PHOTOPERIODIC REGULATION OF THE FEMALE REPRODUCTIVE SYSTEM IN THE SYRIAN HAMSTER (OVARY, PINEAL, HORMONES, PITUITARY).

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Hauser, Ursula Esther

PHOTOPERIODIC REGULATION OF THE FEMALE REPRODUCTIVE SYSTEM IN THE SYRIAN HAMSTER

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University Microfilms International

PHOTOPERIODIC REGULATION OF THE FEMALE REPRODUCTIVE SYSTEM IN THE SYRIAN HAMSTER

by Ursula Esther Hauser

A Dissertation Submitted to the Faculty of the DEPARTMENT OF ANATOMY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA

1 986

Copyright 1986 Ursula Esther Hauser THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have read the dissertation prepared by ------Ursula Esther Hauser entitled Photoperiodic Regulation of the Female Reproductive System in the Syrian Hamster

and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy ------~~------

d I 9-' 0, 118':...t

Date ; l 7PI/rr{.. Date

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's sUbmission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

D~~r~on Director Date~ STATEMENT BY AUTHOR

This dissertation has been subm·itted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allow­ able without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder.

SIGNED: t.L.rS~ e '#0' ~~ ACKNOWLEDGEMENTS

I would like to express my deep gratitude to my advisor, Dr. Bryant Benson. His guidance, great patience and generosity made my graduate education a very special and invaluable experience. I also would like to thank Dr. Philip H. Krutzsch, Dr. David E. Blask and Dr. Christopher A. Leadem for their service on my committee, individual contributions to my education as a researcher, and last but not least, for their friendship, which I cherish and which helped me greatly in the persuit of my goal. Finally, I would like to thank the many members of the Department of Anatomy who contributed to my education, provided technical assistance, or in other ways made my life as a graduate student more enjoyable.

iii

... - ...__ ... _.•. _------_._-_._._...... - TABLE OF CONTENTS

Page LIST OF .I LLUSTRATI ONS . vii LIST OF TABLES ...... xi ABSTRACT ...... • xii 1. INTRODUCTION AND BACKGROUND. 1 Origin of the Hamster as an Experimental Animal 1 The Female Reproductive System • ...... 3 Estrous Cycle •.••.•.•••.••. 3 Follicular Development and Steroidogenesis 7 Follicular Atresia • • • • • • • • • • 10 Interstitium . . • • . . . • . • . . . .. 12 Neural Control of the Female Reproductive System . • . • • • . • . . • • . . . • .. 13 Neuroregulation of Gonadotropin Secretion. 13 Gonadotropin Regulation in the Female Hamster • . • • . • •. ••••.. 21 Control of Gonadotropins at the Pituitary Level...... 25 Regulation of Prolactin • . • • • • • . • . 28 Suprachiasmatic Nucleus and Circadian Rhythms • . • . . . • . . • • . . . 32 Seasonal Breeding and the Pineal Gland . . . . 37 PhotoperioG and the Female Hamster • • •• 41 2. STATEMENT OF THE PROBLEM AND SPECIFIC AIMS 47 3. EXPERIMENT I: A HISTOCHEMICAL STUDY OF THE SITES OF STEROID SYNTHESID IN BLINDED, FEMALE HAMSTERS •.. 49 Introduction • • • • • • • . • 49 Materials and Methods. . • • . . . . • •. 50 Observations ••• . • . • • . • . . . .. 52 Discussion . . • • . . • • • • • • • . 55 Pl ates . . . • • . . • • • . . •• 59

iy v

TABLE OF CONTENTS--Continued

Page 4. EXPERIMENT II: RAPID CESSATION OF ESTROUS CYCLICITY AND DEPRESSED CASTRATION RESPONSE IN SHORT-PHOTOPERIOD TREATED INBRED LSH/SsLak HAMSTERS. 63 Introduction ..•••••••. 63 Materials and Methods 63 Results ••••••. 66 Discussion ....•..•. 67 Figures ••...•...... 73 5. EXPERIMENT III: EARLY SHORT PHOTOPERIOD EFFECTS IN FEMALE LSH/SsLak GOLDEN SYRIAN HAMSTERS • • . • . . • • • . . 80 Introduction .•.... • . . . . •• 80 Materials and Methods. • • • • • • • . 81 Results. . . . • . . • • . 83 Discussion .••••• .•• ••. 85 Table 1...... 92 Figures . • . . • • . . . • . . 93 6. EXPERIMENT IV: HORMONAL CHANGES PRECEEDING THE SHORT PHOTOPERIOD-INDUCED ANOVULATORY CONDITION IN LSH/SsLak HAMSTERS . . • . • • . . • • . 101 Introduction .•. •. 101 Materials and Methods. • • • • • • • • 102 Results ..• • .•• . . . • • • 106 Discussion •...... 110 Table 1...... • 117 Figures ...••••.•• · 118 7. EXPERIMENT V: SHORT PHOTOPERIOD DEPRESSES CASTRATION RESPONSE AND MAY ALTER STEROID FEEDBACK SENSITIVITY IN FEMALE LSH/SsLak HAMSTERS .•. 131 Introduction •••••. · 131 Materials and Methods. • 132 vi

TABLE OF CONTENTS--Continued

Page Results ...... • 134 .Discussion ••• • 136 Table 1. .... • • • • 141 Figures •••••••••••••• • • • . 142 8. SUMMARY DISCUSSION . . . . • 148 9. REFERENCES 164 LIST OF ILLUSTRATIONS

Page Experiment I. Plate 1. NADH Diphorase Activity in the Ovary of a Control Hamster on Diestrus II •• . . . 59 2. Glucose-6-Phosphate-Dehydrogenase Activity as Seen on Proestrus in a Control Ovary • 59 3. 3-Beta-Hydroxysteroid Dehydrogenase Activity Observed on Proestrus in the Ovary of a Control Animal .••• •.••••• 60 4. NADH Diaphorase Activity in the Ovary of a Blinded Acyclic Animal •••••••. 60

5. Glucose-6-Phosphate-Dehydrogenase A~tivity Seen in the Ovary of a Blind Animal .. 61 6. 3-Beta-Hydroxysteroid Dehydrogenase Activity Seen in the Ovary of a Blind Hamster 61 7. Hand E Section of an Ovary from an Animal on Proestrus Day. • . . • • • • • • . • . . . • 62 8. Hand E Section from the Ovary of a Blinded Animal ...•.....•••.. 62 Experiment II. Figure 1. Effects of SP on Estrous Activity ••••. 73 2. Serum LH Profiles of SP- and LP- Exposed Ovariectomized Animals During the First and Second Postcannulation Day •••••••• 74 3. Pituitary LH Levels in SP- and LP-Treated Ovariectomized Animals •.•..••••• 75 vii viii

LIST OF ILLUSTRATIONS--Continued

Page 4. Serum FSH Patterns in LP- and SP-Treated Ovariectomized Hamsters Over the First and Second Postcannulation Day ••• 76 5. Pituitary FSH Levels in LP- Versus SP- Ovariectomized Treated Hamsters •.•••.• 77 6. Pituitary PRL Content in LP- and SP­ Treated Ovariectomized Animals • • • • 78 7. Serum PRL Levels in SP- and LP-Treated Ovariectomized Animals Measured at the Time of Sacrifice On the Third Post- cannulation Day ...•.....••.... 79 Exper iment II I. Figure 1. Serum PRL Levels of LP and SP-Treated Cycling Animals and SP-Induced Acyclic Animals are Shown • • • • •••••••••• '. • • . • • 93 2. Pituitary PRL Levels are Shown for LP Controls, SP-Treated Cyclic and Acyclic Animals •.•• 94 3. Secondary FSH Surge Levels of LP- and SP- Treated Cycling Animals and Levels Seen in Acyclic Animals ...••..••• . 95 4. Pituitary FSH Levels are Shown for LP Controls, SP Cyclic and Acyclic Animals ••••.•.• 96 5. Serum LH Levels in LP and SP-Treated Cycling and SP-Induced Acyclic Animals •.•. 97 6. Pituitary LH Levels are Depicted for LP and SP Cycling and SP-Induced Acyclic Animals .•. 98 7. Pituitary Weights of LP and SP-Cycling and SP­ Treated Acyclic Animals. . . • . • . • • . • 99 ix

LIST OF ILLUSTRATIONS--Continued

Page 8. uterine Weights in LP and SP-Cycling and SP Acyclic Animals. . ••••..•• 100 Experiment IV. Figure 1. Effect of SP Treatment on Morning Serum LH Levels on Each Day of the Estrous Cycle in SP­ Treated Cyclic (Left) and Acyclic (Right) Animals ••.•....••••••..... 118 2. Effect of SP Treatment on Pituitary LH Content on Each Day of the Estrous Cycle in Acyclic Animals ••••••..•• •. 119 3. SP Effects on Serum FSH Levels in Cycling and Acyclic Animals. . . • . . . • • .• .. 120 4. Effect of SP Treatment on Pituitary FSH Content. 121 5. Serum PRL Levels in LP- and SP-Treated Cycling Animals ...... ••••••...... 122 6. Pituitary PRL Levels in LP-Treated Cyclic and SP-Treated Cyclic and Acyclic Animals.. 123 7. Correlation Between Pituitary PRL Contents and Number of Day~ After Last Observed Ovulation 124 8. Relationship Between Pituitary Weights and Pituitary PRL Contents ..••••.• • 125 9. Close Correlation Between Pituitary Weights and Days an Animal is Anovulatory. • • •• . 126 10. The Effect of SP Treatment on Serum Progesterone Levels in SP-Cyclic and SP-Acyclic Animals . 127 11. Ovarian Estrogen Contents in LP-Treated, SP- Cyclic and SP-Acyclic Animals ..••.•.• 128

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LIST OF ILLUSTRATIONS--Continued---.-- Page 12. SP Effects on MBH LHRH Content of LP- and SP­ Treated Cycling Animals and SP-Treated Acyclic Hamsters ••••••• . •• •. 129 13. Effect of SP on POA LHRH Concentration in Animals Displaying Estrous Cycles and SP-Treated Acyclic Animals ••••.• 130 Experiment v. Figure 1. The Effect of SP on Serum LH Levels in Ovariectomized Untreated (Controls) and EB­ Treated Animals in Exp. I (I), and 17-Beta­ Estradiol (E-2) Treated and Control Animals in EXp. II (II) ...... • 142 2. SP Effects on Pituitary LH Content in Controls, EB and E-2 Treated Hamsters • . . • • . . . . 143 3. The Effects of SP Treatment on Serum FSH Levels in Controls, EB and E-2 Treated Hamsters •• 144 4. The Effects of SP on Pituitary FSH Contents in Controls, EB and E-2 Treated Hamsters .. 145 5. SP Effects on Serum PRL Levels in Controls, EB and E-2 Treated Animals are Depicted ...• 146 6. The Effects of SP Exposure on Pituitary PRL Content in EB, E-2 Treated and Control Animals are Represented •.•.••••... 147 LIST OF TABLES

Page Exper iment II 1. Table 1. Quantitation of Ovarian Changes in SP-Exposed Female Hamsters . . . • • . . • . . . . . 92 Experiment IV. Table 1. Short Photoperiod Effects on Organ Weights in LSH/SsLak Female Hamsters •.•••...•• 117 Experiment V. Table 1. Short Photoperiod Effects on Organ Weights in Ovariectomized Hamsters ••.••.••.•• 141

xi

------_._---_._------_. • ABSTRACT

Female golden Syrian hamsters are seasonally breeding animals, capable of maintaining continous e~trous cycles when the daylength is 12.5 hrs. or longer. In shorter photoperiod (SP) the ovaries of anovulatory animals are chara.. C't'e-rized by few small growing follicles, an absence of corpora lutea and extensive hypertrophied interstitium.

Ste~oid-histochemical studies revealed that enzymes related to steroidogenesis show intense activity in the interstitial tissue of SP-exposed animals. The major objectives of these studies were to examine SP-induced hormonal and ovarian changes which occur prior to onset of the acyclic condition in inbred LSH/SsLak hamsters. Other experiments explored hormonal changes in the absence of ovarian hormones and the interaction of SP and steroids. Initial results revealed that the LSH/SsLak hamster ceased

estrous cyclicity between 14 and 31 days of SP exposure t a response far more uniform than generally seen in outbred hamsters. Experiments carried out in SP-exposed cyclic animals indicated that the secondary FSH surge and follicular recruitment were not affected by SP treatment, and that no major changes in gonadotropin levels and ovarian steroids were present on individual days of the estrous cycle. Once the animals were anestrous, daily gonadotropin xi i xiii

surges were present and pituitary gonadotropin contents increased. Serum PRL levels showed a slight, yet significant, decrease in SP cycling animals followed by a further reduction in pituitary and serum levels after animals ceased cycling. Medial basal hypothalamic LHRH contents did change in SP, yet there was a significant increase in the preoptic area, and LHRH became significantly elevated in both areas after the animals became anestrous. Ovarian histology revealed fewer corpora lutea and a slight shift from healthy to atretic antral follicles. Experiments carried out in ovariectomized SP-treated animals showed that serum gonadotropin levels were significantly reduced, and that estrogen treatment was either equally or less effective in reducing levels in SP animals. In contrast, PRL levels did not change and responded in a dose dependent way to estrogen treatment. Although the studies yielded no definite proof, the result suggest that SP impairs the maintenance of follicular growth leading eventually to the acyclic state. CHAPTER 1

INTRODUCTION AND BACKGROUND

Origin of the Hamster as an Experimental Animal According to the review by Murphy (1985) the Golden Syrian hamster was first described in 1797 by the physician and naturalist Alexander Russel in the second edition of The Natural History of Aleppo. Being unaware that this species was different from the "common" European hamster, Russel did not claim to have discovered a new species. In April 1839, George Robert Waterhouse, who at that time was curator of the London Zoological Society, presented the new species he had received from Aleppo at the Society·s meeting and named it Cricetus auratus Waterhouse (the genus name Mesocricetus was a later modification). Ninety-one years later, Saul Adler a parasitologist at the Hebrew University of Jerusalem became interested in the Syrian hamster for practical reasons. In his research on the prevalent disease leishmaniasis, he had used the Chinese hamster as an animal model. Since he was unable to breed this species in his laboratory, he had to rely on shipments from China, a situation which became more and more 2 unacceptable. These difficulties led him to request that zoologist Israel Aharoni look for the Syrian hamster on one of his extensive field trips. The request led to a special expedition in April, 1930 on which, after several hours of digging by a few laborers hired by the local sheik and partial destruction of a wheat field, one complete hamster nest containing a mother and eleven young was raised from a depth of eight feet. Disturbed, the mother started to attack her young, but she was destroyed and the young were saved. The young, measuring less than 2.5 cm, painstakingly raised by Aharoni and his wife, were given to Heim Ben-Menachem, the founder and head of the Hebrew University and of the animal facilities. Placed in a cage with a wooden floor, the animals chewed their way out, and only three females and one male were recaptured. According to recorded history, Ben-Menachen who was known as a tough man and freedom fighter, was completely devastated by this event. These remaining four animals soon made up for the loss, and within one year the colony multiplied to 150 animals. Eight years after the initial capture, golden Syrian hamsters were imported to the United Ste.·tesfar pUY'poses of'resea'r'ch,b'u't also soon became favorite animals in pet stores. Over the last two decades the Syrian hamster has been used in a wide variety of experiments and has become a special model in studies designed to yield information about the physiological role of the pineal gland. Experiments 3

concerning the pineal have been especially productive and have led to the currently well-accepted role of this gland as a mediator of environmental stimuli on the endocrine system, especially the effects of photoperiod on reproduction. Male hamsters have been used extensively in these studies and are better understood,whereas females have been the subject of less experimental concern. Nevertheless, the following summary considers the information known regarding the female reproductive events.

The Female Reproductive System

A. The Estrous Cycle. Adult female hamsters display regular four-day ovulatory cycles, easily monitored by observing the conspi­ cuous, opaque, and viscous vaginal discharge on the morning of estrus, often referred to as day one; ovulation has taken place on this day. Five-day may cycles occur occasionally, but are, in fact, an exception to the rule. The major events taking place each day of the estrous cycle are as follows: 1. Estrus (Day One). Antral follicles which matured in the previous cycle ovulate between midnight and approxi­ mately 0200 h. The secondary (estrus) FSH surge which began on the evening of proestrus, reaches its highest levels between 0400 and 0800 h (peak values within the same range as the proestrous surge) and drops sharply until about 1600 h 4

(Bast and Greenwald, 1974; Bex and Goldman, 1975). In response to the FSH surge, ten to twelve preantral follicles

(250 ~m in diameter) with six ~o seven layers of granulosa cells are recruited in each ovary (Greenwald, 1961, 1974; Greenwald and Siegel, 1982). By the completion of the cycle, half of these commited follicles will either ovulate or undergo atresia. The cells of the newly formed corpora lutea start to luteinize and begin to synthesize progesterone (Leavitt and Blaha, 1970; Lukaszewska and Greenwald, 1970). 2. Diestrus I (Day Two). The recruited follicles

(267 - 392 ~m in diameter), begin to form antral cavities and start to synthesize estrogen (Baranczuk and Greenwald, 1973; Saidapur and Greenwald, 1978a,b). The corpora lutea are enlarged, fully luteinized, and progesterone synthesis is at its peak. The large size of the corpora lutea is due to granulosa cell hypertrophy, with cell division occurring only in the newly formed vasculature (Chatterjee and Greenwald, 1976). Between diestrus I and II (cycle day two and three respectively) corpora lutea begin to regress, resulting in a

luteal-follicular.~ or a shift from predomi~antely proges­ terone to estrogen synthesis and accelerated follicular growth (Terranova and Greenwald, 1978). 3. Diestrus II (Day Three). All recruited follicles have reached the antral stage and measure between 393 and 476

~m. Progesterone synthesis by corpora lutea is greatly diminished and these structures start to show signs of

_._ ...... _- ... - ..... _. - ._ ... _--_ ... _. __ .. _.- ...... - --.- ._---_. . -- ._ ... __ .------._------.- - 5

involution. At this time the antral follicles are well developed and synthesize high amounts of estrogen. 4. Proestrus (Day Four) Morning. Between diestrus two and proestrus, approximately half of the antral follicles undergo atretic changes (Greenwald, 1961). An average of seven healthy large antral follicles, measuring between 477 and 560 ~m in diameter, remain on the morning of proestrus and will ovulate in response to the proestrous surge. Corpora lutea are in an advanced state of involution and unable to synthesize progesterone (Leavitt et al., 1973; Saidapur and Greenwald, 1978a); antral follicles continue to synthesize estrogen. 5. Proestrus Afternoon. The timing of the proestrous surge is closely related to the light schedule. If lights are on between 0600 and 2000 h, the peak gonadotropin elevation occurs around 1600 h and will last for about three hours (Turgeon and Greenwald, 1972). During the surge, LH levels will increase about ten to twenty fold while FSH levels are elevated five fold (Bex and Goldman, 1975). The proestrous gonadotropin peak results in a rise of estrogen and testosterone levels which lasts for about one hour and is followed by a switch to mainly progesterone synthesis (Saidapur and Greenwald, 1979a; Terranova and Garza, 1983). The inhibitory effects on estrogen and androgen synthesis appear to be mediated by LH-stimulated progesterone pro­ duction (Saidapur and Greenwald, 1979b); the latter 6 originates largely from the interstitial tissue, although there is a small contribution from antral theca and granulosa cells (Lukaszewska and Greenwald, 1970). LH levels remain at baseline throughout the cycle except during the proestrous surge. FSH levels rise during the proestrous surge, decrease to baseline during the evening hours and then rise once again for the extended secondary surge which lasts well into the day of estrus (Bex and Goldman, 1975; Bast and Greenwald, 1974). On diestrus I, FSH levels are elevated about two-fold over baseline seen on diestrus II and proestrus. PRL levels display a daily rhythm, highest during the afternoon hours and lowest during the dark period. On day four, a surge in PRL can be observed coincident with the proestrous gonadotropin surge (Talamantes et a1., 1984). The hamster estrous cycle differs from that observed in rats in many respects, of which only some of the more obvious differences will be mentioned. In the rat, corpora 1utea remain functional over several cycles, whereas in the hamster corpora 1utea start to involute during diestrus II, and are practically non-functional on proestrus. These short life spans are responsible for the precisely timed four day cycle. Another obvious difference is the duration and timing of the proestrous surge. In the rat, this surge lasts for about six hours, whereas in the hamster one observes a sharp peak lasting for only two to three hours. In addition, the 7 secondary FSH surge of the rat is not clearly separated from that of the proestrous peak, a feature typical of the hamster.

