W W \ \ 1| WHHUNIIW )3 é NW!lll‘xlllmfli 734 w- . : . :fiflflsn flaw ‘- 4’1 6“? I. This is to certify that the thesis entitled EFFECT OF CHARCOAL TREATED BOVINE FOLLICULAR FLUID ON SERUM FOLLICLE-ST‘IMULATING HORMONE AND LUTElNlZlNG HORMONE IN CASTRATED RATS AND HEIFERS: A DEMONSTRATION OF INHlBlN-AC‘TIVITY presented by ANDREA DAY CURATO has been accepted towards fulfillment of the requirements for JA§T£RQ degree in AMMALSCLENCE Date MAY I i , [982 0-7639 MS U is an Mnnative Action/Equal Opportunity Institution MSU LIBRARIES “- RETURNING MATERIALS: Piace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped beiow. A \- r'fVU'V , m i we r v EFFECT OF CHARCOAL-TREATED BOVINE POLLI- CULAR FLUIDCHQSERUM FOLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE IN CASTRATED RATS AND HEIFERS: A DEMONSTRATION OF INHIBIN-ACTIVITY BY Andrea Day Curato A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1982 ABSTRACT EFFECT OF CHARCOAL-TREATED BOVINE FOLLI- CULAR FLUIDCflJSERUM FOLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE IN CASTRATED RATS AND HEIFERS: A DEMONSTRATION OF INHIBIN-ACTIVITY BY Andrea Day Curato The objective of these studies was to determine if charcoal-treated bovine follicular fluid selectively suppressed the secretion of follicle-stimulating hormone in castrate rats and heifers. To meet this objective follicular fluid was collected from ovarian follicles of cattle. Steroids were removed from follicular fluid with charcoal, and charcoal—treated follicular fluid was injected into heifers ovariectomized three days earlier and into castrated male and female rats. It was demon- strated (1) that bovine follicular fluid lowered concen- trations of follicle-stimulating hormone, but not luteiniz- ing hormone in heifers and female rats; and (2) that bovine follicular fluid blocked the castration rise in follicle-stimulating hormone but not luteinizing hormone in male rats. Since total amounts of estradiol and progesterone were low in bovine follicular fluid after Andrea Day Curato charcoal extraction, factors other than these steroids, perhaps inhibin, altered secretion of follicle-stimulating hormone in heifers and rats. .....In dedication to hOpes and dreams.... ii ACKNOWLEDGMENTS I would like to thank all of the faculty, graduate students, and staff of the Department of Animal Science at Michigan State University for all the times you shared your time with me to teach, console, encourage, assist, talk, and most of all just for being there. I would also like to thank my family for the continued love and sup- port that was always there. I would also like to eXpress special thanks to my major professor, Dr. James Ireland, for all the knowledge he so freely gave and the guidance through the completion of this thesis. I thank the department heads, Dr. Harold Hafs and Dr. Ron Nelson for their continued encouragement and financial support. I would like to also thank the other members of my graduate committee, Dr. Edward Convey and Dr. Richard Dukelow for their guidance through the completion of my thesis. Special thanks go to Larry Chapin, Dr. Clyde Anderson, Dr. John Gill, Jim Liesman, and Manuel Villarreal for their advise and assistance in the statistical analy- sis of my results and to Dr. Rosemary Grady, Dr. Neena Schwartz, Dr. Vincent Hylka, and Dr. William Sonntag for iii their assistance in the completion of my thesis work. Also thanks go to members of the Animal Reproduction Lab and Neuroendocrine Lab of Michigan State University for any assitance given me in the work necessary for the completion of my Masters degree and to John Wilson for his diligent assitance in the main project of my thesis. I extend my deepest gratitude to Dr. Limin Kung, Dr. Vasantha Padmadaban, Dr. Edward Convey, and Dr. Roy Fogwell for their continued encouragement, but most of all for their listening ears and understanding through difficult times. Lastly, I wish to eXpress how much I cherish the friendships I've made here at Michigan State and the lasting memories given to me by friends and visitors alike. This means as much, if not more, to me than the completion of my Masters degree. I look forward to the years ahead when we can meet again and reminisce. iv TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . Vii LIST OF FIGURES . . . . . . . . . . . viii LIST OF ABBREVIATIONS . . . . . . . . . ix INTRODUCTION . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . 3 Definition of Inhibin . . . . . . . 3 Sources and Origin of Inhibin . . . . . 5 Measurement of Inhibin-Activity . . . . 6 Detection of Inhibin-Activity Using Cultures of Pituitary Cells . . . . 6 In Vivo Detection of Inhibin . . . . 7 Radioligand Assays for Detecting Inhibin . . . . . . . . . . 8 Chemical Characteristics of Inhibin . . . 9 The Nature of Inhibin . . . . . . . 9 Purification and Characterization of Inhibin . . . . . . . . . . 10 Sites of Inhibin Action . . . . . 11 The Putuitary as a Site of Inhibin Action . . . . 11 The Hypothalamus as a Site of Inhibin Action . . . . 12 The Gonad as a Site of Inhibin Action . 13 The Endocrine Role of Inhibin in Domestic Animals . . . . . . . . . . . 14 MATERIALS AND METHODS . . . . . . . . . 18 Experiment 1: Effect of Charcoal-Treated Bovine Follicular Fluid on Serum FSH in Rats Ovariectomized at Metestrus . . . 20 Experimental Design . . . . . . . 20 Statistical Analysis . . . . . . . 21 Results . . . . . . . . . . . 