholesterol to pregnenolone in the theca cells

by way of activating the rate limiting cytochrome P-450

cholesterol side chain cleavage (P-450scc) enzyme in

mitochondria (Evans et al., 1981). Luteinizing hormone also

promotes the conversion of pregnenolone to progesterone in the

theca cell endoplasmic reticulum by activating 3B-

hydroxysteroid dehydrogenase (38-HSD) (Ireland, 1987).

Progesterone is then converted to the androgens,

androstenedione and testosterone, through the activation of

17,20 lyase and 17B-dehydrogenase enzymes, respectively (Evans

et al., 1981). The lipid soluble androstenedione and

testosterone can enter the systemic circulation and bind to

carrier protein globulins or they can freely diffuse from the

theca to the granulosa cell and be converted to estrogens by

the granulosa cell aromatase enzyme (Baird, 1977; Dorrington

and Armstrong, 1979; Leung and Armstrong, 1980). In contrast,

prior to the Graafian follicle stage of development, granulosa

cells have only FSH receptors. Follicle stimulating hormone

is the gonadotropin responsible for stimulating the conversion

11

of cholesterol to progesterone in the granulosa cells. Like

LH, FSH activates protein kinases through stimulation of

adenylate cyclase. In addition to stimulating the synthesis

of enzymes necessary for converting cholesterol to

progesterone, the FSH-stimulated protein kinases within the

granulosa cell activate an aromatase enzyme that catalyzes

the conversion of androgens to estrogens (Fortune and

Armstrong, 1978). Estrogens can in turn act back on the

follicle to stimulate further cell proliferation as well as

induce or inhibit (depending on the feedback pathway) the

synthesis of more gonadotropin and steroid receptors.

STEROID HORMONE TRANSPORT

Steroid hormones are, for the most part, transported

throughout the body bound to serum proteins. However, a

relatively smaller fraction may circulate in an unbound or

free form. More specifically, estrogens and androgens are

bound to sex steroid hormone binding globulin (SHBG), while

progesterone is bound to progesterone binding globulin (PBG).

Both of these binding globulins are comparatively high

affinity, low capacity carriers. Alternatively, these

steroids may also circulate throughout the body bound to low

affinity, high capacity serum albumins. Only approximately

2% of steroids actually circulate in the free form. The

mechanism by which the steroid is able to dissociate from its

carrier and thus freely diffuse through the plasma membrane

12

of the target cell is unclear. However, Avvakumov et al.

(1986) found the steroid binding globulin in complex with the

plasma membrane of human decidual endometrial tissue. These

investigators hypothesized that the binding of the protein

binding globulin to the plasma membrane may send some kind of

a signal which then causes dissociation of the steroid and

binding globulin, thus allowing simple diffusion of the

steroid across the plasma membrane. It is also conceivable

that the entire steroid and binding globulin complex may enter

the cell by way of endocytosis where the steroid is then

liberated by lysosomal degradation of the protein globulin.

STEROID HORMONE RECEPTORS

In any biological system, receptors function by

recognizing a specific hormone from all other molecules in

the cellular environment and then upon binding of the hormone,

transmit a signal thereby creating a biological response. The

magnitude of a hormonal response depends on at least three

factors; these include the hormone concentration, the number

of available receptors and the affinity of the hormone for the

receptor. Steroid hormone receptors belong to a family of

regulatory proteins whose ability to control gene expression

is dependent on the binding of their ligand. Binding of the

hormone to its receptor stimulates the synthesis of specific

proteins by altering the rate of transcription of specific

genes (Mueller et al., 1957; Norman and Litwack, 1987).

13

Over the years, the cellular localization of the steroid

hormone receptors has been a source of controversy. It was

once thought and accepted that the unoccupied steroid receptor

was localized in the cytoplasmic fraction of the target cell

(Gorski et al., 1968; Jensen et al., 1968). Upon binding of

the receptor to its specific hormone the extranuclear steroid

hormone-receptor complex was believed to be translocated to

the nuclear compartment where it attached to the DNA to exert

stimulatory effects on gene transcription (Gorski et al.,

1968; Jensen et al., 1968). The foundation for this central

dogma was based on studies in which cells not previously

exposed to a given steroid were homogenized and the nuclear

and cytosolic fractions separated by means of differential

centrifugation. After incubation of these separate fractions

with a radiolabeled steroid the steroid receptors were

predominately found in the cytosol. However, when cells

previously exposed to a radiolabeled steroid were homogenized

and separated by centrifugation, the bound steroid receptors

were found in the nuclear fraction. Recently, studies by

independent laboratories revealed that occupied as well as

unoccupied steroid receptors resided predominantly in the

nucleus. Results of these studies indicated that the presence

of steroid receptors in the cytosolic fraction of cells as

detected in earlier studies was an artifact brought about by

their extraction from the nucleus after cell breakage

(Welshons et al., 1984).

14

With the recent availability of monoclonal antibodies

specific for a number of steroid receptors and the technique

of immunocytochemistry, it is now widely accepted that both

the unoccupied and occupied steroid receptors are

predominately localized in the nucleus (King and Greene, 1984;

Welshons et al., 1984). King and Greene (1984) found that

treatment of cells or tissues in vivo or in vitro with

[3H]estradiol -178 altered the intensity of staining of the

estrogen receptor but not the distribution of the receptor.

After passage across the plasma membrane of its target cell,

the lipophilic steroid hormone may or may not bind to low

affinity binding sites in the cytoplasm (Mercer et al., 1981).

The steroid migrates through the cytoplasm and diffuses across

the nuclear envelope where temperature dependent noncovalent

binding to its specific unoccupied receptor occurs resulting

in the formation of the activated steroid-receptor complex

(Miller et al., 1985; Greene and Press, 1986). The steroid-

receptor complex undergoes a conformational change which

stimulates an increase in its affinity for chromatin and

becomes tightly associated with nuclear acceptor sites on the

chromatin. With respect to the estrogen receptor, ligand

binding and activation may involve the dimerization of two

identical estrogen receptor subunits. The dimerization may

be critically involved in the mechanism by which the estrogen

receptor complexes promote changes in gene transcription

(Sabbah et al., 1989). The binding of the steroid-receptor

15

complex to the chromatin may cause an unwinding of a specific

portion of the DNA strand thus facilitating the binding of

ribonucleic acid (RNA) polymerases, which stimulate

transcription of specific genes into messenger RNA (mRNA)

(reviewed by Yamamato, 1985). The mRNA's then translocate to

the cytoplasm where they bind to ribosomes and are translated

into specific proteins such as enzymes, receptors, low

affinity binders and protein hormones, which in turn

facilitate the cellular and metabolic effects on the target

tissue characteristic of the actions of the steroid hormone

in question.

Interestingly, recent immunocytochemical (Berthois et

al., 1986) and fluorescence (Parikh et al., 1987) studies have

revealed the presence of steroid receptors in the cytoplasm

of steroid target cells. Yamamoto (1985), explained this

occurrence by hypothesizing that the nuclear and cytoplasmic

distribution of steroid receptors may depend solely on genomic

sites. Because of the enormous amount of DNA that exists in

mammalian nuclei this equilibrium would naturally favor that

of a nuclear distribution of receptors in intact cells

including the unbound receptors. Thus the majority of unbound

receptors would reside in the nucleus while only a minor

fraction of the free receptors would be transiently

cytoplasmic. These receptors could bind incoming hormone and

translocate to the nucleus where modulation of gene expression

would then take place.

16

Estrogen and progesterone receptor concentrations are

regulated by both estrogen and progesterone. Specifically,

estrogen stimulates the synthesis of both estrogen and

progesterone receptors thereby enhancing the sensitivity of

target tissues to each of these hormones (Zelinski et al.,

1980; DeHertogh et al., 1986; Ekka et al., 1987). On the

other hand, progesterone causes a down-regulation of both

progesterone and estrogen receptors (Koligian and Stormshak,

1977b; West et al., 1986; Selcer and Leavitt, 1988), by

interfering with the replenishment or de novo synthesis of

the receptor (Hsueh et al., 1976; Pavlik and Coulson, 1976).

17

THE ESTROUS CYCLE

FOLLICULAR PHASE

During the follicular phase of the estrous or menstrual

cycle, FSH and LH-induced maturation of the follicle increases

the production and release of estrogens into the blood (Karsch

et al., 1979; Hansel, 1983). Just prior to ovulation high

levels of estrogen positively feedback to activate the release

of gonadotropin-releasing hormone (GnRH) from the hypothalamus

(Karsch et al., 1979). This in turn stimulates the release

of LH and FSH from the pituitary gland. In the ewe, estradiol

secretion begins to rise as the follicle develops and matures.

An increase in estradiol production of 5 to 10-fold then

occurs over the next few days. Estradiol peaks at the

beginning of the preovulatory LH surge and then rapidly

declines to basal levels (Hauger et al., 1977). Some

researchers suggest that a second estradiol peak occurs in

cows (Glencross et al., 1973; Hansel et al., 1973), and ewes

(Cox et al., 1971). It is presumed this second low amplitude

peak of estrogen stems from renewed follicular development in

preparation for a new cycle. To date, the occurrence and

source of the second estradiol peak remains equivocal. A

surge of LH, along with increased levels of FSH precipitate

ovulation. Initiation of the preovulatory LH surge and

subsequent ovulation requires high frequency, low amplitude

18

LH pulses. These pulses stem from the rising levels of

estrogen that act on the hypothalamus to speed up a GnRH pulse

generator (reviewed by Karsch, 1987). The frequent,

estradiol-enhanced GnRH pulses during the follicular phase

stimulate the LH surge and ovulation. Subsequent to

ovulation, estrogen secretion declines and LH then stimulates

the transformation of the ruptured follicle into a CL as the

thecal and granulosa cells of the follicle undergo rapid

mitosis and hypertrophy.

LUTEAL PHASE

The main function of the CL is to secrete progesterone

in order to establish the intrauterine environment necessary

for successful implantation of an embryo and maintenance of

early pregnancy in the ewe, mare and primate (Casida and

Warwick, 1945; Ginther and First, 1971). The CL is the major

source of progesterone for pregnancy maintenance to term in

the rat, cow, sow and goat (Bazer et al., 1982). Like the

follicle, the CL is composed of two cell types; small and

large luteal cells. Small luteal cells arise from the

luteinization of theca cells and large cells are derivatives

of luteinized granulosa cells (Lemon and Loir, 1977; Uresley

and Leymarie, 1979; Rodgers and O'shea, 1982; Niswender et

al., 1985).

In the cow (Donaldson et al., 1965), ewe (Kaltenbach et

al., 1968), mare (Ginther, 1979) and primate (Moudgal et al.,

19

1971; Mais et al., 1986) LH is the primary luteotropin for

maintaining continued progesterone secretion from the CL.

This was demonstrated when administration of LH to cows on day

5, 10, 15 and 20 of the estrous cycle (Schomberg et al.,

1967), or ewes on day 9 (Niswender et al., 1976) increased the

synthesis and secretion of progesterone. In the ewe, systemic

concentrations of progesterone increase from day 3-4 (first

day of detected estrus = day 0) to a maximum by day 7-8 of the

estrous cycle. Rising levels of progesterone from the CL

block the preovulatory LH surge by suppressing the frequency

of the GnRH pulse generator and thereby reduce the frequency

and amplitude of LH secretory pulses from the pituitary gland

(Yen et al., 1972; Foster et al., 1975; Baird, 1978).

Progesterone levels remain high until day 14-15 of the cycle,

after which time they decline to basal levels 1-2 days prior

to the ensuing estrus as a new cycle is initiated (Baird et

al., 1976; Pant et al., 1977). Concomitant to declining

estrogen and increasing progesterone levels during the luteal

phase, an increase in the follicular protein hormone inhibin

occurs. Inhibin feeds back at the level of the hypothalamus

and pituitary inhibiting further FSH release (Schwartz and

Channing, 1977).

Just as gonadotropins and steroid hormones interact in

dynamic synchrony with one another, so to is the complexity

of interaction between ovarian steroids and uterine

endometrium during the estrous cycle and pregnancy (Stone et

al., 1978).

LUTEOLYSIS

20

In the absence of fertilization of an ovum from the

ruptured follicle, many laboratory and domestic animals will

continue to exhibit estrous cycles. In most non-primate

mammals such as the rat, guinea pig, ruminants and sow, the

uterus exerts a local luteolytic action on the ipsilateral

corpus luteum of the non-pregnant animal to bring about luteal

regression and decreased progesterone secretion (Niswender et

al., 1985). This first became evident when excision of the

uterus was found to prolong luteal maintenance in the sow (du

Mesnil du Buisson, 1961), guinea pig (Fischer, 1965),

pseudopregnant rat (Barley et al., 1966) and ewe (Inskeep and

Butcher, 1966). Upon completion of a previous cycle and

initiation of a new cycle, the uterine endometrium secretes

the luteolysin prostaglandin Fla (McCracken et al., 1970;

Goding, 1974). The secretion of prostaglandin Fla (PGF2a) is

under the influence of ovarian progesterone, estradiol -178,

and oxytocin (Baird, 1978; Sheldrick et al., 1980). In the

ewe, PGF2a levels increase from day 12 of the cycle, reaching

maximum concentrations 1-2 days prior to the subsequent estrus

(Baird et al., 1976). This luteolytic factor enters the ovary

by diffusing across the wall of the uterine vein into the

ovarian artery. The close anatomical proximity of the uterine

veins with the convoluted ovarian artery permits the diffusion

21

of substances such as PGF2a directly into the ovarian artery

by a countercurrent diffusion mechanism. They thus bypass the

general circulation and avoid rapid destruction in the lungs

(McCracken et al., 1970). Once in the ovary PGF2a targets

the progesterone secreting luteal cells of the corpus luteum

to cause luteolysis (McCracken et al., 1970). The mechanism

by which PGF2a exerts its luteolytic action on the CL is not

known. However, Niswender et al. (1975) suggest that the

effects of PGF2a may be via a vascular component because a

significant decrease in capillary blood flow to the CL is

associated with, but does not precede luteolysis. Thus, it

is doubtful that PGF2a causes luteal regression by interfering

with blood flow to the CL.