B. Follicular Development and Steroidogenesis Very little is known about early follicular development and initiation of growth from the primordial pool. Gonadotropin and estrogen levels do not seem to affect growth initiation (Peters, Byskov and Faber, 1973), yet the size of the pool of primordial follicles has an apparent influence, i.e. the larger the pool size, the more follicles start to develop (Krohn, 1967; Krarup, Petersen and Faber, 1969). Although growing follicles can develop up to six or seven layers of granulosa c~lls in the absence of gonado­ tropins as shown in studies using hypophysectomized hamsters (Moore and Greenwald, 1974), once initiated, growth can be accelerated by gonadotropins and estrogen. In fact, after a follicle develops six to seven layers of granulosa cells its further development becomes dependent on adequate gonado­ tropin levels. During follicular development the thecal layer, which originates from interstitial tissue (sometimes also considered ovarian stroma), is stimulated by the growing follicle to organize around the latter. In small follicles, the thecal cells are elongated, have the appearance of connective tissue cells and remain inactive. By the time antral formation takes place, the theca interna cells take on 8 a polyhedral shape and become active in steroidogenesis (Knigge and Leathem, 1956). Over the process of follicular growth and differen­ tiation gonadotropin receptor number and type change in the granulosa and theca compartments. The exact sequence of events is not fully understood and several hypotheses are presented (Richards, 1979). Yet, it is generally accepted that small growing follicles contain FSH receptors on granulosa cells and LH receptors on theca cells. During antral formation and the beginning of estrogen synthesis, estrogen appears to induce its own receptors and to maintain and further increase the number of FSH receptors on granulosa cells. In the last stage of development FSH appears to initiate granulosa cell LH receptors, and to prepare the follicle for ovulation (Oxberry and Greenwald, 1982). Enzymatic activity related to steroidogenesis is histochemically not visible in preantral follicles (Saidapur and Greenwald, 1978a), correlating with low levels of steroidogenesis measured in vitro (Terranova, Connor and Greenwald, 1978). Of all follicle types steroidogenesis is most active in antral follicles. Granulosa cells of antral follicles are the major source of estrogen, which is syn­ thesized predominantly through synergism with the cells of the theca interna. The latter provide aromatizable androgen precursors, of which the major form in the hamster is androstenedione (Makris and Ryan, 1975, 1977). The 9

androgens, which appear to diffuse passively through the basement membrane, are taken up by granulosa cells and aromatized to form estrogen. For full follicular development and ovulation certain LH and FSH levels are necessary. High FSH levels, as seen during the estrous surge, are required for growth initiation and coordination of follicular development, but lower maintenance levels are also necessary for the continuation of healthy follicular growth (Moore and Greenwald, 1974). One specific function of FSH is its induction of granulosa cell aromatase activity (Erickson and Hsueh, 1978). The physio­ logical role of LH during folliculogenesis appears mainly to be the stimulation of theca androgen synthesis, the pre­ cursors required for the estrogen production by granulosa cells. The physiological role, if any, PRL has in folliculo­ genesis is not established. One study carried ciut in hypophysectomized hamsters treated with gonadotropins and PRL did not reveal any effects of PRL on follicular growth, yet prolonged maintenance of the corpora lutea was seen (Kim and Greenwald, 1984). Other studies in rats have shown a PRL-induced decrease in follicular estrogen production (Uilenbroek et al., 1982; Uilenbroek and Van Der Linden, 1984; Kalison, Warshaw and Gibori, 1985), which was thought to be mediated by inhibition of FSH-induced aromatase activity (Dorrington and Gore-Langton, 1981; Tsai-Morris et

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a1., 1983). However, whether these observations which were made mostly in rats can be extrapolated to the hamstsr is uncertain. In addition to pituitary control of folliculogenesis, many intra-ovarian processes of regulation take place, of which progesterone effects are probably the best known due to their antiestrogenic activity. The switch from follicular estrogen and androgen production to progesterone synthesis one hour after beginning of the'proestrous gonadotropin surge is thought to be due to the LH-mediated increase in proges­ terone production. As shown by the administration of progesterone to cyclic animals, elevated levels appear to be able to retard folliculogenesis and extend the cycle length (Labhsetwar, 1975; Greenwald, 1977, 1978). Progesterone is known to act at the level of the hypothalamo-pituitary axis, but there is also evidence to suggest that it interfers directly with ovarian androgen and estrogen synthesis (Garza and Terranova, 1984). One known mode of action involves the suppression of FSH-induced aromatase activity on granulosa cells (Fortune and Vincent, 1983).

c. Follicular Atresia Follicular atresia is an ongoing process, affecting follicles at any stage of development; in fact, more follicles undergo degeneration than ovulate. Except in small preantral follicles, granulosa cells degenerate and

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disappear, yet the theca interna, which is organized from the interstitial tissue does not show degenerative changes. During the atretic process of small growing follicles, the interstitial tissue surrounding the degenerating structure becomes activated to form a thick, compact, theca-like band considered a "pseudotheca" (Knigge and Leathem, 1956). This region will be temporarily active in progesterone synthesis, but will eventually regress. The causes for follicular atresia are not completely understood. Favorite models used to study atresia in the hamster are the delay of ovulation over several days by phenobarbital administration, or the treatment of hypophy­ sectomized hamsters with pregnant mare serum (PMS) followed by anti-PMS to obtain a synchronous population of atretic follicles. These studies revealed that withdrawal of gonadotropins or delayed ovulation result in a drop in FSH receptors on granulosa cells, a slower decline of granulosa

LH receptors and no changes in L~ binding to theca and interstitial cells (Shaha and Greenwald, 1982; U;lenbrook, Woutersen and van der Schoot, 1980; Yo1 Na, Garza and Terranova, 1985). Steroidogenesis by theca cells in dege­ nerating follicles switches from androgens to progesterone (Bill II and Greenwald, 1981; Terranova, 1980; Terranova, Martin and Chien, 1982; Hubbard and Greenwald, 1983). The decrease of granulosa cell estrogen production ;s initially caused by a lack of androgen precursors by theca cells and

- - ...... --.... _...... _.. _ ...... • - ..•. _- .... -_ ...... __._------12

secondarily by the slower loss of aromatase activity • (Terranova, 1981; Silavin and Greenwald, 1984). That the lack of adequate FSH levels might be responsible for folli­ cular atresia was shown in a study carried out in intact hamsters. By performing unilateral ovariectomy on diestrus II, which results in increased serum FSH levels, degenerative changes which are usually observed in one-half to one-third of the follicles in intact animals could be prevented (Greenwald, 1961; Bex and Goldman, 1975).

D. Interstitium The interstitium is an important structural and functional component of the hamster ovary. During folliculo­ genesis, theca cells are organized from this tissue, and after follicular degeneration, theca cells revert back to interstitial tissue (Knigge and Leathem, 1956). The inter­ stitial component of the ovary contains LH receptors which are gonadotropin independent since they are maintained after hypophysectomy (Shaha and Greenwald, 1982). When stimulated, the interstitial compartment has a high capacity for progesterone synthesis (Taya and Greenwald, 1980; Taya, Saidapur and Greenwald, 1980). In the cyclic animal the source of progesterone in response to the proestrous surge comes mainly from this tissue, since corpora lutea are defunct at that stage (Saidapur and Greenwald, 1978a). This tissue is highly stimulated during lactation, at certain 13

stages during the onset of puberty and in short photoperiod induced acyclic animals (Donham, DiPinto and Stetson, 1984; Greenwald and Peppler, 1968; Bridges and Goldman, 1975). In these conditions animals show daily gonadotropin surges which stimulate interstitial progesterone synthesis, and probably control the maintenance of the acyclic condition.

Neural Control of the Female Reproductive System

A. Neuroregu1ation of Gonadotropin Secretion Understanding of gonadotropin regulation has increased greatly in recent years, even though there are still many missing links in the proposed hypothesis and many suggested releasing and inhibiting factors await further clarification before their physiological involvement in the gonadotropin control mechanisms can be confirmed or discarded. This review will cover the more established controlling factors, including the luteinizing hormone releasing hormone (LHRH) system and its modulation by steroids, catecholamines, serotonin and opioids. The survey will deal mostly with findings in the rat since only limited data is available for the hamster. Studies carried out in female hamsters will be covered in a separate short section at the end of this review. 1. LHRH Neuronal Network. Than.ks to improved immunocytochemical techniques incorporating antibodies with high specificity it is possible to label the LHRH neuronal

..•...... '."'-'-'.'_._'" ...... _._ ...... __ .... --•... ------14 network more accurately than in early attempts. The following description is based on studies by Hoffman (1983), in which the neuronal networks in the rat, hamster and mouse were compared. LHRH neurons are predominantly found in the preoptic/septal area and medial surface of the olfactory peduncle. In the septum they are associated with the median septal nucleus or the nucleus triangularis septi, the medial septal nucleus (particular at its border with the diagonal band of Broca) and in the medial surface of the olfactory peduncle where they are dispersed along the medial olfactory tract. In the hamster and mouse, the LHRH cell distribution extends into the olfactory bulb as well. More caudally, the LHRH cells lie within the rostral preoptic periventricular nucleus. From there they are found laterally, generally located between the medial and lateral POA. In the anterior hypothalamus cells are situated lateral to the suprachi­ asmatic nucleus (SCN). The most caudal cells are found in the retrochiasmatic area where they are scattered in the ventrolateral hypothalamic area and interspersed with fibers of the medial forebrain bundle (MFB). Compared with the rat and the mouse, LHRH cells in the hamsters are far more scattered in all areas. Concommitant labeling of the vascular system reveals that many cells or their processes rest upon vessel walls, separated only by a basement membrane. Moreover, the cell proces·ses have a tendency to 15

form an extensive network by contact with each other through dentritic or somatic bridges, or other ~eans. Three LHRH pathways to the median eminence arise from a triangular area within the preoptic periventricular area whose apex lies in the caudal septum and its base in the preoptic area rostral to the organum vasculosum lamina terminalis (OVLT). One tract traverses the floor of the third ventricle, a second courses under the optic chiasma and a third runs along the lateral wall of the third ventricle. Cells originating in the MPOA and lateral to the SCN form a fourth tract which courses with the fibers of the MFB. As the fibers course toward the median eminence (ME), they start to branch and merge in the retrochiasmatic area. In the OVLT, fibers loop around vessels and form terminals en passante The LHRH network gives off projections to different limbic areas. Those to the medial and cortical amygdaloid nuclei were among the first e~~rahypothalamic projections to be observed. The nuclei send fibers back to regions where LHRH neurons are located. Lesion studies indicate that these amygdaloid areas are important in the control of estrous cyclicity and reproductive behavior (Kevetter and Winans, 1981). Other LHRH fibers project to the hippocampus, the cingulate cortex, mammillary nuclei and, in the hamster, also to the raphe complex. As a general rule, LHRH axons appear

------~ - 16

to project to areas of the brain in which gonadal steroid concentrating cells are located. 2. LHRH Release Patterns and Its Modulation by Steroids. LHRH cells appear to have the intrinsic ability to release their product in a pulsatile fashion (Ferin, 1983). This pulsatile release can be altered by steroid treatment in

regard to am~litude and frequency. Estrogen (E2) is able to supress the magnitude of LH pulses in gonadectomized animals (Goodman, 1983), whereas E2 and progesterone (P) combined depress frequency and amplitude (Kalra and Kalra, 1984). This steroid modulation appears to be responsible for the distinct episodic LH discharge pattern seen during the different phases of the estrous cycle (Gallo, 1981; Kalra and Kalra, 1983). Changes in the characteristics of pulsatile LH secretion can also be achieved by directly implanting estradiol in the POA or MBH area (Akema, Tadokoro and Kawakami, 1983). The relationship between steroids and LHRH synthesis is not clear, however, some studies have demon­ strated that steroids can stimulate the accumulation of LHRH in the MBH. This appears to be largely due to new LHRH as shown before the proestrous surge, or in surges evoked by the administration of progesterone in estrogen-primed animals (Kalra and Kalra, 1983). The origin of the increased LHRH accumulation could either be the result of increased syn­ thesis or the liberation of immunoreactive hormone from a

-_ ... _._---_ .... __ .- ._----_._-_._----... -_ ....._------_.- ....- ... - ._.-----_. ------17

high molecular weight precursor (Gautron, Pattou and Kordon, 1981). 3. Interactions Between the LHRH and the Adrenergic System. A large body of evidence shows that noradrenaline (NA) and epinephrine (E) producing neurons located in the brain stem (Fuxe et al., 1976) are capable of evoking LHRH release (Gallo, 1980). Adrenergic neurons have a tendency to terminate close to dendrites, perikarya and axons of pep­ tidergic neurons (Jennes et al., 1982). The release is evoked by activating alpha-adrenergic receptors in the preoptic-tuberal pathway (Jennes et al., 1982; Kalra and Kalra, 1983). Although episodic discharges appear to be intrinsic to the LHRH neurons (Kalra and Kalra, 1984), it is suggested that the NA system might provide a permissive intrahypothalamic environment for allowing LHRH neurons to discharge their product in accordance to their intrinsic rhythmicity, or that imposed by gonadal steroids (Kalra and Kalra, 1984). Studies examining the role of NA demonstrated that blockage of NA synthesis with dopamine beta-hydroxylase suppressed median eminence-arcuate nucleus (ME-ARC) LHRH levels preceeding the LH surge (Kalra et al., 1978). Moreover, drastic reductions in hypothalamic NA turnover are observed following pentobarbital supressed LHRH levels (Kalra, Simpkins and Kalra, 1980). Steroids have the capacity to modulate catecholamine turnover. Estrogen appears to have an inhibitory or

------_ .. --_.-----. ------18

stimulatory effect depending on the time of the day. In the morning hours, estrogen exerts an inhibitory action on NA turnover in the MBH and POA areas, whereas during the afternoon hours and coinciding with the timing of the afternoon surge its action is stimulatory (Hiemke et a1., 1984). Progesterone exerts a short stimulatory action followed by inhibition, yet estrogen-priming is essential for both its effects. Experiments demonstrate that progesterone has the capacity to increase LHRH release in vitro (Leadem and Ka1ra, 1984), by directly stimulating NA turnover (Simpkins, Ka1ra and Kalra, 1980; Kalra, Simpkins and Kalra, 1981). The last observation may explain how progesterone is able to override the phenobarbital-blocked proestrous or estrogen-induced gonadotropin surge, as long as the neural pathways between the MPOA and MBH area are intact. 4. Opioid-Adrenergic-Steroid Connection. In recent 'years naturally occurring morphine-like substances have been discovered 1~ the proximity of LHR~~ 5teroid-cancentrati~g and catecholaminergic neurons. More and more evidence is accumulating to show that these endogenous opioid peptides (EOP) might have a physiological role in the reproductive axis. EOP exert a profound inhibitory influence on the frequency and amplitude of LH discharges in ovariectomized rats. This inhibition is believed to be mediated through the exertion of an inhibitory action on the catecholaminergic system, since blockage of opiate receptors by naloxone 19 promptly activates LHRH release, noradrenaline and epine­ phrine turnover (Adler and Crowley, 1984; Kalra and Kalra, 1984) . There is much evidence to demonstrate that the inhibitory activity of steroids might be mediated by EOP. Beta-endorphin levels in the portal plasma and hypothalamus fluctuate according to the steroid milieu (Kumar, Chen and Muther, 1979; Weherenberg et al., 1982). The involvement of steroids in hypothalamic EOP is also revealed by the admini­ stration of naloxone into gonadectomized animals, in which it is shown that naloxone is only effective in the presence of gonadal steroids and its action is eliminated a few days after gonadectomy. Some studies indicate that the ability of opioid administration to depress gonadotropin levels may be disrupted in longterm ovariectomized animals, but can be reinstated by steroid treatment (Bhanot and Wilkinson, 1984; Cicero, Meyer and Schmoecker, 1982). Yet other studies carried out in longterm ovariectomized rats do now show a change in responsiveness to opioids (Kalra, Simpkins and Chen, 1980; Leadem and Ka1ra, 1985). Further evidence for an interrelationship between EOP and steroids is demonstrated by the ability of naloxone to reinstate the pulsatile secretion of LH supressed by the administration of estrogen and progesterone (Sylvester et al., 1982). 5. Serotonin and Dopamine. The involvement of the serotonergic system in the reproductive axis is not clear 20 since different experimental protocols give conflicting results. The principal source of serotonergic fibers innervating the septal-preoptic-tuberal pathway originates from neuronal groups in the dorsal and median midbrain raphe nuclei (Kalra and Kalra, 1983). Studies reveal that sero­ tonin might have an inhibitory action on tonic levels of gonadotropins and stimulatory effects on surge levels. One recent study suggested that the progesterone-mediated surge elicited in ovariectomized estrogen-primed rats might be mediated by the serotonergic system (Iyengar and Rabii, 1983). Intraventricular injections of serotonin or stimu­ lation of raphe nuclei to activate the serotonergic system led to a depression of LH release (Schneider and McCann, 1970; Arendash and Gallo, 1978). Other experimental evidence indicates the effects of estrogen might be mediated by stimulating the opioid system, which in turn would activate serotonergic projections to the medial' preoptic area inhibi­ tory to LHRH release (Johnson and Crowley, 1984). Data on the action of dopamine (DA) is even more controversial. Incubation of hypothalamic fragments with DA stimulated LHRH release (Schneider and McCann, 1969). Other

~ vivo studies in which DA was given to estrogen-primed rats also increased LH secretion (Vijayan and McCann, 1978). Yet there is also a large body of findings pointing to an inhibitory role of DA on LH release (Kalra and Kalra, 1983), and in two studies in which DA turnover was increased by PRL 21 without affecting NA turnover, an inhibitory action on serum gonadotropin levels was observed (Gudelsky et al., 1976; Selmanoff, 1981).

B. Gonadotropin Regulation in the Female Hamster Available information about neural regulation of gonadotropin in the estrous cycle of the hamster is very limited. Only a few studies have been carried out using lesions or steroid implants placed in different areas of the hypothalamus in attempts to define those areas most sensitive for disruption of the estrous cyclicity. Studies using steroid implant techniques to localize neural sites sensitive to estrogen feedback (Lisk and Ferguson, 1973) showed that the arcuate nucleus and surrounding area were most sensitive and steroid placement in this area resulted in the disruption of estrous cyclicity. The next most sensitive area were the mammillary nuclei, in which implants caused irregular cyclicity or disruption of estrous cyclicity in 66% of the animals. In another study it was shown that small proges­ terone implants were able to disrupt cyclicity only when placed in the medial preoptic area (Reuter and Lisk, 1973).

Lesion studies shnwed furthe~ that lateral and posterior cuts did not have any effect ~n estrous cyclicity, but ovulation was permanently blocked by total deafferen­ tation of the medial basal hypothalamus (Norman, Blake and Sawyer, 1972), causing decreased uterine weights, low LH 22

levels and high pituitary LH contents. In contrast, animals with extended frontal cuts disrupting connections between the POA and MBH had increased uterine weights and ovaries full of

1a r ge c y s tic f 0 1 1 i c 1e s, and we r e una b 1e tot rig g e r the ovulatory surge. Studies using more refined techniques revealed that intact connections between the medial preoptic area and medial basal hypothalamus are necessary ·to elicit the positive feedback action of estrogen resulting in the proestrous surge (Norman and Spies, 1974). More recent studies in which monosodium glutamate (MSG) was given on day eight of neonatal life to destroy the arcuate nucleus, and in some animals this treatment combined with experimental lesions placed after two months, revealed that the anterior hypothalamus and arcuate nucleus worked together to regulate tonic LH secretion, whereas the arcuate nucleus region is important in the tonic regulation of FSH (Lamperti and Baldwin, 1983). Although interesting, the validity of

this stud.y appear·sto ·lJe questionab~e since chemical lesio[1s placed during the postnatal period could affect ovarian steroid synthesis and secondarily affect the development of the neural pathways (Clough and Ridriguez-Sierra, 1982; Handa and Gorski, 1985). Two interesting experiments were carried out by Printz and Greenwald (1970, 1971). In the first study it was seen that starvation resulted in the cessation of cyclicity, and ovaries showed a similar appearance to those seen in light

.... "" ."."_ ... _ .. " .. _. " ...- _._ .... _----" .. _- "".--" .. "".- "-"--_ .... "-_ .... -._------23

deprived acyclic animals. In the second study, lesions were

placed in the mammillary ~uclei and different ar~as of the brainstem, including the ventral tegmental nuclei. Lesions in the mammillary nuclei caused similar effects on the estrous cycle as seen in the study on starvation. Yet, in animals with lesions in the ventral tegmental nuclei in which the animals ceased eating, estrous cyclicity continued. Putting the two experiments together they proposed that the ventral tegmental nuclei might become activated by starvation and send inhibitory impulses to the mammillary nuclei which appear to promote FSH release. The origin of the secondary or estrous FSH surge remains unclear. Lesions made after the proestrous surge show that the arcuate-median eminence region is involved in the FSH release at estrus and remains unaffected by discon­ nection of the medial preoptic area (Chappel, Norman and Spies, 1977). Yet a follow-up study by the same investi­ gators (1979) demonstrated that the neural connection between the rostral and basal hypothalamus had to be intact during the time the proestrous surge proceeded for a full expression of the estrous FSH surge, even when ovulation and resulting steroid changes were induced by the administration of LH. This observation would imply that the proestrous and estrous events are neurologically somehow connected, and once the program starts the events carry through.

---_.-..... --.--. -.- -- .. - -._._------.. --_.-.._ ... _------24

A different study showed that suppression of the proestrous FSH surge by phenobarbital reduced, but did not eliminate, the surge, yet when estrogen and progesterone were given in addition during the time the estrous surge was in progress, the surge was completely prevented (Siegel, Bast and Greenwald, 1976). In a further study in which the relationship between follicular recruitment and the pro­ estrous and estrous FSH surges were explored, results suggested that the estrous FSH surge is responsible for follicular recruitment (Greenwald and Siegel, 1982). In all the studies which have attempted to unravel the periovulatory events, changes in inhibin levels were ignored. Since this peptide is thought to be synthesized in large antral follicles and is able to selectively supress FSH synthesis and release, its elimination during the ovulatory process might also have an impact on the magnitude of estrous FSH levels. One recent study examined the pulsatile gonadotropin pattern and its regulation in longterm ovariectomized hamsters (Chappel, Miller and Hyland, 1984). FSH and LH release occurred in pulsatile fashion with a pulse interval of about forty minutes. Although pulse frequency of both gonadotropins was similar, LH and FSH pulses could not be superimposed. Injections of alpha-methyl-para-tyrosine suppressed the pulsatile release of LH, but did not affect FSH release. Injections of metoclopramine, a dopamine 25 antagonist, resulted in an abrupt increase in LH pulse amplitude without affecting FSH. On the other hand, anti-LHRH injections completely eliminated LH release and retarded the frequency of FSH release, indicating that FSH appears to be under additional neural control.

D. Control of Gonadotropins at the Pituitary Level. Many important regulatory and modulatory mechanisms affect gonadotropin release at the pituitary level. The data described in this section are drawn from experiments carried out in the hamster and rat. Hypothalamic LHRH appears to control LH release, whereas FSH release appears to be governed by additional factors not yet completely identified (Chappel, Miller and Hyland, 1984) • LHRH is released in a pulsatile fashion (Ferin, 1983) , yet its pattern of release, which affects the ratio of LH/FSH, is modulated by steroids (Wildt, et al., 1981). The magnitude of the response is largely dependent on the concentration of pituitary LHRH receptors which, among other factors, is affected by the frequency of LHRH pulses. LHRH "upregulates" its own receptors and, therefore, the higher the pulse frequency the more effective the hormone release (Katt et al., 1985; Pickering and Fink, 1976, 1977; Waring and Turgeon, 1983) an apparent "downregulation" occurs only after continuous administration (Liu and Jackson, 1984). 26

Although there are also data to the contrary, the preponderance of evidence reveals that estrogen treatment increases LHRH pituitary receptors and, as a result, respon­ siveness to LHRH ~ vitro (Menon, Peegel and Katta, 1985).