22 Experiment II: Effect of Charcoal-Treated Bovine Follicular Fluid on tion Rise in Serum FSH and LH Rats . . . . . . Experimental Design . Statistical Analysis . Results . . . . . Experiment III: Effect of Charcoal-Tre Bovine Follicular Fluid on Serum FSH the Castra- LH in Ovariectomized Heifers Experimental Design . Statistical Analysis . Results . . . . . DISCUSSION . . . . . . . LITERATURE CITED . . . . . vi in Male ated and Page 22 22 24 24 25 25 27 28 34 39 Table LIST OF TABLES Concentrations of Steroids before and after Charcoal Extration of Steroids from Bovine Follicular Fluid . . . . . . . . . Concentrations of Protein before and after Charcoal Extraction of Steroids from Bovine Follicular Fluid . . . . . . . . . Effects of Bovine and Porcine Follicular Fluid on Castration Levels of FSH in Female Rats Ovariectomized at Metestrus . . . . Effect of Charcoal-Treated Bovine Follicular Fluid or Serum from an Ovariectomized Cow on Concentration of Follicle-Stimulating Hormone and Luteinizing Hormone in Male Rats after Castration . . . . . . . . . vii Page 19 19 23 26 Figure LIST OF FIGURES Page Serum follicle-stimulating hormone in ovariectomized heifers given three intra- venous injections of serum from an ovariectomized cow, or charcoal-treated follicular fluid from cattle . . . . . 30 Serum luteinizing hormone in ovariec- tomized heifers given three intravenous injections of serum from an ovariec- tomized cow, or charcoal-treated follicu- lar fluid from cattle . . . . . . . 32 viii ABBREVIATIONS bFF............bovine follicular fluid OC.............degrees centrigrade cm.............centimeter(s) E2.............estradiol FF.............follicular fluid FSH............follicle-stimulating hormone g..............gram(s) GnRH...........gonadotropin-releasing hormone h..............hour(s) i.v. ..........intravenous kg.............kilogram(s) LH.............luteinizing hormone mg.............milligram(s) min............minutes pl.............microliter(s) ml.............milliliter(s) mm.............millimeter(s) n..............number of animals per treatment group NIAMDD.........National Institutes of Arthritis, Metabolic, and Digestive Diseases ovx............ovariectomized P..............progesterone pFF............porcine follicular fluid PGF............prostaglandin F2a ps.............pig serum PBS............phosphate buffered saline RIA............radioimmunoassay RPl............reference preparation-l T..............testosterone TSH............thyroid-stimulating hormone x g............times gravity ix INTRODUCTION The hypothalamus synthesizes and releases gonado- tropin-releasing hormone (GnRH). GnRH acts on the pitui- tary to stimulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonado- trOpins are transported via the blood to the gonads where FSH and LH regulate spermatogenesis, folliculogenesis, steroidogenesis, and ovulation. Increasing output of estradiol (E2) and progesterone (P) from the gonad feeds back on the hypothalamus to partially regulate pituitary secretion of gonadotropins. Although E2 and P exert a negative feedback effect on LH, these steroids do not fully account for the negative feedback control of FSH. A gonadal factor called inhibin has been implicated in the control of secretion of FSH from the pituitary gland. The objective of my research was two fold: first, to demonstrate the presence or absence of an inhibin-like substance in bovine follicular fluid (bFF) and second, to determine if bFF could selectively inhibit the secretion of FSH in ovariectomized (ovx) heifers. My first objec- tive was studied by employing two documented in 3139 assay systems for the detection of inhibin-activity (female rats Marder et al., 1977; male rats, Hermans et al., 1980). My second objective was measured by injecting bFF into ovx heifers and measuring changes in concen- trations of LH and FSH in serum. REVIEW OF LITERATURE Definition of Inhibin The concept of inhibin had its beginning in 1923 when Mottram and Cramer described the effects of X-irradiating testes. Seminiferous tubules were shrunken and disorganized while interstitial cells appeared normal, and the pituitary showed histological changes resembling those after castration. Based on these data, Mottram and Cramer postulated that a hormone from the seminiferous tubles was a major inhibitor of pituitary function and this hormone had a role in the regulation of sperm pro- duction. It was not until 1932 that the term inhibin was used by McCullagh to describe a water soluble testicular extract, which prevented hyperfunction of the pituitary gland in castrated rats. McCullagh also described a fat soluble entity, separate from inhibin, known today as androgen or testosterone, that was responsible for the maintenance of the secondary sex glands. Although the structure of testosterone was reported in 1935 (David et a1.), the structure of inhibin remains unknown. Recent interest in inhibin occurred with the demonstration that a non-steroidal inhibitor of FSH was present in bull semen (Franchimont, 1972) and in ovine rete testis fluid (Setchell and Sirinathsinghji, 1972). Main et a1. (1979) stated the inhibition hypothe- sis as two pituitary gonadotropins acting on the testes, FSH stimulating the seminiferous tubles, and LH stimu- lating the Leydig cells. The Leydig cells produce andro- gens (particularly testosterone) which control LH secre- tion by negative feedback, and the seminiferous tubles produce inhibin which controls FSH secretion by negative feedback. This hypothesis is often challenged by the "androgen only" hypothesis. Here, gonadotropin control is believed regulated only by androgen feedback and not inhibin (see Setchell et al., 1977, for review). However, in contrast to preparations of inhibin, neither androgens nor other steroids lower FSH in castrated animals to normal levels without either suppressing LH or causing hypertrophy of secondary sex organs. There has been increasing evidence which supports the presence of a specific non-steroidal inhibitor of FSH in follicular fluid. This inhibitor of FSH secretion was first detected in bovine follicular fluid (FF; de Jong and Sharpe, 1976) and subsequently in porcine (Marder et al., 1977; Schwartz and Channing, 1977; Welchen et al., 1977), equine (Miller et al., 1979b), and human FF (Chari et al., 1979; Chappel et al., 1980b). Although inhibin is defined as a water-soluble gonadal substance that exerts a specific inhibition of FSH release from the pituitary gland (deJong, 1979a), LH release from the pituitary gland can also be affected (Legace et al., 1978; Shander et al., 1980; Scott and Burger, 1981; Rush et al., 1980). This suggests that