Inhibins, activins and follistatins

J. steroid Biochem. Vol. 33, No. 4B, pp. 705-713, Printed in Great Britain. All rights reserved

INHIBINS,

1989 Copyright

ACTIVINS &IAO-YAO

Laboratories for Neuroendocrinology,

0

0022-4731/89 $3.00 + 0.00 1989 Pergamon Press plc

AND FOLLISTATINS YING*

The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92031, U.S.A.

Summary-Inhibins are proteins consisting of two subunits, 18 K alpha- and 14 K beta-subunits, linked by disulfide bonds. Two forms of inhibins A and B consisting of a common alpha-subunit and a similar but distinguishable beta-subunit specifically suppress the secretion and cell content of FSH in a delayed action. The production of inhibin is regulated mainly by FSH; therefore, a reciprocal relation between FSH and inhibin is established. Each subunit (alpha-, betaA- or betaB-) is encoded by a different mRNA species. Inhibin secreted in response to FSH from the pituitary originates primarily in the granulosa cells of the ovary and the Sertoli cells or testes. Two beta-subunits, betaAbetaA, betaAbetaB, or betaBbetaB form a new molecule, activin, that has opposite endocrine function to inhibin, but in the presence of inhibin, the activity of inhibin overrode

that of activin. Follistatin, a single-chain polypeptide with no structurally similarity to inhibin, also specifically inhibits the release of FSH, approximately one third potency of inhibin. This probably is due to the fact that inhibin suppresses both FSH secretion and cell content of FSH whereas follistatin primarily inhibits the release of FSH. In addition to the endocrine functfon, these gonadal proteins also exert paracrine function on gonadotropin-mediated steroidogenesis. Outside the reproductive system, inhibin and activin also play a role in hemoglobin production, erythroid cell differentiation; all three proteins, together with TGFbeta, are involved in immunosuppresion.

INTRODUCTION

The concept that a water-soluble, non-steroidal protein of gonadal origin specifically inhibits the secretion of follicle stimulating hormone (FSH) from the pituitary is an old one, but its isolation, the elucidation of its chemical structure, and its dual function controlling the basal secretion of FSH are new. With the knowledge of the structure of inhibin, a totally novel protein, as a result of the rearrangement of the gene products of inhibin subunit precursor, was isolated from follicular fluid and found to be structurally related to inhibin, but functionally opposite to that of inhibin; we named it activin. A third protein, which has inhibin-like activity but structurally has no homology to inhibin, was also isolated from porcine follicular fluid using the same bioassay as inhibin. We called this new molecule follistatin. In addition, inhibin, activin and follistatin not only regulate the secretion of follicle stimulating hormone (FSH) (endocrine action) and modulates the gonadotropin mediated steroidogenesis (paracrine/autocrine action) but also play a role in the general system, such as erythropoiesis and the immune response, possibly as part of the body’s defense against degenerative processes-disease and aging. *Present address: Department of Anatomy and Cell Biology, University of Southern California, Medical School, (BMT-401), 1333 San Pablo Street, Los Angeles, CA 90033, U.S.A. Proceedings of the International Symposium: Recent Advances in Gonadotropins (Structure, Biogenesis, Regulation, Mechanism of Action, and Therapeutic Applications) (Paris, 2(r22 April 1988).

In 1923, Mottram and Cramer [l] reported pituitary hypertrophy as determined by the appearance of so-called castration cells in the anterior lobe of pituitary after treatment of the testis with radium. Meanwhile, Martins and Rocha [2] have observed, in parabiosis experiments, complete prevention of this type of castration effects by injection of extracts of bull testes. In 1932, McCullagh [3] named it inhibin for this water-soluble substance which prevents the pituitary hypertrophy after castration or damage of seminiferous tubules. However, this idea of preferential inhibition of the release of FSH by gonadal aqueous extracts was not rekindled until about a decade ago. Interests in this water-soluble substance of gonadal origin regulating the secretion of FSH dwindled for the following reasons. First, when testosterone was isolated, it was demonstrated that this steroid plays an important role in regulating the secretion of gonadotropin and the production of spermatozoa is under the influence of gonadotropins and of testosterone itself, implying that inhibin might not be essential for the control of gonadotropin secretion [4]. A second reason was the utilization-hypothesis, proposed to account for the inhibitory effects of the gonad on gonadotropin secretion. In other words, the damaged germinal epithelium is supposed not be able to utilize or inactivate circulating FSH, resulting in increased urinary FSH levels [5]. Again, this suggests that inhibin might be irrelevant in regulation of gonadotropin secretion. The third reason was the fact that the bioassay for FSH as determined by ovarian weight increments was not sensitive enough to reflect FSH levels in