~ vivo data also show that short term estrogen treatment is able to augment LH release in response to LHRH administration by increasing LHRH receptors, but after longterm estrogen administration these effects are often obscured by the decreased pulse frequency of LHRH release, which in turn lowers the receptor number (Marchetti, et al., 1982). In vitro and ~ vivo experiments also show that estrogen, besides increasing the number of LHRH receptors, appears to have direct stimulatory effects on LHRH-induced LH release, which in many experiments is preceeded by a short inhibitory phase (Libertun, Orias and McCann, 1974; Drouin, Lagace and Labrie, 1976; Baldwin, Bourne and Marshall, 1984). Progesterone generally counteracts the effects of estrogen, causing a "downregulation" of LHRH receptors seen after the proestrous gonadotropin surge or after progesterone administration (Witcher, Nearhoof and Freeman, 1984). This phenomena is also seen during the other days of the estrous cycle, and corresponds well with the pituitary responsiveness to LHRH during the different days of the cycle (Gordon and Reichlin, 1974; Savoy-Moore and Schwartz, 1980). Further anti-estrogenic activities of progesterone which result in a decrease in pituitary responsiveness to LHRH, appear to 27 involve a progesterone-mediated decrease in pituitary estrogen receptor binding (Blaustein and Brown, 1984; Smanik et al., 1983). Although the above studies have been carried out in rats, it does appear that the female hamster responds in a comparable manner. One study carried out in intact cycling and ovariectomized hamsters, of which some were steroid treated, demonstrated that the LH release in response to LHRH injections is greatest during proestrus when estrogen levels are highest, and declines after the ovulatory surge. Ovariectomy resulted in a 75% decrease in LH release and an even greater drop in FSH after an LHRH challenge. Yet the gonadotropin response to the LHRH challenge could be rein­ stated to levels seen in intact proestrous animals by treating the animals with estrogen (Shander and Goldman, 1978) • In recent years more and more evidence has accumulated to show that the putative ovarian hormone inhibin specifi­ cally suppresses FSH levels. This protein is synthesized by granulosa cells of antral follicles and its suppressive activity is specific for FSH (Ledwitz-Rigby and Rigby, 1983). Experiments carried out in vitro reveal that the inhibitory activity is effected through the suppression of FSH synthesis and increased degradation (van der Schaaf-Verdonk, et al., 1984). In addition, inhibin is supressive to basal FSH release and re'duces

------._---. - 28

LHRH-stimulated FSH release in in vivo and in vitro situations (Massicotte et al., 1984; Charlesworth et al., 1984). Studies in the golden hamster confirm that this FSH inhibiting activity is present in hamster ovaries and fluctuates during the estrous cycle, with highest activity found in ovaries harvested on the day of proestrus (Chappel, 1979). A newly suggested control mechanism, paracrine control, has recently received attention. Paracrine regu­ lation implies that the activity of one cell population directly impacts the action of neighboring cells. These effects have been demonstrated by cO-incubating different ratios of gonadotropin and PRL cells, with the result in one study that gonadotrophs can activate the secretory activity or PRL (Denef and Andries, 1983). If further studies can support the importance of this type of control mechanism, it might have interesting implications in reproductive states (i.e., pregnancy, lactation) and might force us to do some re-thinking and re-evaluating.

D. Regulation of Prolactin Prolactin synthesis and release is regulated by complex interactions of gonadal steroids, neurotransmitters and peptides. The activity of lactotrophs and their control are sexually dimorphic, therefore, the discussion will focus on the literature dealing with female animals and in vitro 29 experiments. Since there is a lack of knowledge with regard to the control of PRL in the hamster, the review will mostly deal with findings in the rat, yet one has to be aware that the rat is different from the hamster in many respects, and that these findings might not always apply to the hamster. Two major factors governing PRL synthesis and release are estrogen and dopamine (DA). As in vivo and in vitro data show, estrogen is the only gonadal steroid with potent stimulatory effects on PRL biosynthesis and release. In vitro studies demonstrate that estrogen increases synthesis of PRL by the induction of gene transcription, followed by increased ribonucleic acid activity, PRL synthesis and release (Vician, Shupnik and Gorski, 1979; Maurer, 1982, 1982; Gorski et al., 1985). Parts of the stimulatory action of estrogen on increasing PRL release involve a reduction of the inhibitory effects of dopamine (DA) on PRL cells. DA is considered a pituitary PRL inhibitory factor, and one way estrogen is thought to promote increased PRL release is by reduction of the capacity of PRL cells to incorporate dopamine into secretory granula, thus decreasing the effec­ tiveness of DA (Ferland et al., 1979; Gudelsky, Nansel and Porter, 1981). Longterm administration of estrogen has been shown to increase DA secretion of the tuberoinfundibular neurons (TIDA) into the portal blood. This supressive effect has been shown to be indirectly induced by increased PRL 30

levels, which in turn stimulate the activity of the dopa­ minergic system (Demarest and Moore, 1980). Many studies have demonstrated that the interaction between PRL and the activity of the TIDA neurons self­ regulate each other to a large extent, with TIDA neurons showing enhanced activity in response to increased PRL secretion (I-Iokfelt and Fuxe, 1972; Gudelsky et al., 1976; Gudelsky and Porter, 1980; Demarest, Riegle and Moore, 1984). Progesterone counteracts estrogen activity in several ways. Administration of the steroid into animals ovariec­ tomized on proestrus and sacrificed one day later, increases DA release into the hypophysial portal blood (Cramer, Parker and Porter, 1979). In vitro studies show that progesterone indirectly reduces PRL synthesis perhaps by causing a reduction in estrogen receptor synthesis (Haug, 1979). Progesterone given to estrogen primed rats induces a short facilitory, effect on ,PRL release'whichlasts for three hours and is followed by inhibitory activity (Caligaris, Astrada

and Taleisnik J 1974). Steroid interactions on PRL control are also reflected during the estrous cycle, revealing lowest DA binding sites on proestrus and highest numbers on diestrus I (Pasqualini et al., 1984). TRH is known to have stimulatory activity on PRL release. The mechanism of action is not completely eluci­ dated, but the activity appears to be related to estrogen levels and the activity of the serotonergic system. TRH 31

challenges during the different days of the estrous cycle elevate PRL levels tenfold higher on proestrus when estrogen levels are high compared to diestrus I when progesterone levels dominate. The responses appear to be indirectly related to estrogen levels, since estrogen is known to induce pituitary TRH receptors (De Lean et al., 1977a,b). Coincu­ bation of pituitaries with DA and TRH show that DA severely depresses the TRH induced stimulatory effect on PRL release, although with low dosages a stimulatory effect can still be seen (Fagin and Neill, 1981). On the other hand, there are also suggestions that TRH, when given intraventricularly, can have an inhibitory action by activating the DA system (Ohta et al., 1985). It is well documented that serotonergic activity which can be enhanced by estrogen when given centrally, results in an increase of PRL secretion (Johnson and Crowley, 1983). How this enhanced secretion is mediated is far from being clear. Data suggest that increased serotonergic activity decreases DA concentration in the portal blood, whereas decreased serotonergic activity reduces TRH release (De Greef et al., 1985). Still another study suggests that increased serotonergic activity can enhance secretion of vasoactive intestinal peptide (VIP), a putative PRF, into the hypophysial portal blood (Kato et al., 1978; Ruberg et al., 1978; Shaar, Clemens and Dininger, 1979; Shimatsu et al., 1982). 32

The mechanism of action of morphine and opiate-like peptides in stimulating PRL release is better established. Morphine administration results in a marked reduction of OA release into the portal blood. This reduction is achieved by suppressing OA biosynthesis and inhibiting OA release into the portal blood (Reymond and Porter, 1983). Since many factors controlling PRL release are also involved in the control of gonadotropin secretion, many interactions between the two systems exist, making it difficult to dissect out the individual mechanism of action.

E. Suprachiasmatic Nucleus and Circadian Rhythms The suprachiasmatic nucleus (SCN) is considered a true internal clock, coordinating and controlling many behavioral and hormonal rhythms in the absence of environmental cues. The clock is set by the daily light regimen, and in constant light or darkness is "free running", with an endogenous rhythm generally slightly longer than twenty-four hours (Morin et al., 1977; Elliott, 1981). Besides many other rhythms, the SCN controls activity onset, drinking rhythm, and in female hamsters or rats the timing of the proestrous surge and estrous behavior (Morin et al., 1977; Turek, Swann and Earnest, 1984). The proestrous surge, daily afternoon gonadotropin surge in ovariectomized hamsters, or surges seen in lactating or short photoperiod­ induced acyclic animals all originate from the same neural 33

trigger and can be supressed by the administration of phenobarbital, destruction of the SCN, appropriate steroid treatment or ablation of the connection between the preoptic area (POA) and the medial basal hypothalamus (MBH) (Norman and Spies, 1974; Alleva et al., 1975; Stetson and Watson-Whitmyre, 1976, 1977; Morin et a1., 1977; Stetson, Watson-Whitmyre and Matt, 1978; Stetson et al., 1981). This "Clock-like" rhythm is clearly demonstrated by the suppression of the proestrous surge with phenobarbital, which although the drug wears off within hours, only reappears twenty-four hours later (Stetson and Watson-Whitmyre, 1977). The magnitude of the daily gonadotropin surge can be changed or completely suppressed by the administration of various steroids. Exposure of an animal to estrogen for at least twenty-four hours elicits a positive feedback response. In hamsters, which display a daily surge in the absence of steroids, estrogen will augment the surge (Stetson, Watson-Whitmyre and Matt, 1978), whereas in the rat estrogen treatment allows the expression of a surge (Legan, Coon and Karsch, 1975; Legan and Karsch, 1975). On the other hand, progesterone given alone and without pre-treatment with estrogen has no effect on the surge, but if animals are first pre-treated with estrogen, progesterone augments the estrogen-induced surge on the day of administration, and is able to induce surges in animals in which the surge is 34

blocked by phenobarbital (Norman, 1975). The latter effect is thought to be mediated by the ability of progeseerone to directly activate the noradrenergic system by increasing cAMP activity (Ramirez, Kim and Dluzen, 1985). In contrast to the short facilitory effects of progesterone, the combination of estrogen and progesterone treatment for more than twenty-four hours completely supresses daily surges (Norman, Blake and Sawyer, 1973; Norman, 1975). The latter observation explains how, in animals displaying estrous cycles, surge levels are expressed only on proestrus, at a time when estrogen levels are high and progesterone levels either low or undetectable. The timing of the proestrous LH surge is tightly coupled to locomotor activity, with peak levels occurring three to four hours prior to activity onset (Moline et al., 1981; Swann and Turek. 1985). The coupling of these two events is clearly demonstrated in animals exposed to constant light, in which animals displaying split activity rhythm also have two LH surges, each one preceeding the onset of loco­ motor activity (Turek, Swann and Earnest, 1984). Cyclic LH release which can be augmented by estrogen treatment occurs only in the absence of androgen exposure during the postnatal period. Thus, males or androgenized females are not able to display daily surges; on the other hand, normal females or males castrated at birth will display a positive estrogen response and a surge pattern. Adult females treated with androgens during the postnatal period

. ~ -~.-- .. -- ... _. _. _.------_.---- ._-- -- ._--._---_. ---+------35

remain in persistent estrus, unable to elicit an ovulatory LH surge. On the other hand, males castrated during the postnatal period and implanted with ovaries are able to display ovulatory cycles (Swanson, 1970). This sexual dimorphism can explain why daily gonadotropin surges are seen in female hamsters acyclic as a result of short photoperiod (SP) exposure. In this state the ovaries synthesize mainly progesterone which by itself is unable to suppress the cyclic center. Since males do not possess a cyclic center, no diurnal pattern is seen in SP-exposed males (Goldman and Brown, 1979; Albers, Moline and Moore-Ede, 1984). This sex dependent response is also observed in regards to the circadian clock, which is advanced by estrogen treatment in females and remains unaffected by steroids in males (Zucker, Fitzgerald and Morin, 1980). As alluded to above, there is much evidence that the noradrenergic system is responsible for triggering the proestrous or daily surge, a system which does not function in androgenized females or intact males (Buhl, Norman and Resko, 1978; Lookingland and Barraclough, 1982; Lookingland, Wise and Barraclough, 1982). The circadian system is entrained by photic input via retinohypothalamic fibers reaching the SCN (Morin et al., 1977; Pickard and Silverman, 1981). In the absence of light, this internal clock is free-running, but in the presence of a light-dark cycle reset daily to a 24 hour rhythm. The SCN nuclei are also part of the pathway mediating light input to

------36

the pineal gland and as such is part of the system controlling seasonal reproduction. Elliott. Stetson and Menaker (1972) first established a circadian basis for photoperiodic time measurement in the

hamster. Their experiment was stimulated by the B~nning hypothesis which is based on observations in birds and postulates a circadian rhythm of sensitivity to light (Elliott, 1981). According to this model, each twenty-four hour day is divided into approximately equal phases in which the organism is either sensitive or insensitive to light. In this scheme light entrains the photosensitive rhythm and also acts as an inducer, e.g. light stimulates photoperiodc effects when it extends into the photosensitive phase of the circadian cycle. Subsequent studies showed that in the hamster the photosensitive phase begins shortly prior to the activity onset and lasts for about 11.5 hrs after activity onset (Elliott. 1974). Resonance experiments further supported the application of the BUnning hypothesis in the hamster (Elliott, Stetson and Menaker, 1972; Elliott, 1976). In these experiments animals were exposed to light dark (LD) cycles of different durations, and it was demonstrated that as little as one hour of light could maintain testicular activity provided it coincided with the photosensitive phase. The above experiment also explains the observation by Gaston and Menaker (1967) in which it was shown that male

------37 hamsters required a minimum of 12.5 hrs of light per day to maintain reproductive competence.

Seasonal Breeding and the Pineal Gland

A. General The survival of an animal or plant species is largely dependent on its ability to adapt to changing environmental conditions, ability to conserve energy and its success in propagation. One way to optimize the survival of the young is to bear them at a time of the year when food is plentiful and the environmental conditions favorable. Since daylength is nature's most reliable "Zeitgeber", the majority of plants and animals rely largely on this environmental cue to synchronize reproduction with the time of the season. Depending on the species, decreasing daylength may serve either as the stimulus for the beginning of a breeding season or the signal to shut down reproductive activity. Deer, goats and sheep are good examples of "short day breeders" in which mating occurs during late summer, resulting in the birth of the young during spring time. Rodents with short gestation periods have evolved as "long day breeders", and for them day length below a critical value causes gonadal involution. In most animals which are seasonal breeders, photo­ periodic effects on the reproductive system are thought to be transduced by the pineal gland, the activity of which is 38 altered by short photoperiods. Depending on whether an animal is a long day or short day breeder, pineal activity "stimulated" by short photoperiod can have pro- or antigo­ nadotropic effects. There is also evidence in the ferret, Djungarian hamster and the vole, that the pineal gland can participate in conveying effects of long photoperiod exposure stimulatory to the reproductive system. Since the pineal gland can have anti- and progonadotrophic activity, its role appears to be to translate the photoperiodic message into a humeral response, thus enabling an animal to synchronize reproduction with the most appropriate time of the year. The role of the pineal gland has been most extensively explored in the golden Syrian hamster (Mesocricetus auratus) which, as stated earlier, is a seasonal breeder under natural photoperiodic conditions. These animals start to undergo gonadal involution around the fall equinox, remain reproductively quiescent during the winter months, and undergo spontaneous recrudescence before the spring equinox. Gaston and Menaker (1967) established that in male hamsters the critical amount of light required to maintain repro­ ductive competence is 12.5 hrs. Although detailed studies were not carried out in females, experiments show that their light requirements are most likely comparable. In his studies on hamsters Czyba et al. (1964) was the first to demonstrate a functional link between the pineal gland and the reproductive system of mammals. That study was

.. -_ .... ------39

the beginning of an intensive search for the IIpineal hormone ll and its mechanism of action - a search still going on now although the indoleamine melatonin has assumed a major role in this regard in recent years (Cardinali, 1984). The hamster pineal gland perceives photic information from the eyes through multisynaptic connections. The retinohypothalamic tract provides a direct pathway between the retina and the suprachiasmatic nucleus (SCN) (Pickard and Silverman, 1981; Pickard and Turek, 1983). Two major projections emerge from the SCN forming tuberal projection and a periventricular system. Based on findings from lesion studies, the periventricular system, which projects from the SCN into the parvocellular region of the paraventricular nucleus (PVN), is responsible for relaying photic information to the pineal gland. It has been shown recently that lesions of the PVN can prevent pineal-mediated photoperiodic effects without interrupting the circadian system (Pickard and Turek, 1983; Bartness, Bittman and Wade, 1985). Although a mono­ synaptic connection between the PVN and the preganglionic neurons of the intermediolateral cell column (IMLCC) exists (Pickard and Turek, 1983; Sladek, verbal communication), to my knowledge no studies have been carried out to determine whether photic information is transmitted by this system or by a multisynaptic pathway. The cells originating from the IMLCC form the preganglionic sympathetic chain and make synaptic connections in the superior cervical ganglia (SCG) •

. .•. _. -- ._-.- ...... __ .. _.------40

Postganglionic fibers originating in that ganglia ride on the internal carotid artery, enter the cranium and give off the nervii conari which innervate the pineal gland directly and are necessary for the maintenance of its activity. Since synaptic connections to the IMLCC are thought to be inhi­ bitory, increased light impulses decrease the activity of the pineal. Based on many experiments, it is postulated that hamsters are able to interpret photoperiodic condition with the assistance of an internal circadian clock which is divided into a light-sensitive and light-insensitive phase. Details of the interesting experiments which led to this hypothesis are covered in Section E dealing with circadian rhythms. The mechanism of action through which the pineal mediates its effects on the reproductive axis are not fully established. The most intensively studied and most widely discussed pineal hormone is melatonin, which is synthesized in the pineal gland and is able to replicate SP effects on the reproductive axis when administered at the appropriate time of the day (Cardinali, 1984). In addition, there are a whole host of other compounds found in the pineal gland, many of which have known structures and others with anti- or progonadotropic properties which await further characteri­ zation (Ebels and Benson, 1978; Vaughan, 1981). 41

B. Photoperiod and the Female Hamster

The first observations on the effects of li~ht deprivation in female hamsters were made in 1966 by Hoffman and Reiter. After four weeks of exposure to one hour of light per twenty-four hours, the decreased uterine weights observed could be prevented by pinealectomy prior to SP exposure. One year later, Reiter showed that follicular development and ovarian and uterine weights in blinded animals could be stimulated with the administration of LH and FSH (Reiter, 1967). A further experiment carried out by Reiter (1968) revealed that cold exposure was able to suppress the reproductive system, and was additive to the effects of blinding. Although these effects were similar in appearance, the effects of blinding and cold exposure appear to be under different controls since pinealectomy only reversed the effects of blinding. In the same experiment, Reiter showed that removal of both eyes was necessary to induce suppressive effects on the reproductive system. One year later, an experiment revealed that nine weeks after optic enucleation female hamsters had uteri one-third the size of intact animals, and ovaries which generally showed a lack of corpora lutea, few small growing follicles and marked interstitial hypertrophy (Reiter, 1969). Some animals in the above study were observed for an extended period of time and were seen to undergo spontaneous recrudescence after twenty to twenty-seven weeks. 42

Estrous cycles of light-deprived hamsters were carefully monitored for the first time by Sorrentino and Reiter in 1970. It was seen that as long as estrous cycli­ city persisted, vaginal smears of blinded animals were comparable to those seen in intact or blinded pinealectomized cycling animals. But once acyclic, the smears took on a similar appearance to those seen in ovariectomized animals. A further, interesting observation made in that experiment was the significantly increased pituitary weights developed by blinded animals after ovariectomy (usually seen in intact animals), revealing that, although it was assumed that there was no obvious ovarian activity, blinded animals were apparently still synthesizing steroids or other ovarian factors able to affect pituitary function. After optic enucleation was employed experimentally to activate the pineal gland, it was later confirmed that exposure of female hamsters to natural photoperiods during the winter months also caused gonadal regression unless prevented by prior pinealectomy (Reiter, 1974). An addi­ tional study by Reiter (1973/74) further revealed that, although pinealectomized hamsters exposed to natural photo­ periods were able to breed, litter sizes were reduced during January, and pups were cannibalized during that and part of the following month. Since cold exposure also suppresses reproduction (Reiter, 1968) and animals were kept in unheated rooms, it is impossible to determine whet~er the reduced 43

litter size and the aberrant maternal behavior was the result of pinealectomy or cold exposure. As radioimmunoassay techniques became available, serum and pituitary levels of LH and PRL were determined in blinded acyclic, intact cycling, blinded pinealectomized and blinded superior cervical ganglionectomized animals (Reiter and Johnson, 1974a,b; Reiter, 1975). The results showed that blinded acyclic animals had increased pituitary LH contents and reduced PRL contents compared to other control groups, and further revealed that superior cervical ganglionectomy was as effective as pinealectomy in the prevention of the effects of light deprivation. An additional object of the above study was to stimulate ovarian function with LHRH injections. Although injections were given twice a day over a ten to thirty day period, they were ineffective in stimu­ lating any changes in ovarian histology or uterine weights in spite of elevated serum gonadotropin levels and decreased pituitary contents. A new series of experiments started when Seegal and Goldman (1975) exposed female hamsters to ten hours of illumination, which resulted in the acyclic condition after six weeks. Hormonal profiles were determined in the acyclic animals and it was found that they displayed daily gonado­ tropin surges, with low levels seen in the morning and surge levels during afternoon hours. Ovariectomy did not increase morning LH levels, but magnified the afternoon surge.