a non-steroidal inhibitor of LH secretion is present in the gonads. Sources and Origin of Inhibin In males, inhibin-activity is found in different components of semen, spermatozoa (Fachini et al., 1963; Lugaro et al., 1969; 1973), seminal plasma (Franchimont et al., 1975a, 1977; Chari et al., 1978b), epididymal homogenates (LeLannou and Chambon, 1977), rete testes fluid (Setchell and Sarinathsinghji, 1972; Setchell and Jacks, 1974; Davies et al., 1976, 1978), and testicular lymph (Baker et al., 1976, 1978). Ovine (Lee et al., 1974; Nandinini et al., 1976), bovine (Keogh et al., 1976), and rat (Moodbidri et al., 1980) testicular tissue also contain inhibin. The Sertoli cell is believed directly involved in the snythesis and secretion of inhibin (Steinberger and Steinberger, 1976). However, Sertoli cells may require the presence of germinal cells for the induction of inhibin secretion (Rich and DeKretser, 1977). In females, inhibin-activity is found in ovarian FF (de Jong and Sharpe, 1976; Hopkinson et al., 1977; Marder et al., 1977; Schwartz and Channing, 1977; Welchen et al., 1977; Channing et al., 1980), ovarian homogenates (Chappel, 1979), and ovarian venous plasma (DePaulo, et al., 1979; Shander et al., 1980; Channing et al., 1980). Granulosa cells in the ovary may be the source of inhibin in females (de Jong and Sharpe, 1976). For example, when media (charcoal extracted to remove steroids) from cul- tured granulosa cells is added to a pituitary cell culture system, concentration of FSH in media is reduced (Welchen et al., 1977; Erickson and Hsueh, 1978). Media from undifferentiated granulosa cells also reduces secretion of FSH from cultured pituitary cells (Erickson and Hsueh, 1978). Thus, granulosa cells acquire the ability to pro- duce inhibin early in the development of a follicle. Measurement of Inhibin-Activity Detection of Inhibin-Activity Using4Cultures of Pituitary Cells Baker et a1. (1976) developed the first monolayer pituitary cell culture to assay for inhibin-activity. Either basal concentrations of GnRH-stimulated release of gonadotrOpins are measured after addition of prepara— tions containing suspected inhibin-activity. Culture systems differ in sex and age of rats used, concentration of pituitary cells in culture, composition of culture media, and amount of time of eXposure to the test mate- rial (Erickson and Hsueh, 1978; Franchimont, 1979; de Jong et al., 1979b; Steinberger, 1979; DePaulo et al., 1979; Scott et al., 1980; Scott and Burger, 1981). A pituitary cell culture is a very sensitive method of detecting inhibin-activity. As little as 0.5 p1 of charcoal-treated bovine FF (Franchimont et al., 1979) or 1.0 ul of charcoal-treated porcine FF (Shander et al., 1980b) reduces the capacity of pituitary cells in culture to release FSH. Shander and coworkers (1980) have demonstrated in pituitary cell cultures that E2, P, and testosterone (T), either alone or in combination have no selective negative effects on the release of FSH. In Vivo Detection of Inhibin The suppression or blockage of a post-castration rise in concentration of FSH in castrated animals or sup- pression of FSH in intact animals is taken as direct evidence of inhibin-activity. The model system of Marder et a1. (1977) is one of the most sensitive and repeatable in yiyg_methods for detection of inhibin. On the morning of metestrus, as determined by vaginal cytology, rats are bilaterally ovx and injected intravenously (tail vein) 3.5 h later with the test substance, such as FF. Rats are killed 5.5 h following injection to obtain trunk blood for quantification of LH and FSH. Concentrations of LH are unaffected by injection of follicular fluid whereas FSH is suppressed in a dose dependent fashion. For example, 23 pl of bovine FF has been shown to suppress concentrations of FSH in the serum 50 percent below con- trol levels (Grady et al., 1982). Radioligand Assays for Detecting Inhibin Antibodies to semipurified inhibin has been pro- duced in rabbits (Franchimont et al., 1975c; Vaze et al., 1979; Shashidhara Murtly et al., 1979; Sairam et al., 1978). Adult intact rats exhibit a dose-dependent increase in serum FSH when injected with increasing amounts of inhibin antisera. This suggests that endogen- ous inhibin is neutralized by the antibody and therefore incapable of negative feedback control of FSH secretion (Franchimont et al., 1975c; Vaze et al., 1979). Franchimont et al. (1975c) developed a radioimmunoassay (RIA) for ovine rete testis fluid and Vaze et a1. (1979) developed an RIA for human seminal plasma. Antibodies to the ovine fluid do not cross react with GnRH, somatos- tatin, or pituitary hormones, but do cross react with bovine testicular extract and bovine follicular fluid inhibin preparations. Antibodies to human seminal plasma inhibin do not cross react with prolactin, LH, or FSH from humans, sheep, and rats, thyrotrOpin stimulating hormone (TSH), GnRH, or steroids from humans, ram testi- cular extracts, or bull semen. However, the antibody of Vaze et a1. does cross react with monkey semen, rat serum, and FF from cows, sheep, and pigs. The above assays were not developed to measure inhibin in serum, but the assay of Vaze et a1. is sensitive enough to measure inhibin in the peripheral circulation of rats, providing evidence that inhibin does enter blood circula— tion. However, one universal inhibin molecule has not been found, therefore bioassays are necessary to monitor attempted purification regimens and refinement of RIAs for inhibin. Chemical Characteristics of Inhibin The Nature of Inhibin Testosterone (T), E2, dihydrotestosterone, and P in semipurified inhibin preparations are not present in sufficient amounts to eXplain the inhibitory effect on FSH 99 3939 or 99 39939 (Franchimont et al., 1975a, 1979; Hopkinson et al., 1977). Low doses of androgen prefer- entially suppress serum LH over FSH (Franchimont et al., 1975b). However, androgens are capable of suppressing the GnRH-stimulated release of both FSH and LH (Eddie et al., 1978, 1979). The dose response