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the blood [6]. This was eventually improved by the development of radioimmunoassays (RIA) for both FSH [7] and LH [8]. In addition, the establishment of a pituitary monolayer culture system facilitated the determination of direct effect of secretagogues on the secretion of gonadotropins [9]. The combination of RIA and in vitro bioassay drastically improved the sensitivity of the measurement of FSH secretion. Using this improved bioassay system, a decapeptide that regulates the secretion of gonadotropin by the pituitary was isolated and characterized from the hypothalamus [lo, 111. Thus, the control of gonadotropin secretion by the anterior lobe of pituitary has been convincingly demonstrated to be influenced by the decapeptide secreted by the hypothalamus. This small peptide, originally designated as LRF (luteinizing hormone releasing factor) or LHRH (luteinizing hormone releasing hormone), when isolated and characterized, was found to stimulate the secretion of both FSH and LH. Further, hundreds of analogs were synthesized; not a single one, either agonist or antagonist, has been shown to release FSH or LH selectively. All analogs were found to influence the secretion of both FSH and LH. For this reason, it was renamed GnRH (gonadotropin releasing hormone). However, there is abundant evidence, experimentally and clinically, to suggest a selective secretion of FSH by the pituitary. For example, urine FSH levels are increased in men with oligozoospermia or azoospermia [ 121. Although the concept of inhibin was originally developed to explain a phenomenon in males, there are many experimental conditions implicating the existence of inhibin in females as well. During the early part of normal estrous or menstrual cycle, there is a secondary FSH, but not LH surge; that is, immediately before follicular development, there is an increase in the FSH to LH ratio which is responsible for the maturation of follicles that leads to the shedding of a mature egg(s) for fertilization [13]. Prior to ovulation, there is a sudden, large, and rapid rise in serum LH and FSH in the rat estrous cycle, known as the pre-ovulatory surge of gonadotropins as a result of a release of hypothalamic GnRH, which can be blocked by drugs such as Phenobarbital. However, the secondary surge of FSH cannot be blocked [14]. This secondary surge of FSH can be induced by the exogenous injection of LH, LRF or human chorionic gonadotropin, which in essence mimics the preovulatory surge of gonadotropins. This surge is thus dependent on the gonads, and not central nervous system [14]. Such a differential regulation of FSH release cannot be accounted for by the interaction between steroids and GnRH. Furthermore, immediately after bilateral ovariectomy in female rats, there is a rapid increase in serum FSH which is not accompanied by a rise in serum LH [15]. Similarly, unilateral ovariectomy and orchiectomy in the rat not only result in a transient increase in serum FSH, but also lead to compensatory hypertrophy of the re-