_.- .... '-'---"'-~-- 44

Compared with LP-exposed ovariectomized animals, morning levels were significantly depressed, but afternoon surges increased. On the other hand, morning and afternoon FSH levels showed a castration response in SP-exposed ovari­ ectomized animals, although still several-fold lower than levels seen in LP ovariectomized controls. These observations were followed up by another interesting study by Bridges and Goldman (1975). Knowing that lactating hamsters displayed similar ovarian histology to that of light deprived hamsters, and that the interstitial tissue of the hamster ovary is capable of progesterone synthesis, they felt that the hormonal profile of gonado­ tropins might also be similar in these two conditions, and thus made a direct comparison. The results verified their notion and confirmed the results of the previous experiment. In addition, they measured daily afternoon progesterone surges in both treatment groups which could be eliminated by ovariectomy. Follow-up experiments confirmed that the ovaries were the source of progesterone, and that the adrenal glands were not responsible for the reduction of the cast­ ration response in SP-exposed animals (Bittman and Zucker, 1977; Bittman and Goldman, 1979). Goldman and Brown (1979) demonstrated that the daily surge pattern observed in SP-exposed, acyclic females was not seen in males and was, therefore, sex dependent. They confirmed their hypothesis by showing that males castrated at birth also showed afternoon 45

surges in response to SP, whereas androgenized females displayed a hormone pattern similar to that seen in intact males. A somewhat different approach was taken by Little and Benson (1978) who tested the ability of ovaries from blinded hamsters to undergo compensatory hypertrophy after unilateral ovariectomy. Their findings revealed that hamsters blinded for three weeks demonstrated a reduced ability for compen­ satory folliculogenesis and 32p uptake in the remaining ovary, pointing out that reduced FSH levels might be" respon­ sible for the acyclic condit.ion. In 1981, Widmaier and Campbell examined whether SP treatment could reduce PRL serum levels in the absence of the ovary, and alter the interaction of estrogen and PRL. These were the first experiments to show that SP treatment can reduce serum PRL levels after four weeks in the absence of the ovaries, and that although estrogen treatment can increase PRL levels in both treatment groups, it was less able to do so in SP-exposed animals. One drawback of this study was that only a heterologous radioimmunoassay (not especially sensitive) was available to determine PRL levels. In the same investigation, serum estrogen levels in the SP-exposed acyclic animals were compared with levels of intact animals and found to be low, even lower than values seen during any stage of the estrous cycle. This finding, although novel, was not surprising considering the size of

...... - .- -. -----_._------._-_._----,-".---,.",,-.------46

the uteri and the appearance of the ovarian histology. Kumar et al., (1982) added to our knowledge with the finding of an increase in the dopamine depletion rate and LHRH content in the medial basal hypothalamus of noncyclic SP-exposed hamsters. This was the first study to show changes in hypothalamic catecholaminergic activity in female hamsters, and since dopamine is generally believed to be inhibitory to PRL and gonadotropin release, if proven correct could explain changes induced by SP conditions. To summarize the major findings at the time experi­ ments were designed for this dissertation project: female hamsters had been observed to cease the display of ovarian cyclicity after three to twelve weeks of either SP exposure or blinding. In the acyclic condition the uteri were shown to be involuted, yet the ovaries were either the same size or enlarged. Histologically, the ovaries of anestrous hamsters were characterized by the absence of corpora lutea and a paucity of growing follicles, of which many were seen to. undergo atretic changes. Yet the most prominent feature of these ovaries that had been reported was the marked inters­ titial hypertrophy and rich blood supply. The hormonal profile of acyclic animals was characterized by low gonado­ tropin levels in the morning, daily afternoon gonadotropin surges and elevated pituitary gonadotropin contents, which was in marked contrast to the observations of low pituitary and serum PRL levels. CHAPTER 2

STATEMENT OF THE PROBLEM AND SPECIFIC AIMS

A small body of knowledge is available in regards to the acyclic condition induced in hamsters by light deprivation, and little is known about the events leading to this condition. Specifically, the hormonal and histological changes which take place prior to the onset of the anovulatory condition are not known •. Experiments were designed to examine critical events during the estrous cycle which, if altered by SP, could render an animal acyclic. Other experiments examined possible SP-induced changes of hormonal levels either in the absence of ovarian steroids or in the presence of known amounts of estrogen. It was hoped that the combination of these two approaches would provide an understanding of events causative to the anestrous state. The specific aims of the dissertation project were: - To eyaluate with histochemical methods the steroid synthetic capacity of the ovaries from animals rendered acyclic through light deprivation. - To determine the response of the neuroendocrine-repro­ ductive axis of the LSH/SsLak golden hamster to SP and to evaluate their usefulness for other proposed experiments. - To evaluate early hypothalamic, pituitary and ovarian

47

------'- - 48

hormonal responses to abrupt reductions in photoperiod which are known to lead to the anestrous condition. - To determine the short term effects of reduced photoperiod on pituitary hormones in the absence of gonadal steroids. CHAPTER 3

EXPERIMENT I. A HISTOCHEMICAL STUDY OF THE SITES OF STEROID SYNTHESIS IN THE OVARIES OF BLINDED, FEMALE HAMSTERS

Introduction Ovaries from hamsters which have become anovulatory as a result of optic enucleation or short photoperiod (SP) exposure are characterized by few small and mostly atretic follicular elements, the absence of corpora lutea and pronounced interstitial tissue hypertrophy (Reiter, 1968, 1969; Sorrentino and Reiter, 1970; Reiter and Johnson, 1974a,b) • This study was designed to examine histochemically the ovaries of hamsters induced into the anovulatory conditions by blinding. Based on the hypertrophic condition of the interstitial tissue we expected to find a histo­ chemical picture indicative of altered activity iri steroid synthesis. An attempt was made to determine the localization and relative activities of enzymes related to steroid production, and to compare these activities with observations made in the ovaries of hamsters displaying regular estrous

cycles. The following e~zymes were studied: 3-beta­ hydroxysteroid dehydrogenase (3-beta-HSDH), an enzyme necessary for the synthesis of all biologically active steroids yet in the ovary generally related to progesterone

49

...- ._-- -_ .•... _---_._-----_._-_ .. -.- ---- .._._- _.- .. -- -_._._------50 synthesis; NADH and NADPH diaphorases, which are enzymes required for NAD- and NADP-linked dehydrogenases; and glucose-6-phosphate dehydrogenase (G-6-PDH), an important NADPH generator for the hydroxylation steps. The localization and intensity of the enzymatic activity was visualized by formazan precipitation from tetrazolium salt over areas of dehydrogenase activity (Wattenberg, 1958).

Material and Methods Young adult female golden Syrian hamsters were obtained from Lakeview Hamster Colony (Newfield, NJ) and accl imatized under long photoperiod (LP) (14L: 10D); 1 ights on at 0600 h) with food and water provided ad libitum. Animals were monitored for regular estrous cyclicity according to the method described by Orsini (1961). Nine animals were blinded by bilateral optic enucleation under ether anesthesia. After nine to thirteen weeks three intact hamsters from each day of the estrous cycle (a total of twelve), and blinded acyclic animals were sacrificed by decapitation. The experimental procedures were such that only four to five ovaries could be successfully processed on one day. Therefore, the data were collected over five different days, including always ovaries from intact and blinded animals on anyone day. One ovary from each animal was embedded in OCT compound and rapidly frozen over dry ice, 14 ~m sections cut in a cryostat maintained at -18 0 to -20 0 C, and the tissue 51 sections mounted on microscope slides. Incubation rings were coated on the attaching surface with silicone grease and placed around the tissue sections. The liquid-proof tub so formed was filled with one of the following appropriate incubation media and incubated at 37°·for a period to demonstrate the following enzyme activity: 1) 3 beta-HSDH activity - 1.0 mg/m1 of a steroid substrate was dissolved in dimethylformamide and added to 0.1 M phosphate buffer (pH 7.4) containing 1.5 mg/ml beta-NAD and 1.0 mg/ml Nitro-Blue Tetrazolium (Nitro-BT). Pregnenolone, dehydropregnenolone and 17 alpha-hydroxypregnenolone were used as substrate. 2) G-6-PDH activity - 1.5 mg/ml D-Glucose- 6-phosphate, 0.5 mg/ml NADP and 0.5 mg/ml Nitro-BT were dissolved in the phosphate buffer. 3) NADPH and NADH diaphorase activity - 0.5 mg/ml of the nucleotides and 0.5 mg/ml Nitro-BT were dissolved in the phosphate buffer. Control sections were processed for all incubation media by leaving out the substrates (e.g. steroid precursors, D-Glucose-6-phosphate, NADH and NADPH). Sections for the demonstration of diaphorase activity were incubated for 15 minutes, whereas all other sections were incubated for two hours. Following incubation the rings were removed and the sections were rinsed with distilled water, fixed in 10% neutral formalin for ten minutes, dehydrated and stained with 52

eosin. The histochemical staining methods were adapted from Saidapur and Greenwald (1978b). The second ovary from intact and blinded animals was fixed in Bouin's solution for 24 hours followed by dehydration and embedding in paraplast. Sections of 7 llm thickness were cut on a microtome, attached to glass slides and stained with hematoxylin and eosin (H and E).

Observations Intact cycling animals - as seen in Plate 1, NADH activity was weak in preantral follicles but increased in antral follicles in which the strongest reaction was noted over the theca interna, Atretic follicles and corpora lutea also showed a strong reaction product. The reaction intensity of the corpora lutea changed over the different phases of the cycle and was strongest on the second day after ovulation, the time steroidogenesis is at a maximum. In the interstitial tissue intense activity was observed on all days of the cycle. NADPH and G-6-PDH showed a similar pattern (Plate 2), with most intensity noted in antral follicles, especially over the granulosa cell layers and in corpora lutea, Only a weak reaction product was noted in the interstitial tissue. Ovarian sections processed for 3-beta-HSDH activity (Plate 3) resulted in a weaker reaction product, but the general distribution was similar to the one seen for NADH 53

diaphorase, with reaction products noted in antral follicles, corpora lutea, interstitial tissue and some atretic follicles. Anovulatory animals - NADH diaphorase showed strong activity throughout the ovary (Plate 4), but was most intense around remnants of atretic follicles in which the granulosa cells were degenerating or had disappeared and the surrounding theca interna had hypertrophied, forming a thick band around former follicles. Healthy primary follicles showed only weak reaction products. NADPH diaphorase and G-6-PDH had similar distributions with highest activities noted in cortical regions around follicles in different stages of atresia (Plate 5). Generally, all steroid substrates used to demonstrate 3-beta-HSDH activity gave similar reaction intensity (Plate 6). The distribution of this enzyme was similar to that seen for NADH diaphorase, but the reaction product weaker. Compared with the activity in the ovary of cycling animals, the reaction intensity of diaphorase and G-6-PDH in the interstitial tissue of anestrus hamsters appeared to be more intense. 3-beta-HSDH activity also seemed to be as intense or slightly increased. Microscopic evaluation of the stained ovarian sections confirmed the cyclic stage of each intact animal. On the day of estrus large preantral follicles with approximately eight to nine layers of granulosa cells were the most dominant 54

feature of the ovaries. The newly formed corpora lutea, although in the process of luteinization, still demonstrated the features of collapsed follicles. On diestrus I the recruited follicles were fully luteinized, large and highly vascularized. On diestrus II all recruited follicles possessed large antral cavities and generally had a healthy ·appearance. Signs of regression were visible in the corpora lutea and its blood supply diminished. On proestrus (Plate 7) some of the large antral follicles started to show degenerative changes, and the corpora lutea were regressed and invaded by macrophages and white blood cells. The appearance of the interstitial tissue compartment did not change over the different days of the cycle. Ovarian histology was drastically different in blinded animals, and did not resemble any stage of the estrous cycle in intact controls. Follicular development was nearly absent, and most of the ovaries consisted mainly of interstitial tissue (Plate 8). Direct comparison of the interstitium from the two treatment groups revealed dramatic differences in its amount and cellular morphology. In addition to the increase in amounts of interstitial tissue in blinded animals, a rich blood supply was evident. This apparent increase is at least partly due to the increased size of individual cells, which took on a polyhedral appearance and stained strongly eosinophilic, an appearance which corresponded well with the increased enzymatic activity observed in the frozen

. _...... _.. _- ~ .. - _. _. -.~ ---.-.---~-., ...... -- ..• - ._. __ .. - ... --.. ~ .. - _._-_ .. _----- 55

sections. Follicular development was sparse and few follicles had more than six to seven layers of granulosa cells. The few follicles which occasionally developed further became atretic at the beginning of antral formation; atresia of small preantral follicles was a common feature. Most frequently, only a few degenerating granulosa cells or remnants of the zona pellucida remained, surrounded by a broad band of pseudotheca. The pseudotheca, which is named for its resemblance to the theca interna of antral follicles, is interstitial tissue surrounding the degenerating follicle and becomes temporarily activated by the latter to produce progesterone (Knigge and Leathem, 1956). In the steroid­ histochemical study the enzymatic activity of this tissue was clearly seen as patches of increased activity around remnants of follicles.

Discussion The histochemical observations in control animals displaying estrous cycles were generally in agreement with observations made by Saidapur and Greenwald (1978b). Ovaries of blinded acyclic hamsters have not been examined with these techniques previously and the results were revealing. The hypertrophic interstitial tissue, which, in this reproductive condition makes up most of the ovary, was shown to be highly active enzymatically, and potentially capable of synthesizing large amounts of progesterone. Light microscopic 56 observations (H and E sections) of these ovaries revealed large eosinophilic interstitial cells surrounded by a rich blood supply. The histochemical observations in this experiment support the hypothesis that ovaries from acyclic hamsters are active in steroidogenesis, and show additionally that the most active interstitial tissue was found in areas in which preantral follicles were undergoing or had recently undergone degeneration. Atresia of preantral follicles was also present in animals displaying estrous cycles although to a far lesser extent. Relating this information to the hormonal profile seen in light-deprived acyclic animals helps to explain the obser­ vations. In the acyclic condition animals have low LH and FSH levels in the morning, and display daily afternoon gonadotropin surges (Bridges and Goldman, 1975; Seegal and Goldman, 1975; Bittman and Goldman, 1979). Since the inte~stitial tissue contains LH receptors and has the capacity for steroidogenesis (Silavin and Greenwald, 1984), daily LH surges may activate the tissue, causing hypertrophic and probably hyperplastic changes and the activation of progesterone synthesis. In cycling animals, the interstitial tissue contri­ butes significant amounts of progesterone only in response to the proestrous gonadotropin surge (Saidapur and Greenwald, 1979a), yet as a recent experiment showed (Exp. IV) it may be capable of the synthesis of large amounts of progesterone. 57

When progesterone levels of cycling animals were compared directly with those seen in SP-induced acyclic females, it was seen that morning levels of acyclic animals were comparable to levels seen on the day of estrus, and levels measured during the afternoon LH surge reached values comparable to those found on diestrus 1, the day progesterone levels reach highest values during the estrus cycle. High levels of interstitial progesterone production might explain the severe depression of folliculogenesis even though daily FSH surges occur in anestrous hamsters. Progesterone is often considered an anti-estrogen, inter­ fering with estrogen activity in various ways at hypothalamic and pituitary levels, and directly on the ovary as well. If given during the estrous cycle, progesterone retards folli­ cular growth and extends the length of the cycle (Greenwald, 1977). In hypophysectomized PMS-treated hamsters progesterone severly interferes with follicular development (Moore and Greenwald, 1974). Part of its mechanism of action appears to involve interference with FSH-induced aromatase activity on granulosa cells, and the inhibition of theca cell androstenedione production necessary for estrogen synthesis (Fortune and Vincent, 1983; Garza and Terranova, 1984). In acyclic animals follicles appear to be stimulated to grow, but in the presence of high progesterone levels are usually not able to develop beyond the multilayered stage without becoming atretic. Only a significantly increased release of 58

FSH, as for example that which occurs at the termination of lactation or during the pubertal stage (Greenwald, 1965; Donham, DiPinto and stetson, 1984), and most likely during the period of recrudescence in the annual reproduction cycle, appears to be able to override this futile cycle. In summary, although reproductive activity ceases in light-deprived female hamsters, the ovaries are highly active enzymatically with regards to steroidogenesis. This activity is indicative of high progesterone synthesis which probably further assists the suppression of folliculogenesis and maintenance of a quiescence reproductive state. 59

Plate 1. NADH Diaphorase Activity in the Ovary of a Control Hamster on Diestrus II.

Plate 2. Glucose-6-Phosphate-Dehydrogenase Activity as Seen on Proestrus in a Control Ovary. 60

Plate 3. 3 Beta-Hydroxysteroid Dehydrogenase Activity Observed on Proestrus in the Ovary of a Control Animal.

Plate 4. NADH Diaphorase Activity in the Ovary of a Blinded Acyclic Animal 61

Plate 5. Glucose-6-Phosphate-Dehydrogenase Activity Seen in the Ovary of a Blind Animal

Plate 6. 3 Beta-Hydroxysteroid Dehydrogenase Activity Seen in the Ovary of a Blind Hamster 62

Plate 7. Hand E Section of an Ovary from an Animal On Proestrus Day,

Plate 8. Hand E Section from the Ovary of a Blinded Animal. CHAPTER 4

EXPERIMENT II. RAPID CESSATION OF ESTROUS CYCLICITY AND DEPRESSED CASTRATION RESPONSE IN SHORT PHOTOPERIOD-TREATED, INBRED LSH/SsLak HAMSTERS

Introduction Although there is extensive information about the SP-induced acyclic hamster, little is known about the events leading to the cessation of ovarian cyclicity. The diverse response to SP treatment of outbred hamsters, and the dynamic histological and hormonal changes occurring throughout the four day cycle, complicate any straightforward experimental approach. We attempted to reduce the first problem by incorporating a strain of inbred hamsters whose response to SP were postulated to be more homogeneous. Therefore, the primary object of the experiment outlined below was to explore the temporal aspects of the response of LSH/SsLak hamsters with regard to cessation of estrous cyclicity. In addition, we examined the effects of SP exposure on the castration response in these inbred animals in regard to morning levels and timing and magnitude of the afternoon gonadotropin surge.

Materials and Methods Forty-six, eight to ten week old, LSH/SsLak female hamsters were obtained from Charles River breeding colony

63 64

(Wilmington, MA). The animals were housed in clear plastic cages (5-6 animals/cage) in a long photoperiod (LP) (L/D = 14/10 h; lights on at 0600 h M.S.T.) with food and water provided ad libitum. All animals were monitored for regular estrous cyclicity over four to five cycles according to the method of Orsini (1961), and animals displaying irregular cyclicity were excluded from the experiment. After this initial period, 26 animals were transferred into specially constructed light-tight and well-ventilated metal chambers in which a short photoperiod (SP) (L/D = 8/16 h; lights on at 0600 h) was maintained. The light source consisted of fluorescent GE light bulbs (#F20T12-CW) mounted above each shelf, and air flow was maintained at approximately 32 cu ft/minute. All animals were checked daily for vaginal discharges and, on the first day after the first missed estrous dis­ charge (five days after last ovulation), the acyclic animals were bilaterally ovariectomized along with a LP control in diestrus one. The ovaries were fixed in Bouin's solution and processed for histological (light microscopic) observations. To determine the optimum time for the measurement of afternoon surges at 29 to 33 days post-ovariectomy, intra­ atrial silastic cannulae (Dow Corning #602-155) were inserted via the jugular vein under Nembutal anesthesi~ (70 mg/kg, ip) between 0600 hand 1000 h and filled with physiological saline containing heparin (100 U/m1). Over the following two 65

days, 0.6 ml blood samples were collected via the cannulae at 0700 h, 1200 h, 1400 h, and 1600 h in SP animals, and at 0700 h, 1400 h, 1600 hand 1800 h in LP controls. After each sampling time, blood cells were separated from serum by centrifugation, resuspended in 0.9% saline and the suspension returned through the cannulae immediately following removal of the next blood sample. The animals were sacrificed by decapitation on the third postcannulation day, either in the morning between

0800 ~ and 0900 h or in the afternoon (.SP animals at 1300 h, LP animals at 1500 h). The ovaries were embedded in para­ plast, serially sectioned and every tenth section mounted on a glass slide and stained with hematoxylin and eosin for histological (light microscopic) evaluation. Pituitaries were weighed, sonicated in 500 ul O.OIM PBS (pH 7.6) and stored at -20 0 C for hormonal determination. Blood components were separated by centrifugation and the sera stored at -20 0 C. FSH and LH levels were measured in all samples with NIADDK radioimmunoassay kits which included rFSH-RP-2 and rLH-RP-2 as reference standards. PRL levels were measured in the samples taken at the time of sacrifice with the homo­ logous radioimmunoassay developed by Soares, Colosi and Talamantes (1983). Student's t-test and one-way analysis of variance were applied for statistical analysis of the data.

------. - --- - . 66

Results All SP-treated animals ceased ovulatory cycles between 14 and 34 days after transfer to the short photoperiod (mean

+ S.E. = 23.2 ~ 0.8 days; median 22.5 days), whereas LP animals maintained regular estrous cyclicity (Fig. 1). Interestingly, 25% of these animals displayed one five day cycle prior to the last ovulation. Histological observations confirmed the absence of recent ovulations; the ovaries contained no new corpora lutea, numerous atretic follicles, many of which had reached a large antral stage and few small healthy follicles. The interstitial tissue was stimulated to different degrees. Generally, more stimulation was observed when fewer atretic antral follicles were present or when follicles were in an advanced state of atresia. Pituitary weights were significantly reduced by

SP treatment, with means of 4.62 ~ 0.21 mg in LP-treated vs. 3.51 +,0.072 mg in SP-treated animals (P < 0.001). This difference was also apparent when pituitary weights were adjusted for body weight. In Fig. 2 it is seen that tonic LH levels were significantly lowered in SP (P < 0.001), and the daily surges, advanced by about two hours, were reduced compared with LP animals. It was noted that the magnitude of the afternoon surges in SP-treated hamsters was lower on day one and significantly decreased on day two compared with LP

.. _ .... __ ._._------_._---_ .. __ .-_._-_. __ ._------_...... - .. 67

animals (P < 0.1 on day one; P < 0.001 on day two). Pitui­ tary LH content (Fig. 3) was also significantly lowered in

SP-tr~ated animals compared with LP controls (P < 0.001). Fig. 4 shows that serum FSH levels in SP-treated animals were consistently lower than those of LP-treated hamsters (P < 0.001), and exhibited afternoon surges which paralleled the LH pattern (P < 0.001). On the other hand, serum FSH levels were consistently high in LP controls and did not demonstrate distinct surges. Similar to LH content, pituitary FSH content was significantly reduced by SP treatment to a value which was 21% of that for controls (P < 0.001) (Fig. 5). Both pituitary and serum levels of PRL were similarly significantly reduced (P < 0.001) in response to SP treatment (Figs. 6 and 7).