lines differ 10 from responses obtained with inhibin. Small asmounts of E2 and androgens stimulate the secretion of FSH from rat pituitary cells in culture (Labrie et al., 1978), while inhibin suppresses the secretion of FSH. Follicular fluid preparations (steroid free) retain their ability to selec- tively suppress FSH both 99 3939 and 99 39939 (Steinberger and Steinberger, 1976; Erickson and Hsueh, 1978; Welchen et al., 1977; de Jong et al., 1978; Setchell et al., 1977), suggesting a nonsteroidal factor is responsible for the suppression of FSH. However, steroids do have roles in regulation of LH and FSH secretion (see review, Barraclough et al., 1979). Purification and Character— ization of Inhibin Many attempts have been made to purify the inhi- bin molecule (see de Jong et al., 1981). Various methods consist of precipitation, ultrafiltration and dialysis, gel filtration and electrOphoresis, ion exchange chroma- atography, isotachOphoresis, precipitation with antibodies, and affinity chromatography. The biochemical data obtained so far, do not agree on a single molecular weight for inhibin. Estimates range from 1,000 to 90,000 daltons. The large number of inhibin sources and the variety of methods used for purification have resulted in confusion with respect to the characterization of inhibin and its ll biological similarities across species. de Jong et a1. (1981) reported that most results suggest a molecular weight between 15,000 and 25,000 daltons and that inhibin may be a hydrophobic glycoprotein with an isoelectric point between pH 5.0 and 6.0. Sites of Inhibin Action The Pituitary as a Site of Inhibin Action It is difficult to make conclusive arguments for or against any one inhibin target site considering that the sourcecfiforigin and chemical nature are questionable. Nevertheless, there is increasing evidence that the pituitary is the site of action. Release and synthesis of FSH, but not LH, is reduced when charcoal-treated media from granulosa (Erickson and Hsueh, 1978) or Sertoli cell cultures (Chowdhury et al., 1978), is added to pituitary cells in culture. This provides evidence for direct action of inhibin on the pituitary. A GnRH-induced release of FSH, but not LH, in rats is reduced with injections of charcoal-treated porcine FF (Rush and Lipner, 1979; De Paulo et al., 1979). Similar results are demonstrable 99 39339 (Shander et al., 1980b). However, inhibin is unable to reduce concentrations of FSH in the face of high concentrations of GnRH suggesting that inhibin 12 activity can be overcome with GnRH at the pituitary (Rush and Lipner, 1979). The Hypothalamus as a Site of Inhibin Action Since the release of LH and FSH is mediated by GnRH, the possibility exists for a hypothalamic site of inhibin action. Media from rat testes organ culture reduces the amount of GnRH released from rat hypothalami in culture (Demoulin et al., 1980). Six hours after an injection of semi-purified inhibin into the third ventri- cle, release of FSH is maximally suppressed in castrated rats (Lumpkin et al., 1981). However, the pituitary responds to GnRH stimulation at this time, suggesting that an inhibin-hypothalamic interaction was responsible for reduced FSH secretion from the pituitary. Reduced secretion of FSH may be mediated by lowered secretion of GnRH and differences in threshold responsiveness to GnRH for the secretion of FSH and LH. This threshold hypothesis is supported with the finding that a 10-fold greater concentration of synthetic GnRH is required to stimulate a half maximal response in the release of FSH over that required for half maximal response in release of LH (Leung and Padmadabhan, personal communication). Thus it is possible that lowered GnRH reduces the release of FSH, but not LH, because the pituitary is more sensi— tive to GnRH for the release of LH. 13 The Gonad as a Site of Inhibin Action Spermatogenesis is negatively effected by inhibin 99‘39339 and 99 3939 (Demoulin et al., 1980). In imma- ture rats, inhibin injections resulted in atresia of follicles (Chari et al., 1981). Atresia may be a result of binding inhibition of FSH to its receptors on granu- losa cells (Sato et al., 1980). Oocyte maturation inhibitor (OMI; Jagiello et al., 1974; Tsafirri and Channing, 1975) and an inhibitor of luteinization (Ledwitz- Rigby et al., 1977) are found in FF. These non-steroidal substances could be responsible in part for gonadal effects following treatment of animals with FF. Folli- cular fluid also contains a component capable of inhibit- ing the adenylyl cyclase system, inhibiting progesterone secretion, inhibiting luteinization without affecting the steroidogenic enzyme systems (Rigby et al., 1980; Ledwitz and Rigby, 1980) and reducing prostaglandin F20 (PGF) from theca and granulosa cells (Kraiem et al., 1978). Preovulatory follicles lack this PGF inhibitor, support— ing the idea that a decline in inhibitory action may be necessary for follicular maturation and, if not, atresia may result. In summary, follicular fluid contains components that affect the hypothalamus, pituitary, and gonad. The components may be separate entities or a complex family 14 of regulators required to maintain the cyclic waves of folliculogenesis. Inhibin is a possible member of this family. The Endocrine Role of Inhibin in Domestic Animals Most of the evidence in support of inhibin has come from laboratory animal studies; however, inhibin- activity has been demonstrated in domestic animal species. Bovine testicular extracts, when infused over a 24 h period, reduce concentrations of FSH within the next 24 h period in castrate rams (Keogh et al., 1976; Baker et al., 1976). Concentrations of LH increased slightly. Concentrations of FSH, but not LH in blood are reduced following the infusion of human follicular fluid from the follicular but not luteal phase of the menstrual cycle into the anterior pituitary gland of ovariectomized mon- keys (Chappel et al., 1980b). Reductions in the concen- tration of serum FSH ranged from 30 to 70 percent. This variation may