AN ABSTRACT OF THE THESIS OF

Patrice L. Prater for the degree of Master ofScience in Animal Science presented on November 14. 1990

Title: Physiological Factors Affecting Ovine Uterine Estrogen and Progesterone ReceptorConcentrations Redacted for Privacy Abstract Approved: w-A Fredrick Stormshak

Two experiments were conducted todetermine whether in ewes uterine concentrationsof estrogen and progesterone receptors are affected by the presence of aconceptus or by the hormonal milieu associated withextremes in photoperiod to which ewes are exposed.

In Exp.1, nine mature ewes were unilaterally ovariectomized by removing an ovary bearing the corpusluteum

(CL). The ipsilateral uterine horn wasligated at the external bifurcation and a portion of theanterior ipsilateral

uterine horn was removed and assayed forendometrial nuclear and cytosolic concentrations of estrogenreceptor (ER) and

progesterone receptor (PR) by exchange assays. After a recovery estrous cycle, ewes werebred to a fertile ram. On

day 18 of gestation a 10 ml jugular bloodsample was collected

for measurement of serum concentrationsof 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 asystemic 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 removedand assayed for endometrial cytosolic and nuclear ER. This procedure was repeated on the remaining five ewes during the height of the summer solstice (lateJune). Endometrialnuclear 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 endometrialnuclearandcytosolic 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 E2during 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 ewesat 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) anddid not differ in ewes prior to and after E2 treatment at the winter and summersolstice

(p>.05). These data indicate that physiological factors and(or) hormones such as prolactin and melatonin secretedin response to extremes in photoperiod do not appearto influence uterine concentrations of ER in ovariectomized ewes. Physiological Factors Affecting Ovine Uterine

Estrogen and Progesterone Receptor Concentrations

by

Patrice L. Prater

A THESIS submitted to Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Completed November 14, 1990

Commencement June, 1991 APPROVED:

Redacted for Privacy Professor of Animal Science and Biochemistry andBiophysics in charge of the major

Redacted for Privacy Head of Department of Animal Science

Redacted for Privacy

Dean of C

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 thetail 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 56 Nuclear and Cytosolic Estrogen Receptor Assay 57 TABLE OF CONTENTS (CONT.)

Page

DNA Determination 57 Serum Progesterone and Estradiol Radioimmunoassay 58 Statistical Analysis 58 Results 58 Discussion 61

BIBLIOGRAPHY 64 LIST OF FIGURES

Figure Page

1. Specific binding ofClflestradio1-178 to nuclear and cytosolic estrogen receptors (mean ± SE) in endometria of ewes on day 10 of the estrous cycle and in endometria of gravid and non-gravid uterine horns of ewes on day 18 of gestation. Mean nuclear and cytosolic concentrations of estrogen receptors, day 10 vs day 18 gravid and nongravid horns differ (p<.05). 51

2. Specifically bound (3H)R5020 to nuclear progesterone receptors (mean ± SE) in endometria of ewes on day 10 of the estrouscycle and in endometria of the gravid and nongravid uterine horns of ewes on day 18 of gestation. Day 10 vs day 18 gravid and nongravid horns differ in concentrations of progesterone receptors (p<.05). 51

3. Specifically bound [3H]estradio1-178 to nuclear and cytosolic estrogen receptors (mean ± SE) in nonstimulated and exogenous estradiol-stimulated endometria of ewes during the winter and summer solstice. Stimulated vs nonstimulated endometrium; nuclear (p<.05), cytosolic (p<.01). 60 PHYSIOLOGICAL FACTORS AFFECTING OVINE UTERINE ESTROGEN AND PROGESTERONE RECEPTOR CONCENTRATIONS

REVIEW OF LITERATURE

INTRODUCTION

The mechanisms of steroid hormone action havelong been an area of fascination andscientific inquiry. In the female, estrogen and progesterone play a primary rolein promoting the development of an intrauterine environment necessaryfor

successful procreation. Prior to 1960 most research on estrogens and progesterone

entailed studies with various mammalian species toelucidate

the physiological effects of these steroids onthe development

of the uterus and mammary glands. During the 1960's interest in the effects of these hormones shifted fromphysiological

to biochemical. Indeed, it was during the late 1960's that experimental evidence was first presented to suggest thatthe

biological responses evoked by estrogens were the consequence of this steroid being bound within the targetcell by a

macromolecular receptor. Subsequently, research demonstrated the existence of target tissue receptors for othersteroids,

including progesterone. Because the availability of estrogen andprogesterone

receptors directly affects the biologicalactivity of these

steroids, much research hasbeenconductedtoquantify 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 statesin domestic animals.