Discussion The response of LSH/SsLak hamsters to SP treatment was surprisingly rapid and uniform, in marked contrast to that for outbred animals in which three to nine weeks of SP exposure or binding is required to induce the acyclic state (Bridges and Goldman, 1975; Bittman and Zucker, 1977; Hauser and Benson, unpublished data). This homogeneous response would appear to make these inbred hamsters highly suited for studies designed to provide information about the neuro­ endocrine events which take place early after transfer to SP. 68

In contrast to the ovaries of long-term acyclic animals, the ovaries of SP-treated animals, removed only six days after their last ovulation, contained numerous large antral follicles in different stages of atresia. The presence of large antral follicles suggests that these animals had a normal secondary FSH surge six days earlier, but that the maintenance of follicular growth was impaired, resulting. either in estrogen levels which were insufficient to elicit a proestrous gonadotropin surge, or in atretic follicles unable to respond to such a surge. Based on the observation that 25% of the animals displayed one five-day cycle prior to becoming acyclic, it appears more likely that the former defect is present, i.e., that follicular develop­ ment becomes impaired to the extent that insufficient estrogen is synthesized to elicit a proestrous gonadotropin surge. The interstitial tissue showed different degrees of stimulation, but, at five days after last ovulation, was not developed to the extent seen in animals anovulatory for weeks (Reiter, 1968, 1969, 1974; Sorrentino and Reiter, 1970;

Rei~er and Johnson, 1974a,b; Hauser et a1., 1982, EXp. I). These observations agree with a recent report in which the presence of gonadotropin surges was correlated with ovaries characterized by stimulated interstitial tissue and a lack of antral follicles, while ovaries containing large antral follicles and little stimulation of the interstitium were observed in animals showing no afternoon surges (Jorgenson 69

and Schwartz, 1985). In addition, consistent gonadotropin surges were only present 16 days after last observed ovu­ lation. These observations strongly suggest that gonadotropin surges develop as a result of a deficiency of the combination of ovarian hormones able to suppress daily surges. In hamsters, daily surges in gonadotropin levels are present in the absence of the ovaries (Stetson, Watson-Whitmyre and Matt, 1978; Stetson et al., 1981), are magnified by estrogen, do not seem to be affected by pro­ gesterone alone and can be suppressed only by the combination of estrogen and progesterone (Norman, Blake and Sawyer, 1973; Norman, 1975). In the ovaries of acyclic animals, small amounts of estrogen (Widmaier and Campbell, 1981; Exp. IV) and high levels of progesterone are present (Bridges and Goldman, 1975; EXp. IV); this combination may be unable to suppress the daily trigger of gonadotropin surges. These daily surges, which stimulate the interstitium to produce high levels of progesterone (Bridges and Goldman, 1975; Donham, DiPinto and Stetson, 1984; Silavin and Greenwald, 1984; Taya Saidapur and Greenwald, 1980; Exp. IV) might secondarily impair follicular development and promote the acyclic condition. The LH profile seen in ovariectomized LP- and SP-treated hamsters has been reported previously (Goldman and Brown, 1979; Seega1 and Goldman, 1975; Bittman and Goldman,

------70

1979; Jorgenson and Schwarz, 1984) and the observed suppres­ sion of tonic LH levels by SP is in agreement with these findings. Yet the daily surge levels seen in the present experiment were decreased by SP treatment, in contrast to previous findings in which surges were increased by SP treatment. These differences might be due to sampling times, different time spans between ovariectomy and blood collection and/or a more pronounced photoperiodic response of these inbred animals. The differences seen in FSH levels between the two treatment groups were even more dramatic than the differences in LH values. Whereas LP-treated ovariectomized animals had no discernable afternoon FSH surges, they had significantly higher serum FSH levels at all times than SP-exposed ovariec­ tomized animals, which showed a clear surge pattern. This pattern has been reported previously (citations), and it might indicate that SP treatment affects tonic and surge levels differently. Perhaps one of the most important, early changes which results in the anovulatory condition is the drastic reduction in FSH release. FSH is known to be of critical importance in follicular recruitment, induction of aromatase activity and general maintenance of follicular growth (Erickson and Hsueh, 1978; Greenwald and Siegel, 1982; Kim and Greenwald, 1984); changes in any of these events could interrupt the estrous cycle. 71

To our knowledge, the reduction in pituitary gonado­ tropin content by SP treatment has not been reported pre­ viously for ovariectomized female hamsters. This reduction could result either from prolonged decreased hormonal release, over a period of time, decreased synthesis or increased degradation. An alternate, but less attractive hypothesis, since there is little evidence, is that of a direct photoperiod-mediated effect on the pituitary gland. The observed reduction of PRL has been seen in previous experiments in which heterologous radioimmunoassays were employed. Two experiments by Reiter and Johnson (1974a,b) showed decreased pituitary PRL content, yet unchanged serum levels in SP-exposed acyclic animals. Since estrogen stimulates PRL synthesis and release (Meites et al., 1972: Maurer and Gorski, 1977; Vician, Shupnik, and Gorski, 1979), and acyclic animals synthesize low to undetectable levels of this steroid (Widmaier and Campbell, 1981; Jorgenson and Schwartz, 1985; Exp. IV) it is not clear whether the reduction in pituitary PRL seen in intact acyclic animals is a direct consequence of SP exposure or results from reduced circulating levels of estrogen in anestrous animals. The study by Widmaier and Campbell (1981) and the present study support the idea that reduced PRL may be caused by the combination of both. They studied SP effects on serum PRL levels in ovariectomized hamsters implanted with either estrogen-filled or empty capsules, and demonstrated that,

- .- _ .... - _._------_._-----_ .. _ ...._-_._- -_._._------_._-_ ... _------72

indeed, PRL levels were affected by both treatments in opposite ways, i.e., stimulated by estrogen and suppressed by SP treatment. Overall, the photoperiodic response of the LSH/SsLak inbred species is similar to that seen in outbred animals, yet its response is more rapid and uniform. These characteristics make the LSH/SsLak female hamster well-suited for exploration of the changes which occur prior to the cessation of cyclicity. The information gained by this experiment should allow us to design future experiments which focus on events occurring prior to the cessation of cyclicity and which are causative in the production of the acyclic condition.

------_._------.. _- .' .-- .. _._ ...... _--_._------73

100

Cl C

(J >. (.)

I/) -\U E c < ~ 20 10 0 4 e 12 16 20 24 28 32 36 Days after transfer to SP ( LI 0=6/1 8 )

Figure 1. Effects of SP on Estrous Activity. The vertical axis depicts the .percentage of cycling animals while the horizontal axis shows the numer of days of SP exposure. 74

_ Short Photoperiod (L/D:8/16) 800 ______Long Photoperiod (LlC=14/10) 700 I, I '

:: sao I \ e ,I: \1 .... I CI L , I \ I I I \ \ .5 500 I f I I . ! ,I I I I I , I I :

a .'1 ILlC:14/101 I t.::::~~=~.~~Ii L/C= 8/1 st.::::::::=:1 •••• 08.0011 10.0011 14.0011 18.0011 22.0011 08.0011 10.0011 14.00h 18.0011 22.0011 DAY I DAY II

Figure 2. Serum LH Profiles of SP- and LP-exposed Ovariectomized Animals During the First and Second Post-cannulation Da~ Each time point is a mean + S.E. of 8 to 15 samples. Tonic levels were significantly reduced by SP at all time points (P < 0.001). LH surges, which occurred at approximately 1400 h in SP animals and at 1600 h in LP controls, were also depressed by SP treatment (P < 0.1 on postcannulation day I and P < 0.001 on day II). 75

3

"Cl ...... ::l.. 10 "Cl c: E -G) .... Cl -c: 2 ::l.. 0 ...... (J J: J: -I -I >-... >-... as as 5 ~- -:I a::- *** a.-

o o Photoperiod: long short

Figure 3. Pituitary LH Levels in SP- and LP-treated Ovariectomized Animals. Open bars depict total content, hatched bars tissue concen­ trations. Means + S.E. are indicated. *** = P < 0.001. 76

_ Short Photoperiod ( L/D-8/16) 40 ______Long Photoperiod (L/D-14/10)

~ 30 ...... sCI :: ~------+--t/~ ,'~,,'~ ,~ 20 ~------i" . E 2 enCD

LID • 14/10 t==-=::;JJ __II c.I ::::::::=~!lII.. LID - 8/16 . 06.00 10.00 14.00 18.00 II22.00h 06.00 10.00 14.00 18.00 22.00h DAY I DAY II'

Figure 4. Serum FSH Patterns in LP- and SP-treated, Ovariectomized Hamsters Over the First and Second Post-cannulation Day.

Each time point is a mean + S.E. of 8 to 15 samples. Serum levels of FSH were significantly depressed in SP animals at all time points (P < 0.001). 77

400

...... Cl c 1500 ~ ...... c 300 Cl -Q) .....E -c Cl 0 C (,) ~ 1000 :I: :I: CI) 200 CI) u. u. ..>- >- as ..as -:::l '5- 500 ••• ii:- 100 a.-

o o Photoperiod: long short

Figure 5. Pituitary FSH Levels in LP- Versus SP-treated Ovariectomized Hamsters. Open bars represent mean + S.E. of total content; hatched bars tissue concentrations. *** = P < 0.001. 78

6.

..... 5 CI ...... ::l. .J 4 ..... a: CI a...... E » CI as~ ...... ::1. .J -::I a: 10 a.. a.. 2 as ••• 0 ... 1

(14)( 13) 0 Photoperiod: long short

Figure 6. Pituitary PRL Content in LP- and sP-treated Ovariectomized Animals. Mean total content is depicted by open bars, tissue concen­ trations by hatched bars. *** = P < 0.001. 79

16

14

12 ....E Cl ....,c 10 ..J a: 0- 8 E ...::J 6 enCD 4

2 •••

( 1 4) 0 Photoperiod: long short

Figure 7. Serum PRL Levels in SP- and LP-Treated Ovariectomized Animals Measured at the Time of Sacrifice on the Third Postcannulation Day. *** = P < 0.001. CHAPTER 5

EXPERIMENT III. EARLY SHORT PHOTOPERIOD EFFECTS IN FEMALE LSH/SsLak GOLDEN SYRIAN HAMSTERS

Introduction In this study we examined the response of regularly cycling animals to SP exposure over various periods of time. The focus was on the morning of the day of estrus (day one) for several reasons: the secondary FSH surge which occurs on this day is a pivotal event in the four day cycle, since it is thought to be responsible for the recruit­ ment of follicles destined to either ovulate or become atretic three days later (Greenwald and Siegel, 1982). A depression of this surge could lead to a reduction of follicular growth, the disruption of the estrous cycle and eventually to the acyclic condition. Light microscopic evaluation of the ovaries at this stage of the cycle would, in addition, provide information about the number of fol­ licles which were able to respond to this surge, and the destiny of follicles which were recruited in the previous cycle. An additional objective of this study was to measure gonadotropin levels in the morning and during the time of the expected afternoon surge in animals which had become acyclic as a result of SP exposure. The acquisition of these data 80 81 provided a direct comparison with levels observed in the SP-treated cycling animals, and with values seen in SP-treated ovariectomized inbred hamsters in a previously reported experiment (Hauser and Benson, 1986).

Materi~1s and Methods Fifty, eight- to ten-week-old LSH/SsLak inbred female hamsters were obtained from Charles River Breeding Laboratory (Wilmington, MA) and acclimatized for a period of four weeks in LP (14L:I0D; lights on at 0600 h). The animals were housed in clear plastic cages (five/cage) in a temperature­ controlled room (22 0 C) with water and food provided ad libitum. Regular estrous cyclicity was monitored over four to five cycles by the method of Orsini (1961). After this initial period of acclimatization, 40 animals were transferred individually on day one (estrus) of the ovulatory cycle into specially constructed, light-tight and well-ventilated metal chambers (air flow approximately 32 cu ft/minute) located in the same room. SP (8L:16D; lights on at 0600 h) was provided in these chambers. The light source consisted of fluorescent GE light bulbs (#F20T12-CW) mounted approximately 25 cm above the cages. After transfer to SP all animals were examined regularly for the presence of estrous discharges. Groups of six to eight animals were sacrificed by rapid decapitation between 0630 hand 0730 h on the day of estrus after four, eight, twelve or sixteen days 82 of SP exposure, i.e., after one, two, three or four four-day estrous cycles. In the last group, only six of the remaining animals displayed an estrous discharge and could therefore be included. Ten Lp-treated control animals were sacrificed at the same time of day on day one of the cycle. Trunk blood was allowed to clot at 40 C and the serum separated by centrifugation and stored at -20 0 C. Pituitaries were sonicated in 500 III O.OIM PBS (pH 7.6) and stored at -20 0 C for determination of FSH, LH and prolactin. Ovarian and uterine weights were recorded, and one ovary from each animal was fixed in Bouin's solution for histological evaluation. The ovaries were embedded in paraplast and serially sectioned at a thickness of 7.0 llm. Every sixth section was mounted on a microscope slide stained with hematoxylin and eosin, and healty growing follicles (225 llm and above in diameter), corpora lutea, and atretic antral follicles counted by double blind methods. Morning and afternoon gonadotropin levels were measured in ten intact females which had become acyclic two to three weeks before, after 8 to 16 days of SP treatment. The timing of the afternoon surge in SP was established by gonadotropin determinations in repetitive blood samples obtained at frequent intervals in a previous experiment. Silastic cannulae (Dow Corning #602-155) were fitted under Nembutal anesthesia (70 mg/kg) into the right atrium by insertion through the right jugular vein. On the first

----_.---_... - .... 83

postoperative day, 0.7 ml blood samples were withdrawn into

heparinized syringes at 0800 and 1400 h; the following d~y, an additional sample was withdrawn at 1400 h. Sera were separated by centrifugation after each blood collection, and the blood cells resuspended in 0.9% saline and re-injected through the cannulae following withdrawal of subsequent blood samples. On the third day, the animals were sacrificed by decapitation at 0900 h and the tissues treated in a manner similar to that described above.

Radioimmunoassay for Gonadotropins and Prolactin LH and FSH levels were determined with double-antibody RIA kits supplied by NIADDK, incorporating LH/FSH-RP-2 as reference standards. The homologous RIA developed by Soares, Colosi and Talamantes (1983) was employed for measurement of hamster prolactin (PRL).

Statistics Significant differences between treatment and control groups were determined by the two-tailed Student's t-test and one-way analysis of variance (ANOVA) followed by the Newman-Keul's multiple range test. Except where noted, significant differences are based on ANOVA.

Results Animals transferred from LP responded rapidly to SP treatment. By the sixteenth day of SP exposure, only six of 84 sixteen animals continued the display of estrous cycles, whereas all control hamsters in LP continued to cycle regularly. Serum PRL levels diminished as early as eight days after transfer into SP, but were significantly depressed after 16 days of SP treatment in cycling animals (P < 0.05), and underwent an additional reduction after ovarian cyclicity ceased (P < 0.005vs. day 16, Student's t test) (Fig. 1). Pituitary PRL content, on the other hand, was not affected while the animals were displaying regular estrous cycles during the period of SP exposure, but fell markedly once the animals reached the acyclic state (P < 0.01) (Fig. 2). As seen in Fig. 3 and 4, secondary surge levels of FSH and pituitary FSH content did not differ for hamsters maintained in the two photoperiods. Yet once the animals were anovulatory, they displayed low morning serum levels followed by daily aft~rnoon FSH surges,· and had significantly elevated FSH pituitary contents (P ~ 0.025, Student's t test). Similar to FSH levels, LH concentrations in sera and pituitaries (shown in Figs. 5 and 6) were not altered in the cycling SP-treated animals. On the other hand, once the animals ceased ovulation, daily afternoon LH surges appeared, and pituitary LH contents became elevated (P < 0.01). As depicted in Fig. 7, pituitary weights became reduced after 16 days of SP treatment (P <0.01) and underwent an additional reduction (P < 0.01) after estrous cyclicity 85 terminated. Uterine weights (Fig. 8) showed similar trends, becoming significantly reduced after 16 days of SP treatment (P < 0.01 for total weights; P < 0.01 for adjusted weights) and dropping drastically after cessation of ovarian cyclicity (P < 0.01). Ovarian weights did not show significant dif­ ferences in any of the treatment groups. The overall appearance of the ovaries revealed no striking dissimilarities between those of SP-treated, cycling hamsters and LP-treated animals, yet the tabulation of major ovarian structures showed some differences (Table 1). As can be seen, the number of recruited follicles was similar in all groups, correlating with the observation that the secondary FSH surge was not altered throughout the 16 days of SP treatment. In contrast to this finding, the number of newly formed corpora lutea was significantly less (P < 0.01, day 16) in the SP animals, and atretic antral follicles were apparently increased. Microscopic observations of the ovaries of acyclic hamsters confirmed the anovulatory condition, and revealed a highly stimulated interstitial tissue, few, mostly atretic, follicular elements and no corpora lutea.

Discussion In this study the response of the inbred hamsters to SP treatment was rapid; in fact, the induction of the acyclic state was faster than that seen in a previous experiment

------.------86

(Hauser and Benson, 1986; Exp. II). Considering this marked response, it was quite surprising to learn that, at the particular stage of the estrous cycle studied, only minor changes were observable in the SP-treated animals before the cessation of cyclicity. Thus, the termination of cyclicity appears to be a sudden event which may be difficult to detect. The secondary FSH surge was of equal magnitude in cycling, SP-treated hamsters and LP-treated controls, an observation which is in agreement with a recent finding in outbred hamsters (Jorgenson and Schwartz, 1985). Although FSH was measured at only one time point during a surge which lasts from the evening of proestrus until late in the morning on the day of estrus, the follicular counts support the conclusion that the secondary FSH surge and follicular recruitment were unimpaired during the period of SP treatment. These observations also indicate that an approximately equal number of follicles are able to respond to the secondary FSH surge, suggesting that follicular FSH receptor number and affinity are not altered by SP treatment. The observed reduction of serum PRL levels in SP-exposed hamsters displaying estrous cycles is to our knowledge a new finding. Low pituitary and serum levels have been observed previously in SP-induced acyclic hamsters (Reiter and Johnson, 1974a,b; alask et al., 1985). However,

. _._._ .. _------87

in the latter condition the ovaries synthesize scant to undetectable amounts of estrogen (Jorgenson and Schwartz, 1985; Widmaier and Campbell, 1981; Hauser and Benson, Exp. IV), and since PRL synthesis and release are strongly stimulated by this steroid (Meites et al., 1972; Maurer and Gorski, 1977; Massa, 1986), low PRL levels seen in the acyclic condition might occur secondary to reduction in ovarian estrogen secretion rather than as a primary result of SP exposure. The reduction of serum PRL levels in SP-treated cycling hamsters, observed both in this and in an additional study in which a comparable approach was used (Exp. IV), could be due to direct SP-mediated effects or indirectly as a result of reduced TRH release seen in SP-exposed hamsters (Vriend, 1983). The latter notion is supported by a study demonstrating that hypothalami from blinded acyclic hamsters resulted in more PRL release than hypothalami from intact animals (Orstead and Blask, 1985). The reduction of serum PRL levels could also be as a result of an increased clearance rate in response to SP exposure. Although the above observations are interesting, it is not clear what, if anything, depressed PRL levels signify to the animals. The physiological role PRL plays in cycling female hamsters is not yet resolved; one study in female hamsters in which its effect on folliculogenesis was examined (Kim and Greenwald, 1984) did not show a relationship. 88

Maybe more revealing than the hormonal levels were the observed changes in organ weights and ovarian histology. Although the content of pituitary hormones did not change in the SP-treated, cycling animals, pituitary weights were reduced significantly on day 16 of SP treatment, indicating that some other hormonal or histological change(s) not measured in this experiment must take place. On the other hand, the secondary drop in pituitary weight observed in acyclic animals may relate directly to the dramatic reduction of PRL content (Exp. IV). The observed reduction in uterine weight in SP-treated, cycling animals most likely reflects reduced ovarian estrogen synthesis. This notion is supported by the fact that fewer corpora lutea and a greater number of large atretic antral follicles were found in the SP-treated animals compared to the LP control animals. These combined observations suggest that, although equal numbers of fol­ licles undergo recruitment, maintenance of their growth may be impaired by SP treatment and consequently fewer follicles are able to complete ovulation. The ovarian histology suggests that SP might mediate its effects by interfering with the maintenance of follicular development. One possible mechanism suggested, based on studies in male hamsters and ewes (Tamarkin, Hutchison and Goldman, 1976; Turek, 1977, 1979; Bittman, Karsch and Hopkins, 1983), is that SP treatment increases the steroid feedback sensitivity of the hypothalamo-pituitary axis.

- -. ------.-.-----.. -_ ...._. --_. __ ._._._--.. _._---.------89

However, recent experiments (Hauser and Benson, 1985a; Exp. V) showed clearly that SP-exposed, ovariectomized female hamsters respond either similarly to, or are less sensitive to, steroid feedback interaction compared with LP control animals. Other experiments in this study showed that SP exposure decreases serum gonadotropin levels in the absence of gonadal steroids (Exp. V). The latter two changes might be difficult to detect in the intact cycling animal due to the interactions of ovarian steroids and peptides at a variety of levels in the control mechanism of gonadotropins. Certainly, when conclusions are drawn from the data presented, one should be reminded that the current study examined only one time frame out of a dynamic four-day cycle. Clearly, many major changes take place once the SP-treated animals cease ovarian cyclicity and these are more readily documented. Compared with cycling animals, pituitary and serum PRL levels become significantly reduced, most probably due to marked attenuation or termination of estrogen synthesis in the ovaries of acyclic animals (Widmaier and Campbell, 1981; Exp. IV). The observed increase in pituitary gonadotropin content in these acyclic animals is in agreement with previous findings (Reiter and Johnson, 1974a,b) and correlates with the higher hypothalamic LHRH content, possibly due to decreased release (Kumar et al., 1982; Exp. IV).

._------.. - _ ..- - .... 90

Animals which were allowed to become acyclic in SP displayed daily gonadotropin surges which were similar to those seen in outbred animals (Bridges and Goldman, 1975; Seegal and Goldman, 1975; Bittman and Goldman, 1979; Goldman and Brown, 1979; Jorgenson and Schwartz, 1985). Comparing

these levels with values seen in an experiment using 3~-day ovariectomized hamsters from the same inbred strain (Hauser and Benson, 1986; Exp. II), we found LH levels within the same range; however, tonic and surge levels of FSH (determined in the same RIA) were significantly lower in the

intact SP-treated acyclic animals (2.54 ~ 0.23 vs. 3.86 ~

0.20 at 0800 h, P < 0.001; 4. 92 ~ 0.45 vs. 8. 27 ~ 0.58 at 1400 h, P < 0.01). The latter indicates that the ovaries of the acyclic animals, which produce low to undetectable levels of estrogen, large amounts of progesterone (Bittman and Zucker, 1977; Jorgenson and Schwartz, 1985; Widmaier and

Cam pbe 11, 1981; Ex P . I V) and po s sib 1y 0 the r s t e r 0 ids and peptides, can nonetheless suppress FSH release either at pituitary or hypothalamic levels. To summarize, this study showed that SP treatment does not affect the secondary FSH surge and follicular recruit­ ment, but might interfere with the maintenance of follicular growth, thus leading to fewer follicles available to undergo ovulation and an increase in follicular atresia. Further­ more, the cessation of estrous cyclicity appears to be an abrupt event, maybe detectable only over one or two cycles.