be due to differences in individual folli- cular fluids tested or differences in responses between monkeys. Charcoal-treated porcine FF injected early or midway in the menstrual cycle of monkeys reduces circu- lating serum concentrations of FSH, but not LH (Channing et al., 1981). Steroids may interact with inhibin. Removal of steroids with ethanol extraction or destruction 15 of proteins by heating (100°C, 2 min) diminished the ability of equine follicular fluid to lower concentra- tions of FSH in ovariectomized mares as compared to the response to whole FF (Miller et al., 1979b). In fact, when charcoal-treated equine FF is injected in combination with E2 (1 mg) the decrease in FSH is greater than that induced by either treatment alone (Miller et al., 1981). Gonadotropin secretion is regulated by feedback of ovarian products. Basal concentrations of LH are regu- lated by E2 and P in rats (Grady et al., 1981), sheep (Goodman et al., 1981), and cattle (Kesner et al., 1981). Although E2 and P are involved in the regulation of basal FSH secretion, some other ovarian factor, possibly inhibin, is required for complete control (Chappel, 1980a,kn Goodman et al., 1981; Grady et al., 1981). The preovulatory surge of gonadotrOpins in cattle is a result of increasing E2, decreasing P, and a possible increase of GnRH (Kesner et al., 1981). Concentrations of LH remain at baseline throughout the bovine estrous cycle, with the exception of the periovulatory period. However, concentrations of FSH increase and then return to baseline following ovulation (Dobson et al., 1978; Roche and Ireland, 1981). Inhibin may be present during both releases of FSH. The GnRH surge may overcome inhibin's 16 ability to suppress FSH during the periovulatory period, while the gonadotrOpin surge may shut down the ovary so that its products are not secreted or are secreted at very low levels. Therefore, FSH could be released in the absence of negative feedback from a combination of steroids and inhibin. Inhibin's presence throughout the remainder of the cycle may act in concert with E2 and P to keep concentrations of FSH at baseline. Gonadotropin stimulation of the ovary is necessary for the initiation and maintenance of folliculogenesis (Richards, 1978). Systemic administration of bovine follicular fluid to sheep and cattle following prosta- glandin synchronization reduces the total number of sur- face follicles and reduces the size of the largest folli- cle (Miller et al., 1179a). Concentrations of circulating gonadotropins were not measured, therefore, events cannot be attributed to reduced concentrations of FSH. However, in monkeys, porcine FF reduces concentrations of FSH dur- ing the early follicular phase and reduces recruitment and selection of the ovulatory follicle. Reduced con- centrations of FSH during the late follicular phase results in atresia of the pre-existing dominant follicle (Dizerega et al., 1981). These results suggest that grow- ing follicles require continued FSH support throughout the cycle. Lowering FSH by injecting FF, therefore, results 17 in interrupted folliculogenesis. Preantral follicles may enter the growing pool of follicles following the preovulatory surge of FSH (Matton et al., 1981) or following the cyclic four-day increases in FSH reported by Schams and Schallenberger (1976). Since FSH controls growth of ovarian follicles, a better understanding of the role of inhibin on negative feedback control of FSH secretion will improve our ability to regulate estrous cycles in cattle. However, no stud- ies have examined the effects of inhibin on secretion of FSH in cattle. Thus, we proposed to determine whether FF from bovine follicles contains inhibin-activity using two 99 3939 bioassays and to examine whether FF from bovine follicles suppressed FSH secretion in heifers. MATERIALS AND METHODS Bovine ovaries were collected in pairs from non- pregnant cows at a local slaughterhouse and stage of the estrous cycle was estimated based on gross appearance of the corpus luteum (Ireland at al., 1980). Follicular fluid was aspirated from follicles greater than 10 mm in diameter and assigned to one of the following pools: preovulatory (day 18-1), postovulatory (day 2-6), or mid- cycle (day 7-17). Follicular fluid was centrifuged (5°C, 10 min, 248 x g) to remove follicular cells and frozen (-20°C). After thawing, FF was stirred with 25 mg of washed Norit charcoal per m1 at 37°C for 20 min and cen- trifuged at 105,700 x g for 30 min. This supernatant was used for all subsequent experiments. Charcoal treat- ment of bovine follicular fluid (bFF) reduced steroid content 99 percent (Table 1). Steroids were quantified by single antibody radioimmunoassay (RIA) as previously described for estradiol (Wetteman et al., 1972), proges- terone (Convey et al., 1976), and testosterone (Haynes et al., 1977). Protein in follicular fluid was quanti- fied using the method of Lowry et a1. (1951) before and after charcoal extraction of steroids (Table 2). Porcine FF contained-75.40 mg protein per ml of fluid. 18 19 TABLE 1. Concentrations of Steroids (ng/ml) before and after Charcoal Extraction of Steroids from Bovine Follicular Fluid E2 P T Before After Before After Before After Preovula- tory 108.09 0.45 312.00 1.33 17.64 * Post- ovulatory 127.14 0.26 171.00 1.47 18.62 * Midcycle 98.51 0.50 226.00 1.82 14.32 * *Concentrations of testosterone were not detectable (lower limit of assay, 0.5 ng/ml). TABLE 2. Concentrations of Protein (mg/ml) before and after Charcoal Extraction of Steroids from Bovine Follicular Fluid Before After Preovulatory 97.81 96.04 Postovulatory 94.71 96.48 Midcycle 96.04 95.59 20 Experiment 1: Effect of Charcoal-Treated Bovine Follicular Fluid on Serum FSH in Rats Ovariectomized at Metestrus This study was done in collaboration with Dr. Rosemary Grady and Dr. Neena Schwartz, Northwestern Uni- versity, Evanston, Illinois. Portions of the Grady et a1. (1982) results have previously been reported. EXperimental Design This eXperiment was designed to determine if the pools