One oftheleadingcausesofreduced reproductive efficiency in many species is embryonic mortality. Most embryos are lost before or soon after implantation. Many factorsinfluencetheabilityof an embryoto 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 progesteroneisunknown. 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 andwinter 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 decreaseand 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 concentrationsof prolactinor melatonin canalterthe responsiveness of the uterus of ewes to steroids. These hormones have been implicated as having a direct effect onthe 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 butwhen 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 severalstages of maturation. In the initial stages of folliculogenesis, each oocyte is surrounded by a single layer ofgranulosa cells, together forming a primordial follicle (Ireland, 1987). Just prior to or following birth the primordialfollicle enters and remains in a resting state until theappropriate hormonal signals cue it to resume maturation inpreparation

for ovulation or atresia (Ireland, 1987). Follicular growth and maturation occurin a series of events whereby the primordial follicle first developsinto aprimary, then secondary follicle, followed by formation of the fluidfilled 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 nonpregnantanimal. Similarly, once the CL has formed it also becomes a major

source of steroid synthesis with progesterone being themajor 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 accountsforonly a minor fraction ofthe total estrogen and progesteronein the systemic circulation. Without steroid synthesis and secretion

thecomplexand highlysynchronizedevents thatpermit reproduction could not occur.

PHYSIOLOGICAL RESPONSES TO ESTROGENS

Depending upon the mammalian species, there are several

forms of estrogens that are synthesizedand circulate

throughoutthe body. However, the most importantand 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 inhigh concentrations in primates during gestation.In general, estriol is considered the leastpotent of these three estrogenic forms (Hisaw, 1959). During the estrous or menstrual cycle, estrogens areresponsible for stimulating cellular proliferation and growth of thefemale

reproductive tract, regulation of utero-ovarianfunction and intrauterine environment, and development andmaintenance of

secondary sex characteristics. The following discussion will focus on the role of estrogens inregulating utero-ovarian

function and the intrauterine environment.

Estrogens act on the uterine myometrium toincrease both

thefrequencyandamplitudeofcontractions, which are requisite for sperm and accelerated ovum transport,and in

some species, migration andspacing of embryos (Pope et al., 1982). Estrogens initiate endometrial stromaledema (Astwood,

1938) as a result of increaseduterine capillary and venule permeability (Halkerston et al., 1960; Martin etal., 1973;

Espey, 1980). In response to estrogens, the glandsof the endometrium become increasingly tortuous as thenuclear RNA

polymerases and mRNA activate and facilitate protein synthesis, cell hypertrophy and cell hyperplasia. Estrogens

are also responsible for increasing uterineblood flow (Williams, 1948; Greiss and Anderson,1970),which has been

suggested to be a result of hormone-inducedvasodilation 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 productof 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 hormonessynthesized and secreted by the hypothalamus, pituitary, placenta and by the CLitself.

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 severalfactors, 8 namely, follicle maturation,selection and luteinization. Changes in steroidogenesis associated with folliculogenesis and luteal development are dependent initially, on the amount of folliclestimulating hormone (FSH) availabletothe maturing follicle, and later the ratio of FSH and luteinizing hormone (LH) during the preovulatory and luteal phase ofthe 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 lowdensity 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 vesiclecontaining

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 denovo from small

molecule precursorssuch as acetate. Thispathwayof synthesis is activated when cells are deprived of LDL or HDL.

Cholesterol is transported to the mitochondria where itis

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 thissteroid are secreted into the extracellular space to ultimately enterthe blood stream (Miller and Turner, 1963;Niswender and Nett,

1988) . Ovarian steroid synthesis and secretion isdynamically regulated by the anterior pituitary gonadotropins FSHand 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 andsecretion of steroids in the ovary by binding tohigh-affinity, low capacity plasma membrane receptors. The formation of the hormone-receptorcomplex activates the enzyme adenylate cyclase which in turn catalyzes the formationof 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 otherproteins necessary for the conversion of cholesterol into steroidsvia 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 basementmembrane of the follicle, and granulosa cells that lie alongthe inner surfaceofthe basement membrane. Theinteraction 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; Liuand 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 lipidsoluble androstenedioneand 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).

Bothof these binding globulinsarecomparatively high affinity, lowcapacity 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 causesdissociation 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 thesteroid 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 thehormone, 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 forthe

receptor. Steroid hormone receptors belong to a family of regulatory proteins whose ability to control geneexpression

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

hormone to its receptor stimulates the synthesis ofspecific proteins by altering the rate of transcription of specific

genes (Mueller et al., 1957; Norman andLitwack, 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 unoccupiedsteroid receptor was localized in the cytoplasmic fraction ofthe target cell

(Gorski et al., 1968; Jensen et al., 1968). Upon binding of the receptor to its specific hormone the extranuclearsteroid 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 radiolabeledsteroidthesteroid 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 theirextraction fromthe nucleusafter 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 changesin 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 proteinssuchasenzymes, 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 nuclearand 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, estrogenstimulates thesynthesis of both estrogenand 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 ormenstrual cycle, FSH and LH-induced maturationof the follicle increases the production and release ofestrogens into the blood (Karsch et al., 1979; Hansel, 1983). Just prior to ovulation high levels of estrogen positively feedbackto 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 pituitarygland. In the ewe, estradiol secretion begins to rise as the follicledevelops and matures.