The protein hormone oxytocin is currently believed to

also play an essential role in the events leading to

luteolysis (Mitchell et al., 1975; Flint and Sheldrick, 1986).

Uterine PGF2a exerts its effect in a local manner, targeting

the ipsilateral ovary and thus causing both inhibition of

luteal progesterone secretion and stimulation of luteal

oxytocin release. This was demonstrated by Walters et al.

(1983) and Sheldrick and Flint, (1983) who found that oxytocin

secretion was stimulated by an intramuscular injection of the

PGF2a analog, cloprostenol in ewes. Similiarly,

Schallenberger et al. (1984) found the same phenomenon to

occur in cows. In ewes, oxytocin triggers the release of more

uterine PGF2a and the positive feedback cycle continues until

22

luteolysis is complete (Mitchell et al., 1975; Flint and

Sheldrick, 1986). Oxytocin concentrations in both ovine

(Sheldrick and Flint, 1983) and bovine (Abdelgadir et al.,

1987) luteal tissue is maximal during the early luteal phase.

Stormshak et al. (1969) found that injection of exogenous

estradiol into ewes on days 10-11 of the estrous cycle caused

premature luteal regression. It is believed that estradiol

also acts to increase uterine PGF2cf secretion thereby

promoting luteal regression (Ford et al., 1975).

The mechanism involved in regression of the primate CL

is not completely understood. Shutt et al. (1976) found that

removal of the primate uterus did not prolong the maintenance

of the CL. It is believed that an intra-ovarian prostaglandin

may be involved, because the ovary is a significant source of

PGF2cc in primates (Shutt et al., 1976). In primates, the

reduction in progesterone secretion that occurs with luteal

regression precipitates atrophy of the endometrial epithelium

and invasion of the endometrium by lymphocytes, causing

stromal degradation, exposure of arterioles and mensus, or

shedding of the uterine endometrial lining (Knobil, 1973).

With a decrease in both progesterone and ovarian secretion of

inhibin, GnRH and the gonadotropins FSH and LH are released

and a new menstrual cycle is initiated.

23

EARLY GESTATION

In order to ensure normal embryonic development and

differentiation a complex communication network between the

embryo and maternal tissues must be established. During the

preimplantation or preattachment stage of gestation conceptus

secretory proteins and steroids as well as hormones of

maternal origin act upon the uterus and ovaries to ensure

maintenance of pregnancy. In particular, factors of conceptus

origin are important for stimulation of vasodilation and

angiogenesis, which are necessary in order to increase uterine

blood flow, stimulate transfer of nutrients into the uterine

lumen, protect the conceptus from immunological rejection, and

maintain luteal function.

MATERNAL RECOGNITION OF PREGNANCY

Moor and Rowson (1966b) found that the critical period

for maternal recognition of pregnancy in the ewe was day 13

of gestation. Maternal recognition of pregnancy occurs when

the presence of an embryo negates the luteolytic action of

the uterus on the CL prior to trophoblastic attachment (Short,

1969). It was determined that this antiluteolytic action

occurred locally, as demonstrated by the fact that a viable

conceptus, confined to one uterine horn was able to maintain

only the ipsilateral corpus luteum in ewes (Moor and Rowson,

24

1966b) .

Following fertilization and implantation, an endocrine

signal, presumably from the conceptus, maintains the function

of the CL by blocking the release of PGF2a from the uterus so

that progesterone secretion continues (Northey and French,

1980; Fincher et al., 1986). In many mammals, including the

rat, sow, cow and goat, continued secretion of progesterone

from the CL is essential for the maintenance of pregnancy to

term (Bazer and First, 1983). In other mammals, such as the

human, mare and ewe, the CL is the primary source of

progesterone initially, but later in gestation (day 56, day

200 and day 60 in the human, mare and ewe, respectively) the

placenta produces progesterone or progestins necessary for

intrauterine maintenance of the embryo (Ginther and First,

1971; Bazer and First, 1983). Godkin et al. (1984b) found

that proteins released by cultured day 15-16 conceptuses

prolonged luteal maintenance when introduced into the uterine

lumen of cyclic ewes. One protein involved in the maternal

recognition of pregnancy in the ewe is ovine trophoblast

protein-1 (Godkin et al., 1984a; Anthony et al., 1988). Ovine

trophoblast protein-1 (oTP-1) is a major polypeptide

synthesized by the ovine conceptus between days 13 and 21 of

gestation. It directly acts on the uterine endometrium to

suppress uterine PGF2a release thereby extending luteal

maintenance. This was demonstrated when oTP-1 infused into

the uterine lumen of ewes prolonged the life span of the CL

25

by reducing the production of uterine PGF2a (Godkin et al.,

1984a). Interestingly, oTP-1 and the antiviral,

immunoregulatory factor a-interferon have been found to be

structurally similar (Farin et al., 1990). Both oTP-1 and a-

interferon in fact bind to endometrial a-interferon receptors

(Stewart et al., 1987). However, it is presently unknown if

oTP-1 also plays an immunoregulatory or antiviral role during

gestation. It is conceivable that oTP-1 constitutes just one

complex, and that other conceptus secretory proteins make up

the remainder of this complex (Martal et al., 1979; Klopper,

1983). A functionally similar and structurally related

antiluteolytic bovine trophoblast protein (bTP-1) also exists

in cattle (Knickerbocker et al., 1986; Anthony et al., 1988).

Prostaglandins may also play a role in mediating the

local endometrial vascular response and transformation of

stromal to decidual cells, both of which are necessary for

embryonic development. Synthesis of prostaglandins by the

endometrium, as well as by the blastocyst, has been reported

for the mouse (Chepenik and Smith, 1980), rabbit (Harper et

al., 1983), ewe (Hyland et al., 1982) and human (Tsang and

Ooi, 1982). Uterine PGF2a as previously discussed, is

responsible for luteal regression (Ford, 1982) during the

estrous cycle. However, uterine and embryonic secretion of

prostaglandin E2 (PGE2) occurs during early gestation (Marcus,

1981; Silvia et al., 1984). Prostaglandin E2 (PGE2) is

vasodilatory and non-luteolytic, and may be responsible for

26

luteal resistance to the luteolytic effect of PGF2a.

Prostaglandin E2 has been demonstrated to protect luteal

tissue from the actions of PGF2cz both in vivo and in vitro.

Treatment of ewes with PGE2 delayed luteal regression in

nonpregnant ewes (Pratt et al., 1977), and increased

progesterone secretion by large luteal cells (Silvia et al.,

1984). It has been suggested that oTP-1 may induce

endometrial synthesis and secretion of PGE2 and thus prevent

luteolysis.

IMPLANTATION OR ATTACHMENT

Among the most critical stages of pregnancy is

implantation or in the case of domestic animals, attachment.

Because the conceptus of the sow, cow and ewe has a non-

invasive trophoblast and diffuse epitheliochorial

placentation, the focus of this discussion will be with

respect to attachment rather than implantation of trophoblast

unless otherwise indicated. In ewes the blastocyst attaches

on day 16 of gestation. Progesterone is essential for

implantation/attachment in most species. In species such as

the mouse and rat, (species that experience a phase of

embryonic diapause or delayed implantation when the mother is

lactating), estrogen has been found to be a requirement for

implantation (Aitken et al., 1977). Successful implantation

in these species requires an estradiol-primed uterus during

proestrus to up-regulate estrogen and progesterone receptors

27

thus sensitizing the uterus to these hormones. This is

followed by progesterone conditioning during the first 4 days

of gestation in order to inhibit myometrial contractility and

embryo expulsion. A preimplantation estrogen peak occurs on

day 4 to establish the endometrial vascular permeability

necessary for implantation and nutrient transport. As

pregnancy progresses secretion of progesterone again increases

and then remains relatively constant until parturition

(Nalbandov, 1971; Aitken, 1979). In species such as the sheep

(Cumming et al., 1974), which do not exhibit embryonic

diapause, progesterone alone will allow attachment to occur

if the proliferative effects of estrogens on the endometrium

have already been achieved. In order for successful

attachment to occur in the ewe, the intrauterine environment

must be primed with estrogen secreted during estrus. This

steroid enhances endometrial and myometrial vascularization

and proliferation of various cell types in the uterus (Moor

and Rowson, 1959; Bindon, 1971; Miller and Moore, 1976).

Progesterone can function successfully if cells of the

reproductive organ have been exposed to estradiol, which

induces the formation of progesterone receptors (Muldoon,

1981). Together with estradiol and the action of anterior

pituitary hormones on the CL, progesterone acts to transform

the uterine endometrium from a proliferative to a secretory

state. Subsequent to estrus, serum concentrations of

progesterone increase as the CL develops accompanied by a

28

decline in serum concentrations of estrogen (Moor and Rowson,

1959; Bindon, 1971; Miller and Moore, 1976). Although

estrogen is not obligatory for implantation in some species,

it has been found to promote increased placental development

(Dalton and Knight, 1983) and litter size (Wilde et al., 1976)

in gilts. Preimplantation blastocysts in the gilt or sow

(Fischer et al., 1985) are able to convert androgens and

progestins to estrogens (Perry et al., 1973; Gadsby et al.,

1980; Fischer et al., 1985), which are essential for migration

and spacing of embryos within the uterine horns. In addition,

estrogen of conceptus origin ensures maintenance of the CL

because in the gilt this steroid is luteotropic (Gardner et

al., 1963). Because of this phenomenon, it is presumed that

estrogen of maternal origin is not necessary for attachment

of embryos in this species. It has been demonstrated that

significant amounts of estrogens are present in the systemic

circulation prior to embryo attachment in the cow (Eley et

al., 1979; Shemesh et al., 1979), ewe (Ellinwood, 1978;

Reynolds et al., 1982) and mare (Zavy et al., 1979). However,

questions remain about the origin of these estrogens in the

indicated species.

Steroid metabolizing enzymes such as 38-hydroxysteroid

dehydrogenase, 1713 -hydroxysteroid dehydrogenase and aromatase

have been detected in rabbit, mouse, rat and hamster

preimplantation blastocysts (George and Wilson, 1978; Singh

and Booth, 1979; Wu, 1985) establishing the possibility for

29

embryonic steroid synthesis. Evidence which supports the

concept that the preimplantation embryo is able to synthesize

and metabolize steroids is complicated by the fact that some

species (rat and mouse) require maternal estrogen for

implantation while other species (pig, sheep, rabbit, hamster

and guinea pig) require only maternal progesterone. These

differences among species may exist because rat and mouse

blastocysts are unable to supply sufficient estrogen compared

with those of the pig, sheep, rabbit, hamster and guinea pig.

Once established, the fetoplacental unit is able to metabolize

and interconvert steroids, and thus possesses the ability to

potentially influence events such as uterine secretory

activity, nutrient transport and uterine blood flow (Singh and

Booth, 1979; Wu, 1985).

30

SEASONAL REPRODUCTION

Reproduction in some species of animals is regulated by

environmental cues such as availability of food or

photoperiod. Animals whose reproductive activity coincides

with changes in photoperiod are commonly referred to as

seasonal breeders. Traditionally these animals have been

referred to as long- or short-day breeders but in actuality

these designations denote the hours of daylight to which the

animal is exposed. Changes in circannual photoperiod

perceived by the animal are transduced into neuroendocrine

signals that ultimately effect gonadal activity. This

photoperiod-induced regulation of reproductive activity

ensures, at least in part, that parturition occurs at a time

when environmental factors are most beneficial to permit

survival of newborn offspring. Species such as hamsters,

ferrets, mink, horses and birds are long-day breeders, with

mating occurring during the spring and summer (Gwinner and

Dorka, 1976; Thorpe and Herbert, 1976). Sheep, deer, and

certain other species of wildlife are short-day breeders and

mate during the fall or winter (Karsch et al., 1984; Bittman

et al., 1985).

31

MELATONIN

Melatonin is a hormone whose secretion is characterized

by circadian and circannual rhythms (Karsch et al., 1984).

This hormone is believed to play an essential role in

regulating seasonal reproduction. Melatonin secretion is

maximal during darkness. Thus, duration of secretion, and

hence, daily quantity of melatonin secreted, is greater during

the seasons of the year when hours of daylight are the

shortest (fall and winter). In essence, the circadian rhythm

of melatonin secretion synchronizes the circannual rhythm of

reproduction.

The mechanism by which photoperiodic signals regulate

melatonin secretion involves various components of the central

nervous system. Light entering the eye stimulates the retina

to produce neurotransmitters, which in turn activate the

retinohypothalamic nerve fibers that terminate in the

suprachiasmatic nucleus (SCN) of the hypothalamus. From the

SCN, nerves descend through the lateral hypothalamus to

synapse in the superior cervical ganglia with post-ganglionic

autonomic nerves that terminate in the pineal gland. The

stimulus evoked by light suppresses the release of a

neurotransmitter (norepinephrine) in the pineal gland (Moore

and Klein, 1974; Stetson and Watson-Whitmyre, 1976; Karsch et

al., 1984). Absence of light results in the release of this

neurotransmitter, which then binds to B-adrenergic receptors

32

located in the plasma membrane of pinealocytes. Binding of

norepinephrine to its receptor promotes activation of

adenylate cyclase, which catalyzes the conversion of ATP to

cAMP. This nucleotide then binds to protein kinase to

activate its catalytic subunit which then stimulates the

activity of N-acetyltransferase, the rate limiting enzyme

involved in the synthesis of melatonin. In species such as

sheep, melatonin is believed to act back upon the hypothalamus

to regulate the pulsatile secretion of LH by modulating the

frequency of the gonadotropin releasing hormone (GnRH) pulse

generator. This action of melatonin is accomplished by

sensitizing the GnRH pulse generator to the feedback effect

of estrogen (Karsch et al., 1980; Goodman et al., 1982; Karsch

et al., 1984).