of charcoal-treated bFF were capable of exhibiting inhibin-activity. A colony of female Sprague-Dawley rats were housed under 14 h light, 10 h dark with feed and water 99 libitum. Rat vaginal smears were examined daily to determine the length and stage of cycle. Rats having regular 4-5 day cycles were bilaterally ovariec- tomized under ether anesthesia at metestrus. Three- and-one-half hours following ovx, rats received bFF, porcine FF (pFF) or porcine serum (PS) via a tail vein at one of the following levels of protein, 2.25, 4.50, or 9.00 mg. Volume equivalents of these fluids were 23, 47, or 94 ul of bFF, 30, 60, or 119 ul of pFF, and 90 ul of PS (9.00 mg protein). Porcine FF and PS have been pre- viously characterized and shown to selectively suppress concentrations of FSH in ovx rats (Marden et al., 1977; Lorenzen et al., 1978) and therefore served as reference standards (n = 3 or 4 rats). Preovulatory, postovulatory 21 and midcycle bFF were the unknown test fluids (n = 3 or 4 rats). The doses chosen were previously determined in a pFF dose-response trial using PS as a control fluid. Level of protein as no effect on concentrations of gona- dotropins when PS is injected so the highest level served as a control in this study. At 5.5 h after treatment the rats were decapitated and trunk blood collected. Plasma was obtained by centrifugation (5°C, 20 min, 1548 x g) and frozen (~20°C) until assayed. RIA was used to detrmine concentration of FSH in plasma. NIAMDD kit instructions were followed and NIAMDD-FSH-RPl was used as the reference standard. Concentrations of LH in serum of rats at metestrus do not rise withn 9 h post ovariectomy (Marder et al., 1977; Campbell et al., 1977) and Lorenzen et a1. (1978) found that procine FF has no effect on LH. Therefore, concentrations of LH were not assayed for in the metes- trous rat model system. Statistical Analysis The hormone data were found heterogeneous in vari- ance and was not correctable by transformation. There— fore, specific comparisons were made using Behren's test statistic (Gill, 1978). Level of significant difference was tested against Bonferroni-t values since all compari- sons were non-orthogonal (Gill, 1978). 22 Results Injections of PS control do not effect castration concentrations of FSH in plasma (Marder et al., 1977; Lorenzen et al., 1978). Injections of PS result in con- centrations of FSH similar to those obtained following injection of phosphate buffered saline (PBS) injections containing the same concentration of protein (unpublished data). ReSponses to the lowest does of pFF and bFF, were not significantly different from the PS control with the exception.<1f postovulatory bFF which reduced concentra- tions of FSH (P < 0.05; Table 3). As level of protein increased in injectionscflprF and bFF, concentrations of FSH in plasma decreased. Postovulatory bFF had the greatest negative effect on concentrations of FSH and bFF was more potent than pFF at the two lower doses. Experiment II: Effect of Charcoal-Treated Bovine Follicular Fluid on the Castration Rise in Serum FSH and LH in Male Rats Experimental Des9gn The assay of Hermans et a1. (1980) was employed to examine effects of bFF on the postcastration increase in FSH in 50-day-old male Sprague-Dawley rats (200-250 gm) housed under 12 h light, 12 h dark. Rats were bilaterally castrated or sham Operated under ether anesthesia. Immediately following surgery each rat received a subcutaneous injection of 500 p1 per 100 g 23 .maouucoo mm ou pmummEoo mm Ho.o v m u o .mo.o v m u n .oa.o v m u m .cm>flw :Hmuoum mam 29:3 mmonu ou umHfiEflm 2mm mo wcoflumuucmocoo ca muHSmmH mam>ma Hm3oa pm mm mo macauomncH .mmwmcucmumm a“ Houycoo mm ou pmummfioo mm coflmmmummam ucwoummp .Esumm CH mmm mo 0: Auouum pumpcmum “V cmmzo .UmCHEHmumc #0: U 92 .GHus uwHDUHHHOM mafi>on n mmn .pflsam HmHDOHHHOw mcflouom n mmm .Esumw mcflonom mmn .cwmuoum mo mamnmflaaflzm o.m~.~.m “m.nma 61mmck.mafih.mam 1am.¢.~vfim.aom mausocae olmncm.s “¢.mma n1mo.4.aflwo.opa n.0m1a.m H¢.Hmm spoumas>oumom 31mm.a.vaflo.mma n.5mca.m “H.mam «Amvvo.amwm.mam suoumHs>omum man olmecm.MHHm.ovH mlsvcm.s Hm.mo~ mlom.o.mmfis.mqm mum ~.~oam.oom oz oz Houunoo. mmm oo.m om.v Hm~.~ ucmEpmmuB. Enumm Ho pagan umHSOflHHOM pmummuunamooumno mo ucDOEd monummumz um pmNflEouomHum>o mumm mHmEmm ca AHE\0:. mmm mo mam>wq coflumuummo co pagan “masowaaom mcflouom paw mafi>om mo muommmm .m mqm¢e 24 body weight of one of the following: preovulatory bFF (n = 10), postovulatory 01 10), midcycle bFF (n = 9), 13) as a control fluid. or serum from an ovx cow (n Follicular fluid was collected and prepared as described earlier. va serum was charcoal-treated as described for bFF. Rats were killed and trunk blood collected 8 h following treatment. Plasma was collected after centri- fugation (5°C, 20 min, 1548 x g) and frozen (-20°C) until assayed for gonadotropin content. Concentration of FSH and LH were determined by RIA (NIAMDD kit directions; Niswender et al., 1968) with the exception that the second antibody was replaced by 2% Protein A (IgSLlO, The Enzyme Center, Boston, MA.) in 1% bovine serum albumin in PBS to separate free from bound hormone. On the third day of incubation, 150 p1 of 2% Protein A was added to each assay tube and incubated at room temperature for 30 min. Sodium chloride solution (2 ml, 0.9%) was added to terminate the separation reaction. Statistical Analysis Hormone data were heterogeneous in variance and was not correctable by transformation. Therefore, the same procedure as discussed for Experiment 1 was used. Results Serum LH increased (P < 0.01) from 32 ng/ml in the sham operated group (N = 10) to 103 ng/ml in the 25 castrated control group (n = 10) (Table 4). Concentra-. tions of LH in the group receiving control serum were not different from the castrate group. The concentration of FSH increased (P < 0.01) from 424 ng/ml of serum in the sham operated grouptx>555 ng/ml of serum in the castrate control group. The concentration of FSH in the group receiving control serum were not different from the