An increase in estradiolproduction of 5 to 10-fold then occurs over the nextfew days. Estradiol peaks at the beginning of the preovulatory LH surgeand then rapidly declines to basallevels (Haugeretal., 1977). Some researchers suggest that a secondestradiol peak occurs in

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

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

peak of estrogen stems from renewedfollicular development in preparation for a new cycle. To date, the occurrence and

source of the secondestradiol peak remains equivocal. A

surge of LH, alongwith increased levels of FSH precipitate

ovulation. Initiation of the preovulatory LH surgeand subsequent ovulation requireshigh 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 surgeand ovulation. Subsequent to ovulation, estrogen secretion declines and LH then stimulates the transformation of the ruptured follicle into a CL asthe 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 pregnancymaintenance 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. Smallluteal 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 luteotropinfor 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 etal.,

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 ofthe estrous cycle. Rising levels of progesterone from the CL block the preovulatory LH surge by suppressing thefrequency of the GnRH pulse generator and thereby reduce the frequency and amplitude of LH secretory pulses from thepituitary gland

(Yenetal., 1972; Foster et al., 1975; Baird, 1978). Progesterone levels remain high until day 14-15 of thecycle, after which time they decline to basal levels 1-2 daysprior to the ensuing estrus as a new cycle isinitiated (Baird et al., 1976; Pant et al., 1977). Concomitant to declining estrogen and increasing progesterone levels during theluteal phase, an increase in the follicular protein hormoneinhibin

occurs. Inhibin feeds back at the level of the hypothalamus and pituitary inhibiting further FSH release (Schwartzand

Channing, 1977). Just as gonadotropins and steroid hormonesinteract 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 20 al., 1978). LUTEOLYSIS

In the absence of fertilization of an ovumfrom the ruptured follicle, many laboratory anddomestic animals will continue to exhibit estrous cycles. In most non-primate mammals such as the rat, guinea pig, ruminantsand sow, the uterus exerts a local luteolytic action on theipsilateral corpus luteum of the non-pregnantanimal 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 lutealmaintenance in the sow (du

Mesnil du Buisson, 1961), guineapig (Fischer, 1965), pseudopregnant rat (Barley et al., 1966) and ewe(Inskeep and

Butcher,1966). Upon completion of a previous cycleand initiation of a new cycle, the uterine endometriumsecretes

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 fromday 12 of the cycle, reaching maximum concentrations 1-2 days prior to the subsequentestrus

(Baird et al., 1976).This luteolytic factor enters the ovary by diffusing across the wall ofthe uterine vein into the

ovarian artery.The close anatomical proximity of theuterine

veins with the convoluted ovarian arterypermits the diffusion 21 of substances such as PGF2a directlyinto the ovarian artery by a countercurrent diffusionmechanism. They thus bypass the general circulation and avoidrapid destruction in the lungs

(McCracken et al., 1970). Once in the ovary PGF2a targets the progesterone secreting lutealcells of the corpus luteum to cause luteolysis (McCracken etal., 1970). The mechanism by which PGF2a exerts itsluteolytic action on the CL is not known. However, Niswender et al. (1975) suggest that the effects of PGF2a may be via a vascularcomponent because a significant decrease in capillary blood flowto the CL is associated with, but does not precedeluteolysis. Thus, it is doubtful that PGF2a causes lutealregression by interfering with blood flow to the CL. The protein hormone oxytocin iscurrently believed to also playan essentialrole in theeventsleading to luteolysis (Mitchell et al., 1975; Flint andSheldrick, 1986).

Uterine PGF2a exerts its effect in a local manner,targeting the ipsilateral ovary and thuscausing both inhibition of lutealprogesteronesecretionandstimulation 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 intramuscularinjection 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 releaseof more uterine PGF2a and the positive feedbackcycle 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 etal.,

1987) luteal tissue is maximal during the earlyluteal phase.

Stormshak et al. (1969) found that injection of exogenous estradiol into ewes on days 10-11 of the estrous cyclecaused premature luteal regression. It is believed that estradiol

alsoacts to increaseuterine PGF2cf secretionthereby promoting luteal regression (Ford et al., 1975). The mechanism involved in regression of theprimate CL

is not completely understood.Shutt et al. (1976) found that removal of the primate uterus did not prolong themaintenance

of the CL. It is believed that an intra-ovarianprostaglandin

may be involved, because the ovaryis a significant source of

PGF2cc in primates (Shutt et al., 1976). In primates, the reduction in progesterone secretion that occurswith luteal

regression precipitates atrophy of the endometrialepithelium

andinvasion of the endometrium bylymphocytes, causing stromal degradation, exposure of arterioles and mensus, or

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

With a decrease in both progesterone and ovariansecretion of

inhibin, GnRHand the gonadotropins FSH and LH are released

and a new menstrual cycle is initiated. 23

EARLY GESTATION

In order to ensure normal embryonic developmentand 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 steroidsas wellas hormones of maternal origin act upon the uterus andovaries to ensure maintenance of pregnancy.In particular, factors of conceptus origin are important for stimulation of vasodilationand angiogenesis, which are necessary in order to increaseuterine blood flow, stimulate transfer of nutrients into theuterine lumen, protect the conceptus from immunologicalrejection, and maintain luteal function.