maining gonad in both males and females, as well as compensatory ovulation in the female. Interestingly, some of these preferential increases in serum FSH cannot be blocked by estrogen or testosterone suggesting a “missing-link” in the pituitary gonadal feedback mechanism. All of this evidence suggests the existence of a regulatory factor other than GnRH, therefore, there must be another substance or substances that selectively regulate the FSH secretion. To this day, the long-proposed hypothalamic FRF (FSH releasing factor) has not yet been isolated and characterized. Although initial efforts to purify inhibin employed material from reproductive organs such as seminal plasma, rete testes fluid, and extracts of spermatozoa and testes, it was ovarian follicular fluid that ultimately provided material with high biological inhibin activity and made possible the first successful isolation and characterization of inhibin. Again, it is worthwhile to note that the earlier observations of de Jong and Sharpe[ 161,and Schwartz and Channing[ 171 that a similar inhibin-like substance in the follicular fluid have provided readily available and abundant source of starting materials for purification of inhibin. This rekindled the interests in inhibin research, and led to its isolation and characterization. Isolation and Characterization of Inhibin, Activin and Follistatin In 1985, four laboratories were successful at isolating and characterizing inhibin from procine or bovine follicular fluid [ 18-231. Coincidentally, these laboratories used a universally accepted bioassay to monitor the biological activities and removed a large portion of inert protein in the follicular fluid, therefore, the biological active inhibin can be further purified. Inhibin molecules have a tendency to aggregate to form very large molecules, for this reason, a denaturing agent such as 6 M guinadine, urea, or 30% acetic acid is essential to prevent the aggregation of inhibin during the process of isolation. In our laboratories, we have used a six-step procedure to isolate two forms of inhibin (Inhibins A and B) from porcine follicular fluid [20]. The first step of purification is heparin-sepharose affinity chromatography. It serves to concentrate the inhibin activity for further purification and removing the large inert proteins. The next steps of purification are two Sephacryl S-200 superfine gel filtrations, four reversed-phase high performance liquid chromatographies (HPLC), and SDS/PAGE electrophoresis. At each stage of this purification procedure, the activities of inhibin were monitored by an in vitro bioassay [24]. Pituitary cells of immature female rats were enzymatically dispersed and the cells plated. On the second day of culture, the medium was removed and sample tested. After a further 48-h incubation, the medium was harvested and the LH and FSH present were determined by radioimmunoassays (RIAs). A dose-response curve was determined with crude porcine follicular fluid,

Inhibins, activins and follistatins which only inhibits the basal secretion of FSH, but LH was unaffected. It is important to realize that the bioassay used to monitor biological activity during the process of isolation of a protein inevitably predetermines the chemical entities to be isolated. Using different bioassays to monitor biological activities may lead to different molecules with different biological activities. Furthermore, the same pituitary tissue incubated differently may also lead to the isolation of different molecules. This is true when Li et aZ.[25] and Sheath et al. [see 261 isolated and characterized alpha-inhibin and beta-inhibin from human seminal plasma (two single-chain polypeptides of 92 and 94 amino acids, respectively). They used a bioassay different from the pituitary monolayer culture system described above. These two seminal plasma polypeptides, or synthetic fragments containing the putative biologically active region of either alpha-inhibin or beta-inhibin are not effective in the bioassay described here [26,27]. Furthermore alpha-inhibin is identical to the major degradation product of the gel-forming protein in seminal plasma and that beta-inhibin derived from a sperm-coating antigen synthesized in prostate tissue [28-291. Thus, these two polypeptides isolated from human seminal plasma are substances different from the inhibin described here. Even when the same bioassay was used to monitor inhibin activities, different molecules having similar inhibin activities or activities opposite to that of inhibin have been obtained. In addition, using different bioassays, the same molecule was isolated and characterized as described later. Therefore, different bioassays will lead to isolation of different molecules; the same bioassay can result in the isolation of different molecules of similar or opposite biological activities; and occasionally, different bioassays may yield a similar or identical molecule as in the case of activin and erythroid differentiation factor (EDF). After the second Sephacryl S-200 gel filtration, the small molecular weight inhibin eluted was further purified [20]. The biological active fractions were submitted to the first HPLC column. There are two separate areas of inhibin activity; they are designated A and B. In addition, this chromatography reveals the FSH-enhancing activities and an additional FSHsuppressing activities which were later isolated and characterized as two forms of activins [30-321 and follistatins [33]. Both inhibins A and B were submitted to three additional systems of HPLC and SDS/PAGE electrophoresis and microsequencing. The amino acid sequence was determined and we found two N-terminal sequences. Inhibins A and B have mol. wts of 32 K and each is shown to be a dimer composed of an 18 K common alpha-subunit and one of two distinct, but highly homologous beta-subunits. The alpha-subunit and beta-subunit are linked by disulfide bridges. SDS/PAGE analysis of the purified inhibins A and B under non-reducing conditions showed bands at 32 K, however, separated