Anestrus in most breeds of sheep in the northern

hemisphere begins during late winter and extends through late

August or early September. This reproductively quiescent

period is characterized by a comparatively low daily

production of melatonin (due to extended daylight) and reduced

secretion of ovarian estradiol. The limited daily production

of melatonin presumably decreases the sensitivity of the

hypothalamic GnRH pulse generator to the feedback action of

estradiol. Hence, the pulsatile release of GnRH is suppressed

resulting in a corresponding reduction in LH secretion. As

hours of daylight decrease during late summer and fall the

increased daily production of melatonin increases the

33

sensitivity of the hypothalamus to the feedback action of

estradiol. Consequently, increased activity of the GnRH pulse

generator stimulates increased frequency of LH secretion with

a resultant stimulation of additional estradiol production by

the ovarian follicles. This hypothalamic-hypophyseal-gonadal

interrelationship ultimately leads to the preovulatory surge

of LH secretion, which induces the first ovulation of the

breeding season.

In contrast, the pineal gland of long-day photoperiodic

breeders such as the hamster and ferret, secretes low levels

of melatonin during the breeding season and high levels of

the hormone during the anestrous period. In this case

melatonin is inhibitory to the hypothalamic secretion of GnRH

and the subsequent secretion of gonadotropins, thus causing

a decrease in ovarian steroidogenesis and inhibition of

ovulation. Melatonin infusions into male hamsters equally

induced gonadal atrophy in both sham and SCN-lesioned animals;

demonstrating that the SCN may not be an essential component

of the neural circuits that measure the melatonin signal in

long-day photoperiod breeders (Maywood et al., 1990).

Prolactin, another non-ovarian, seasonally regulated

hormone may also be involved in mediating the pivotal

neuroendocrine changes that occur during the breeding and non-

breeding season. In fact, secretion of melatonin and prolactin

have been shown to be inversely related in a number of

species. Melatonin inhibits prolactin release (Clark, 1980;

34

Glass and Lynch, 1983), as demonstrated by an increase in

prolactin secretion after pinealectomy.

PROLACTIN

Prolactin secretion is depressed in hamsters (long-day

breeders) exposed to shortened hours of daylight and exogenous

administration of this hormone can induce gonadal growth and

steroidogenesis. This effect of prolactin may be due to an

increase in LH receptor numbers (Holt et al., 1976; Advis et

al., 1981). Of particular interest with respect to this

thesis is the fact that the secretion of prolactin has been

demonstrated to be influenced by photoperiod in goats,

(Buttle, 1974), hamsters (Bex et al., 1978), heifers (Schams

and Karg, 1974; Peters and Tucker, 1978) rats (Williams et

al., 1978) and ewes (Posner et al., 1974; Webster and

Haresign, 1983; Kennaway et al., 1983). In general, a

positive correlation between increasing daylight and serum

prolactin was detected in each of these species. Because

secretion of prolactin and melatonin occurs in response to

changes in photoperiod it is conceivable that they are

necessary components in regulating seasonal reproduction.

Prolactin is a protein hormone (MW=24,000) whose

structure and activity are similar to those of growth hormone.

Prolactin is secreted from the lactotropes of the

adenohypophysis (Nicoll, 1974). Interestingly, prolactin mRNA

is found in many tissues throughout the body indicating a

35

potential for synthesis. However, in many of these tissues,

synthesis, secretion and the action of this hormone are at

present unknown. The classical function of prolactin is to

promote the development of the mammary gland during gestation

and postpartum lactation (Nicoll, 1974). This stimulation is

essential for milk production and ultimate survival of

offspring.

However, prolactin may also play far reaching and

essential roles in regulating the reproductive cycle of many

mammals through its influence on gonadotropic and

steroidogenic responses. In fact, the presence of prolactin

receptors found widely distributed in a number of non-mammary

tissues from several different species (Posner et al., 1974;

Rolland et al., 1976; Rose et al., 1986) indicates the

potential diversity of actions this hormone may possess.

During pregnancy, prolactin can also be found in the amniotic

fluid and extraembryonic membranes of the developing fetus as

well as the maternal endometrium (Riddick, 1985). The

interaction of prolactin with its specific target cell

receptor triggers a cascade of molecular processes culminating

in a series of diverse responses, including, the synthesis of

its own receptor (Djiane and Durand, 1977; Huhtaniemi and

Catt, 1981; Muldoon, 1981). However, the sequence of

molecular events by which prolactin expresses its action on

target cells is not completely understood.

Estrogen stimulates the synthesis and release of

36

prolactin in rabbits (Tindal, 1969), rats (Neill, 1974; Brann

et al., 1988) and women (Vekemans and Robyn, 1975). It is

therefore not surprising that prolactin concentrations are

greatest during proestrus, estrus, the end of gestation, and

early lactation when estrogen levels are the highest (Sar and

Meites, 1968; Swanson et al., 1972; Boyer et al., 1975;

Webster and Haresign, 1983). In turn, it appears that

prolactin inhibits estrogen production. This occurrence has

been observed in several species including the rat and mink

(Wang et al., 1980; Dorrington and Gore-Langton, 1981; Wang

and Chan, 1982; Slayden, 1990). One explanation of this

phenomenon as demonstrated by Tsai-Morris et al. (1983) is

the ability of prolactin to prevent estrogen synthesis in rats

by inhibiting granulosa cell aromatase activity. Inhibition

of aromatase by prolactin may be due to the ability of the

hormone to stimulate production of intracellular factors that

suppress the activity of the enzyme. Alternatively, such

factors may interfere with the ability of gonadotropins to

maintain normal enzyme activity. Interestingly, Chilton and

Daniel (1987) found that treatment of ovariectomized rabbits

with prolactin caused an increase in the uterine endometrial

surface area as well as stimulation of both uterine estrogen

and progesterone receptors.

Prolactin plays an essential role in promoting luteal

function in rodents (Greenwald and Rothchild, 1968). In these

animals, prolactin is the primary luteotropin responsible for

37

luteinization of granulosa cells necessary for continued

progesterone secretion (Grota and Eik-Nes, 1967). It is

believed that prolactin accomplishes this role by increasing

LH receptor levels (Advis et al., 1981) as well as by

preventing catabolism of newly synthesized progesterone (Advis

et al., 1981). This process enhances the effectiveness of LH

to stimulate the steroidogenic biosynthetic pathway. In

addition prolactin also increases the activity of cholesterol

esterase and 3B-hydroxysteroid dehydrogenase, the enzyme

required for converting pregnenolone to progesterone. The

increased levels of prolactin-stimulated progesterone

secretion (Advis et al., 1981) in turn causes a down-

regulation and inhibition of synthesis of progesterone

receptors in the rabbit, hamster and mink (Djiane and Durand,

1977; Bex et al., 1978; Slayden, 1990, respectively).

Prolactin has also been implicated in stimulating progesterone

secretion in non-rodent species (Advis et al., 1981; Slayden,

1990), however, the mechanism remains unclear.

38

EXPERIMENT 1

INTRODUCTION

There is a paucity of information about the fluctuations

that occur in uterine estrogen and progesterone receptor

concentrations during the estrous cycle and early pregnancy

in the ewe. The presence of a conceptus in one uterine horn

ensures maintenance of the corpus luteum in the ipsilateral

ovary (Moor and Rowson, 1966a), suppresses the synthesis and

secretion of prostaglandin Fla by the uterine endometrium

(Moor and Rowson, 1966b) and undoubtedly alters other aspects

of uterine metabolism and function. However, it has not yet

been determined whether the conceptus influences uterine

estrogen and progesterone receptor concentrations and whether

such an effect of the conceptus would be localized or

systemic.

An understanding of how uterine steroid receptor

concentrations vary between pregnant and nonpregnant animals

may provide insight regarding the role of steroids in

regulating luteolysis during the estrous cycle and the

establishment of maternal recognition of pregnancy. Because

embryonic mortality is one of the most prevalent reasons for

reduced reproductive efficiency it is important that research

be conducted that may explain these embryonic losses.

Certainly, inappropriate levels of estrogen and(or)

39

progesterone receptors during early gestation could result in

desensitization or overstimulation of the intrauterine

environment at inappropriate times thereby causing embryonic

loss.

The present experiment was conducted to determine whether

the presence of a conceptus would alter endometrial

concentrations of estrogen and progesterone receptors in the

gravid and nongravid uterine horns compared with those present

in uteri of nonpregnant ewes.

MATERIALS AND METHODS

Animals

Nine mature Polypay ewes were checked for estrus twice

daily with a vasectomized ram until all had exhibited an

estrous cycle of normal duration (16.3 ± 0.9 days). On day

10 of a subsequent cycle ewes were anesthetized by intravenous

injection of 2% thiamylal sodium and inhalation of a mixture

of oxygen and halothane. A midventral laparotomy was

performed through which the uterus and ovaries were

exteriorized. All ewes were unilaterally ovariectomized by

removing an ovary bearing CL, and the ipsilateral (adjacent)

uterine horn was ligated with #2 surgical silk, 1 cm distal

to the external bifurcation. Approximately one-third to one-

half of the anterior ipsilateral uterine horn was removed and

immediately placed on ice for determination of endometrial

40

concentrations of progesterone and estrogen receptors. Tissue

was transported to the laboratory within 90 min of surgical

removal. All ewes were allowed to complete one full recovery

estrous cycle and at the ensuing estrus, were bred twice to

a fertile ram. The first breeding occurred at the time of

detected estrus (day 0) and the second breeding occurred 8 h

later if estrus was first detected in the morning and 12 h

later if estrus was first detected in the afternoon. On day

18 of gestation 10 ml of blood were collected by jugular

venipuncture for subsequent analyses of serum concentrations

of estrogen and progesterone. Ewes were relaparotomized and

the remaining portion of the ipsilateral and the entire

contralateral uterine horns were removed for analyses of

endometrial estrogen and progesterone receptors. Pregnancy

was verified by the presence of an embryo in saline flushings

of the contralateral uterine horn.

Blood samples that were collected prior to surgeries were

allowed to clot at room temperature (20 °C) for approximately

4 h and then stored overnight at 4°C. Blood samples were then

centrifuged at 500 x g for 15 min at 4°C and the resultant

sera were decanted into vials and stored at -20°C until

assayed for concentrations of estrogen and progesterone.

Nuclear Estrogen Receptor Assay

Intercaruncular uterine endometrium was carefully

dissected from the lumen of the uterine horn. Unless

41

otherwise indicated, all procedures were performed at 4°C

using acid-washed, siliconized glassware. Approximately 300

± 5 mg of endometrium were weighed and homogenized with an all

glass tissue grinder in 2 ml of ice cold TE buffer (0.01M

Tris-0.0015M Na2EDTA, pH 7.4, at 25°C) and centrifuged at 1000

x g for 15 min. The supernatant containing cytosol was

decanted into a tube and saved for analysis of estrogen

receptor concentrations. The remaining nuclear pellet was

washed three times with cold TE buffer and then resuspended

with buffer to an equivalent of 50 mg of endometrium/ml. The

nuclear fraction was vortexed and 1 ml aliquots were pipetted

into each of four tubes. Nuclear concentrations of estrogen

receptor were determined by exchange and binding assay as

described by Koligian and Stormshak (1977a). The exchange was

performed in duplicate using 6,7[3H]- estradiol -17B (specific

activity 1.0 mCi/mmol, NEN Research Products, Boston, MA)

at a concentration of 2 x 10-8M for determination of total

binding and labeled estradiol -178 plus a 100-fold excess of

diethylstilbestrol (DES) for determination of nonspecific

binding. Samples were incubated for 30 min at 37°C.

Following this exchange period, samples were placed on ice for

10 min to terminate the exchange process, and centrifuged for

10 min at 1000 x g. Nuclear pellets were then washed four

times with 2 ml of cold TE buffer to remove any unbound

ligand. The nuclear pellets were then extracted with 2 ml of

redistilled absolute ethanol overnight at 4°C. Following

42

extraction, the samples were vortexed, centrifuged (1000 x g),

and the ethanolic supernatants were decanted into

scintillation vials. Ten milliliters of scintillation fluid

(0.7% 2,5-diphenyloxazole (PPO) and 0.01% p-bis[2-(5-

phenyloxazoly1)]-benzene (POPOP) dissolved in toluene and

Triton X-100 (2:1)) were added and samples were then counted.