MATERNAL RECOGNITION OF PREGNANCY

Moor and Rowson (1966b) found that the criticalperiod for maternal recognition of pregnancy in the ewe wasday 13 of gestation. Maternal recognition of pregnancy occurs when the presence of an embryo negates the luteolyticaction of the uterus on the CL prior to trophoblastic attachment(Short,

1969). It was determined that this antiluteolyticaction occurred locally, as demonstrated by the fact that aviable

conceptus, confined to one uterine horn was able tomaintain

only the ipsilateral corpus luteum in ewes (Moorand 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, mareand ewe, theCL isthe primary sourceof 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

suppressuterine PGF2a releasethereby 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 foundto be

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

interferon in fact bind to endometrial a-interferonreceptors

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

oTP-1 also plays an immunoregulatory orantiviral role during

gestation. It is conceivable that oTP-1 constitutesjust one complex, and that other conceptus secretoryproteins make up

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

1983). A functionally similar and structurallyrelated antiluteolytic bovine trophoblast protein (bTP-1)also exists

in cattle (Knickerbocker et al., 1986; Anthonyet al., 1988). Prostaglandins may also play a role in mediating the

local endometrial vascular response andtransformation of stromal to decidual cells, both of which are necessaryfor

embryonic development. Synthesis of prostaglandins by the endometrium, as well as by the blastocyst, has beenreported

for the mouse (Chepenik and Smith, 1980),rabbit (Harper et al., 1983), ewe (Hyland et al., 1982) andhuman (Tsang and

Ooi, 1982). UterinePGF2aas previously discussed, is responsible for luteal regression (Ford,1982)during the

estrous cycle. However, uterine and embryonicsecretion of prostaglandin E2 (PGE2) occurs during early gestation (Marcus,

1981;Silvia et al., 1984). Prostaglandin E2 (PGE2) is vasodilatory and non-luteolytic, and may beresponsible for 26 luteal resistance to the luteolytic effect of PGF2a.

ProstaglandinE2has been demonstrated to protect luteal tissue from the actions of PGF2cz both in vivoand in vitro. Treatment of ewes with PGE2 delayed lutealregression in nonpregnant ewes (Pratt et al., 1977), and increased progesterone secretion by large luteal cells(Silvia et al.,

1984). It has been suggested thatoTP-1 may induce endometrial synthesis and secretion of PGE2 and thusprevent

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, cowand ewe has a non-

invasive trophoblast and diffuse epitheliochorial

placentation,the focus of this discussion will bewith

respect to attachment rather thanimplantation of trophoblast

unless otherwise indicated. In ewes the blastocyst attaches

on day16 of gestation. Progesteroneis 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 themother is

lactating), estrogen has been found to be arequirement for

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

in these species requires an estradiol-primeduterus during

proestrus to up-regulate estrogen and progesteronereceptors 27 thus sensitizing the uterus to these hormones. This is followed by progesterone conditioning during thefirst 4 days of gestation in order to inhibitmyometrial contractility and embryo expulsion. A preimplantation estrogen peak occurs on day 4 to establish the endometrial vascularpermeability necessary forimplantation andnutrienttransport. As pregnancy progresses secretion ofprogesterone again increases and then remainsrelatively constantuntil 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 attachmentto occur if the proliferative effects of estrogens on theendometrium have alreadybeen achieved. In order for successful attachment to occur in the ewe, theintrauterine environment must be primed with estrogen secretedduring estrus. This steroid enhances endometrial and myometrialvascularization and proliferation of various cell typesin the uterus (Moor

and Rowson,1959; Bindon,1971; Miller and Moore, 1976). Progesteronecan function successfully if cellsof the reproductive organ have been exposed to estradiol,which induces the formation of progesterone receptors(Muldoon,

1981). Together with estradiol and the action ofanterior pituitary hormones on the CL, progesterone acts totransform

the uterine endometrium from aproliferative to a secretory

state. Subsequent to estrus, serumconcentrations of progesterone increase as the CL developsaccompanied 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 formigration 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 beendetected in rabbit, mouse, rat andhamster 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 ableto synthesize and metabolize steroids is complicated by thefact that some species (rat andmouse) requirematernal 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 andguinea pig.

Once established, the fetoplacental unitis able to metabolize and interconvert steroids, and thus possesses theability to potentially influence events suchas 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 changesin photoperiod are commonly referred to as seasonal breeders. Traditionally these animals have been referred to as long- or short-day breeders but inactuality these designations denote the hours of daylight towhich the animal is exposed. Changes in circannual photoperiod perceived by the animal are transduced into neuroendocrine signals that ultimately effect gonadalactivity. This photoperiod-induced regulation of reproductive activity ensures, at least in part, thatparturition occurs at a time when environmental factors are most beneficial topermit 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 secretionis 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 hoursof daylight are the shortest (fall and winter). In essence, the circadian rhythm of melatonin secretion synchronizes thecircannual rhythm of reproduction. The mechanism by which photoperiodicsignals regulate melatonin secretion involves various componentsof the central

nervous system.Light entering the eye stimulates theretina 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 thelateral hypothalamus to

synapse in the superiorcervical ganglia with post-ganglionic autonomic nerves that terminate in thepineal gland. The

stimulusevoked by lightsuppresses the release of a neurotransmitter (norepinephrine) in thepineal gland (Moore