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bands at 18 K and approximately 14 K, indicating inhibins A and B are proteins comprised of two polypeptides linked by disulfide bond(s). Amino acid analysis of these isolated molecules indicates that inhibins A and B are closely related, but not identical. With these partial amino terminal sequence of inhibin, we have collaborated with Mason, HayfIick, Niall and Seeberg in Genentech and deduced the sequences of the precursors of inhibin subunits from complementary DNA sequences[34]. Each form of inhibin (A or B) comprised two subunits: mol. wt 18 K (alpha-subunit) and 14 K (beta-subunit), crosslinked by one or more disulfide bridges. The alphasubunit is a common chain of 134 amino acid residues with 7 cysteine; the beta-subunits of inhibins A and B are similar, but not identical. Beta-A (beta-subunit of inhibin A) has 116 amino acids; beta-B-(betasubunit of inhibin B) has 115 residues. The betasubunits show 70% homology and have 9 cysteine residues, with no glycosylation site. This 9 cysteine sequence distribution pattern is similar to that of transforming growth factor-beta (TGFbeta), which was originally isolated from platelets based on its anchorage-independent growth [35]. In addition, this pattern is also found in the carboxyl portion of Miillerian Inhibiting Substance (MIS) which is a protein of testicular origin that suppresses the development of Miillerian ducts [36], in a protein encoded in the decapentaplegic gene complex in Drosophila [37], and in cartilage growth factors [38]. In addition, human, porcine, bovine and murine inhibin are closely related in structure and highly conserved [39-43]. There are additional forms of inhibin in the porcine follicular fluid. During the isolation of inhibin, we also observed inhibin activity at two positions of >32 K and a 32 K inhibin that is less retarded than inhibins A and B in the first HPLC separation [20]. This was later isolated and characterized as a singlechain polypeptide, follistatin. An Australian group has isolated and characterized from bovine follicular fluid a 58 K inhibin that is formed by 44 K alphasubunit and a 14 K beta-subunit [18]. The 44-K alpha-subunit has 5 consecutive arginine residues and can be processed proteolytically to form a 18 K alpha-subunit, resulting in 32 K inhibin. Similar and additional pairs of basic residues were found in precursors of both alpha- and beta-subunits, suggesting potential proteolytic cleavage sites. This 58 K inhibin is probably further by proteolytic cleavage to the 32 K form of inhibin similar to those isolated from porcine follicular fluid and bovine follicular fluid. Similarly, the precursor of the alpha-subunit as reported by Mason et a1.[34], consists of a pair of arginines preceding the carboxyl-terminal 134 residues, probably the protolytic release site for mature 32 K inhibins. In addition, there are arginine pairs at positions 55-56, 59-60 and 68869 of the pro-region of the inhibin precursor, suggesting other potential forms in inhibin. Similarly, five consecutive