Cytosolic Estrogen Receptor Assay

The cytosolic supernatant that was collected after

homogenization and centrifugation of uterine endometrium was

brought to a volume of 50 mg/ml with cold TE buffer. One-

hundred microliters of dextran-coated charcoal (0.5%

dextran:5% charcoal) were added and incubated for 5 min to

remove any free ligand. The sample was centrifuged for 10

min at 1000 x g and the supernatant decanted into a clean

tube. This procedure was repeated a second time to ensure

all charcoal had been removed. One milliliter aliquots were

pipetted into each of four tubes to which were added 617[3]fl-

estradio1-1713 at a concentration of 2 x 104IM (total binding

tubes) or labeled estradio1-178 plus a 100-fold excess of DES

(nonspecific binding tubes). Samples were incubated for 30

min at 37°C. After completion of the exchange process, the

cytoplasmic fraction was subjected to 500 Al of

hydroxylapatite (60% solution v/v of hydroxylapatite in TE

buffer) for adsorption and incubated at 4°C for 1 h with 15

min intermittent vortexing. Following incubation the

43

hydroxylapatite to which the estrogen-receptor complex was

adsorbed was washed four-times with 2 ml of TE buffer and then

subjected to extraction with 2 ml of redistilled absolute

ethanol at 4°C overnight. After centrifugation the

supernatant was decanted into scintillation vials containing

10 ml of scintillation fluid as described above, and counted.

Nuclear Progesterone Receptor Assay

Aliquots of intercaruncular endometrium were homogenized

in ice cold TEM buffer (10mM Tris-HC1, 1mM Na2EDTA, 12mM

monothioglycerol, 30% v/v glycerol, 5mM Na molybdate, pH 7.4

at 25°C), and centrifuged at 1000 x g for 10 min. The

supernatant was decanted into tubes for assay of cytosolic

concentrations of progesterone receptors. The nuclear pellet

was washed four-times with TEM buffer and then resuspended to

a final volume of 100 mg endometrium/ml of buffer. Samples

were vortexed and aliquots of 1 ml were pipetted into each of

four tubes. The nuclear exchange assay for progesterone

receptors was performed as described by West et al. (1986).

The exchange was carried out by adding [3Hjpromegestone

(17,21-dimethy1-19-nor-4,9-pregnadiene-3,20-dione,alsoknown

as R5020, specific activity 1 mCi/mmol, NEN Research Products,

Boston, MA) at a concentration of 1 x 10-6M to the total

binding tubes. The synthetic glucocorticoid dexamethasone was

added (2 x 10-4M) to prevent any progesterone from binding to

the glucocorticoid receptors. Similarly the labeled ligand

44

and dexamethasone were added to non-specific binding tubes as

described above with an additional 100-fold excess of

unlabeled promegestone. Samples were incubated overnight at

4°C to complete the exchange process. After completion of the

exchange, the nuclear pellet was washed four-times with TEM

buffer and extracted with 2 ml of absolute ethanol at 4°C

overnight. Following extraction, the supernatant was decanted

into scintillation vials containing 10 ml of scintillation

fluid and counted.

Cytosolic Progesterone Receptor Assay

After homogenization and centrifugation, the cytosolic

supernatant was also subjected to a progesterone receptor

exchange assay. The supernatant was brought to a final volume

of 100 mg equivalent of endometrium/ml of TEM buffer. One-

hundred microliters of dextran-coated charcoal (0.5%

dextran:5% charcoal in TE buffer) were added to the

supernatant to remove any free ligand, centrifuged (1000 x g)

and decanted into new a tube. This washing procedure was

repeated an additional time to ensure that all charcoal was

removed from the supernatant. The cytosolic sample were then

aliquoted into 1 ml fractions and subjected to the same

binding exchange procedure as described for the nuclear

fraction. After the overnight incubation at 4°C, 500 Al of

dextran-coated charcoal (0.05:0.5% in phosphate buffered

saline, pH 7.4) were added. Samples were vortexed at 5 min

45

intervals during a 10 min incubation period and then

centrifuged at 1500 x g for 10 min. Five-hundred microliters

of supernatant were pipetted into scintillation vials

containing 10 ml of scintillation fluid and counted.

DNA Determination

Deoxyribonucleic acid concentration in the endometrial

tissue were measured using the procedures described by Burton

(1956). In brief, the resultant nuclear pellet from the

exchange assay was stored at 4°C in 0.5M perchloric acid (PCA)

until analyzed for DNA. Standards containing a known amount

of calf thymus DNA (0, 50, 100, 150, 200, 250, 300, 350, 400,

450 and 500 µg /ml dissolved in 5mM sodium hydroxide solution),

were pipetted (100 Al) in duplicate. Bovine serum albumin (1

ml of a 0.1% solution) and 1 ml of 0.5M PCA was then added to

the standard tubes to precipitate the protein and DNA. All

tubes were centrifuged at 1000 x g for 10 min after 1 h of 4°C

incubation and the resultant supernatant was discarded.

Perchloric acid was added to all tubes (2 ml of 0.5M PCA)

which were then capped with marbles and placed in a 90°C

water-bath for 30 min in order to hydrolyze the DNA. Tubes

were then cooled at 4°C for 30 min and subsequently

centrifuged at 1000 x g for 10 min. The resultant supernatant

was pipetted (500 Al) into fresh tubes and colored for 16 h

with 1 ml of diphenylamine reagent (1.5 g diphenylamine

dissolved in 1.5 ml of 96% concentrated sulfuric acid and 100

46

ml of concentrated glacial acetic acid). Samples were then

read using a spectrophotometer set at 600 Am wavelength.

Concentration of estrogen and progesterone receptors were

based on radioactive counts converted to fmol/Ag DNA.

Serum Progesterone Radioimmunoassay

Serum concentrations of progesterone were measured

according to procedures described by Koligian and Stormshak

(1977a). In brief, duplicate 100 Al aliquots of serum were

extracted with 2 ml of benzene:hexane (2:1 v/v) by vortexing.

Separation of organic solvent from the serum was accomplished

by subjecting tubes to -20°C until the serum was frozen and

then decanting the benzene:hexane into tubes. Subsequently

the benzene:hexane was evaporated using a gentle stream of

air. One-hundred microliters of progesterone antibody (#337

antiprogesterone-11-BSA, Gordon Niswender, Colorado State

University) diluted 1:2400 with 0.1% gelatin phosphate

buffered saline (GPBS), were added to tubes containing the

extracted progesterone or quantities of standards (10, 25, 50,

100, 200, 400, 500, 800, 1000 and 2000 pg of progesterone/100

Al) and allowed to equilibrate for >30 min. Following

equilibration, 100 Al of competitor 1,2,6,7[3H]-progesterone

(12 x 103 dpm; 115 Ci/mmol; New England Nuclear [NEN], Boston,

MA), were added to all tubes and the mixture was allowed to

incubate at 4°C overnight. After overnight incubation 1 ml

dextran-coated charcoal (0.25% dextran:2.5% charcoal in 0.01

47

M NaH2PO4(H20) buffered saline) was added and the samples

incubated at 4°C for 15 min to remove any free ligand.

Samples were then centrifuged at 1700 x g for 10 min. The

resultant supernatant was decanted into scintillation vials

and counted. The sensitivity of the assay was 10 pg/assay

tube (100 Al). All serum samples for the progesterone

radioimmunoassay were analyzed in a single assay where the

intraassay coefficient of variation was 8.7 ± 0.37% and the

extraction efficiency was 91.5 ± 3.8%.

Serum Estradiol Radioimmunoassay

Serum estradiol was measured using the extraction

procedures described by Hotchkiss et al. (1971) and the

radioimmunoassay described by Butcher (1977). Briefly, 200

Al of sample serum were pipetted in triplicate into 10 ml

centrifuge tubes with screw caps. Every third tube received

100 Al of 2,4,6,7(3H]- estradiol -178 (diluted to 3500 cpm/200

Al in 0.1% GPBS, NEN) for determination of extraction

efficiency. Anesthetic grade anhydrous diethyl ether (3 ml)

was added and samples were vortexed for 30 sec. In order to

ensure complete separation of organic solvent from the serum,

samples were centrifuged at 1000 x g for 10 min. The sample

tubes were then snap frozen in a dry ice and ethanol bath.

The resultant ether layer was decanted into 12 x 75 mm tubes

and evaporated to dryness under light air in a 37°C water

bath. The frozen pellets from the original tubes were allowed

48

to thaw and the above procedure was repeated. Sample tubes

containing the dried extractant were then stored at 4°C until

the estradiol RIA was conducted.

Standards containing 0, 0.5, 1, 5, 10, 25, 50, 100, 200

and 500 pg estradiol - 17!3/100 Al redistilled absolute methanol

were pipetted in duplicate and dried under light air in a 37°C

water-bath while extractant tubes were allowed to come to room

temperature. To all except blank tubes, 100 Al of 0.3%

gelatin phosphate buffered saline (GPBS) was added [0.3% Knox

gelatin, 0.01M Na2HPO4(H20), 0.15M NaC1, 0.01% merthiolate, pH

7.0]. Blank tubes received 200 Al of 0.3% GPBS. Tubes were

vortexed and allowed to equilibrate for 15 min in order for

the GPBS to take up the ligand. All tubes excluding the

blanks received 100A1 of antiestradiol -178 (anti-E2-

hemisuccinate-BSA diluted to a concentration of 1:10,000 in

0.1% GPBS). In addition, 200 Al of 2,4,6,7[3H)- estradiol -178

(3500 cpm/200 Al) were added to all tubes, blanks included,

and vortexed. Samples were then placed in a 37°C water-bath

for 5 min for the binding process to occur. After incubation

tubes were again vortexed and placed in a 4 °C ice bath for 40

min. Rapidly, 800 Al of ice cold dextran coated charcoal (1%

dextran T-70 from Sigma Chemical, St. Louis, MO, 0.25% Neutral

Norit from Pharmecia Fine Chemicals, Piscataway, NJ) were

added to tubes and vortexed. After 10 min on ice, tubes were

then centrifuged at 2500 x g for 10 min and the resultant

supernatant was decanted into vials containing 10 ml of

49

scintillation fluid and counted. All serum samples for

estradiol radioimmunoassay were analyzed in a single assay,

with a sensitivity of 2.3 pg/tube. The coefficient of

variation was 10 ± 0.43% and extraction efficiency was 95 ±

2%.

Statistical Analysis

Data on nuclear and cytosolic concentrations of estrogen

and progesterone receptors were subjected to log

transformation because of heterogeneity of variances. The

transformed data were analyzed by least squares analysis of

variance for an experiment of completely randomized block,

repeated measures design. Differences among means were tested

for significance by use of Students t-test. For purposes of

presentation, data are presented as nontransformed means.

RESULTS

Endometrial nuclear concentrations of estrogen receptor

in uterine horns of ewes on day 10 of the estrous cycle were

significantly greater (1.21 ± 0.22 fmol/Ag DNA, p<.01) than

in the pregnant and nonpregnant horns of ewes on day 18 of

gestation (0.13 ± 0.23 and 0.22 ± 0.25 fmol/Ag DNA,

respectively; Figure 1). Similarly, cytosolic concentrations

of estrogen receptor in endometrium on day 10 of the estrous

cycle were significantly greater (4.90 ± 0.56 fmol/Ag DNA,

p<.01) than in endometrium of pregnant and nonpregnant horns

50

of ewes on day 18 of gestation (1.25 ± 0.58 and 1.10 ± 0.64

fmol/gg DNA, respectively).

Nuclear concentrations of progesterone receptor in

endometrium of ewes on day 10 of the estrous cycle were

significantly higher (0.63 ± 0.21 fmol/Ag DNA) than in

endometrium of pregnant and nonpregnant uterine horns of ewes

on day 18 of gestation (0.18 ± 0.21, p<.05 and 0.13 ± 0.21

fmol/Ag DNA, p<.01 respectively; Figure 2). Cytosolic

concentrations of progesterone receptor on day 10 of the

estrous cycle were determined to be 1.14 ± 1.03 fmol/Ag DNA.

Data on cytosolic concentrations of progesterone receptor in

both the pregnant and non-pregnant horns of ewes on day 18 of

gestation were invalidated by excessive nonspecific binding

(NSB) of the ligand (77.5 ± 8.6%). Serum concentrations of

estradiol -1713 on day 18 of gestation as determined by

radioimmunoassay were 16.56 ± 2.43 pg/ml. Mean serum

concentration of progesterone on day 18 of gestation was 1.74

± 0.57 ng/ml.

,6.00

4.805

?3.60

2.40

7N 120

0.00

INN NUCLEAR CYTOSOL

d 10 OF CYCLE d18 PREG.HORN d 18 NPREG.HOFts1

Fig. 1. Specific binding of [ flestradiol-178 to nuclear and cytosolic estrogenreceptors (mean ± SE) in endometria of ewes onday 10 of the estrous cycle and in endometriaof gravid and nongravid uterine horns of eweson day 18 of gestation. Mean nuclear andcytosolic concentrations of estrogenreceptors, day 10 vs day 18 gravid andnongravid horns differ (p<.05).

1.00

0.80

3.1

0.60

0.400011.

0.20

A

0.00d10 OF CYCLE d18 PREGHORN d18 NPFEG.HORN

Fig. 2. Specifically bound [ 4]115020 tonuclear progesterone receptors (mean ± SE) inendometria of ewes on day 10 of the estrouscycle and in endometria of the gravid andnongravid uterine horns of ewes on day 18 ofgestation. Day 10 vs day 18 gravid andnongravid horns differ in concentrations ofprogesterone receptors (p<.05).

51

52

DISCUSSION

Results of Exp. 1 indicate that the endometrium of the

nongravid horn of ewes on day 10 of the estrous cycle contains

greater concentrations of nuclear and cytosolic estrogen

receptors than the endometrium of gravid and nongravid horns

of ewes on day 18 of gestation. Similiarly, endometrial

nuclear concentrations of progesterone receptor were greatest

on day 10 of the cycle than in gravid and nongravid uterine

horns of ewes on day 18 of gestation. These changes in

uterine estrogen and progesterone receptor are consistent with

the reported ability of progesterone to down-regulate the

estrogen receptor as well as its own receptor (Koligian and

Stormshak, 1977a; Selcer and Leavitt, 1988). The greater

concentration of these receptors on day 10 of the cycle most

likely reflects the gradual change in the concentrations of

these receptors that occurs from the time of estrus.