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

al., 1984). Absence of light results in the release ofthis neurotransmitter, which then binds toB-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 inmostbreeds of sheep in thenorthern 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 LHsecretion with a resultant stimulation ofadditional estradiol production by the ovarian follicles. Thishypothalamic-hypophyseal-gonadal interrelationship ultimately leads to the preovulatory surge of LH secretion, which induces the firstovulation of the breeding season. In contrast, the pineal gland of long-dayphotoperiodic breeders such as the hamster and ferret, secreteslow levels of melatonin during the breeding season andhigh levels of the hormone during the anestrous period. In this case melatonin is inhibitory to the hypothalamicsecretion of GnRH and the subsequent secretion of gonadotropins,thus causing a decreasein ovarian steroidogenesis and inhibition of

ovulation. Melatonin infusions into male hamsters equally induced gonadal atrophy in both sham and SCN-lesionedanimals;

demonstrating that the SCN may not be an essential component

of the neural circuits that measure themelatonin signal in long-day photoperiod breeders (Maywood et al., 1990). Prolactin, another non-ovarian, seasonally regulated

hormone may also beinvolvedin mediating the pivotal neuroendocrine changes that occur during the breeding and non-

breeding season. In fact, secretion of melatonin andprolactin have been shown to be inversely relatedin 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 tobeinfluencedby photoperiodin goats, (Buttle, 1974), hamsters (Bex et al., 1978), heifers (Schams and Karg, 1974; Peters and Tucker, 1978) rats (Williams et al., 1978) and ewes (Posneretal., 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 changesin photoperiodit isconceivable that they are necessary components in regulating seasonalreproduction.

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 formilk productionandultimatesurvival of offspring.

However, prolactin mayalsoplayfarreachingand 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

interactionofprolactin with its specifictarget 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, thesequenceof 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 forcontinued progesterone secretion (Grota and Eik-Nes, 1967). It is believed that prolactin accomplishes this role byincreasing

LH receptor levels(Advis et al., 1981) as well as by preventing catabolism of newly synthesizedprogesterone (Advis et al., 1981).This process enhances the effectiveness of LH to stimulate the steroidogenicbiosynthetic pathway. In addition prolactin also increases theactivity of cholesterol esterase and3B-hydroxysteroid dehydrogenase,the enzyme required for converting pregnenolone toprogesterone. The increased levels of prolactin-stimulated progesterone secretion (Advisetal., 1981) in turn causesadown- regulationand inhibition of synthesis of progesterone receptors in the rabbit, hamster andmink (Djiane and Durand,

1977; Bex et al., 1978; Slayden, 1990, respectively). Prolactin has also been implicated instimulating progesterone secretion in non-rodent species (Advis et al., 1981;Slayden,

1990), however, the mechanism remainsunclear. 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 luteumin the ipsilateral ovary (Moor and Rowson, 1966a), suppresses thesynthesis 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 effectof theconceptus would belocalized or systemic. An understanding of how uterine steroid receptor concentrations vary between pregnant and nonpregnant animals may provide insightregardingtheroleof steroids in regulatingluteolysisduringthe estrouscycleand 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 conductedthat may explain theseembryonic 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 byintravenous injection of 2% thiamylal sodium and inhalation of a mixture of oxygenandhalothane. A midventrallaparotomy 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 laboratorywithin 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 byjugular venipuncture for subsequent analyses of serum concentrations of estrogen and progesterone. Ewes were relaparotomized and the remaining portion of the ipsilateral and theentire contralateral uterine horns were removed for analyses of endometrial estrogen and progesterone receptors. Pregnancy was verified by the presence of an embryoin 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°Cuntil assayed for concentrations of estrogen and progesterone.

Nuclear Estrogen Receptor Assay

Intercaruncular uterine endometrium was carefully dissected from thelumenof 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 andhomogenized with an all glass tissue grinder in 2 ml of ice cold TEbuffer (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 analysisof estrogen receptor concentrations. The remaining nuclear pellet was washed three times with cold TE buffer and thenresuspended with buffer to an equivalent of 50 mg of endometrium/ml.The nuclear fraction was vortexed and 1 ml aliquots werepipetted

into each of four tubes. Nuclear concentrations of estrogen receptor were determined by exchange andbinding assay as described by Koligian and Stormshak (1977a).The exchange was performed in duplicate using6,7[3H]- estradiol -17B (specific

activity 1.0 mCi/mmol, NENResearch 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. Sampleswereincubated for 30 minat 37°C. Following this exchange period, samples were placed on ice for

10 min to terminate the exchange process, andcentrifuged for

10 min at 1000 x g. Nuclear pellets were then washed four times with 2 ml of cold TE buffer to remove anyunbound

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 scintillationfluid

(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 werethen counted.

Cytosolic Estrogen Receptor Assay

The cytosolicsupernatant that was collected after homogenization and centrifugation of uterineendometrium was brought to a volume of 50 mg/ml withcold TE buffer. One-

hundred microliters of dextran-coated charcoal (0.5% dextran:5% charcoal) were added andincubated for 5 min to

remove any free ligand. The sample was centrifuged for 10 min at 1000 x g and the supernatant decantedinto a clean

tube. This procedure was repeated a second timeto ensure all charcoal had been removed. Onemilliliter aliquots were pipetted into each of four tubes to which were added617[3]fl-

estradio1-1713 at a concentration of 2 x104IM (total binding

tubes) or labeled estradio1-178 plus a100-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 hydroxylapatitein TE

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

min intermittent vortexing. Following incubation the 43 hydroxylapatite to which the estrogen-receptorcomplex was adsorbed was washed four-timeswith 2 ml of TE buffer and then subjected to extraction with 2 mlof redistilled absolute the ethanol at 4°C overnight. After centrifugation supernatant was decanted intoscintillation vials containing

10 ml of scintillationfluid as described above, and counted.