------~---- - 91

It is clear that most drastic changes in ovarian histology and hormonal levels occur after the cessation of ovarian cyclicity and that PRL levels appear to be more dependent on circulating estrogen levels than on photoperiodic conditions. In general, the results also reinforce our notion that studies of female hamsters already rendered acyclic by SP do not allow an adequate reconstruction of events which lead to the acyclic state in the first place. 92

Table 1. Quantitation of Ovarian Changes in SP-Exposed Female Hamsters

Days of SP Treatment Ovarian Structures 0 8 12 16 Corpora Lutea 7.1+0.45 *5.25+0.36 *5.28+0.47 *5.0+0.25 Recruited Follicles 24.5+1.5 22.7+3.1 29.3+1.7 27.8+2.5 ( 225 ~m in Diameter) Large Atretric 2.4+0.5 1. 5+0.42 3.3+0.61 3.2+0.40 Antral Follicles

* = p < 0.02 vs. LP controls. 93

20

..J a: Q, e to ••• ..::I ~ en

5 •••

a (to) (7) (7) (8) (6) "LP SP SP SP SP SP Control 40 80 t20 t60 Acyclic

Figure 1. Serum PRL Levels of LP and SP-treated Cycling Animals and SP-induced Acyclic Animals are Shown. Samples were taken between 0630 and 0730 h on estrus after 0 days (LP controls), 4 days (40), 8 days (80), 12 days (120) and 16 days (160) of SP exposure and in animals acyclic for two to three weeks. Significant differences are indicated by P values derived by the Student's t test. Bars represent means and S.E.M. *** = P < 0.001. 94

60

-Cl 50 ~ C CD C 40 Q U ...I a: a. 30 >­.. CD ..>- 6 = CD :I

:I 20 a. 4

10 2

(8) (7) (8) (7) (8) (8) (6) (6) o LP SP SP SP SP Control 40 80 120 160 Acyclic

Figure 2. Pituitary PRL Levels are Shown for LP Controls, SP-treated Cyclic and Acyclic Animals. Open bars represent total contents, cross-hatched bars tissue concentrations. *** = P < 0.001.

------95

10

:J: en II. e ..:::I CD tIJ

•• 2

o (10) (7) (8) (7) (6) (10) (9) (9) LP SP SP SP SP , 4.00h 14.00h OS.OOh Control 40 80 120 160 Day 1 Day 2 Day 3

Figure 3. Secondary FSH Surge Levels of LP and SP-treated Cycling Animals and Levels Seen in Acyclic Animals. Cycling animals are represented by open bars and cross­ hatched bars represent A.M. and P.M. values for acyclic animals bearing intra-atrial cannulae. ** = P < 0.01 vs. all other groups. 96

400 125

.... QI oS 100 a 300 ~ C.. QI C oS 0 (J :t 75 ~ til II. II. ~ 200 >- ! ! ] ] 50 a:: a::

100 25

a (8) (8) (8) (8) (6) (6) (9) (9) a loP SP SP SP SP SP Control 40 80 120 160 Acyclic i

Figure 4. Pituitary FSH Levels are Shown for LP Controls, SP Cyclic and Acyclic Animals. Open bars represent total content; hatched bars indicate tissue concentrations. *** = P < 0.001; * = P < 0.025 vs. LP controls. 97

•..

••• -e ~ 300 J: ...J E 2 enIII

(9) (9) 14.00h 08.00h Control Day 1 Day 2 Day 3 Postcannulation Day

Figure 5. Serum LH Levels in LP and SP-treated Cycling and SP-induced Acyclic Animals. Open bars represent values in the cycling animals; cross­ hatched bars indicate levels in acyclic animals bearing intra-atrial cannulae. *** = P < 0.001 vs. all other groups. 98

20

,... 01 .... ::. 01 - 15 ~ C 01 ! ::. c .... a (J 40 :I:.... 10 ! >- ca.. a.. ;:, Ii: 5

10

(10) (10) (8) (8) (9) (9) 0 o LP SP SP SP SP SP Control 40 80 120 160 Acyclic

Figure 6. Pituitary LH Levels are Depicted for LP and SP Cycling and SP-induced Acyclic Animals. Open bars represent total contents; cross-hatched bars indicate tissue concentrations. *** = P < 0.001 vs. cycling animals.

~ .. ~--~-.~--~--.~-~ ~-~ ---- ~ .. -.--- ... ~. ------~--.------99

5

~ .... III CI ....e 4

OJ .s::- ...- CI ! Gl 3 3 .s:: :: CI ell :..... :: III :.. -; ... 2 2 III Q. :I

Q.

o o LP SP SP SP SP Control 40 80 120 160 Acyclic

Figure 7. Pituitary Weights of LP and SP-cycling and SP-treated Acyclic Animals. Total (open bars) and adjusted (cross-hatched bars) pituitary weights (mg/100g BW) are shown for LP and SP cycling and SP-induced acyclic animals. * = P < 0.05; *** = p < 0.001 vs. LP control animals. 100

400 400

-~ a ai E ... 300 300 E ....co II co ~ co E

o o Lf' Control 40 120 180 Acyclic

Figure 8. Uterine Weights in LP and SP-cyc1ing and SP Acyclic Animals. Total and adjusted uterine weights (mg/100g BW) are depicted for LP and SP cycling and SP-induced acyclic animals. *** = P < 0.001; ** = P < 0.01 vs. LP controls •

...... _._- ...... __ ...... _._._------CHAPTER 6

EXPERIMENT IV. HORMONAL CHANGES PRECEEDING THE SHORT PHOTOPERIOD-INDUCED ANOVULATORY CONDITION IN LSH/SsLak HAMSTER

Introduction The previous study explored short photoperiod (SP) induced changes in the cycling animal on the day of estrus. The results did not show any significant changes in the secondary FSH surge or follicular recruitment, yet ovarian histology revealed a tendency for fewer corpora lutea, and an increase in large atretic follicles in SP-treated animals, indicating SP-exposure might impair the maintenance of follicular development. These observations prompted us to compare hormonal levels and ovarian histology of long photoperiod- (LP) and SP-treated LSH/SsLak females on each day of the estrous cycle, and to compare these values with observations in the SP-induced acyclic hamsters. Based on the previous studies, it was postulated that follicular development and gonadotropin levels might be diminished on day three and four. The information derived should provide indications about mechanisms through which SP acts to achieve eventual quiescence of the female reproductive system.

101 102

Materials and Methods Eighty-six, ten- to twelve-week-old female golden Syrian hamsters of the LSH/SsLak strain were purchased from Charles River Breeding Laboratories (Wilmington, MA) and acclimatized to conditions of LP (14L:10D, lights on at 0600 h M.S.T.). The animals were housed in clear plastic cages with food and water provided ad libitum. All animals were monitored for regular estrous cyclicity over a minimum of four to five cycles according to the method of Orsini (1961), and three animals not displaying regular four-day cycles were excluded from the experiment. After this initial period, 54 hamsters were transferred individually to SP (8L:16D, lights on at 0600 h) on day one (estrus) of the cycle. This lighting regimen was maintained in specially constructed light-tight and well-ventilated metal chambers. The light source consisted of fluorescent light bulbs (#F20T12-CW) mounted approximately 25 cm above each cage, and air flow was controlled at an average of 32 cu ft/minute in order to maintain ambient temperature at 22 0 C. All animals were monitored for regular estrous cyclicity, and groups of SP-treated animals (eight animals/group) were sacrificed by rapid decapitation between

0900 hand 1000 h on day 15 (diestrus II) J day 16 (pro-estrus), day 17 (estrus) and day 18 (diestrus I) of SP treatment. Equal numbers of LP control animals were sacri­ ficed on each day of the estrous cycle between 1100 and 103

1200 h. Twenty-two SP-treated animals which were anovulatory for 6 to 34 days were sacrificed either between 0900 and 1100 h, or 1415 and 1430 h. Trunk blood was allowed to clot overnight at 40 C, the sera separated by centrifugation and stored at -20 0 C. The weights of the anterior pituitaries were recorded, the glands sonicated in 500 111 0.01 M PBS (pH 7.6) and stored at -20 0 C. The excised uteri were split, blotted dry, and their weights recorded. One ovary from each animal was immersed in Bouin's fixative and prepared for light microscopy. Serial paraffin

sections of 8 l.lm thickness were cut, every seventh section mounted on a microscope slide and stained with hematoxylin and eosin. The second ovary was suspended in 400 111 of 95% ethanol and kept frozen on dry ice. At the end of each collection period, 600 111 of distilled water was added, the tissue homogenized in a glass homogenizer and stored at -20 0 C for steroid determinations. Brains were removed, quick-frozen in liquid nitrogen and stored over dry ice until further dissection. The medial basal hypothalamus (MBH) and preoptic area (POA) blocks were isolated as follows: For the MBH, one transverse cut was made anterior to the median eminence, a second cut anterior to the mammillary bodies, two lateral cuts followed the hypothalamic sulci, and the horizontal cut was approxi­ mately 1.5 mm deep, resulting in a tissue block weighing an average of 11 mg. For the POA, the rostral cut was made 104

anterior to the optic chiasma, the lateral cuts along the hypothalamic sulci, and the horizontal cut was made at approximately the level of the anterior commissure. The tissues were weighed, immersed in 500 III of 0.1 N HC1, immediately sonicated and stored at -20 0 C for LHRH deter­ mination.

Evaluation of Ovarian Histology Four ovaries from each day of the estrous cycle were evaluated from animals maintained in each photoperiod. Corpora lutea, growing follicles and atretic antral fol­ licles were counted by double blind methods. Atretic follicles were divided into three stages: stage I. Only a few granulosa cells adjacent to the antral cavity undergoing atretic changes and occasionally the theca interna showing slight hypertrophy; stage II. Increased numbers of granulosa cells undergoing degeneration, WBC and macrophages starting to invade the antral cavity, the theca interna showing beginning hypertrophy and occasional ova beginning to degenerate; Stage III. All later stages of atresia in which an antral cavity is still discernable. Healthy follicles were counted and their sizes determined by measuring diameters. On cycle days one and two, all follicles with at least eight layers of granu­

losa cells (> 250 ~m) were included; on days three and four only antral follicles were counted. 105

Radioimmunoassays Prolactin levels were determined by the homologous radioimmunoassay (RIA) developed by Soares, Colosi and Talamantes (1983). LH and FSH measurements were made with the NIADDK kits employing LH-RP-2 and FSH-RP-2 as reference standards. Prior to the LHRH assay, the acid extracts were neutralized with 1 N NaOH, centrifuged twice and the pellets discarded. Hormonal determinations were made by routine RIA procedures, with the exception that alcohol precipitation was used instead of a second antibody. The antiserum was supplied by Dr. V. D. Ramirez, and the synthetic LHRH which was used for iodination and for the standard curve was purchased from Peninsula Laboratories (Belmont, CAl. All samples were processed in the same assay. Steroids were extracted from serum with petroleum ether, and their levels determined by RIA. The antibody employed for progesterone was provided by Hazelton Laboratories (under contract with Dr. Gary Hodgen at Eastern Virginia Medical School) and an incubation time of 24 hours at 40 C used. Samples were processed in three different assays and, to avoid differences caused by interassay variations, the same number of samples from each treatment group was included in each assay. Due to an inadequate amount of serum, estrogen levels were determined in ovarian homogenates. Steroids were extracted with ethyl ether and the estradiol antibody (E2TG-K) was kindly provided by 106

Or. Del Collins at Emory University. Incubation time and division of samples was similar to that described for the progesterone assay.

Statistics Significant differences between treatment and control groups were determined by Student's t-test or two-way analysis of variance (ANOVA) followed by Newman-Keuls Multi-range test. Except where noted, Student's t-test was used for comparison of group means. A P value of less than 0.05 was considered significant.

Results Animals which were allowed to become acyclic displayed their last estrous smear at an average of 21 days after SP exposure. SP treatment did not affect serum LH levels in cycling animals, yet once the animals became acyclic, daily afternoon surges appeared (Fig. 1). Pituitary LH contents (Fig. 2) fluctuated during the estrous cycle, yet no photo­ periodic effects were superimposed. Serum FSH levels (Fig. 3) indicate that on day one, the secondary FSH surge was slightly, but significantly, reduced in SP-treated animals (P < 0.01; Two-way ANOVA). Day two and three levels were within the same range in all treatment groups, while on day four there appeared to be a slight elevation in SP-exposed animals (P < 0.05; Two-way ANOVA). Once the animals were anovulatory, daily afternoon 107

surges were measurable. Pituitary FSH contents (Fig. 4) changed throughout the four day cycle, without apparent photoperiod effects except on day four, at which time SP-treated animals had slightly, yet significantly, increased levels (P < 0.01). Contents in the acyclic animals were in the same range as in cycling animals, yet showed a signi­ ficant decrease after the afternoon surge started

(P < .001). Serum PRL levels (measured only in cycling animals) were generally depressed in SP animals and significantly reduced (P < 0.05) on day one and day four (Fig. 5). Comparison of all SP and LP groups using Two-way ANOVA demonstrated that there was a significant difference (P < 0.001) between these photoperiod-exposed groups. On the other hand, pituitary PRL contents (Fig. 6) did not change in the cycling animals, but became significantly depressed after cyclicity ceased (P < 0.001). Since there were strong indications that there might be a correlation between pituitary weights, pituitary PRL contents and number of days an animal was anovulatory (based on last observed estrous discharge), correlation coefficients were calculated and the relationships graphically repre­ sented. The correlation coefficients for PRL vs. the number of days after last ovulation (Fig. 7) was 0.8171 (P < 0.001), adjusted pituitary weights vs. acyclic days (Fig. 8) 0.8187 108

(P < 0.001) and adjusted pituitary weights vs. pituitary PRL contents (Fig. 9) 0.7554 (P < 0.001). Fig. 10 depicts serum progesterone levels during the four day cycle and in anovulatory animals. A difference was seen in the SP-treated, cyclic animals on day one (after 17 days of SP exposure), at which time progesterone levels were slightly depressed (P < 0.05). In the acyclic animals, progesterone levels were low in the morning and approximately doubled (P < 0.001) in the afternoon. Total ovarian estrogen contents demonstrate no differences between LP- and SP-treated, cycling animals (Fig. 11). In the acyclic groups ovarian estrogen contents (morning and afternoon groups were combined since levels were not different) were below levels measured during any day of the cycle (P < 0.001). Since there was no correlation between the size of the dissected hypothalamic area and hormonal concentrations, MBH LHRH levels (shown in Fig. l2) are expressed as total contents. Morning levels of LHRH increased significantly between day one and day four of the estrous cycle (P < 0.001; Two-way ANOVA), but were not affected by reduced photo­ periodic conditions. Once the animals ceased ovulations, MBH LHRH contents (morning and afternoon levels combined since there was no difference) increased significantly (P < 0.001). LHRH values in the POA are expressed as concen­ tration per mg tissue since the dissected areas varied 109

in size, and a close correlation was found between sizes of dissected areas and hormone levels (P < 0.001). In Fig. 13 it is seen that the concentration of LHRH in the POA was increased in SP-treated cycling animals on day two, three and four (day two P < 0.005), and was further elevated once the animals ceased cyclicity. When the values for all cycling LP- and SP-treated animals were compared with Two-way ANOVA, a significant difference (P < 0.001) was noted as well. The organ weights are summarized in Table 1. Total and adjusted pituitary weights (weights/lOOg aW) were consistently decreased in SP cycling animals. This decrease became significant when all cycling SP- vs. Lp-treated animals were compared (P < 0.001 for adjusted weights; P < 0.05 for total weights). Once the SP-treated animals ceased ovarian cycles, a further large reduction in pituitary weights resulted (P < 0.001). Compared with LP controls, uterine weights showed a slight decrease in cycling, SP-trea­ ted animals (day three P < 0.025 for adjusted weights) and dropped significantly after cessation of ovarian cyclicity. Ovarian weights in cycling animals were affected neither by the cyclic stage nor oy photoperiodic conditions, yet were significantly increased in anovulatory animals. Overall, SP treatment did not induce significant differences in sizes and number of ovarian structures on the different days of the estrous cycle, although there was a tendency for fewer corpora lutea and an increase of atretic 110

follicles in SP-exposed animals. Also, three of the sixteen ovaries examined from SP-exposed cycling animals revealed marked changes in the number of growing follicles (described in greater detail in the discussion), changes which would have made it unlikely that the animals could have gone through another ovarian cycle.

Discussion Animals which were allowed to become acyclic ceased ovulation between 6 and 34 days of SP exposure (mean = 21 days). The timing of this photoperiod response is comparable with earlier observations in this inbred strain (Hauser and Benson, 1986; Exp. II), yet slower than in a most recent experiment (Hauser and Benson, 1984; EXp. III) in which the majority of the animals had ceased ovulatory cycles after 16 days of SP. Considering that these animals may have been only one to two weeks away from becoming acyclic, it was surprising to find so few differences between LP and SP cycling animals. Gonadotropin levels remained essentially unchanged in SP-treated animals, although FSH levels showed a slight, yet significant, reduction in the estrous surge and an increase on day four of the estrous cycle. There are several reasons to doubt that the observed decrease in the estrous surge reflects a real reduction: at 0900h, the time the blood samples were taken, the FSH surge levels drop sharply (Rani

------111 and Mougal, 1977), and any slight changes in blood sampling time or timing of the estrous surge could result in differences. In addition, the magnitude of the estrous FSH surge was unchanged by SP treatment in previous experiments using LSH/SsLak hamsters in which samples were taken at 0630 h at the peak of the surge (Hauser and Benson, 1984; Exp. III). This was also true in similar experiments reported by other investigators using outbred hamsters (Jorgenson and Schwartz, 1985). The apparent increases in serum and pituitary FSH levels on day four are more difficult to explain, especially since steroid levels and ovarian histology do not show significant differences. These findings are contrary to what one might expect if the hypothesis (based on observations in male hamsters) is correct that SP treatment increases the steroid feedback sensitivity of the hypothalamo-pituitary axis (Tamarkin, Hutchison and Goldman, 1976; Turek, 1977, 1979; Ellis and Turek, 1979). On the other hand, the above. observations support recent findings in this and another laboratory. In one of these recent studies, LSH/SsLak hamsters were ovariectomized and simultaneously treated with either empty or estradiol-benzoate (EB) filled, S.C. implanted capsules. After exposure to either LP or SP for twenty days, EB treatment was more effective in sup­ pressing serum gonadotropin levels in LP- than SP-exposed animals (Hauser and Benson, 1985a; Exp. V). A second study, 112

which was similar except that 17-beta-estradiol was used

ins tea d 0 fEB, g a ve com par a b 1 ere s u 1 t s ( Ex p • V). An ex p e­ riment using a similar approach in outbred ovariectomized hamsters (Jorgenson and Schwartz, 1984) also did not give any indication that increases in steroid feedback sensitivity were induced by SP. Another avenue, which is to our knowledge unexplored, is the possibility of an alteration in inhibin feedback on hypothalamic and/or pituitary levels. Inhibin activity has

be~n demonstrated in hamster ovaries and these animals respond to follicular fluid injections with a reduction of FSH levels (Chappel, 1979; Norman and Spies, 1980). It is feasible that levels of inhibin, and consequently FSH control, might be altered by SP treatment. Serum PRL levels showed a slight, but consistent, reduction in the SP-treated cycling animals compared with LP cycling animals, although pituitary PRL concentrations did not change while the SP-treated animals displayed estrous cycles. Similar observations were made in a previous study (Hauser and Benson, 1984; EXp. III) in which the SP-treated animals were also sacrificed before they ceased estrous cyclicity. In that experiment it was suspected that the reduction in PRL might be due to reduced estrogen synthesis, since uterine weights were significantly depressed before the animals ceased ovulatory cycles. The present experiment does not support this idea, since ovarian estradiol levels were

------113 found to be within the same range in all animals. As previously proposed, the decreased PRL release could be a direct pineal-mediated photoperiod effect (Reiter and Johnson, 1974a,b), or occur as a result of reduced TRH release (Vriend, 1983). Studies carried out in LSH/SsLak ovariectomized hamsters give the following indications: twenty days of SP-treatment, adequate to decrease serum gonadotropin levels,only suppressed PRL levels in one out of three studies (Hauser and Benson, 1985b), and estrogens were consistently stimulatory to pituitary and serum PRL levels (Hauser and Benson, 1985a; Exp. V). An experiment utilizing outbred, ovariectomized hamsters, in which SP treatment was extended for four weeks, showed significant depressions in serum PRL levels (Widmaier and Campbell, 1981), indicating that a longer period of SP treatment might be required for ovariectomized animals to achieve a reduction than that required for intact animals. The above described studies also d~monstrate the importance of estrogen with regard to PRL levels, and agree with the observations made in the present experiment in which a significant correlation was seen between pituitary PRL levels and the number of days an animal was anovulatory. They also point out the importance of taking secretion of ovarian hormones into consideration when evaluating PRL levels. MBH LHRH content increased significantly between day one and day four of the estrous cycle but was not altered by 114

SP exposure. A significant increase occurred only after the animals failed to ovulate and might therefore be due to the altered steroid milieu in anovulatory animals. To our knowledge no one has reported changes in hypothalamic LHRH in intact, SP-treated female hamsters. Kumar et a1. (1982) observed increased MBH LHRH content in SP-induced acyclic hamsters which were subsequently ovariectomized. The discrepancy between these observations and ours might be due to different experimental designs. LHRH concentrations in the POA did not appear to fluctuate during the estrous cycle, yet were higher in the SP-treated cycling animals and underwent another increase after cyclicity ceased. If real, the increase in POA LHRH in SP-cycling animals could be mediated by the pineal gland or through a direct photo­ periodic effect mediated by retinohypothalamic fibers (Pickard and Silverman, 1981). The latter notions are based on several findings. The pro-estrous surge in intact cyclic animals and the daily afternoon surge in ovariectomized or acyclic animals appear to be timed and triggered by the suprachiasmatic nucleus (Stetson and Watson-Whitmyre, 1976). The magnitude of the pro-estrous LH surge can also be modulated by photoperiod conditions (Moline et a1., 1981; Campbell and Schwartz, 1980). In a recent finding, increased afternoon LH and progesterone levels, coincident with the timing of the pro-estrous surge, were observed in SP-treated animals on the day of estrus (Jorgenson and Schwartz, 1985). 115

This increase occurred early after SP exposure and weeks before the animals be~ame acyclic. Direct photoperiodic input might also provide a reason for the observation that pinealectomy does not always totally reverse the effects of SP or blinding in female hamsters (B1ask et a1., 1985). The analysis of ovarian histology, which revealed no striking differences between the two treatment groups, supports the observations of hormone, and especially steroid, levels. Comparing these findings with a previous experiment, the results were not surprising. In that experiment, animals responded exceptionally rapidly to SP exposure, and at the time of sacrifice (day 16 of SP treatment) the majority of the animals were acyclic (Hauser and Benson, 1984; Exp. III). In spite of that fact, ovarian histology showed only a small, though significant reduction of corpora 1utea and a slight increase in atretic follicles. In the present experiment, the trends, e.g., fewer corpora lutea and increased atresia, were similar but less pronounced. Both experiments strongly suggest that the transition between ovarian cyclicity and acyc1icity is a sudden event, possibly only detectable over one or two cycles. This view is corroborated by the histological observations, in which three of the 16 ovaries examined from SP-exposed animals revealed drastic changes. One ovary had only four newly recruited follicles and the ovaries of two other animals had few, and smaller than normal, antral follicles accompanied by low 116 estrogen levels, making it unlikely that another cycle could have been completed. The results obtained in acyclic animals were of no surprise, and in agreement with previous experiments in this and other laboratories. All animals, even six days after their last ovulation, showed daily afternoon gonadotropin and progesterone surges and low to nondetectable ovarian estrogen levels. These findings deviate slightly from the obser­ vations by Jorgenson and Schwartz (1985), in which consistent gonadotropin surges were seen only after about 14 days of acyc1icity. The difference might be due to the more uniform and rapid photoperiodic response displayed by LSH/SsLak hamsters in this and previous studies. In conclusion, few detectable hormonal changes occur in the cyclic, SP-exposed hamster. Cessation of ovarian cyclicity appears to occur abruptly, and the lack of major changes may be due to hormonal feedback control at a local, pituitary and/or hypothalamic level. The increase in POA LHRH content seen in this experiment could be the result of decreased release, but there is also the possibility of a combination of direct photoperiodic (through retinohypo­ thalamic fibers) and pineal (humorally-mediated) photo­ periodic effects. Such dual and perhaps opposing systems of control could balance each other over specific periods of time until changes in one predominate, leading finally to the acyclic condition. Table 1. Short Photoperiod Effects on Organ Weights in LSH/SsLak Female Hamsters

Pituitary wt (mg) Ovarian Wt (mg) Uterine Wt (mg) Body Wt (g)

Total Adjusted Total Adjusted Total Adjusted

Day 1 LP 5.2+0.1 4.1+0.1 37.2+1.6-- 29.2+0.7 336.8+12.0 264.4+4.6 127.4+4.5 SP 4.7+0.2 3.6+0.1 38.1+0.8-- 29.8+0.6 324.0+11.6 250.7+5.9 128.1+3.9 Day 2 LP 5.0+0.3 3.8+0.2 37.0+1.9 27.8+0.6 258.7+11.4 195.2+7.0 133.0+5.9 SP 4.8+0.3 3.6+0.1 38.4+1.4 28.8+0.5 258.4+15.2 192.6+6.9 133.6+6.0 Day 3 LP 5.0+0.3 3.9+0.2 35.0+1.5 27.3+1.8 306.5+17.2 235.8+8.5 129.6+5.3 SP 4.5+0.2 3.4+0.1 35.5+1.5 27.2+0.9 273.3+12.4 208.6+6.4c 131.3+5.9 Day 4 LP 4.8+0.2 3.9+0.2 29.4+1.8 23.3+1.9 418.1+20.8 333.5+9.1 125.7+5.6 SP 4.6+0.2 3.6+0.1 32.8+1.2 25.9+1.0 376.1+23.6 298.7+20.6 126.7+5.2 Acyclic 3.7+0.1a 2.5+0.1a 47.7+1.9a 32.8+0.8a 118.1+6.2a 81.4+4.1a 144.4+3.7b,c a = P < 0.001 vs cycling animals; b = P < 0.01 vs LP and SP day 1; c = P < 0.02 vs LP and SP day 4. Adjusted = organ weights/100 g BW.