Endometrial concentrations of nuclear and cytosolic estrogen

receptors have been reported to be maximal at estrus and to

decrease during the luteal phase of the cycle of the ewe

(Koligian and Stormshak, 1977a; Miller et al., 1977), and cow

(Zelinski et al., 1982) as progesterone secretion increases.

Few data are available regarding uterine concentrations of

steroid receptors during early gestation in ruminants.

However, Senior (1975) reported low levels of uterine

cytosolic estradiol binding sites on day 20 of gestation in

53

cows. Continued production of progesterone by the corpus

luteum whose maintenance is ensured by the conceptus, as well

as low production of ovarian estrogen may be surmised to be

responsible for the reduced concentrations of progesterone and

estrogen receptors observed in this study. Indeed, the low

concentrations of estrogen and progesterone receptors observed

in the uteri of ewes on day 18 of gestation most likely

represent the minimal levels of these receptors necessary to

ensure basal function of the uterus. Logically, greater

concentrations of receptor than detected during early

gestation, especially for estrogen, would not be consistent

with the ability of the uterus to remain in a comparatively

quiescent state necessary for maintenance of pregnancy. It

is possible that some other factor (perhaps of conceptus

origin) besides progesterone may also operate to suppress the

uterine concentrations of steroid receptors. This possibility

is supported in part by the observation that endometrial

concentrations of nuclear and cytosolic estrogen and nuclear

progesterone receptors did not differ between pregnant and

nonpregnant uterine horns on day 18 of gestation. These

latter data indicate that the release of a conceptus secretory

factor may influence uterine concentrations of estrogen and

progesterone receptors in a systemic rather than local manner.

This is further supported by the fact that vascular

connections between horns were blocked by a ligature at the

external bifurcation.

54

Unfortunately, quantification of levels of cytosolic

progesterone receptor concentrations on day 18 of gestation,

as determined by receptor exchange assay, was confounded by

extremely high nonspecific binding. One explanation for this

phenomenon may be the increase in systemic binding proteins

such as progesterone binding globulin (PSG) during early

gestation. Progesterone binding globulin has a high affinity

for progesterone and has been shown to increase in

concentration during gestation in several species (Westphal,

1983). Similiarly, increased levels of systemic binding

globulins that bind estrogens (such as sex steroid hormone

binding globulin) may have contributed to nonspecific binding

in the assay for estrogen receptor, but to a lesser extent.

It is conceivable that the problems encountered by high

nonspecific binding could have been circumvented by using a

substantially greater quantity of unlabeled ligand in the

assay.

These data indicate that the concentrations of estrogen

and progesterone receptors during early gestation in the ewe

are significantly lower than during the midluteal phase of

the estrous cycle when progesterone concentrations are

maximal. Additionally, results of this experiment can be

interpreted as suggesting a systemic effect of the conceptus

on uterine concentrations of estrogen and progesterone

receptors.

55

EXPERIMENT 2

INTRODUCTION

Only recently have researchers identified the hormonal

mechanisms by which varying photoperiod functions to control

seasonal breeding of ewes. In the northern hemisphere,

exposure of ewes to reduced hours of daylight in the fall

results in increased duration of melatonin secretion during

the lengthening hours of darkness (Karsch et al., 1984;

Bittman et al., 1985). It has been proposed that increased

daily secretion of melatonin relieves the sensitivity of the

hypothalamic GnRH pulse regulator to the negative feedback

action of estrogen (Karsch et al., 1980; Goodman et al., 1982;

Karsch et al., 1984). Thus this pineal hormone appears to be

essential for promoting continuity of estrous cycles once

initiated. In addition, melatonin has been found to suppress

the secretion of prolactin in a number of species (Clark,

1980; Glass and Lynch, 1983). This is consistent with data

demonstrating that secretion of prolactin is maximal during

spring and summer anestrus when ewes are exposed to long hours

of daylight. Thus, the patterns of secretion of these two

hormones are inversely related. With the exception of its

effect on mammary gland function, the role(s) of prolactin in

the ewe is not known. During seasons of maximal secretion of

melatonin and prolactin these hormones may act on distant

target organs such as the uterus. Evidence has been presented

56

that melatonin can alter the uterine estrogen receptor

concentrations in the hamster (Danforth et al., 1983) and

human breast cancer cells (Danforth and Lippman, 1981).

However, these conclusions remain equivocal.

This experiment was conducted to determine whether

concentrations of estrogen receptor in control and estradiol-

stimulated uteri of ovariectomized ewes differ between fall

and summer. A detected difference in this uterine

characteristic would suggest the possibility that either

melatonin or prolactin was acting either directly or

indirectly to alter uterine function.

MATERIALS AND METHODS

Animals

Ten mature Polypay ewes were checked twice daily for

estrus to ensure all ewes exhibited an estrous cycle of normal

duration. Ewes were then bilaterally ovariectomized in

October and allowed 4-6 weeks for recovery. At the height of

the winter solstice, a 10 ml blood sample was collected by

jugular venipuncture from each of five ewes for analyses of

serum concentrations of estrogen and progesterone. These five

ewes were then anesthetized and a midventral laparotomy was

performed. Approximately one-third to one-half of a uterine

horn was removed and placed on ice. The contralateral uterine

horn was ligated at the external bifurcation and estradiol-

57

1713 (10 gg/3ml of physiological saline) was injected into the

lumen of the contralateral horn to stimulate synthesis of

estrogen receptors. This dose of estradiol -178 was chosen

because results of a preliminary experiment indicated lower

doses of this steroid to be nonstimulatory. It was assumed

that a dose of 10 leg would not, or at least be minimally

effective, in stimulating release of prolactin. After 48 h

ewes were relaparotomized and a section of the stimulated

uterine horn was removed and placed on ice. Endometrium (50

mg) was assayed within 90 min of removal for concentrations

of estrogen receptors using the estrogen nuclear and cytosolic

exchange methods. The above procedure was repeated during

the height of the summer solstice on the remaining ewes (n=5).

Comparisons were made between the concentration of estrogen

receptors found in the endometrium of ewes during the winter

and summer solstices, as well as between stimulated and

nonstimulated endometrium.

Nuclear and Cytosolic Estrogen Receptor Assay

The assay of nuclear and cytosolic estrogen receptors

was performed as described in experiment 1.

DNA Determination

Deoxyribonucleic acid concentration in the endometrial

tissue was measured as described in experiment 1.

58

Serum Progesterone and Estradiol Radioimmunoassay

Systemic concentrations of progesterone and estradiol-

1713 were measured as described in experiment 1.

Statistical Analysis

Data on endometrial nuclear and cytosolic concentrations

were analyzed by analysis of variance for an experiment of 2

x 2 factorial design. The factors or main effects were season

(winter and summer) and exogenous estradiol (0 and 10 Ag).

RESULTS

Changes in endometrial nuclear and cytosolic

concentrations of estrogen receptor before and after estradiol

treatment at the winter and summer solstice are depicted in

figure 3. Nuclear concentrations of estrogen receptor in the

endometrium of chronically ovariectomized ewes prior to

exogenous estradiol stimulation during the time of the winter

solstice were not different (p>.05) from those prior to

estradiol stimulation during the summer solstice (0.46 ± 0.10

vs 0.46 ± 0.07 fmol/Ag DNA, respectively). Similarly,

cytosolic concentrations of estrogen receptor in endometrium

of ovariectomized ewes prior to exogenous estradiol

stimulation were not different at the winter and summer

solstice (2.64 ± 1.45 vs 3.27 ± 1.08 fmol/Ag DNA,

respectively).

59

However, at both the winter and summer solstice,

treatment of ewes with estradiol resulted in a significant

increase in endometrial concentrations of nuclear and

cytosolic estrogen receptors compared with concentrations

present prior to treatment (p<.05). Nuclear concentrations

of estrogen receptor in endometrium of ovariectomized ewes 48

h after estradiol stimulation were 0.804 ± 0.20 fmol/Ag DNA

during the winter solstice and 1.18 ± 0.46 fmol/Ag DNA during

the summer solstice. These mean nuclear concentrations of

estrogen receptor were not significantly different.

Similarly, there was no significant difference detected in

endometrial cytosolic concentrations of estrogen receptor of

estradiol-treated ewes at the winter and summer solstice (6.09

± 2.22 fmol/Ag DNA vs 8.29 ± 1.17 fmol/Ag DNA; p>.05).

Serum concentrations of estradiol prior to treatment of

ewes with estradiol during the winter solstice and summer

solstice did not differ significantly (16.55 ± 4.05 vs 16.00

± 3.00 pg/ml of serum, respectively; p>.05). Similiarly,

serum levels of estradiol 48 h after administration of

estradiol did not differ significantly among ewes at the

winter and summer solstice (18.75 ± 2.40 vs 18.65 ± 1.65

pg/ml, respectively).

Serum levels of progesterone were basal (<0.10 ng/ml)

confirming successful and complete ovariectomy of ewes.

60

ill WINTER SUMMER

-E2 NUC -E2 CYT +E2 NUC +E2 CYT

Fig. 3. Specifically bound Offlestradiol-178 to nuclear and cytosolic estrogenreceptors (mean ± SE) in nonstimulated andexogenous estradiol-stimulated endometria ofewes during the winter and summer solstice.Stimulated vs nonstimulated endometrium;nuclear (p<.05); cytosolic (p<.01).

61

DISCUSSION

Unlike the rabbit uterus, which becomes refractory to

ovarian steroids when the interval between ovariectomy and

hormone replacement is prolonged (Daniel, 1984), results of

this experiment demonstrate that the uterus of the ewe is

responsive to exogenous steroids as long as 9 mo after

ovariectomy. This is reflected by the fact that injection of

10 Ag of estradio1-1713 directly into the uterine lumen at 3

and 9 mo after ovariectomy induced increases in endometrial

estrogen receptors compared with concentrations in control

tissue.

No differences in concentrations of estrogen receptor

were observed between either the estrogen-treated or control

uterine horns of ewes during winter and summer. These data

indicate that there is no nongonadal seasonal effect on

estrogen receptor concentrations in ovariectomized ewes. From

these data, it does not appear that either prolactin (during

summer anestrus), or melatonin (during the fall breeding

season) are able to directly or indirectly alter the

concentration of uterine estrogen receptors in ovariectomized

ewes. This observation is in contrast to those reported by

Danforth et al. (1983) who found that melatonin was able to

induce an increase in estrogen receptor concentrations in

immature hamster uteri in vivo and in vitro. Direct in vitro

62

regulation of estrogen receptor activity by melatonin has also

been shown to occur in human breast cancer cells (Danforth and

Lippman, 1981). Chilton and Daniel (1987) demonstrated that

prolactin treatment of ovariectomized rabbits resulted in an

increase in uterine concentrations of estrogen and

progesterone receptors similar to those detected in the

sexually receptive intact rabbit. Serum concentrations of

estradiol both prior to and after estradiol treatment were not

different from one another indicating that treatment with

estradiol did not increase systemic concentrations of

estradiol and therefore did not likely provoke a release of

prolactin. Serum concentrations of progesterone detected in

the ovariectomized ewes undoubtedly reflect steroid secreted

from the adrenal cortex. Because there was no significant

difference in serum progesterone concentrations among ewes

within season (0 vs 10 Ag estradiol) or between seasons

(winter vs summer) it is unlikely that any slight variation

in progesterone secretion could have accounted for failure to

detect differences in endometrial estrogen receptor

concentrations.

These data indicate that the mechanism by which

seasonality regulates the breeding season is via an indirect

route through the ovary. The secretion of estrogen from the

ovary acts on the uterine endometrium to stimulate the

synthesis of estrogen and progesterone receptors. It appears

that melatonin secreted in greater quantities during the

63

breeding season of the ewe is acting at the level of the

hypothalamus to regulate release of GnRH and subsequently

pituitary LH. Likewise, increased daily secretion of

prolactin during summer anestrus does not appear to act

directly on the uterus to influence synthesis or

concentrations of estrogen receptors and thereby promote this

reproductive state. Collectively, these data indicate that

neither prolactin nor melatonin act directly on the uterine

endometrium to cause fluctuations in the synthesis of estrogen

receptors in the ovariectomized ewe.

64

BIBLIOGRAPHY

Abdelgadir, S.E., Swanson, L.V., Oldfield, J.E. and Stormshak,F. 1987. Prostaglandin Fla- induced release of oxytocinfrom bovine corpora lutea in vitro. Biol. Reprod. 37:550.

Advis, J., Richards, J.S. and Ojeda, S.R. 1981.Hyperprolactinemia-induced precocious puberty: Studieson the mechanism(s) by which prolactin enhances ovarianprogesterone responsiveness to gonadotropins inprepubertal rats. Endocrinology 108:1333.

Aitken, R.J. 1979. The hormonal control of implantation. In:Maternal Recognition of Pregnancy. Ciba Found. Symp., No.64. Excerpta Medica, Amsterdam. p.53.

Aitken, R.J., Bowman, P. and Gauld, I. 1977. The effect ofsynchronous and asynchronous egg transfer on fetal weightin mice selected for large and small body size. J.

Embryol. Exp. Morphol. 37:59.