Nuclear Progesterone Receptor Assay

Aliquots of intercaruncularendometrium were homogenized in ice cold TEM buffer (10mMTris-HC1, 1mM Na2EDTA,12mM monothioglycerol, 30% v/v glycerol, 5mMNa molybdate, pH 7.4

at 25°C),and centrifuged at 1000 x gfor 10 min. The supernatant was decanted intotubes for assay of cytosolic

concentrations of progesterone receptors. The nuclear pellet

was washed four-timeswith TEM buffer and then resuspendedto a final volume of 100 mgendometrium/ml of buffer. Samples

were vortexed andaliquots of 1 ml were pipetted into eachof

four tubes. The nuclear exchange assayfor progesterone receptors was performed asdescribed by West et al. (1986). The exchange was carried outby adding[3Hjpromegestone

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

as R5020, specificactivity 1 mCi/mmol, NEN ResearchProducts,

Boston, MA)at a concentration of 1 x10-6M to the total

binding tubes. The syntheticglucocorticoid dexamethasone was

added (2 x10-4M) to prevent any progesterone from bindingto

the glucocorticoid receptors. Similarly the labeled ligand 44 and dexamethasone were added tonon-specific binding tubes as describedabove with an additional 100-foldexcess of unlabeled promegestone. Samples were incubated overnight at

4°C to complete the exchange process.After completion of the exchange, the nuclear pellet was washedfour-times with TEM buffer and extracted with 2 ml ofabsolute ethanol at 4°C

overnight. Following extraction, the supernatant wasdecanted into scintillation vials containing 10 ml ofscintillation

fluid and counted.

Cytosolic Progesterone Receptor Assay

After homogenization and centrifugation, thecytosolic supernatant was also subjected to a progesteronereceptor

exchange assay.The supernatant was brought to a final volume of 100 mg equivalent of endometrium/ml of TEMbuffer. 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 thatall 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 forthe 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 intervalsduring a 10 min incubationperiod andthen 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 theendometrial tissue were measured using the proceduresdescribed by Burton

(1956). In brief, the resultant nuclear pelletfrom the exchange assay was stored at 4°C in 0.5Mperchloric 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 5mMsodium hydroxide solution), were pipetted (100 Al) induplicate. Bovine serum albumin (1 ml of a 0.1% solution) and 1 ml of 0.5M PCA wasthen added to the standard tubes to precipitate theprotein and DNA. All tubes were centrifuged at 1000 x g for 10min after 1 h of 4°C

incubation andtheresultantsupernatant wasdiscarded. Perchloric acid was added to all tubes (2 ml of 0.5MPCA) which were then capped with marbles and placedin a 90°C water-bath for 30 min in order to hydrolyze the DNA. Tubes

werethen 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 mlof diphenylamine reagent(1.5 g diphenylamine dissolved in 1.5 ml of 96% concentrated sulfuric acidand 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

Serumconcentrations of progesteroneweremeasured according to procedures describedby 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 competitor1,2,6,7[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 freeligand. Samples were then centrifuged at 1700 x g for 10min. The

resultant supernatant was decanted intoscintillation vials

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

tube (100Al). All serum samples for the progesterone radioimmunoassay were analyzed in a single assay wherethe

intraassay coefficient of variation was 8.7 ± 0.37%and the

extraction efficiency was 91.5 ± 3.8%.

Serum Estradiol Radioimmunoassay

Serumestradiol wasmeasured usingthe extraction procedures described by Hotchkiss et al. (1971) and the radioimmunoassay described by Butcher (1977). Briefly, 200 Al of sample serum were pipetted intriplicate into 10 ml

centrifuge tubes with screw caps. Every third tube received

100 Al of2,4,6,7(3H]- estradiol -178 (diluted to 3500 cpm/200

Al in 0.1% GPBS, NEN) fordetermination of extraction

efficiency. Anesthetic grade anhydrous diethyl ether (3 ml) was added and samples werevortexed for 30 sec. In order to

ensure complete separation oforganic solvent from the serum, samples were centrifuged at 1000 x g for 10min. The sample

tubes were then snap frozen in a dryice and ethanol bath.

The resultant ether layer was decantedinto 12 x 75 mm tubes and evaporated to dryness under lightair in a 37°C water

bath. The frozen pellets from the original tubes wereallowed 48 to thaw and the above procedure wasrepeated. Sample tubes containing the dried extractant were then storedat 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 Alredistilled absolute methanol were pipetted in duplicateand dried under light air in a 37°C water-bath while extractant tubes wereallowed to come to room temperature. To all except blank tubes,100 Al of 0.3% gelatin phosphate buffered saline (GPBS) wasadded [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 15min 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 1:10,000in

0.1% GPBS). In addition, 200 Al of2,4,6,7[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 placedin 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 10min and the resultant supernatant was decanted into vialscontaining 10 ml of 49 scintillation fluid and counted. All serum samples for estradiol radioimmunoassay were analyzedin a single assay, with a sensitivity of2.3 pg/tube. The coefficient of variation was 10 ± 0.43% and extractionefficiency was 95 ±

2%.