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arginines preceded the mature 14K beta-subunits, with additional pairs of basic amino acids in the pro-region of the precursor as potential proteolytic cleavage sites. Indeed, biologically active inhibins with a molecular weight estimated to be greater than 58 K have been observed by immuno-affinity chromatography from bovine follicular fluid (120, 108, 88 and 65 K) [44]. Inhibin from the testes has been isolated and characterized and has a primary structure highly identical to that of ovarian follicular fluid 32 K inhibin [45]. Because of the striking similarities between inhibin beta-subunits and TGFbeta, the question was raised whether inhibin has TGFbeta activity and vice versa. Later, we have observed that inhibin showed no TGFbeta activity, but TGFbeta itself has ability to stimulate FSH secretion [46]. This led to the discovery and isolation of novel proteins from porcine follicular fluid that is structurally related to inhibin but possessing biological activities opposite to that of inhibin. Using the same six-step procedure and in vitro bioassay used in inhibin isolation, we purified 2 proteins from the follicular fluid that stimulate FSH secretion. One of the purified proteins showed a single band of apparent mol. wt 24 K on SDS/PAGE analysis under non-reducing conditions, but two separate bands coinciding with the beta-subunits of inhibins A and B under reducing conditions. Microsequencing of the intact molecule reveals the first 32 amino-terminal residues to be identical to the theoretical residues of two beta-subunits of inhibins A and B sequenced simultaneously. Thus, this protein is a heterodimer consisting of the beta-subunits of inhibins A and B, linked by disulfide bonds [30]. The second form of FSH-stimulating protein isolated from follicular fluid found to be homodimer of two beta-subunits of inhibin A joined together by intersubunit disulfide bonds, which we termed activin A [31]. The homodimer of betaAbetaA was also independently isolated and characterized by Vale et aZ.[32]. They called it FRP (FSH releasing protein). This molecule was also isolated and characterized from a human monocyte cell line, THP-1. Eto et af.[47] isolated a protein from the spent medium after THP-1 cells were treated with phorbol ester. They have monitored the biological activity based on its ability to induce differentiation in cultured erythroleukemic cells (Friend cells). They named this protein EDF which turned out to be a homodimer of a mol. wt of 24 K with an N-terminal sequence identical to that of the betaA-subunit of inhibin. Thus, different bioassays yield the same molecule and activin-A or FRP was found to be produced by human cells from outside of the reproductive system. Conceivably, the homodimer of betaB-subunit may also have FSHstimulating activity; thus, betaB and betaB may form activin B. Therefore, a common alpha-subunit and two similar, but distinguishable beta-subunit forms inhibins A

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and B, which specifically suppress FSH secretion; dimers of beta-subunits with similar types of cysteine distribution pattern resulting from rearrangements of gene products possesses biological activities opposite to those of inhibin. Such a rearrangement of gene products resulting in opposite bioactivities may have a profound impact on our understanding of the homeostatic control of biologically active substances. Using the same bioassay, that is pituitary monolayer culture system and the same purification procedure described above, we have isolated a singlechain polypeptide of 35 K. With the partial N-terminal sequence information, we have characterized this molecule by cDNA cloning and DNA sequencing using long synthetic oligo-nucleotide probes. We called this molecule follistatin which is unusually cysteine-rich, containing 36 cysteines in the mature coding sequence of 315 amino acids. It is organized into three homologous domains corresponding to follistatin (66-135) (139-210) and (216287). These domains showed high similarity among themselves and to human pancreatic secretory trypsin inhibitor (PSTI) [47]. The chemical aspects of these three novel gonadal protein hormones modulating the secretion of FSH can be summarized as follows:

(4 Two forms of inhibins A and B consisting of a common alpha-subunit and similar but distinguishable beta-subunits suppress the secretion of FSH. @I Two beta-subunits, betaA betaA, betaA betaB, or betaB betaB form a new molecule, activin, that enhances FSH secretion. (c) TGFbeta, which has a structure similar to that of activin, also facilitates FSH secretion. (d) Follistatin, a single-chain polypeptide with no structurally similarity to inhibin, also inhibits the release of FSH.

Biological Activities of Inhibin, Activin and Follistatin The biological activities of purified inhibin have been demonstrated both in vitro and in vivo as specifically suppressing the FSH secretion but not the LH secretion [18-20,48,49]. The two forms of 32 K inhibin are statistically equipotent as specific inhibitors of the basal secretion of FSH with an ED50 of lO-14M, which is more potent than all hypothalamic regulatory factors isolated and characterized to date. In comparison, activin specifically stimulates the basal secretion of FSH by the pituitary with an ED50 of 1.5 x lo-” M [30-321. When both activin and inhibin are added to the pituitary monolayer system, the activities of inhibin prevail. In the presence of an effective dose of inhibin, the FSHenhancing activities was overriden by inhibin [49]. This is the reason why the activin activity was not observed in the crude preparation of follicular fluid. Only when the inhibin and activin were separated at