Anthony, R.V., Helmer, S.D., Sharif, S.F., Roberts, R.M.,Hansen, P.J., Thatcher, W.W. and Bazer, F.W. 1988.Synthesis and processing of ovine trophoblast protein-1and bovine trophoblast protein-1, conceptus secretoryproteins involved in the maternal recognition ofpregnancy. Endocrinology 123:1274.

Astwood, E.B. 1938. A six hour assay for the quantitativedetermination of estrogen. Endocrinology 23:25.

Auletta, F.J. and Flint, A.P.F. 1988. Mechanisms controllingcorpus luteum function in sheep, cows, nonhuman primates,and women especially in relation to the time ofluteolysis. Endocrine Rev. 9:88.

Avvakumov, G.V., Survilo, L.I., Strel'chenok, O.A. 1986.Interaction of serum sex steroid-binding globulin withcell membranes of human decidual tissue. Biochem. Acad.Sci. U.S.S.R. 50:983.

Baird, D.T. 1977. Evidence in vivo for the two-cell hypothesisof oestrogen synthesis by the sheep Graafian follicle.J. Reprod. Fertil. 50:183.

Baird, D.T. 1978. Local utero-ovarian relationships. In:Crighton, D.B., Haynes, G.R., Foxcroft, G.R. and Lamming,G.E. (Eds.) Control of Ovulation. Butterworths, London.pp. 217-232.

65

Baird, D.T., Land, R.B., Sacaramuzzi, R.J. and Wheeler, A.G.

1976. Endocrine changes associated with luteal regression

in the ewe: The secretion of ovarian oestradiol,

progesterone, androstenedione and uterine prostaglandinFez throughout the oestrous cycle. J. Endocrinol. 69:275.

Barley, D.A., Butcher, R.L. and Inskeep, E.K. 1966. Local

nature of utero-ovarian relationships in the

pseudopregnant rat. Endocrinology. 79:119.

Bazer, F.W. and First, N.L. 1983. Pregnancy and parturition.

J. Anim. Sci. 57(Supp1.2):425.

Bazer, F.W., Geisert, R.D., Thatcher, W.W. and Roberts, R.M.1982. The establishment and maintenance of pregnancy. In:

Cole, D.S.A. and Foxcroft, G.R. (Eds.) Control of PigReproduction. Butterworth Scientific, London.

Berthois, Y., Pourreau-Schneider, N., Gandilhon, P., Mittre,

H. and Tubiana, N. 1986. Estradiol membrane binding sites

on human breast cancer cell lines. Use of a fluorescent

estradiol conjugate to demonstrate plasma membranebinding systems. J. Steroid Biochem. 25:963.

Bex, F., Bartke, A., Goldman, B.D. and Dalterio, S. 1978.

Prolactin, growth hormone, luteinizing hormone receptors

and seasonal changes in testicular activities in thegolden hamster. Endocrinology 103:2069.

Bindon, B.M. 1971. The role of progesterone in implantationin the sheep. Aust. J. Biol. Sci. 24:149.

Bittman, E.L., Kaynard, A.H., Olster, D.H., Robinson, J.E.,

Yellon, S.M. and Karsch, F.J. 1985. Pineal melatoninmediates photoperiodic control of pulsatile luteinizinghormone secretion in the ewe. Neuroendocrinology 40:409.

Bjersing, L. 1967. On the morphology and endocrine functionof granulosa cells in ovarian follicles and corporalutea. Acta Endocrinol. 56(Suppl. 125).

Boyar, R.M., Finkelstein, J.W., Kapen, S. and Hellman, L.

1975. Twenty-four hour prolactin (PRL) secretory patternsduring pregnancy. J. Clin. Endocrinol. Metab. 40:1117.

Brann, D.W., Rao, I.M. and Mahesh, V.B. 1988. Antagonism of

estrogen-induced prolactin release by progesterone. Biol.

Reprod. 39:1067.

Burton, K. 1956. A study of the conditions and mechanism of

the diphenylamine reaction for the colorimetricestimation of deoxyribonucleic acid. Biochem. J. 62:315.

66

Butcher, R.L. 1977. Changes in gonadotropins and steroidsassociated with unilateral ovariectomy in the rat.Endocrinology 101:830.

Buttle, H.L. 1974. Seasonal variation of prolactin in plasmaof male goats. J. Reprod. Fertil. 37:95.

Caffrey, J.L., Fletcher, P.W., Diekman, M.A., O'Callaghan,P.L. and Niswender, G.D. 1979. The activity of ovineluteal cholesterol esterase during several experimentalconditions. Biol. Reprod. 21:601.

Caron, M.G., Goldstein, S., Savard, K. and Marsh, J.M. 1975.Protein kinase stimulation of a reconstituted cholesterolside chain cleavage enzyme system in the bovine corpusluteum. J. Biol. Chem. 250:5137.

Carson, R.S., Findlay, J.K., Burger, H.G. and Trounson, A.O.1979. Gonadotropin receptors of the ovine ovarianfollicle during follicular growth and atresia. Biol.Reprod. 21:75.

Casida, L.E. and Warwick, E.J. 1945. The necessity of thecorpus luteum for maintenance of pregnancy in the ewe.J. Anim. Sci. 4:34.

Chepenik, K.P. and Smith, J.B. 1980. Synthesis ofprostaglandins by mouse embryos. Int. Res. Commun. Syst.Med. Sci. 8:783.

Chilton, B.S. and Daniel, Jr., J.C. 1987. Differences in therabbit uterine response to progesterone as influenced bygrowth hormone or prolactin. J. Reprod. Fertil. 79:581.

Clark, J.H. and Markaverich, B.M. 1988. Actions of ovariansteroid hormones. In: Knobil, E., Neill, J.D., Ewing,L.L., Greenwald, G.S., Markert, C.L. and Pfaff, D.W.(Eds.) The Physiology of Reproduction. Raven Press, NewYork. pp. 675-726.

Clark, J.R. 1980. Physiological problems of seasonal breedingin eutherian animals. Oxford Rev. Reprod. Biol. 2:244.

Cox, R.I., Mattner, P.E. and Thorburn, G.D. 1971. Changes inovarian secretion of oestradiol -178 around oestrus in thesheep. J. Endocrinol. 49:345.

Cumming, I.A., Baxter, R. and Lawson, R.A.S. 1974. Steroidhormone requirement for the maintenance of earlypregnancy in sheep : A study using ovariectomized,adrenalectomized ewes. J. Reprod. Fertil. 40:443.

67

Dalton, D.L. and Knight, J.W. 1983. Effects of exogenousprogesterone and estrone on conceptus development in

swine. J. Anim. Sci. 56:1354.

Danforth, Jr., D.N., Tamarkin, L., Do, R. and Lippman, M.E.1983. Melatonin-induced increase in cytoplasmic estrogenreceptor activity in hamster uteri. Endocrinology 113:81.

Danforth, Jr., D.N. and Lippman, M.E. 1981. Melatonin-inducedincrease in estrogen receptor concentration in MCF-7human breast cancer cells. Abstr. 63rd Annu. Mtg.Endocrine Soc. p. 247, Abstr. 660.

Daniel, J.C., Jetton, A.E. and Chilton, B.S. 1984. Prolactinas a factor in the uterine response to progesterone inrabbits. J. Reprod. Fertil. 72:443.

DeHertogh, R., Ekka, E. and Vanderheyden, I. 1986. Estrogendependent induction of low affinity binding sites in thenuclear fraction of rat uterus. J. Steroid Biochem.24:1171.

Denamur, R., Martinet, J. and Short, R.V. 1973. Pituitarycontrol of the ovine corpus luteum. J. Reprod.Fertil.32:207.

Djiane, J. and Durand, P. 1977. Prolactin-progesteroneantagonism in self regulation of prolactin receptors inthe mammary gland. Nature 266:641.

Donaldson, L.E., Hansel, W. and van Vleck, L.D. 1965.Histological study of bovine corpora lutea. J. DairySci. 48:331.

Dorrington, J.H. and Armstrong, D.T. 1979. Effects of FSH ongonadal function. Rec. Prog. Horm. Res. 35:301.

Dorrington, J. and Gore-Langton, R.E. 1981. Prolactin inhibitsoestrogen synthesis in the ovary. Nature 290:600.

du Mesnil du Buisson, F. 1961. Regression unilaterale descorps jaunes apres hysterectomie partielle chez la truie.Ann. Biol. Anim. Biochem. Biophys. 1:105.

Ekka, E., Vanderheyden, I., Glorieux, B. and DeHertogh, R.1987. Estradiol-induced progesterone receptor synthesisin normal and diabetic ovariectomized rat uterus. J.

Steroid Biochem. 28:61.

Eley, R.M., Thatcher, W.W. and Bazer, F.W. 1979. Hormonal andphysical changes associated with bovine conceptusdevelopment. J. Reprod. Fertil. 55:181.

68

Ellinwood, W.E. 1978. Maternal recognition of pregnancy in theewe and the rabbit. Ph.D Dissertation. Colorado StateUniv., Fort Collins.

Espey, L.L. 1980. Ovulation as an inflammatory reaction--ahypothesis. Biol. Reprod. 22:73.

Evans, G., Dobias, M., King, G.J. and Armstrong, D.T. 1981.Estrogen, androgen and progesterone biosynthesis by theca

and granulosa of preovulatory follicles in the pig. Biol.

Reprod. 25:673.

Falck, B. 1959. Site of production of oestrogen in the ratovary as studied by microimplants. Acta Physiol. Scand.

47:94.

Farin, C.E., Imakawa, K., Hansen, T.R., McDonnell, J.J.,

Murphy, C.N., Farin, P.W. and Roberts, R.M. 1990.

Expression of trophoblastic interferon genes in sheep and

cattle. Biol. Reprod. 43:210.

Fincher, K.B., Bazer, F.W., Hansen, P.J., Thatcher, W.W. andRoberts, R.M. 1986. Ovine conceptus secretory proteinssuppress induction of uterine prostaglandin Fla releaseby oestradiol and oxytocin. J. Reprod. Fertil. 76:425.

Fischer, H.E., Bazer, F.W. and Fields, M.J. 1985. Steroidmetabolism by endometrial and conceptus tissues duringearly pregnancy and pseudopregnancy in gilts. J. Reprod.

Fertil. 75:69.

Fischer, T.V. 1965. Local uterine inhibition of the corpusluteum in the guinea pig. Anat. Rec. 151:350.

Flint, A.P.F. and Sheldrick, E.L. 1986. Ovarian oxytocin andthe maternal recognition of pregnancy. J. Reprod. Fertil.

76:831.

Ford, S.P. 1982. Control of uterine and ovarian blood flowthroughout the estrous cycle and pregnancy of the ewe,

sow and cow. J. Anim. Sci. 55(Suppl. 2):32.

Ford, S.P., Weems, C.W., Pitts, R.E., Pexton, J.E., Butcher,R.L. and Inskeep, E.K. 1975. Effects of estradiol -178 andprogesterone on prostaglandin F in sheep uteri anduterine venous plasma. J. Anim. Sci. 41:1407.

Fortune, J.E. and Armstrong, D.T. 1978. Hormonal control of178-estradiol biosynthesis in proestrous rat follicles:Estradiol production by isolated theca versus granulosa.

Endocrinology 102:227.

69

Foster, D.L., Lemons, J.A., Jaffe, R.B. and Niswender, G.D.1975. Sequential patterns of circulating luteinizinghormone and follicle-stimulating hormone in female sheepfrom early postnatal life through the first estrouscycle. Endocrinology 97:985.

Gadsby, J.E., Heap, R.B. and Burton, R.D. 1980. Oestrogenproduction by blastocyst and early embryonic tissue ofvarious species. J. Reprod. Fertil. 60:409.

Gardner, M.L., First, N.L. and Casida, L.E. 1963. Effect ofexogenous estrogen on corpus luteum maintenance in gilts.J. Anim. Sci. 22:132.

George, F.W. and Wilson, J.D. 1978. Estrogen formation in theearly rabbit embryo. Science 199:200.

Ginther, O.J. 1979. Reproductive Biology of the Mare: Basicand Applied Aspects. McNaughton and Gunn, Inc. Ann Arbor,MI.

Ginther, O.J. and First, N.L. 1971. Maintenance of the corpusluteum in hysterectomized mares. Am. J. Vet. Res.32:1687.

Glass, J.D. and Lynch, G.R. 1983. Melatonin: Identificationof sites of antigonadal action in mouse brain. Science.214:821.

Glencross, R.G., Munro, I.B., Senior, B.E. and Pope, G.S.1973. Concentrations of estradio1-1713, oestrone andprogesterone in jugular venous plasma of cows during theoestrous cycle and in early pregnancy. Acta Endocrinol.73:374.

Goding, J.R. 1974. The demonstration that PGF2a is the uterineluteolysin in the ewe. J. Reprod. Fertil. 38:261.

Godkin, J.D., Bazer, F.W. and Roberts, R.M. 1984a. Ovinetrophoblast protein-1, an early secreted blastocystprotein, binds specifically to uterine endometrium andaffects protein synthesis. Endocrinology 114:120.

Godkin, J.D., Bazer, F.W., Thatcher, W.W. and Roberts, R.M.1984b. Proteins released by cultured day 15-16conceptuses prolong luteal maintenance when introducedinto the uterine lumen of cyclic ewes. J. Reprod. Fertil.71:57.

70

Goodman, R.L, Bittman, E.L., Foster, D.L. and Karsch, F.J.1982. Alterations in the control of luteinizing hormone-pulse frequency underlie the seasonal variation in

estradiol negative feedback in the ewe. Biol. Reprod.

27:580.

Gorski, J., Toft, D., Shyamala, G., Smith, D. and Notides, A.