Statistical Analysis

Data on nuclear and cytosolicconcentrations of estrogen and progesterone receptors were subjected to log transformation because of heterogeneity ofvariances. The transformed data were analyzed by least squaresanalysis of variance for an experiment of completelyrandomized block, repeated measures design. Differences among meanswere tested

for significance by use of Studentst-test. For purposes of presentation, data are presented asnontransformed means.

RESULTS

Endometrial nuclear concentrations of estrogenreceptor

in uterine horns of ewes on day 10 ofthe estrous cycle were significantly greater (1.21 ± 0.22 fmol/AgDNA, p<.01) than

in the pregnant and nonpregnanthorns 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 inendometrium on day 10 of the estrous cycle were significantly greater(4.90 ± 0.56 fmol/Ag DNA,

p<.01) than in endometrium ofpregnant and nonpregnant horns 50 of ewes on day 18 of gestation (1.25 ± 0.58and 1.10 ± 0.64 fmol/gg DNA, respectively).

Nuclearconcentrations of progesteronereceptor in endometrium of ewes on day 10 of the estrouscycle were significantly higher (0.63 ± 0.21fmol/AgDNA) than in endometrium of pregnant and nonpregnantuterine horns of ewes on day 18 of gestation(0.18 ± 0.21, p<.05 and 0.13 ± 0.21 fmol/Ag DNA, p<.01respectively; Figure 2). Cytosolic concentrations of progesterone receptor on day 10of the estrous cycle were determined to be 1.14± 1.03 fmol/Ag DNA.

Data on cytosolic concentrations ofprogesterone receptor in

both the pregnant and non-pregnant hornsof ewes on day 18 of gestation were invalidated by excessivenonspecific binding

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

estradiol -1713 onday 18 of gestation as determinedby

radioimmunoassay were 16.56 ± 2.43 pg/ml. Meanserum concentration of progesterone on day 18 ofgestation was 1.74

± 0.57 ng/ml. 51 INNNUCLEAR CYTOSOL

6.00

4.80 ,5 ? 3.60

2.40

7 N 120

0.00 d 10 OF CYCLE d18 PREG.HORN d 18 NPREG.HOFts1 Fig. 1. Specific binding of [ flestradiol- 178 to nuclear and cytosolic estrogen receptors (mean ± SE) in endometria of ewes on day 10 of the estrous cycle and in endometria of gravid and nongravid uterine horns of ewes on day 18 of gestation. Mean nuclear and cytosolic concentrations of estrogen receptors, day 10 vs day 18 gravid and nongravid horns differ (p<.05).

1.00

0.80

3.1

0.60

0.40 0 0 0.20 11. A

0.00 d10 OF CYCLE d18 PREGHORN d18 NPFEG.HORN Fig. 2. Specifically bound[ 4]115020 to nuclear progesterone receptors (mean ± SE) in endometria of ewes on day 10 of the estrous cycle and in endometria of the gravid and nongravid uterine horns of ewes on day 18 of gestation. Day 10vs day18 gravid and nongravid horns differ in concentrations of progesterone receptors (p<.05). 52

DISCUSSION

Results of Exp. 1 indicate that theendometrium of the nongravid horn of ewes on day 10 of the estrouscycle contains greater concentrations of nuclear andcytosolic estrogen receptors than the endometrium of gravidand nongravid horns of ewes on day 18 of gestation. Similiarly, endometrial nuclear concentrations of progesteronereceptor were greatest on day 10 of the cycle thanin gravid and nongravid uterine horns of ewes on day 18 ofgestation. These changes in uterine estrogen and progesterone receptor areconsistent with the reported ability of progesteroneto down-regulate the estrogen receptor as well as its ownreceptor (Koligian and Stormshak, 1977a; Selcer and Leavitt, 1988). The greater concentration of these receptors on day 10 ofthe cycle most likely reflects the gradual change in theconcentrations of these receptorsthat occurs from the time of estrus. Endometrial concentrations of nuclear andcytosolic estrogen receptors have been reported to bemaximal at estrus and to decrease during the luteal phase ofthe cycle of the ewe

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

(Zelinski et al., 1982) as progesteronesecretion increases.

Few data are available regardinguterine concentrations of

steroidreceptorsduringearlygestation in ruminants.

However, Senior (1975) reported low levels of uterine cytosolic estradiol binding sites on day 20of gestation in 53 cows. Continued production of progesterone by the corpus luteum whose maintenance is ensured by the conceptus, aswell as low production ofovarian estrogen may be surmised to be responsible for the reduced concentrations of progesteroneand 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 mostlikely represent the minimal levels of these receptors necessaryto ensure basal function of the uterus. Logically, greater concentrations of receptor than detected during early gestation, especially for estrogen, would not beconsistent with the ability of the uterus to remain in acomparatively quiescent state necessary for maintenance of pregnancy. It is possible that some other factor (perhaps ofconceptus origin) besides progesterone may also operate to suppressthe uterine concentrations of steroid receptors. Thispossibility is supported in part by the observation thatendometrial concentrations of nuclear and cytosolic estrogen and nuclear progesterone receptors did not differ between pregnantand nonpregnant uterine horns on day 18 of gestation. These latter data indicate that the release of a conceptussecretory factor may influence uterine concentrations of estrogenand progesterone receptors in a systemic rather than local manner.