Inhibins, activins and follistatins the first HPLC two opposite biological activities were clearly demonstrated. Since inhibin is isolated and characterized on the basis of an in vitro bioassay system, it is essential to ascertain the specificity of the molecule both in vitro and in vivo. The specificity of all three gonadal proteins were ascertained in vitro. The purified inhibin only suppresses the secretion of FSH but have no effect on that of LH. Inhibin has no effects on the basal secretion of other pituitary hormones including TSH, and prolactin. Activin shows specific FSHenhancing activities but not that of LH. Again, since both inhibin and follistatin were isolated based on the same bioassay, the biological activities of inhibin and follistatin in vitro were compared. Follistatin is approximately one third potent as inhibin. This probably can be accounted for by the fact that inhibin suppresses both secretion of FSH and the cell content accumulation of FSH but follistatin primarily acts on the inhibition of FSH secretion and has little effect on the cell content of FSH. In the in vitro system, it takes at least 18 h for inhibin to demonstrate the activity, the suppression of FSH secretion. The action of inhibin is clearly demonstrated after 48 h incubation. This is also true with activin and follistatin. This delayed effect is different from that of all hypothalamic releasing factors which only takes minutes to a few hours to act. The biological activity of inhibin in viuo has been demonstrated [49]. Intravenous injections of purified inhibin to acutely ovariectomized metaestrous rats drastically reduce the serum FSH, but not LH. Crude porcine follicular fluid has long been demonstrated to specifically suppress the secretion of FSH in uiuo in several experimental regimens. Given our current knowledge of inhibin, follistatin and activin, however, the effect of crude porcine follicular fluid probably reflects only the overriding of inhibin and follistatin activity in the presence of activin. Antisera raised against the native molecule or synthetic fragments of the alpha-subunit of inhibin have been found to neutralize the biological activities of inhibin in vitro [48-501. Antisera to (Gly26, Tyr27) inhibin-alpha (l-27) infused into female rats during estrous cycle elevate the FSH profile, but not that of LH. After the antiserum was ascertained to read the native molecule of inhibin, a single injection of antiserum to (Gly26, Tyr27) inhibin-alpha (l-27) or (Tyr30) inhibin-alpha (l-30) into metaestrous and diestrous rats resulted in an apparent high serum FSH level but LH level was unaffected. This is best explained by the antiserum neutralizes the endogenous inhibin consequently removes the inhibition of FSH secretion. Using such antisera, the primary site of inhibin production has been localized immunohistochemically to be the granulosa and Sertoli cells [5 11. Inhibin was shown to be produced primarily by

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granulosa cells as determined by bioasssay [16,52], radioimmunoassay for inhibin [53,54], immunohistochemistry [51], in situ hybridization [55], gene expression [56] and incorporation studies. Using a specific, sensitive RIA for inhibin, developed with antiserum against synthetic fragments of the N-terminal portion of alpha-subunit of inhibin, it was demonstrated that the inhibin produced in cultured rat granulosa cells was primarily FSH dosedependent [53,57]. The concentration of FSH added to the culture well is in proportion to the quantity of inhibin measured. The production of inhibin in this system is in proportion to the concentration of FSH added, the density of cells plated and the culture time of the cells. FSH by itself can also stimulate the inhibin production although the presence of androgen or estrogen definitely potentiates the inhibin production. Similarly, FSH stimulates the enriched Sertoli cells to secrete inhibin. The inhibin produced by these cells can be purified by immunoaffinity chromatography and the eluted inhibin are biologically and immunoreactively indistinguishable from the purified inhibin. This process is mediated through a CAMPdependent pathway [57]. Using incorporation studies, Bicsak et al. also demonstrated that the precursor of inhibin alpha-subunit proceed to a 49 K molecule which then further proceeded to the 18 K mature alpha-subunit, while the 14 K beta-subunit is formed directly from the precursor. The 18 K mature alphaand 14 K beta-subunits for the mature 32 K inhibin molecule. In immature rats treated with pregnant mares’ serum gonadotropin (PMSG), the immunoassayable inhibin increases, reflecting follicular maturation. Animals untreated, ovariectomized controls, and ovariectomized PMSG-treated rats show no increase in inhibin levels [48]. Recently, Woodruff et a/.[551 have demonstrated that dynamic changes of ovarian inhibin gene expression and/or secretion of inhibin play an important role in the coordination of hypothalamic- hypophyseal-ovarian axis of female reproductive cycle. Three lines of evidence exist suggesting that inhibin plays a role in the normal reproductive process. First is the reciprocal relationship between FSH secretion and inhibin production [49,53] as well as high immunoassayable inhibin during the luteal phase of human menstrual cycle [50,58]. It can be hypothesized that the inhibin produced by granulosa cells and/or by luteal cells regulates the amount and timing of initial FSH secretion for folhcular recruitment. With the development of a few follicles, adequate amounts of estrogen is produced and induces the positive feedback for more FSH and/or luteining hormone (LH) release from the pituitary for further follicular development. Prior to the preovulatory stage of the cycle, the optimal amount as well as the ratio of estrogen and progesterone triggers the surge of preovulatory LH and FSH. The production of