1968. Hormone receptors: Studies on the interaction ofestrogen with the uterus. Rec. Prog. Horm. Res. 24:45.

Greene, G.L. and Press, M.F. 1986. 1. Steroid receptor

structures (including monoclonal antibodies and newmethods of determination). J. Steroid Biochem. 24:1.

Greenwald, G.S. and Rothchild, I. 1968. Formation and

maintenance of corpora lutea in laboratory animals. J.Anim. Sci. 27(Suppl. 1):139.

Greiss, Jr., F.C., and Anderson, S.G. 1970. Effect of ovarianhormones on the uterine vascular bed. Am. J. Obstet.

Gynecol. 107:829.

Grota, L.J. and Eik-Nes, K.B. 1967. Plasma progesteroneconcentrations during pregnancy and lactation in the rat.

J. Reprod. Fertil. 13:83.

Gwinner, E. and Dorka, V. 1976. Endogenous control of annualreproductive rhythms in birds. In: Proc. 16th Int.

Ornithol. Cong. Austr. Acad. of Sci., Canberra. pp.223.

Hafs, H.D. and Armstrong, D.T. 1968. Corpus luteum growth andprogesterone synthesis during the bovine estrous cycle.

J. Anim. Sci. 27:134.

Halkerston, I.D.K., Eichhorn, J., Feinstein, M., Scully, E.

and Hechter, 0. 1960. Early permeability effects ofestradiol on castrate rat uterus. Proc. Soc. Exp. Biol.

Med. 103:796.

Hansel, W., Concannon, P.W. and Lukaszewska, J.H. 1973.

Corpora lutea of the large domestic animals. Biol.

Reprod. 8:222.

Hansel, W. and Convey, E.M. 1983. Physiology of the estrouscycle. J. Anim. Sci. 57(Suppl. 2):404.

Harper, M.J.K., Norris, C.J. and Rajkumar, V. 1983.

Prostaglandin release by zygotes and endometria of

pregnant rabbits. Biol. Reprod. 28:350.

71

Hauger, R.L., Karsch, F.J. and Foster, D.L. 1977. A newconcept for control of the estrous cycle of the ewe based

on the temporal relationships between luteinizinghormone, estradiol and progesterone in peripheral serumand evidence that progesterone inhibits tonic LH

secretion. Endocrinology 101:807.

Hisaw, Jr., F.L. 1959. Comparative effectiveness of estrogenson fluid imbibition and growth of the rat's uterus.Endocrinology 64:276.

Hixon, J.E. and Clegg, M.T. 1969. Influence of the pituitaryon ovarian progesterone output in the ewe: Effects ofhypophysectomy and gonadotropic hormones. Endocrinology84:828.

Holt, J.A., Richards, J.S., Midgley, Jr., A.R. and ReichertJr., L.E. 1976. Effect of prolactin on LH receptor in ratluteal cells. Endocrinology. 98:1005.

Hotchkiss, J., Atkinson, L.E. and Knobil, E. 1971. Time course

of serum estrogen and luteinizing hormone (LH)

concentrations during the menstrual cycle of the rhesusmonkey. Endocrinology 89:177.

Hsueh, A.J.W., Peck, E.J. and Clark, J.H. 1976. Control ofuterine estrogen receptor levels by progesterone.Endocrinology 98:438.

Huhtaniemi, I.T. and Catt, K.J. 1981. Induction and

maintenance of gonadotropin and lactogen receptors inhypoprolactinemic rats. Endocrinology 109:483.

Hyland, J.H., Manns, J.G. and Humphery, W. D. 1982.Prostaglandin production by ovine embryos and endometriumin vitro. J. Reprod. Fertil. 65:299.

Ichii, S., Forchielli, E. and Dorfman, R.I. 1963. In vitroeffect of gonadotropins on the soluble cholesterol sidechain cleaving enzyme system of bovine corpus luteum.

Steroids 2:631.

Inskeep, E.K. and Butcher, R.L. 1966. Local component ofutero-ovarian relationships in the ewe. J. Anim. Sci.

25:1164.

Ireland, J.J. 1987. Control of follicular growth and

development. J. Reprod. Fertil. Suppl. 34:39.

72

Jensen, E.V., Suzuki, T., Kawashima, T., Stumpf, W.E.,Jungblut, P.W. and DeSombre, E.R. 1968. A two-stepmechanism for the interaction of estradiol with ratuterus. Biochemistry 59:632.

Kaltenbach, C.C., Graber, J.W., Niswender, G.D. and Nalbandov,A.V. 1968. Effect of hypophysectomy on the formation andmaintenance of corpora lutea in the ewe. Endocrinology82:753.

Karsch, F.J. 1987. Central actions of ovarian steroids in thefeedback regulation of pulsatile secretion of luteinizinghormone. Ann. Rev. Physiol. 49:365.

Karsch, F.J., Bittman, E.L., Foster, D.L., Goodman, R.L.,Legan, S.J. and Robinson, J.E. 1984. Neuroendocrine basisof seasonal reproduction. Rec. Prog. Horm. Res. 40:185.

Karsch, F.J., Cook, B., Ellicott, A.R., Foster, D.L., Jackson,G.L. and Nalbandov, A.V. 1971. Failure of infusedprolactin to prolong the life span of the corpus luteumof the ewe. Endocrinology 89:272.

Karsch, F.J., Foster, D.L., Legan, S.J., Ryan, K.D. and Peter,G.K. 1979. Control of the preovulatory endocrine eventsin the ewe: Interrelationship of estradiol, progesteroneand luteinizing hormone. Endocrinology 105:421.

Karsch, F.J., Legan, S.J., Ryan, K.D. and Foster, D.L. 1980.Importance of estradiol and progesterone in regulatingLH secretion and estrous behavior during the sheepestrous cycle. Biol. Reprod. 23:404.

Kennaway, D.J., Dunstan, E.A., Gilmore, T.A. and Seamark, R.F.1984. Effects of pinealectomy, oestradiol and melatoninon plasma prolactin and LH secretion in ovariectomizedsheep. J. Endocrinol. 102:199.

King, W.J. and Greene, G.L. 1984. Monoclonal antibodieslocalize oestrogen receptor in the nuclei of targetcells. Nature 307:745.

Klopper, A. 1983. Specific pregnancy proteins. In: Martin, L.and James, V.H.T. (Eds.) The Endocrinology of Pregnancyand Parturition. Academic Press, New York. p. 127.

Knickerbocker, J.J., Thatcher, W.W., Bazer, F.W., Drost, M.,Barron, D.H., Fincher, K.B. and Roberts, R.M. 1986.Proteins secreted by day 16 to 18 bovine conceptusesextend corpus luteum function in cows. J. Reprod. Fertil.77:381.

73

Knobil, E. 1973. On the regulation of the primate corpusluteum. Biol. Reprod. 8:246.

Koligian, K.B. and Stormshak, F. 1977a. Nuclear and

cytoplasmic estrogen receptors in ovine endometriumduring the estrous cycle. Endocrinology 101:524.

Koligian, K.B. and Stormshak, F. 1977b. Progesteroneinhibition of estrogen receptor replenishment in ovineendometrium. Biol. Reprod. 17:412.

Lemon, M. and Loir, M. 1977. Steroid release in vitro by twoluteal cell types in the corpus luteum of the pregnantsow. J. Endocrinol. 72:351.

Leung, P.C.K. and Armstrong, D.T. 1980. Interactions of

steroids and gonadotropins in the control of

steroidogenesis in the ovarian follicle. Ann. Rev.

Physiol. 42:71.

Ling, W.Y. and Marsh, J.M. 1977. Reevaluation of the role ofcyclic adenosine 3',5'-monophosphate and protein kinasein the stimulation of steroidogenesis by luteinizinghormone in bovine corpus luteum slices. Endocrinology

100:1571.

Liu, Y.-X. and Hsueh, A.J.W. 1986. Synergism between granulosa

and theca-interstitial cells in estrogen biosynthesis bygonadotropin-treated rat ovaries: Studies on the two-celltwo-gonadotropin hypothesis using steroid antisera. Biol.

Reprod. 35:27.

Mais, V., Kazer, R.R., Cetel, N.S., Rivier, J., Vale, W. andYen, S.S.C. 1986. The dependency of folliculogenesis and

corpus luteum function on pulsatile gonadotropinsecretion in cycling women using a gonadotropin-releasinghormone antagonist as a probe. J. Clin. Endocrinol.

Metab. 62:1250.

Marcus, G.J. 1981. Prostaglandin formation by the sheep embryoand endometrium as an indication of maternal recognitionof pregnancy. Biol. Reprod. 25:56.

Marsh, J.M. 1975. The role of cyclic AMP in steroidogenesis.Biol. Reprod. 14:30.

Martal, J.M., LaCroix, M.C., Loudis, C., Saunier, M. and

Winterberger-Torres, S. 1979. Trophoblastin, an

antiluteolytic protein present in early pregnant sheep.

J. Reprod. Fertil. 56:63.

74

Martin, L., Hallowes, R.C., Finn, C.A. and West, D.G. 1973.Involvement of the uterine blood vessels in therefractory state of the uterine stroma which followsoestrogen stimulation in progesterone treated mice. J.Endocrinol. 56:309.

Maywood, E.S., Buttery, R.C., Vance, G.H.S., Herbert, J. andHastings, M.H. 1990. Gonadal responses of the male Syrianhamster to programmed infusions of melatonin aresensitive to signal duration and frequency but not tosignal phase nor to lesions of the suprachiasmaticnuclei. Biol. Reprod. 43:174.

McCracken, J.A., Glew, M.E. and Scaramuzzi, R.J. 1970. Corpusluteum regression induced by prostaglandin F2 a. J. Clin.Endocrinol. Metab. 30:544.

Mercer, W.D., Edwards, D.P., Chamness, G.C. and McGuire, W.L.1981. Failure of estradiol immunofluorescence in MCF-7breast cancer cells to detect estrogen receptors. CancerRes. 41:4644.

Miller, B.G. and Moore, N.W. 1976. Effects of progesterone andoestradiol on RNA and protein metabolism in the genitaltract and on survival of embryos in the ovariectomizedewe. Aust. J. Biol. Sci. 29:565.

Miller, M.A., Mullick, A., Greene, G.L. and Katzenellenbogen,B.S. 1985. Characterization of the subunit nature ofnuclear estrogen receptors by chemical cross linking anddense amino acid labeling. Endocrinology 117:515.

Miller, B.G., Murphy, L. and Stone, G.M. 1977. Hormonereceptor levels and hormone, RNA and protein metabolismin the genital tract during the oestrous cycle of theewe. J. Endocrinol. 73:91.

Miller, W.R. and Turner, C.W. 1963. Biosynthesis of steroidsfrom pregnenolone-16-3H by bovine ovarian tissue in

vitro. Steroids. 2:657.

Mitchell, M.D., Flint, A.P.F. and Turnbull, A.C. 1975.Stimulation by oxytocin of prostaglandin F levels inuterine venous effluent in pregnant and puerperal sheep.Prostaglandins 9:47.

Moor, R.M. 1977. Sites of steroid production in ovine Graafianfollicles in culture. J. Endocrinol. 73:143.

Moor, R.M. and Rowson, L.E.A. 1959. Maintenance of pregnancyin ovariectomized ewes by means of progesterone. Nature184:1410.

75

Moor, R.M. and Rowson, L.E.A. 1966a. Local maintenance of thecorpus luteum in sheep with embryos transferred tovarious isolated portions of the uterus. J. Reprod.Fertil. 12:539.

Moor, R.M. and Rowson, L.E.A. 1966b. The corpus luteum of thesheep: Functional relationships between the embryo andthe corpus luteum. J. Endocrinol. 34:233.

Moore, R.Y. and Klein, D.C. 1974. Visual pathways and thecentral neural control of a circadian rhythm in pinealserotonin N-acetyltransferase activity. Brain Res. 71:17.

Moudgal, N.R., Macdonald, G.J. and Greep, R.O. 1971. Effectof hCG antiserum on ovulation and corpus luteum functionin the monkey. J. Clin. Endocrinol. Metab. 32:579.

Mueller, G.C., Herranen, A.M. and Jervell, K.F. 1957. Studieson the mechanism of action of estrogens. Rec. Prog. Horm.Res. 14:95.

Muldoon, T.G. 1981. Interplay between estradiol and prolactinin the regulation of steroid hormone receptor levels,nature, and functionality in normal mouse mammary tissue.Endocrinology 109:1339.

Nalbandov, A.V. 1971. Endocrine control of implantation. In:Blandau, R.J. (Ed.) The Biology of the Blastocyst.University of Chicago Press, Chicago. Chapter 22.

Neill, J.D. 1974. Prolactin: Its secretion and control.Endocrinology 4:469.

Nicoll, C.S. 1974. Physiological actions of prolactin. In:Greep, R.O. and Astwood, E.B. (Eds.) Handbook ofPhysiology. Am. Physiol. Soc. Washington, D.C. pp. 253-292.

Niswender, G.D., Moore, R.T., Akbar, A.M., Nett, T.M. andDiekman, M.A. 1975. Flow of blood to the ovaries ofcycling and pregnant ewes. Biol. Reprod. 13:38.

Niswender, G.D. and Nett, T.M. 1988. The corpus luteum andits control. In: Knobil, E., Neill, J.D. Ewing, L.L.,Greenwald, G.S., Markert, C.L. and Pfaff, D.W. (Eds.) ThePhysiology of Reproduction. Raven Press, New York. pp.489-525.

Niswender, G.D., Reimers, T.J., Diekman, M.A. and Nett, T.M.1976. Blood flow: A mediator of ovarian function. Biol.Reprod. 14:64.