This is further supportedby the fact thatvascular connections between horns were blocked by a ligature atthe external bifurcation. 54 Unfortunately, quantification of levels of cytosolic progesterone receptor concentrations on day 18 ofgestation, as determined by receptor exchange assay, wasconfounded by extremely high nonspecific binding. One explanation for this phenomenon may be the increase in systemic bindingproteins 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.

Itis 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 thanduring the midluteal phase of

the estrous cyclewhenprogesterone concentrations are

maximal. Additionally, results of this experiment can be interpreted as suggesting a systemic effect of the conceptus

on uterine concentrations of estrogen andprogesterone

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 ofdaylight in the fall results in increased duration of melatonin secretionduring 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 prolactinin

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 canalterthe 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.

Thisexperiment wasconductedtodetermine whether concentrations of estrogen receptor in control andestradiol- stimulated uteri of ovariectomized ewes differ betweenfall and summer. A detected difference in this uterine characteristic would suggest the possibility thateither melatonin or prolactin was acting either directly or indirectly to alter uterine function.

MATERIALS AND METHODS

Animals

Ten mature Polypay ewes were checkedtwice daily for estrus to ensure all ewes exhibited an estrous cycleof normal duration. Ewes were then bilaterally ovariectomizedin October and allowed 4-6 weeks for recovery. At the height of the winter solstice, a 10 ml blood sample wascollected by jugular venipuncture from each of five ewes for analysesof serum concentrations of estrogen andprogesterone. These five ewes were then anesthetized and amidventral 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 externalbifurcation and estradiol- 57

1713 (10 gg/3ml of physiological saline) wasinjected into the lumen of the contralateral horn to stimulatesynthesis 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 asection of the stimulated uterine horn was removed and placed on ice. Endometrium (50 mg) was assayed within 90 min of removal forconcentrations of estrogen receptors using the estrogen nuclear andcytosolic 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 thewinter 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 inexperiment 1.

DNA Determination

Deoxyribonucleic acid concentration in the endometrial tissue was measured as described in experiment 1. 58

Serum Progesterone and EstradiolRadioimmunoassay

Systemic concentrations of progesterone andestradiol-

1713 were measured as described in experiment 1.

Statistical Analysis

Data on endometrial nuclear and cytosolicconcentrations were analyzed by analysis ofvariance 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 afterestradiol

treatment at the winter and summer solstice aredepicted in

figure 3. Nuclear concentrations of estrogen receptor in the endometrium ofchronically ovariectomized ewes priorto exogenous estradiol stimulationduring 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

vs0.46 ± 0.07fmol/Ag DNA, respectively). Similarly, cytosolic concentrations of estrogen receptor inendometrium

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 thewinterand 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 solsticeand summer solstice did not differ significantly (16.55 ± 4.05 vs 16.00

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

serum levelsofestradiol 48h 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 estrogen receptors (mean ±SE) in nonstimulated and exogenous estradiol-stimulated endometria of ewes 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 aslong 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 bymelatonin has also been shown to occur in human breast cancercells (Danforth and

Lippman, 1981). Chilton and Daniel (1987) demonstratedthat prolactin treatment of ovariectomizedrabbits resulted in an increase in uterine concentrations of estrogen and progesterone receptors similar tothose detected inthe sexually receptive intact rabbit. Serum concentrations of estradiol both prior to and afterestradiol treatment were not different from one another indicatingthat treatment with estradiol did not increase systemic concentrations of estradiol and therefore did not likelyprovoke a release of prolactin. Serum concentrations of progesteronedetected in the ovariectomized ewes undoubtedlyreflect steroid secreted from the adrenal cortex. Because there was no significant difference in serum progesteroneconcentrations among ewes within season(0 vs 10 Ag estradiol)or between seasons (winter vs summer) it is unlikely that anyslight variation in progesterone secretion could haveaccounted for failure to detect differences in endometrial estrogen receptor concentrations. These data indicate that the mechanism by which seasonality regulates the breeding seasonis via an indirect

route through the ovary. The secretion of estrogen from the ovary acts on theuterine endometrium to stimulate the

synthesis of estrogen and progesteronereceptors. It appears that melatonin secreted in greaterquantities during the 63 breeding season of the ewe is acting at thelevel of the hypothalamus to regulate release of GnRHand subsequently pituitary LH. Likewise, increaseddailysecretion of prolactin during summer anestrus does not appearto act directly on the uterus to influence synthesis or concentrations of estrogen receptors and therebypromote this reproductive state. Collectively, these data indicate that neither prolactin nor melatonin act directly onthe uterine endometrium to cause fluctuations in the synthesisof estrogen receptors in the ovariectomized ewe. 64

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