castrate group. All bFF pools blocked (P < 0.01) the castration rise in FSH, but were not different in their capacity to suppress FSH. Concentrations of LH were unaffected by bFF as compared with castrate controls. Egperiment III: Effect of Charcoal-Treated Bovine Follicular Fluid on Serum FSH and LH in Ovariectomized Heifers Experimental Design The objective of this experiment was to determine if charcoal—treated bFF would suppress FSH, but not LH, in ovx heifers. Eleven crossbred beef heifers (300-395 kg) were confirmed cycling by rectal palpation. Heifers were offered corn silage and water 99 libitum in loose housing. Heifers were bled via indwelling teflon cannulas (STX053, Becton-Dickinson, Rutherford, N.J.). Cannulas were held in place with a neckwrap of 7.62 cm wide ElastOplast elastic adhesive tape (Beiersdorf, Inc., South Norwalk, CT). Cannulas were filled with a 3.5% sodium citrate solution between sampling to prevent 26 TABLE 4.—-Effect of Charcoal-Treated Bovine Follicular Fluid or Serum from an Ovariectomized Cow on Concentration of Follicle-Stimulating Hormone and Luteinizing Hormone in Male Rats after Castration Treatment LH(ng/m1) FSH(ng/m1) mean i sem mean i sem Sham 32.0: 3.2 424.4:12.2 Castrate 103.2:10.ob 555.7:19.1b va serum 120.4115.0 604.1134.9 Preovulatory bFF 101.3:16.3 422.4:27.7d'e Postovulatory bFF 86.6:14.6 350.8:18.9°'e Midcycle bFF ll6.1il3.7 401.2117.7°'e aRats received 0.5 ml per 100 g body weight of treatment fluids directly after surgery. Trunk blood was collected 8 h < < as as as as following treatment. compared to sham control group. compared to castrate control group. compared to castrate control group. compared to ovx serum control group. 27 coagulation. Blood was drawn every 15 min for 2 h preceeding ovx to determine precastration baseline concentrations of LH and FSH. Heifers were ovx by insert- ing an ecraseur through an incision in the dorsal wall of the vagina to remove the ovaries. Blood samples were then taken at 15 min intervals for 4 h beginning 66 h after ovx (or 4 h before injections of bFF or serum). This series of samples established post castration base- line concentrations of LH and FSH. Heifers received three 20 m1 injections of pools of bFF removed from follicles at different stages of the estrous cycle as described earlier (preovulatory, n = 3 heifers; post- ovulatory, n = 3 heifers, or midcycle, n = 3 heifers) or ovx serum (n = 2 heifers) via jugular cannula at 6 h intervals. The first injection of bFF or control serum was given at 0 h which was 70 h after ovx. Hourly blood samples were drawn from 0 through 48 h. Blood was refrig- erated (4°C) for 6 to 12 h, incubated at 37°C for 2 h, and returned to 4°C before centrifugation (5°C, 30 min, 1548 x g). Serum was decanted and assayed for LH and FSH as previously described by Convey et a1. (1976), and Carruthers et al. (1980), respectively. Statistical Ana1y§is Serum hormone values from hourly blood samples for treatment and control groups were regressed over time. 28 Hormone data were found heterogeneous in variance and were not correctable by transformation. Therefore, spe- cific comparisons were made using Behren's t (Gill, 1978). Level of significant difference was tested against Bonferroni—t values since all comparisons were non- orthogonal (Gill, 1978). The FSH response curves for each treatment were divided into three parts: the decending portion (0 to 12 h after bFF), the flat portion (12 to 26 h after bFF), and the ascending portion (26 to 48 h after bFF) and analyzed by linear regression. Specific slope compari- sons were made using Sheffe's interval (Gill, 1978). Results Mean concentration of FSH in serum increased from a precastration baseline of 53 ng/ml to 116 ng/ml follow- ing castration. Castration increased mean LH in the serum from approximately 1 ng/ml to 3 ng/ml. Mean con- centration of gonadotropins in serum were not affected by injections of control serum (Figures 1 and 2). All groups of heifers responded similarly to the three pools of bFF through 26 h. Concentrations of FSH drOpped as early as four hours following the first injection of bFF. Although the group receiving bFF from preovulatory folliclesluuihigher postcastration concentra- tions of FSH, the descending lepes were not significantly FIGURE 1 . 29 Serum follicle-stimulating hormone (ng/ml) in ovariectomized heifers given three intravenous injections (20 ml) of serum from an ovariectom- ized cow (control:-€F)p or charcoal-treated follicular fluid from cattle. Follicular fluid was obtained from follicles > 10 mm in diameter and pooled based on stages of estrous cycle: postovulatory follicular fluid = days 2 to 6 (43—), midcycle follicular fluid = days 7-l7(-AP), preovulatory follicular fluid = days 18-1 (-¥F)- Arrows indicate time of injections. Bloou samples were taken at hourly intervals. qu- N¢ 1)- m- cm 4r «1:- manor o" .H muomwm 1r 1r- d. L L .oe .99 6mg 6mg (WM/0N) H83 ”“838 FIGURE 2 . 31 Serum luteinizing hormone (ng/ml) in ovariec- tomized heifers given three intravenous injections (20 m1) of serum from an ovariec- tomized cow (control: 49-). or charcoal- treated follicular fluid from cattle. Folli- cular fluid was obtained from follicles 3 10 mm in diameter and pooled based on stages of the estrous cycle: postovulatory folli- cular fluid = days 2-6 (-EF), midcycle folli- cular fluid = days 7-17 (-¢F), preovulatory follicular fluid = days 18-1 (-%%). Arrows indicate time of injections. Blood samples were taken at hourly intervals. Figure 2. 