76

Niswender, G.D., Schwall, R.H., Fritz, T.A., Farin, C.E. andSawyer, H.R. 1985. Regulation of luteal function indomestic ruminants: New concepts. Rec. Prog. Horm. Res.41:101.

Norman, A.W. and Litwack, G.L. 1987. Hormones. Academic Press,San Diego. pp. 101-130.

Northey, D.L. and French, L.R. 1980. Effect of embryo removaland intrauterine infusion of embryonic homogenates on thelifespan of the bovine corpus luteum. J. Anim. Sci.

50:298.

Pant, H.C., Hopkinson, C.R.N. and Fitzpatrick, R.J. 1977.Concentration of oestradiol, progesterone, luteinizinghormone and follicle stimulating hormone in the jugularvenous plasma of ewes during the oestrous cycle. J.

Endocrinol. 73:247.

Parikh, I., Ranjendran, K.G., Su, J.L., Lopez, T. and Sar, M.1987. Are estrogen receptors cytoplasmic or nuclear?Some immunocytochemical and biochemical studies. J.

Steroid Biochem. 27:185.

Pavlik, E.J. and Coulson, P.B. 1976. Modulation of estrogenreceptors in four different target tissues: Differentialeffects of estrogen vs progesterone. J. Steroid. Biochem.7:369.

Perry, J.S., Heap, R.B. and Amoroso, E.C. 1973. Steroidhormone production by pig blastocysts. Nature 245:45.

Peters, R.R. and Tucker, H.A. 1978. Prolactin and growthhormone responses to photoperiod in heifers.Endocrinology 103:229.

Pope, W.F., Maurer, R.R. and Stormshak, F. 1982. Survival ofporcine embryos after asynchronous transfer. Proc. Soc.Exp. Biol. Med. 171:179.

Posner, B.I., Kelly, P.A., Shiu, R.P.0 and Friesen, H.G. 1974.Studies of insulin, growth hormone and prolactin binding;tissue distribution, special variation andcharacterization. Endocrinology 95:521.

Pratt, B.R., Butcher, R.L. and Inskeep, E.K. 1977.Antiluteolytic effect of the conceptus and of PGE2 inewes. J. Anim. Sci. 46:784.

77

Reynolds, L.P., Magness, R.R. and Ford., S.P. 1982. Uterineblood flow (UBF) and uterine arterio-venous steroidconcentration on d 11, 13, and 15 of the estrous cycleand pregnancy in ewes. J. Anim. Sci. 55(Suppl. 1):382.

Riddick, D.H. 1985. Regulation and physiological relevance ofnon-pituitary prolactin. In: MacLeod, R.M., Thomer, M.O.and Scapagrini, U. (Eds.) Prolactin Basic and ClinicalCorrelates. V. Liviana Press, Padova. pp. 463-473.

Roberts, K.D., Bandy, L. and Leiberman, S. 1967. Theconversion of cholesterol-3H-sulfate-35S intopregnenolone-3H-sulfate-35S by sonicated bovine adrenalmitochondria. Biochem. Biophys. Res. Commun. 29:741.

Rodgers, R.J. and O'shea, J.D. 1982. Purification, morphologyand progesterone production and content of three celltypes isolated from the corpus luteum of sheep. Aust. J.

Biol. Sci. 35:441.

Rolland, R., Grusalus, G.L. and Hammond, J.M. 1976.Demonstration of specific binding of prolactin by porcinecorpora lutea. Endocrinology 98:1083.

Rose, J., Oldfield, J.E. and Stormshak, F. 1986. Changes inserum prolactin concentrations and ovarian prolactinreceptors during embryonic diapause in mink. Biol.

Reprod. 34:101.

Rothchild, I. 1981. The regulation of the mammalian corpusluteum. Rec. Prog. Horm. Res. 37:183.

Sabbah, M., Redeuilh, G. and Baulieu, E.E. 1989. Subunitcomposition of the estrogen receptor. Involvement of thehormone-binding domain in the dimeric state. J. Biol.Chem. 264:2397.

Sar, M. and Meites, J. 1968. Effects of suckling on pituitaryrelease of prolactin, GH and TSH in post-partum lactatingrats. Neuroendocrinology 4:25.

Schallenberger, E., Schams, D., Bullermann, B. and Walters,D.L. 1984. Pulsatile secretion of gonadotropins, ovariansteroid and ovarian oxytocin during prostaglandin inducedregression of the corpus luteum in the cow. J. Reprod.Fertil. 71:493.

Schams, D. and Karg, G. 1974. Prolactin release in cattle. J.Reprod. Fertil. 39:463.

78

Schomberg, D.W., Coudert, S.P. and Short, R.V. 1967. Effectsof bovine luteinizing hormone and human chorionicgonadotropin on the bovine corpus luteum in vivo. J.Reprod. Fertil. 14:227.

Schwartz, N.B. and Channing, C.P. 1977. Evidence for ovarian"inhibin" suppression of the secondary rise in serumfollicle stimulating hormone levels in proestrous ratsby injection of porcine follicular fluid. Proc. Natl.Acad. Sci. U.S.A. 74:5721.

Selcer, K.W. and Leavitt, W.W. 1988. Progesterone down-regulation of nuclear estrogen receptor: A fundamentalmechanism in birds and mammals. Gen. Comp. Endocrinol.

72:443.

Senior, B.E. 1975. Cytoplasmic oestradiol-binding sites andtheir relationship to oestradiol content in theendometrium of cattle. J. Reprod. Fertil. 44:501.

Sheldrick, E.L. and Flint, A.P.F. 1983. Regression of thecorpora lutea in sheep in response to cloprostenol is notaffected by loss of luteal oxytocin after hysterectomy.J. Reprod. Fertil. 68:155.

Sheldrick, E.L., Mitchell, M.D. and Flint, A.P.F. 1980.

Delayed luteal regression in ewes immunized againstoxytocin. J. Reprod. Fertil. 59:37.

Shemesh, M., Milaguir, F., Ayalon, N. and Hansel, W. 1979.

Steroidogenesis and prostaglandin synthesis by culturedbovine blastocysts. J. Reprod. Fertil. 56:181.

Short, R.V. 1962. Steroids in the follicular fluid and thecorpus luteum of the mare. A two-cell type theory ofovarian steroid synthesis. J. Endocrinol. 24:59.

Short, R.V. 1969. Implantation and maternal recognition ofpregnancy. In: Wolstenholme, G.E.W. and O'Conner, M.

(Eds.) Fetal Autonomy. Churchill, London. pp. 2-26.

Shutt, D.A., Clark, A.H., Fraser, I.S., Goh, P., McMahon,G.R., Saunders, D.M. and Shearman, R.P. 1976. Changes inconcentration of prostaglandin F and steroids in humancorpora lutea in relation to growth of the corpus luteumand luteolysis. J. Endocrinol. 71:453.

Silvia, W.J., Fitz, T.A., Mayan, M.H. and Niswender, G.D.1984. Cellular and molecular mechanisms involved inluteolysis and maternal recognition of pregnancy in the

ewe. Anim. Reprod. Sci. 7:57.

79

Singh, M.M. and Booth, W.D. 1979. Origin of oestrogen inpreimplantation rabbit blastocysts. J. Steroid Biochem.11:723.

Sirois, J. and Fortune, J.E. 1988. Ovarian follicular dynamicsduring the estrous cycle in heifers monitored by real-time ultrasonography. Biol. Reprod. 39:308.

Slayden, 0. 1990. Endocrine Regulation of Uterine Physiologyin Mink. Ph.D. Dissertation. Oregon State Univ.,Corvallis Oregon.

Smeaton, T.C. and Robertson, H.A. 1971. Studies on the growthand atresia of Graafian follicles in the ovary of thesheep. J. Reprod. Fertil. 25:243.

Stetson, M.H. and Watson-Whitmyre, M. 1976. Nucleus supra-chiasmaticus: The biological clock in hamsters? Science

191:197.

Stewart, H.J., McCann, S.H.E., Barker, P.J., Lee, K.E.,Lamming, G.E. and Flint, A.P.F. 1987. Interferon sequencehomology and receptor binding activity of ovine

trophoblast antiluteolytic protein. J. Endocrinol.115:13.

Stone, G.M., Murphy, L. and Miller, B.G. 1978. Hormonereceptor levels and metabolic activity in the uterus ofthe ewe: Regulation by oestradiol and progesterone. Aust.

J. Biol. Sci. 31:395.

Stormshak, F., Kelley, H.E., and Hawk, H.W. 1969. Suppressionof ovine luteal function by 1713 - estradiol. J. Anim. Sci.

29:476.

Swanson, L.V., Hafs, H.D. and Morrow, D.A. 1972. Ovariancharacteristics and serum LH, prolactin, progesterone andglucocorticoids from first estrus to breeding size in

holstein heifers. J. Anim. Sci. 34:284.

Thorpe, P. and Herbert, J. 1976. Studies on the duration ofthe breeding season and photorefractoriness in femaleferrets pinealectomized or treated with melatonin. J.Endocrinol. 70:255.

Tindal, J.S. 1969. Central pathways involved in prolactinsecretion. In: Reynolds, M. and Folley, S. (Eds.)

Lactogenesis: The Initiation of Milk Secretion atParturition. Univ. of Penn. Press, Philadelphia, PA. pp.253-256.

80

Tsai-Morris, C.-H., Ghosh, M., Hirshfield, A.N., Wise, P.M.and Brodie, A.M.H. 1983. Inhibition of ovarian aromataseby prolactin in vitro. Biol. Reprod. 29:342.

Tsang, B.K. and Ooi, T.C. 1982. Prostaglandin secretion byhuman endometrium in vitro. Am. J. Obstet. Gynecol.142:626.

Turnbull, K.E., Braden, A.W.H. and Mattner, P.E. 1977. Thepattern of follicular growth and atresia in the ovineovary. Aust. J. Biol. Sci. 30:229.

Uresley, J. and Leymarie, P. 1979. Varying response toluteinizing hormone of two cell types isolated frombovine corpus luteum. J. Endocrinol. 83:303.

Vekemans, M. and Robyn, C. 1975. The influence of exogenousestrogen on the circadian periodicity of circulatingprolactin in women. J. Clin. Endocrinol. Metab. 40:886.

Walters, D.L., Schams, D., Bullermann, B. and Schallenberger,E. 1983. Pulsatile secretion of gonadotropins, ovariansteroids and ovarian oxytocin during luteolysis in thecow. Biol. Reprod. 28(Suppl. 1):142.

Wang, C. and Chan, V. 1982. Divergent effects of prolactin onestrogen and progesterone production by granulosa cellsof rat Graafian follicles. Endocrinology 110:1085.

Wang, C., Hsueh, A.J.W. and Erickson, G.F. 1980. Prolactininhibition of estrogen production by cultured ratgranulosa cells. Mol. Cell. Endocrinol. 20:135.

Webster, G.M. and Haresign, W. 1983. Seasonal changes in LHand prolactin concentrations in ewes of two-breeds. J.Reprod. Fertil. 67:465.

Welshons, W.V., Lieberman, M.E. and Gorski, J. 1984. Nuclearlocalization of unoccupied oestrogen receptors. Nature307:747.

West, N.B., Hess, D.L. and Brenner, R.M. 1986. Differentialsuppression of progesterone receptors by progesterone inthe reproductive tract of female macaques. J. SteroidBiochem. 25:497.

Westpahl, U. 1983. Corticosteroid-binding globulin. A reviewof some recent aspects. Mol. Cell. Biochem. 55:145.

81

Wilde, D.E., Culver, A.A., Morcom, C.B. and Dukelow, W.R.1976. Effect of administration of progesterone andestrogen on litter size in pigs. J. Reprod. Fertil.

48:209.

Williams, M.F. 1948. The vascular architecture of the ratuterus as influenced by estrogen and progesterone. Am.J. Anat. 83:247.

Williams, G.H., Hammond, J.M., Weisz, J. and Mortel, R. 1978.Binding sites for lactogenic hormone in the rat uterus.Biol. Reprod. 18:697.

Wu, J.T. 1985. Metabolism of progesterone by hamsterblastocysts and the ontogeny of progesterone metaboliccapability. Biol. Reprod. 33:53.

Yamamoto, K.R. 1985. Steroid receptor regulated transcriptionof specific genes and gene networks. Ann. Rev. Genet.

19:209.

Yen, S.C., Tsai, C.C., Naftolin, F., Vanderberg, G. and

Ajabor, L. 1972. Pulsatile patterns of gonadotropinrelease in subjects with and without ovarian function.J. Clin. Endocrinol. Metab. 34:671.

Zavy, M.T., Mayer, R., Vernon, M.W., Bazer, F.W. and Sharp,

D.C. 1979. An investigation of the uterine luminalenvironment of nonpregnant and pregnant pony mares. J.

Reprod. Fertil. Suppl. 27:403.

Zeleznik, A.J., Midgley, Jr., A.R. and Reichert, Jr., L.E.1974. Granulosa cell maturation in the rat: Increasedbinding of human chorionic gonadotropin followingtreatment with follicle stimulating hormone in vitro.Endocrinology 95:818.

Zelinski, M.B., Hirota, N.A., Keenan, E.J. and Stormshak, F.1980. Influence of exogenous estradiol -178 on endometrialprogesterone and estrogen receptors during the lutealphase of the ovine estrous cycle. Biol. Reprod. 23:743.

Zelinski, M.B., Noel, P., Weber, D.W. and Stormshak, F. 1982.Characterization of cytoplasmic progesterone receptors

in the bovine endometrium during proestrus and diestrus.

J. Anim. Sci. 55:376.