--...... 118

1.00 10.0

0.75 7.5 ..... E..... Cl ••• .s :5 0.50 5.0 ::E ::Ja: w (JJ

0:25 2.5

o ~~~(8~)~(~8)~ __~(~8~)~(8~)~~~(8~)~(~8~)~~(~77.)~(8~)~~~~~~(~1~1)~ __ 0 DAY 1 DAY 2 DAY 3 DAY 4 0900 h 1400 h ACYCLIC

Figure 1. Effect of SP Treatment on Morning Serum LH Levels on Each Day of the Estrous Cycle in SP-treated Cyclic (Left) and Acyclic (Right) Animals. Note that the V-axis has been reduced on the right side. Open bars = LP controls; hatched bars = SP-treated cycling animals; cross hatched bars = SP-treated acyclic animals. *** = P < 0.001. 119

1000 •••

...... CI .s 750

I­ Z W I­ oZ (.) ::I: 500 ..J >-a: < l- S I- a::: 250

o (a) (8) (8) (8) (8) (8) (8) (8) (20) DAY 1 DAY 2 DAY 3 DAY 4 ACYCLIC

Figure 2. Effect of SP Treatment on Pituitary LH Content on Each Day of the Estrous Cycle and in Acyclic Animals. Open bars = LP controls; hatched bars = SP-treated cycling animals; cross-hatched bar = SP-treated acyclic animals. *** = P < 0.001 vs. Day One and Day Two animals.

------_.. - 120

20

15 ••• ...... E ..sCl J: In u. 10 ::E ::l a: w In

5 "''''

o (8) (8) (8) (8) (8) (8) (8) (8) 1 0) 10) DAY 1 DAY 2 DAY 3 DAY 4 0900 h 1400 h ACYCLIC

Figure 3. SP Effects on Serum FSH Levels in Cycling . and Acyclic Animals. Open bars = LP controls; hatched bars = SP-treated cycling animals; cross-hatched bars = SP-treated acyclic animals. ** P = < 0.01 vs. LP controls; *** = P < 0.001 vs. morning levels.

------.. 121

500

.... CI ,.5 400 I- Z W I- oZ " 300 :z: (Jl ** *.* u. > a: ~ 200 :5 l- ii:

100

o (8) (8) (8) (8) (8) (8) 10) DAY 1 DAY 2 DAY 3 DAY 4 0900 h 1400 h ACYCLIC

Figure 4. Effect of SP Treatment on Pituitary FSH Content. Open bars = LP controls; hatched bars = SP-treated cycling animals; cross-hatched bars = SP-treated acyclic animals. ** = P < 0.01 vs. LP controls; *** = P < 0.001 vs. morning samples. 122

20

,.. 15 ....E ..sCI z ;::: * C,,) < 10 ...Jo a: * a. :E ::Ja: w (J) 5

o (8) (8) (8) (8) (8) (7) (8) (8) DAY 1 DAY 2 DAY 3 DAY 4

Figure 5. Serum PRL Levels in LP- and SP-treated Cycling Animals. Open bars = LP controls; hatched bars = SP-treated hamsters. * = P < 0.05 vs. LP controls. 123

100

...... :CI 75 I-z w I- Z 0 "z i= « 50 "...J 0a: a.. "> a:« l- 5 25 ••• a:I-

(8) (7) (8) (8) (8) (8) (8) (8) 20) o Day 1 DAY 2 DAY 3 DAY 4 ACYCLIC

Figure 6. Pituitary PRL Levels in Lp-treated Cyclic and SP-treated Cyclic and Acyclic Animals. Open bars = LP controls; hatched bars = SP-treated cycling animals; cross-hatched bars = SP-induced acyclic animals. *** = P < 0.001 vs. all other groups. 124

.... • Cl .....::I. I- Z W I- Z 0 () ..J 0:: C. >- 0:: 0( l- S l- ii:

0 10 15 20 25 0 35 DAYS AFTER LAST OVULATION

Figure 7. Correlation Between Pituitary PRL Contents and Number of Days After Last Observed Ovulation.

r = 0.8171; P < 0.001.

-_ ... _--_ .. --_...... _.. -- .... __ ._.-.. _--.. -_ .. _-_ .•... _------125

4

"'"Cl o ,..o .... Cl ....,E • :;: 3 ....CO (J) I- .J: C!I iii ~ 2 a: • • < l- S l-e::

40 30 20 PITUITARY PRL CONTENT

Figure 8. Relationship Between Pituitary Weights and Pituitary PRL Contents. r = 0.8187; P < 0.001. 126

4 ,.. 01 0 ....0 "01 E '-' 3 ;: CXI en" I- J: C!' iii ;: 2 • >-a:: c( l- S l-e: 1 5 10 15 20 25 30 35 DAYS AFTER L.AST OVUL.ATION

Figure 9. ,Close Correlation Between Pituitary Weights and Days an Animal is Anovulatory.

r = 0.7554; P < 0.001.

-- ._ .. _-_ .. - .. _. _... __ ._ .. __ . __ ..... _. _.. _._- ._. __ ._---_ .. _-_ .. _ .. _------127

5000

..... 4000 ....E 01 .s * .. * w 5 3000 ex: ....w CfJw oeI ex: Q. ::E .::J ex: w CfJ. 1000

(8) (8) (8) (10) DAY 1 DAY 2 DAY 3 DAYA 0900 h 1400 h ACYCLIC

Figure 10. The Effect of SP Treatment on Serum Progesterone Levels in SP-cyclic and SP-acyclic Animals.

Open bars = LP controls; hatched bars = SP-treated cycling animals; cross-hatched bars = SP-treated acyclic animals. * = P < 0.05 vs. LP controls; *** = P < 0.001 vs. morning levels.

-.- ---.-.----.. -...... _... _---_._---_._---_._- ._------128

1600

!Z1200 w I- oZ o z w c:J 0800 a: I­ CI) oW Z < 0: ~ 400 o

(8) (8) (7) (8) ••• o DAY 1 DAY 2 DAY 3 DAY 4 ACYCLIC

Figure 11. Ovarian Estrogen Contents in LP- and SP-treated cyclic and SP-acyclic Animals. Open bars = LP controls; hatched bars = SP-treated cycling animals. *** = P < 0.001 vs. all other groups.

__ .~ ... - __ .. __ ._ ... _____ ._ ..... __ ._ ... __ .'. __ . ____ .. _-.0. __ .-.. ------129

•••

+­ zw . I- oZ (J ::I: ~ 100 -I ::I: CD ::E

(6) (8) (7) (7) (8) (8) DAY 1 DAY 3 DAY 4 ACYCLIC

Figure 12. SP Effects on MBH LHRH Content of LP- and SP-treated Cycling Animals and SP-treated Acyclic Hamsters. Open bars = LP controls; hatched bars = SP-treated cycling animals; cross-hatched bars = SP-induced acyclic animals. *** = P < 0.001 vs. all groups of cycling animals. 130

12.5 ••• Gi ::l VI !! ~ 10.0 E .e-CI z •• 0 i= 4( c: I- Z W U Z 0 u ::z: c: ::z: ...J 4( 0 Q,

(5) (8) (7) (8) (8) (7) (8) (8) 20 DAY 1 DAY 2 DAY 3 DAY 4 ACYCLIC

Figure 13. Effect of SP on POA LHRH Concentration in Animals Displaying Estrous Cycles and SP-treated Acyclic Animals. Open bars = LP controls; hatched bars = SP-treated cycling animals; cross-hatched bars = SP-induced acyclic animals. ** = P < 0.005 vs. LP controls; *** = P < 0.02 to 0.001 vs. cycling cycling animals. CHAPTER 7

EXPERIMENT V. SHORT PHOTOPERIOD DEPRESSES CASTRATION RESPONSE AND MAY ALTER STEROID FEEDBACK SENSITIVITY IN FEMALE LSH/SsLak HAMSTERS

Introduction The previous experiments explored the effects of short photoperiod (SP) on ovarian cyclicity. The difficulties encountered in detecting early, SP-induced changes in cycling animals, and the limitations posed on the interpretation of results due to extensive feedback interactions between local, pituitary and hypothalamic levels, prompted us to explore the effects of SP treatment in ovariectomized animals. This approach allowed us to examine hormonal changes in the absence of gonadal steroids, and to study the possibility of a SP-induced change in steroid feedback sensitivity of the hypothalamo-pituitary axis, an experimental approach taken in a number of previous studies with male hamsters (Tamarkin, Hutchison and Goldman, 1976; Turek, 1977, 1979; Ellis and Turek, 1979; Turek, Jacobsen and Gorski, 1980), but not carried out extensively in females. It was hoped at the outset that the information derived would increase our understanding of the mechanism through which SP effects the collapse of the female reproductive system. The more complex neuroendocrine system of the female, with its tonic 131 132 and cyclic components, was not expected to respond in an identical manner to that for the male and, the results show this to be the case.

Materials and Methods

Experiment 1 Thirty-seven six to eight week old inbred LSH/SsLak female hamsters were obtained from Charles River Breeding Colony (Wilmington, MA). Following arrival the animals were housed in clear plastic cages (five animals/cage), in a light and temperature controlled room (22 0 C) with food and water provided ~ libitum. During an initial three weeks, all animals were maintained in LP (14L:10D, lights on at 0600 h M.S.T.) and monitored for regular' estrous cyclicity according to the method of Orsini (1961). All animals displayed regular four-day estrous cyclicity. Bilateral ovariectomies were then performed under Nembutal anesthesia (70 mg/kg, ip). Simultaneously, half of the animals received sub­ cutaneous interscapular Silastic implants (# 602-285; ID = 0.062 in, Dow-Corning Corp., Midland, MI) filled with 2.0 mm columns of densly packed crystalline estradiol-3-benzoate (EB). Prior to surgical implantation, the steroid-filled capsules were incubated overnight in normal saline at room temperature. Each control animal received an empty Silastic capsule. On the day following surgery, half of the animals with EB implants, and the same number of controls, were

.....•...... _...... _._ ...- .. __ ..•.... _.... _ ..._._----_. . ..---.-.- 133 transferred into specially constructed light-tight and well-ventilated metal chambers in which SP (8L:16D; lights on at 0600 h) was maintained. The light source consisted of fluorescent GE light bulbs (#F20T12-CW) mounted approximately 25 cm above each shelf. Air flow in the chambers was provided at approximately 32 cu ft/minute in order to maintain ambient temperature at 22 0 C. After twenty days of SP treatment, approximately the average time required for intact inbred females to become anestrus, SP-exposed and LP control animals were sacrificed by decapitation in the morning. Trunk blood was collected and allowed to clot overnight at 40 C, components were separated by ~entrifugation the following day and the sera stored at -20 0 C. Pituitaries were removed, weighed, sonicated in 500 ~l PBS (pH 7.6) and stored at -20 0 C for hormonal determination. The uteri from EB-treated animals were split open, blotted dry on filter paper and their weights recorded.

Experiment II The experimental protocol was similar to Experiment I , except the animals were 15 weeks old when they were ovari- ectomized on 11 Apr i 1 , whereas animals in the first experi- ment were 11 weeks old when the surgery was performed on 2 February. In Experiment II, 56 animals were divided into SP­ or LP-exposed groups; separate subgroups in each photoperiod 134 received silastic capsules which were either empty or filled with 2.0 or 10.0 mm columns of 17-beta-estradiol (E2).

Radioimmunoassay LH and FSH measurements were made with NIADDK kits employing LH-RP-2 and FSH-RP-2 as reference standards. The assays from the two experiments were run separately and samples from a standard serum pool included as an internal control. Interassay variations were 12% for LH levels (lower pool values in Experiment II) and 5% for FSH~ PRL levels were determined with the homologous radioimmunoassay developed by Soares, Colosi and Talamantes (1983). The hormone for iodination, standard and antibodies were kindly provided by Dr. Frank Talamantes (University of California, Santa Cruz, CA). All PRL samples were processed in the same assay.

Statistical Analysis The significance of the data obtained was evaluated statistically by application of the two-tailed Student t-test and two way analysis of variance (ANOVA) followed by Newman-Keuls multiple range test.

Results For purposes of comparison, the results of Experiment I and II are presented together. In both experiments, SP controls (animals bearing empty implants) had significantly 135 decreased serum LH levels compared with LP controls (P < 0.01, ANOVA Exp. I; P < 0.05, Student's t-test Exp. II) (Fig. 1). EB treatment was effective in reducing serum levels in LP-exposed animals, but did not alter the already depressed levels in SP-treated animals. On the other hand, 2.0 mm E2 implants enhanced LH serum levels several fold in both LP- and SP-treated groups in EXp. II (P < 0.01; Student's t-test), whereas 10.0 mm E2 implants resulted in levels similar to the ones seen in LP and SP controls. Pituitary LH contents, shown in Fig. 2., decreased with EB or E2 treatments and were further reduced by SP treatment; this reduction reached significance in EB-treated animals in Exp. I (P < 0.001, Student's t-test) and in hamsters with 10.0 mm implants in Exp. II (P < 0.05, Student's t-test). The effect of SP on serum FSH levels in controls bearing empty capsules (Fig. 3) paralled that for LH showing significant SP-induced depression in both Experiments I and II (P < 0.001 Exp. I; P < 0.025 Exp. II). EB treatment was effective in reducing values in hamsters maintained in LP, but did not further alter the depressed levels seen in SP­ exposed hamsters. On the other hand, 2.0 mm E2 implants augmented serum levels in both treatment groups significantly in Exp. II (P < 0.05 for LP and P < 0.001 for SP, Student's t-test), and 10.0 mm implants gave values within a similar range to those observed in LP- and SP-treated controls. Pituitary FSH contents (Fig. 4) were reduced by steroid 136

treatment, and SP-induced reductions were seen in SP-treated controls (P < 0.05) in Exp. I and in animals with Ie.O mm E2 implants in Exp. II (P < 0.05, Student's t). As depicted in Fig. 5 and 6, serum and pituitary levels of PRL were not significantly changed by SP exposure in both Experiment I and II. EB or E2 implants magnified PRL levels to similar extents in animals exposed to both photoperiods. Organ weights are summarized in Table I. Body weights decreased with steroid treatment, but were not affected by photoperiod length. Pituitary weights were altered by steroid and photoperiod treatment in opposite ways: these increased with EB or E2 implants, but were consistently decreased by SP treatment in steroid-treated and control animals. EB and 10.0 mm E2 implants stimulated uterine tissue weights to levels found between diestrus II and proestrus in this hamster strain, whereas 2.00 mm E2 implants resulted in weights comparable to those seen in diestrus I.

Discussion Animals in Experiments I and II responded similarly to SP treatment, showing significant reductions of gonadotropin levels after only twenty days of SP exposure. Considering that intact animals of this inbred strain cease estrous cyclicity after an average of about twenty days of SP

------_.... 137

treatment (Hauser and Benson, 1986; Exp. II), this rapid response is not surprising. On the other hand, it is still puzzling that this decrease of gonadotropin levels was not seen in a previous experiment in which morning serum levels were measured throughout the estrous cycle after 15 to 18 days of SP exposure (Exp. IV). The lack of observed changes might be due to the extensive hormonal interactions occurring at several levels in the neuroendocrine-reproductive axis. For instance, estrogen has stimulatory effects on gonado­ tropin release on the pituitary, and inhibitory effects on the hypothalamic level. A decrease in this steroid could reduce pituitary gonadotropin release, but may also decrease hypothalamic inhibition, with the result that within a certain range, changes in steroid concentrations might not be reflected in absolute hormonal concentrations and may be detectable only by studying the hormone release pattern. Gonad-independent reduction of serum gonadotropin levels by light deprivation has been reported previously in female hamsters (Seegal and Goldman, 1975; Bittman and Goldman, 1979; Goldman and Brown, 1979; Kumar, 1982; Jorgenson and Schwartz, 1984, Hauser and Benson, 1986), yet in the majority of those experiments, hamsters were ovariec­ tomized only after they became acyclic in response to SP (usually after six to twelve weeks of SP treatment). It appears questionable whether these findings can be compared directly to values for LP-exposed ovariectomized animals. Of 138

these reports only the experimental protocol employed by

~ Jorgenson and Schwartz (1984) was similar to ours, with the exceptions that the animals were derived from an outbred strain and were exposed to SP for 10-13 weeks. An additional aim of the present experiments was to examine the interaction of estrogen with gonadotropin levels in SP-treated animals. The most widely accepted hypothesis states that SP treatment increases the steroid feedback sensitivity of the hypothalamo-pituitary axis; however, this hypothesis is based primarily on observations in male hamsters (Tamarkin, Hutchison and Goldman, 1976; Turek, 1977, 1979; Ellis and Turek, 1979; Turek, Jacobsen and Gorski, 1980) and, to our knowledge this hypothesis has not been tested in female hamsters. It was surprising therefore to find that in Experiment I, EB treatment effectively reduced serum gonadotropin levels in LP controls, but had no impact on serum levels in SP-exposed animals. The increase of gonadotropin levels seen in animals treated with 2.0 mm E2 capsules (Exp. II) may be due to the known positive action exerted at the pituitary level by estrogen, where an increase of LHRH receptors is effected (Drouin, Lagace and Labrie, 1976; Marchetti et a1., 1982; Menon, Peegel and Katta, 1985), coupled with a failure by this small dosage to supress hypothalamic LHRH release. Overall, 10.0 mm E2 implants seemed to be less potent than EB in reducing serum gonado­ tropin levels. These levels, which were similar to those in

...- .. -- -- .. - --- . -_ .. ---_.--_._-- ._------139

LP and SP controls but were accompanied by highly reduced pituitary contents, could be due to a combination of a positive effect on the pituitary and a supressive effect at the hypothalamic level. These data suggest that, contrary to what was expected based on findings in male hamsters, SP does not change, or might even decrease, the sensitivity of the hypothalamo-pituitary axis to steroid feedback interaction in females. Our observations do, however, agree with, and corroborate, the recent finding by Jorgenson and Schwartz (1984) in which single injections of estrogen reduced gonado­ tropin levels to a lesser degree in SP- than in LP-exposed, outbred ovariectomized hamsters. If, as observed, SP exposure decreases the steroid feedback sensitivity, this could explain the absence of readily observed decreasing gonadotropin levels in SP-exposed cycling, animals. Another unexpected finding was the lack of a SP effect on pituitary and serum PRL levels in hamsters with or without steroid treatment. These observations contrast with our findings in SP-treated cycling animals, in which a slight decrease in serum PRL levels can be observed early after SP exposure but preceding any change in estrogen levels (Exp. IV). It is possible that twenty days of SP treatment were not sufficient to induce differences, since Widmaier and Campbell (1981) found significant reductions in serum PRL levels in ovariectomized outbred female hamsters after six weeks of treatment. In addition, PRL levels in

.- .. - ... _ .. -.-_ ... _..... -._-_ ..•...... _- ...... _._._. __ ._-_ .....--_._------140

ovariectomized animals, which are already depressed due to the absence of estrogen, may be more resistent to further reduction than those in intact animals. Body weights were consistently decreased by the various estrogen treatments, but were not affected by photoperiodic conditions. These observations may explain the increased body weights observed in the SP-induced acyclic condition, in which animals synthesize scant amounts of estrogen. Another interesting observation is the small, but consistent, reduction of pituitary weights in SP-treated animals even though gonadotropin and PRL contents remained unchanged. These reductions occurred in both estrogen­ treated and control animals and have also been seen in cycling SP-treated animals (Hauser and Benson, 1984; Exp. III). At this point, however, these observations defy any explanation. To summarize, we have shown that SP treatment reduces gonadotropin levels in the absence of gonadal steroids. The steroid feedback sensitivity of the hypothalamo-pituitary axis appears to be decreased or unchanged by SP treatment. PRL levels and their interaction with estrogen do not appear to change after short term SP exposure •

. . - .. - .-... -_. -_ .. _-_.------_._-_ ... _----_ .. ------.-----_ .... - Table 1. Short Photoperiod Effects on Body and Organ Weights in Ovariectomized Hamsters

Experiment I

LP LP LP SP SP Control Control EB Control EB

Initial 120.8+3.3 120.2+3.2 121.7+3.7 121.0+3.5 104.2+4.2 Body Wt.(gm) Final 131.1+3.9 114.0+3.2 127.3+3.5 114.8+3.8 1 21 .4+ 7 . 1

Absolute 4.9+0.3 6.0+0.2 4.3+0.2 5.6+0.1 4~5+0.2 Pituitary Wt. (mg) Adjusted 4.1+0.2 4.9+0.1 3.7+0.2 *4.6+0.1 4.3+0.2

Absolute 330.3+8.5 322.3+10.6 93.1+3.5 2 Uterine wt. (mg) Adjusted 290.4+9.9 280.6+8.4 87.7+2.8 1 ~

SP and LP animals of otherwise similar treatments are compared with the Student's t test. * = P < 0.025; EB = estradiol-benzoate; E2 = estradiol. Adjusted = organ weight/100 g BW.