32 up (WW/9N) H1 H0838 GP 30 18 HOURS 33 different. Concentrations of FSH continued falling after the second and third injections of bFF, reaching maximal suppression (P < 0.005) 6 to 12 hours following the third injection of bFF (Figure 1). Concentrations on FSH were suppressed to precastration baseline concentrations (53 ng/ml). Concentrations of FSH remained at precastration levels from 12 through 26 h. At 26 h the responses to bFF started to diverge. The ascending portions of the response curves were different (P < 0.01) between the midcycle bFF and the other two pools (pre- and postovula- tory). By 36 h following the last injection of bFF, concentration of FSH in serum returned to castration levels for the groups of heifers treated with pre- and postovulatory bFF. The inhibitory effect of midcycle bFF on concentrations of FSH were more prolonged and concentrations had not reached postcastration levels by the end of blood sampling. Mean serum concentration of LH were unaffected (P > 0.05) by bFF (Figure 2). DISCUSSION Selective suppression of FSH in the rat assay sys- tems confirmed the results of Marder et a1. (1977) in female rats and Hermans et a1. (1980) in male rats, sug- gesting the presence of inhibin-activity in pools of bFF injected into heifers. DePaulo et al. (1979) found increased inhibin-activity in the ovarian venous plasma (OVP) of rats during diestrus, but the highest activity was found during early proestrus. In humans, FF from the follicular phase of the menstrual cycle contains increased inhibin-activity as compared to FF from the luteal stage (Chappel et al., 1980b; Channing et al., 1981). Inaccu- rate staging of ovaries, or overlapping of stage of the estrous cycle within pools of bFF could be responsible for the lack of differences in capacity of inhibin to suppress FSH in either of the rat bioassay systems. How- ever, the possibility of no variation in inhibin-activity throughout the bovine estrous cycle cannot be ruled out. Inhibin-activity may always be present in bFF but selec- tively released into circulation at certain stages of the cycle in response to increased concentrations of FSH. The female rat model was more sensitive than the male rat model, but this may have been a result of route 34 35 of injection since female rats received 23-93 p1 of bFF intravenously while male rats received volumes approach- ing 1,000 pl subcutaneously. In female rats, concentra- tions of FSH were more negatively effected than in male rats. Protein content of pools of bFF were similar (Table 2), however, pFF contained less total protein than bFF and was less potent suggesting that total amount of protein may be an index of inhibin-activity in follicular fluid. Miller et al. (1979b) found the greatest suppres- sion of FSH in mares 12 h following one injection of equine FF. In the present study, three injections of bFF may not have been necessary to reduce concentrations of FSH to precastration concentrations. It appears that the three injections of bFF prolonged the inhibition of FSH. Since Miller et al. (1979b) observed a rebound of FSH to castration levels within 12 h of maximal suppression. Midcycle bFF may have increased inhibindactivity in the cow, since concentrations of FSH were slower to recover when bFF injections were stOpped (Figure 1). These results differ from results in Experiment I and II using rats where preovulatory bFF appeared to have more inhibin- activity. Differences may be attributed to the method of detection. First, bFF may have different effects in rats than in cows. Secondly the rat assays were endpoint 36 determinations of bFF effects while the heifer experiment allowed mapping of effects over time. Steroid concentrations were determined for each pool of bFF (Table l). Charcoal extraction removed 99% of the total steroids in bFF. Total E2 and P were 8 and 30 ng per 20 m1 dose of FF. On a total blood volume basis (60 ml blood per kg of body weight, Altman, 1961), average circulating concentrations of E2 and P would have been 0.4 pg/ml and 1.4 pg/ml, respectively, following each injection of 20 ml of bFF. Actual concentrations of steroids would have been lower if tissue uptake and metabolic clearance rates were taken into account. Thus, concentrations of E2 and P were well below average circu- lating levels. Concentrations of progesterone range from 0.5 to 10 ng/ml serum (Glencross, 1973; Herriman et al., 1978) and estradiol ranges from 2 to 10 pg/ml serum (Glencross, 1972; Dobson and Dean, 1974) throughout the bovine estrous cycle. Roche and Ireland (1981) reported that exogenous administration of progesterone via vaginal coils in the artificial regulation of bovine estrous cycles had no effect on concentrations of FSH, but did affect LH. Kesner and Convey (1982) injected a large dose of E2 (1 mg) that was effective in reducing concentrations of FSH in ovx cows to precastration con- centrations, but lower levels were not tested. Moreover, 37 E2 and P replacement in ovx ewes to normal levels would not reduce concentrations of FSH to precastration base- line levels (Goodman et al., 1981). Therefore, consider- ing normal levels of E2 and P are not capable of con- trolling basal secretion of FSH in sheep, it is doubtful that the low levels of E2 and P injected could be responsi- ble for the reduced concentrations of FSH. The ovarian transplant studies of Uilenbroek et a1. (1978) have ruled out steroidal involvement in the selective suppres— sion of FSH in the rat. Steroids were inactivated by hepatic metabolism, and replacement of E2 and P to normal levels did not result in decreased FSH. Inhibin-activity of bFF has been destroyed with pronase (Hermans et al., 1980) and heat treatment (de Jong et al., 1981), suggest- ing a proteinaceous component. In conclusion, bFF obtained from prevoulatory (day 18-1), postovulatory' (day 2—6), and midcycle (days 7-17) stages of the estrous cycle contained inhibin- activity. However, the possibility of other steroids not accounted for could be responsible for the selective suppression of FSH. Purification and development of an RIA for inhibin are necessary to answer the question of what causes the selective suppression of FSH following the injection of bFF. Follicular fluid was capable of suppressing concentrations of FSH in ovx heifers to 38 precastration baseline concentrations without altering mean serum concentrations of LH. Inhibin-activity was transient, and concentrations of FSH returned to castra- tion levels in the absence of bFF injections. 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