141 an

Experiment I I

LP LP LP SP SP SP SP 10mm E2 .. EB Control 2mm E2 10mm E2 Control 2mm E2

1 .0+3.5 104.2+4.2 109.7+5.4 106.9+3.7 108.9+4.0 110.9+3.6 108.3+3.0

4.8+3.8 121.4+7.1 110.7+5.4 103.1+4.4 127.8+4.8 111 .6+3.7 103.4+3.0

5.6+0.1 4.5+0.2 5.2+0.2 5.2+0.2 4.2+0.2 5.0+0.6 5.0+0.2

L 6+0. 1 4.3+0.2 5.0+0.2 4.8+0.1 3.9+0.2 *4.5+0.2 4.6+0.1

93.1+3.5 210.4+10.0 302.7+6.5 93. 1+2.9 190.0+6.1 300.3+8.6 ~.3+10.6

>. 6+8 . 4 87.7+2.8 192.8+7.8 285.6+2.5 85.8+2.4 173.9+7.9 281.0+10.2

'e : organ

142

4.0 1[ -' I ~ 3.0 OJ c -J: ...J ::E :::> a: 2.0 w en

1.0

(9) 10) CONTROLS EB CONTROLS 2mm E-2 10mm E-2

Figure 1. The Effect of SP on Serum LH Levels in Ovariectomized Untreated (Controls) and EB-treated Animals in Exp. I (I), and 17-Beta-Estradiol (E-2) Treated and Control Animals in Exp. II (II). The bars indicate the mean + SEM; open bars represent values for LP-exposed, hatched bars SP-treated hamsters. The significance of the difference between similarly treated LP and SP animals was determined by the two-tailed Student's t-test. ** = p < 0.02 in Exp. I; * = P < 0.05 and ** = P < 0.01 in Exp. II. 143

3.0 I Cl -:1. -I- m2.0 I­ oZ () :J: ..J >­c: < 1.0 l- S e:l-

o (8) (9) ('9) (9) (9) (9) CONTROLS ES CONTROLS 2mm E-2 10mm E-2

Figure 2. SP Effects on Pituitary LH Content in Controls, EB and E-2 Treated Hamsters. Open bars represent Lp-treated animals; hatched bars SP­ exposed hamsters. III = P < 0.001; ** = P < 0.005. 144

60 I 1[ -E 'C, 40 c -J: (J) u. ~ ::> c: * ~ 20 *

o (9) 8) (9) (10) (9) (9) CONTROLS EB . CONTROLS 2mm E-2 10mm E-2

Figure 3. The Effects of SP Treatment on Serum FSH Levels in Controls, EB and E-2 Treated Hamsters. LP-treated animals are shown by open bars; SP-exposed hamsters by hatched bars. *** = P < 0.001; ** = P < 0.025. 145

1.6

en -~ ][ ;: 1.2 I z w I­ Zo () J: 0.8 en u. >-a: < I- ~0.4 c:

o (8) (8) (9) (10) (9) (9) (9) 10) CONTROLS EB CONTROLS 2mm E-2 10mm E-2

Figure 4. The Effects of SP on Pituitary FSH Contents in Controls, EB and E-2 Treated Hamsters. Open bars represent LP animals; hatched bars SP-treated animals. * = P < 0.05. 146

20 I ]I 15 -E Cl -c: -..J c: 10 D. ::E :::::> c: w en

(9) (9) (9) (10) (8) (11) CONTROLS EB CONTROLS 2mm E-2 10mm E-2

Figure 5. SP Effects on Serum PRL Levels in Controls, EB and E-2 Treated Animals are Depicted. Lp-treated animals are shown by the open bars; SP-treated animals by hatched bars. 147

200

-~150 I I­ Z W I­ oZ u -' 100 ex: Il. ex:~ c( I- ::; 50 !:::: Il.

o (9) (9) (9) (10) (9) (9) CONTROLS EB CONTROLS 2mm E-2 10mm E-2

Figure 6. The Effects of SP Exposure on Pituitary PRL Content in ES, E-2 Treated and Control Animals are Represented. LP animals are shown by open bars; SP animals by hatched bars. CHAPTER 8

SUMMARY DISCUSSION

These studies were designed to explore different aspects of SP-induced changes in the reproductive axis of the female hamster. Based on the results one can draw some general conclusions: In the first experiment it was revealed that, although females become reproductively quiescent after SP treatment, the ovaries are converted into highly active organs, mainly consisting of steroid producing interstitial tissue which demonstrated histochemically the potential for synthesizing large amounts of progesterone. This deduction was reinforced by subsequent steroid radioimmunoassays which revealed high levels of progesterone in blood and low to undetectable quantities of estrogen in the ovaries themselves (Exp. IV). A most exciting result, discovered in EXp. II and confirmed by subsequent experiments, was the finding that the SP-treated inbred LSH/SsLak hamster made the transition from the cyclic to the acyclic state rapidly and uniformly, in great contrast to outbred hamsters. The two experiments which explored hormonal and histological changes in SP-exposed cycling animals (Exp. III

148 149 and IV), added important new observations to our knowledge. They were the first to demonstrate that serum PRL levels show a reduction prior to the cessation of ovarian cyclicity when hamsters are transferred to SP. Yet they also clearly revealed that the major decrease of serum and pituitary PRL levels occurs only after animals cease estrous cyclicity, and this appears to be due to withdrawal of ovarian estrogens in the acyclic condition. These two experiments also showed that, contrary to our hypothesis, the secondary FSH surge and follicular recruitment were unimpaired in SP-exposed animals. These results excluded the secondary FSH surge as a likely target for SP-mediated changes. The ovarian analyses suggested strongly thit SP might impair maintenance of follicular growth, but that this impairment probably occurs suddenly, and perhaps can be detectable only over one or two estrous cycles. Overall, few hormonal and histological changes were revealed before hamsters ceased ovulatory cycles, supporting our notion that the drastic changes in hormonal patterns and ovarian histology observed in the acyclic condition appear to occur as a result, rather than being causative to the acyclic condition. The last experiment (Exp. V) carried out in ovariec­ tomized animals revealed that in the absence of ovarian steroids gonadotropin levels showed a SP-induced depression, yet did not impair PRL levels. These findings add 150 potentially important information to the literature, the implications of which will be discussed in a later section. Probably due to extensive experiments carried out in male hamsters, PRL has received much attention in regards to photoperiod-induced changes. In males, where pituitary and serum PRL levels show a consistent reduction after SP treatment or blinding, there is much support for a direct correlation between gonadal involution and low PRL levels (Reiter and Johnson, 1974c; Klemcke, Bartke and Goldman, 1981; Goldman et al., 1981; steger, Bartke and Goldman, 1982). PRL has the ability to maintain testicular LH receptors (Klemke et al., 1984) and several studies have shown that testicular regression can be partly or completely prevented by either increasing PRL levels with injections or pituitary grafts, or a combination of PRL and LHRH treatment (Bartke, Croft and Dalterio, 1975; Matthews, Benson and Richardson, 1978; Benson and Matthew, 1980; Chen and Reiter, 1980; Blask, Leadem and Stockmeier, 1984). As one study by Bartke et al. (1981) suggests, part of the stimulatory action of pituitary implants might be mediated by an increase in FSH levels. Unfortunately, the control of PRL levels is more complicated in females and therefore findings in males might not necessarily be applicable. One important sexual difference is the absence of an effect of gonadal steroids on PRL levels in the male (Goldman et al, 1981; Bex et al., 151

1978), which is in contrast to the major impact estrogen and progesterone levels have on synthesis and relase of PRL in females. Our findings on PRL in hamsters show many similarities when compared with experiments carried out in blind-anosmic female rats. The combination of blinding and olfactory bulbectomy (anosmia), which activates pineal function in the rat, reduces PRL synthesis and release, an effect which can be prevented by pinealectomy or other manipulations which inactivate the pineal gland (Blask, Reiter and Johnson, 1977; Leadem and Blask, 1981, 1982, 1984). Since blinding and anosmia also supress ovarian function and estrogen synthesis, part of the reduction in . PRL secretion appears to be as a result of gonadal depression, but similar to female hamsters, a gonad-independent reduction of PRL levels is also noted (Leadem and Blask, 1982). One of the problems encountered in the above studies are the inconsistent serum PRL levels in blinded-anosmic and blind-anosmic pinealec- tomized animals. The conflicting results might stem from the free running circadian rhythms induced by blinding. Endogenous rhythms have a tendency to be slightly longer than twenty-four hours, therefore a significant shift in diurnal rhythms can easily occur after many weeks of blinding or constant light. In the hamster PRL levels undergo large diurnal fluctuations with highest levels seen during

------152 afternoon hours (Talmantes, 1984), and one has no way of knowing whether samples are collected at the peak or nadir of the circadian fluctuations in blinded animals, it it is not possible to make complete interpretations from these findings. The comparison of gonadotropin levels of intact or gonadectomized male and female hamsters reveals some interesting differences and also indicates that the response to SP treatment might be sexually dimorphic. In intact females the gonadotropin profile does not appear to undergo drastic or obvious changes before animals become acyclic (Exp. III and IV) however, in the male decreased LH and FSH levels were observed in many studies (Turek et al., 1975; Tamarkin, Hutchison and Goldman, 1976; Jackson et al., 1984). On the other hand, studies by Jorgenson and Schwartz (1984) and our observations (Exp. V) reveal that SP-exposure is able to reduce serum gonadotropin levels in the females in the absence of gonadal steroids. Although Turek hypothesized this to be the case in male hamsters (Ellis and Turek, 1980), his conclusion appears to be questionable. In all experiments in which decreased gonadotropin levels were observed, intact hamsters were exposed to 60 or 80 days of SP and had already undergone gonadal involution before castrations were performed (Turek et al., 1975; Tamarkin, Hutchison and Goldman, 1976; Ellis and Turek, 1980). It does not seem appropriate to take findings in animals which 153 already had undergone gonadal involution prior to castration and compare them directly with observations made in LP-exposed castrated animals. In all experiments in which animals were first castrated and then followed by SP treatment, no gonadal independent reductions of gonadotropin levels were observed (Ellis and Turek, 1979; 1980; Turek, 1979). The hypothesis that SP-treatment increases the steroid feedback sensitivity of the hypothalamo-pituitary axis is better substantiated, and is supported by many experiments (Turek, 1977; 1979; Ellis and Turek, 1979; 1980). This hypothesis can also be utilized to explain the unchanged or decreased gonadotropin levels in intact male hamsters in spite of a reduction in testosterone. The postulated change in steroid feedback sensitivity which has been examined extensively in the male is still largely unexplored in the female. Our observations (Exp. V) and those of Jorgenson and Schwartz (1984) point in the opposite direction to what is suggested for the male, viz. that SP-exposure might decrease steroid feedback sensitivity. Yet since there also appears to be a gonadal independent reduction in gonadotropin levels, it will be hard or impossible to obtain a definite answer. Another difference between males and females might relate to changes in hypothalmic LHRH content after SP exposure. Several studies in the male have shown that LHRH content increases after SP treatment (Pickard and Silverman, 154

1979; Jackson et al., 1984). In Jackson's study in which LHRH levels were measured every three weeks after SP exposure, an increase occured only after nine to eleven weeks, at the time when· testicular regression had occured (Jackson et al., 1984). Pickard and Silverman (1979) only measured levels after thirteen weeks of SP and saw an increase in LHRH at the time values in the former study were back to control levels. These studies show that the increase in LHRH levels might only occur over a specific time after SP treatment. Since the increase was seen after gonadal regression was already appearant, its role in inducing gonadal involution is difficult to evaluate. Observations of increased LHRH levels have also been made in females (Kumar et a1., 1982; Exp. IV), although only after animals ceased ovarian cyclicity. That this increase might be the result of the acyclic condition and not directly caused by SP treatment, was shown in Experiment IV in which LHRH levels were measured in SP-treated cycling and acyclic animals, and were shown to be elevated only after cyclicity ceased. The above data strongly suggest that males and females use different mechanisms in their response to SP exposure. In males, steroid feedback sensitivity appears to be increased to achieve a reduction in gonadotropin levels leading to gonadal involution, whereas in females an ovarian 155 independent reduction of gonadotropin levels and perhaps a decrease in steroid feedback sensitivity appears to be the case. It is not quite clear why no major hormonal changes were detectable in intact SP-exposed cycling animals (Exp. IV), whereas in ovariectomized SP-exposed animals significant reductions in gonadotropin levels were observed (Exp. II and V). One possible explanation is that in ovariectomized animals the steroid feedback control is eliminated and changes might therefore become more visible. Also, assuming that gonadotropin release is decreased and steroid feedback sensitivity reduced by SP treatment as suggested in Exp. IV, the two opposing factors might cancel each other, until one became ~ore pronounced and the acyclic condition induced through the imbalance. Another possibility is that SP-induced effects might occur suddenly, maybe starting only after 15 to 20 days of SP exposure and causing an abrupt transition between the cyclic and acyclic state. An important aspect one has to be aware of when making an interpretation of data from ovariectomized animals is that by removing the steroid source in an animal one creates a artificial system with hyperactive gonadotrophs on one hand and hypotrophic 1actotrophs on the other. These changes in cellular activity might alter the response to SP and not completely mimic the response seen in intact animals. Another problem one might encounter with 156

gonadectomized animals is that the hormonal feedback interaction with the pineal gland may also be affected and could change pineal activity (Karasek and Reiter, 1982; Cardinali, Nagle and Rosner, 1975). The important question is of course: what changes lead to gonadal involution in the first place? The end result appears to be impairment of follicular growth to such an extend that cyclicity cannot be maintained. This conclusion is based on the principle of elimination and deductive reasoning and from experimental data. The major events in an estrous cycle include follicular recruitment, follicular development, and the preovulatory gonadotropin surge. As we have seen (Exp. III and IV) SP does not impair the estrous FSH surge and follicular recruitment. A similar conclusion was reached by Jorgenson and Schwartz (1985). Another potential target, the proestrous surge, is equally unlikely to be altered in such a way as to induce the acyclic condition. Even if the surge levels would be decreased by SP (an idea for which there is no evidence) about 30% of the normal gonadotropin surge levels could still induce ovulatory events. As daily surges in SP-induced acyclic or ovariectomized hamsters suggest (Exp. II and III and numerous aforementioned experiments), the cyclic center appears to be unimpaired by SP exposure. Alternatively, the possibility that daily surges might induce the acyclic condition does not seem 157

likely. Although the proestrous surge might be modified in magnitude and timing by SP, a daily surge does not appear to be expressed before cyclicity ceases and therefore is unlikely to be responsible for inducing the acyclic condition, although once started, such a daily surge might assist in the maintenance of the anestrous state. This conclusion is based on observations made by Jorgenson and Schwartz (1985) who followed afternoon gonadotropin surges after cyclicity ceased and could detect consistent surges only 16 days after the last observed ovulation in longterm SP-treated outbred hamsters. Our studies of ovaries taken shortly before the beginning of the acyclic condition, or a few days after the last observed ovulation (Experiment II, III and IV), did not reveal any evidence of interstitial tissue stimulation indicative of daily surges. Since corpora lutea once formed are independent of gonadotropin levels except during pregnancy, the only other change able to disrupt estrous cyclicity appears to be an interference with the maintenance of follicular development. The impairment of folliculogenesis by SP treatment is supported by many lines of evidence. In Experiment III, uterine weights of animals sacrificed on day 16 of SP-exposure were significantly decreased and the ovaries contained fewer corpora lutea and an increased number of atretic follicles. In Experiment IV similar tendencies were observed, and a few of the ovaries examined demonstrated a 158 drastically reduced number of, and smaller, developing follicles. Another experiment carried out by Little and Benson (1978) demonstrated that blinded animals had a reduced capacity to undergo compensatory follicular development compared with intact animals. Since compensatory ovulation is accomplished by an increase in FSH levels, the reduced ability to undergo compensatory follicular growth might indicate an impairment of FSH release. Comparing our findings with those in blind-anosmic rats, ovarian and uterine weights reported in rats also strongly suggest that follicular development is significantly reduced (Blask, Reiter and Johnson, 1977; Leadem and Blask, 1981, 1982). Since rats do not display daily gonadotropin surges in the absence of estrogen, ovarian weights are actually a much better indicator of ovarian function in regards to folliculogenesis than are ovarian weights from hamsters. An additional advantage is that in the absence of daily surges and elevated progesterone levels the transition between regular cyclicity and the acyclic state is most likely more gradual and might be easier to observe. Since LH and FSH are required for estrogen synthesis in antral follicles, follicular development could become impaired in SP by a decrease of either hormone, although it appears that FSH levels are more criticaL Reductions in 159

gonadotropins which were observed in Exp. V, could be achieved by changes in turnover rates of hypothalamic noradrenaline (NA), dopamine (DA) or some other neurotransmitter, or alterations in one of a number of peptides affecting gonadotropin release. As mentioned in a previous section, our knowledge about neural control of gonadotropin release in female hamsters is sparse and therefore it is difficult to make any more definite speculations. Data obtained on changes in catecholamine turnover in SP-exposed males remains controversial (Steger et al., 1985), and since females appear to respond in different ways it is doubtful that there may be good correlation. The one study carried out in SP-treated females suggests an increase in medial basal hypothalamic dopamine depletion rate (Kumar et al., 1982). Although the conclusions drawn by the authors in this study could be correct, this single study is not convincing evidence, especially since the animals were allowed to first become acyclic before they were ovariectomized and sacrificed two weeks later. The increase in hypothalamic dopamine turnover reported by Kumar et al., (1982) in females is opposite to the results of Steger (1985) who has reported a decrease in DA turnover in male hamsters transferred to SP. There are also some indication that the opioid system might be involved in mediating SP effect on the reproductive system. One study carried out in males demonstrated that 160

naloxone can partly prevent SP-induced testicular involution (Chen et al., 1984). Another study in females showed increased enkephalin levels in the medial basal hypothalamus of SP-induced acyc1 ic hamsters (Kumar et al., 1984). However since steroid levels influence opioid peptides and the steroid milieu is different in acyclic animals, it is impossible to relate this study to the development of the acyclic condition. Although there is no conclusive evidence one way or the other, it is generally assumed that the pineal gland mediates the effect of SP by changing gonadotropin release via the hypothalamus. Yet one has to be aware that there are also extrahypothalamic sites with projections to the hypothalamus that might affect reproductive function and therefore, could represent potential targets for pineal action. Examples include the amygdala, the ascending noradrenergic projections, from the locus coeruleus and serotonergic projections from raphe nuclei. Two loci which provide intriguing possibilities are the ventral tegmental nuclei and the mammillary bodies. In the rat and the hamster the mammillary nuclei are stimulatory to FSH release, and lesions or estrogen implants in this area result in an acyclic condition. Providing that the ventral tegmental nuclei and their connections to the mammillary bodies are intact, starvation results in an anovulatory condition with similar appearance to the one induced by SP exposure (Printz

------161

and Greenwald, 1970, 1971). Therefore it appears that the ventral tegmental nuclei can be activated by starvation or by other means, and could become inhibitory to the activity of the mammillary bodies. Since nerve fibers from the mammillary nuclei also project to hypothalamic areas, the possibility that the activated pineal gland in SP-exposed hamsters might act via this pathway cannot completely be ignored. The experiments presented have brought us closer to an understanding of changes in the reproductive axis induced by SP treatment in a long day seasonal breeder. They also demonstrated that the female hamster most likely incorporates different mechanisms to respond to short photoperiod treatment than the male. Therefore in the future the most fruitful approach might be to look at the female system completely separate and without preconceived ideas. Many additional experiments will be necessary to further our understanding of events leading to the acyclic condition. The area of steroid feedback sensitivity in regards to negative and positive feedback interactions under different lighting conditions has barely been investigated in the female hamster. The relationship between inhibin and the hypothalamo-pituitary feedback sensitivity remains unexplored in photoperiod manipulation of reproduction in female hamsters. Changes in this area, not measured in the 162 present investigation, could easily lead to an acyclic condition since follicles are critically dependent on adequate FSH levels. This dependence on FSH has been shown by studies on compensatory ovulation, which in the hamster can occur until the evening of diestrus II and is accomplished by a decrease in follicular atresia normally occuring between diestrus II and the day of proestrus {Greenwald, 1961}. Inasmuch as follicular atresia increases in SP-exposed hamsters, this avenue needs extensive future. exploration. Another fruitful approach might be to examine pulsatile patterns of gonadotropin release under different lighting conditions, since a change in pulsatile release might occur· which was not detectable by measuring levels at one time point as was gen~rally the case in the present and most studies to date. SP-induced changes in pulsatile patterns of gonadotropin release might have a profound impact on overall reproductive function. Finally, the inbred LSH/SsLak hamster incorporated in these studies has proved highly advantageous in bur investigation of SP-induced changes, since their response to experimental manipulation has provided data useful in unraveling events which may lead to the quiescent state of 163

the reproductive system under photoperiodic influence. this strain of hamsters can and should be used to great advantage in new investigations. It is hoped that the know1ege gained by these experiments will help focus and direct other investigators in their research and thus eventually contribute to a better understanding of the mechanisms through which the influence of environmental changes can be translated into specific biological events which regulate reproduction. Since the sjstem of control is designed to protect animals from inappropriate behavior which could be detrimental not only for the individual. but could endanger the survival of a species as a whole, it is not surprising that the system has evolved into the highly complex one that continues to provide a challenge for the inquisitive reproductive biologist.

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