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inhibin in cycling rats is critical for such a fine tuning for the interrelationship between inhibin and FSH secretion. The second reason that inhibin, activin and follistatin play a role in the female reproductive system is that these proteins also act directly on the gonadal cells to influence steroidogenesis. Inhibin, activin, and follistatin have a direct effect on the aromatase activities in the granulosa cells, inhibin and follistatin prevent, while activin enhances, the aromatase activity in the presence of FSH. Furthermore, activin, like TGFbeta, stimulates the aromatase activity even in the presence of inhibin. We have observed that 32 K porcine inhibin reduced FSH-mediated aromatase activity, although no such effect was observed with 58 K bovine inhibin by Hutchinson et a1.[59]. This discrepancy is probably due to the addition of insulin to the medium used by Hutchinson et al. Insulin is known to enhance aromatase activity in cultured granulosa cells, and, therefore, masked the inhibition of estrogen accumulation by inhibin. Transforming growth factor-beta (TGFbeta) and activin override the effect of inhibin when cells were co-incubated with both. Intragonadal functions of inhibin and activin were demonstrated in the cultured rat Leydig cells by Hsueh et af.[60]. The paracrine effects of these gonadal proteins not only point to a local fine tuning of the regulation of the steroidogenesis essential for gonadrotropin secretion in the normal reproductive cycle but also suggest that these molecules may play a role in degeneration of hypothalamic-hypophyseal-ovarian axis as well as atresia of individual follicles. Again, the role inhibin, activin and follistatin play in the reproductive process may also shed light on the normal process of atresia and its pathology-infertility which may lead to better understanding of the mechanism underlying polycystic ovary syndrome and menopause and, may offer more efficient management of these conditions. These findings further support the concept that a given molecule may have totally different biological activities when acting on different target cells. This concept was further demonstrated with activin A in a biological system totally unrelated to reproductive physiology as described below. The third reason that inhibin plays a role in the female reproductive system is based on the observations that plasma levels of inhibin rise progressively during the early part of estrous and menstrual cycles. In patients preparing for in vitro fertilization treatment with gonadotropins, the maturation of follicles is accompanied by a rise of plasma inhibin [50, 581. There is a close correlation among the levels of plasma inhibin, the number of follicles developed, and the plasma estradiol. Immunoreactive inhibin was also detected in human placental extract [61]. During the menstrual cycle, a similar early rise in plasma inhibin and decrease when the preovulatory surge of gondotropins begins were also observed. There is no detectable

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plasma level of inhibin in post-menopausal women [50, 581. Similarly, in the rat, a single injection of PMSG (pregnant mare’s serum gonadotrophin) stimulates follicular development, estradiol prodution and a rise in immunoreactive inhibin [48]. Removal of the ovaries prior to PMSG injection completely abolishes this rise of plasma inhibin. Cells of the corpus luteum also reveal immunoreactivity with the inhibin antiserum which is in agreement with the detection of mRNA encoding the inhibin-alpha precursor in rat corpora lutea [56]. Incidentally, a given glycoprotein may have totally different biological activities depending on the target cell it affects. In addition to their endocrine and paracrine functions, both inhibin and activin, play an important role in erythoid differentiation and hemoglobin production. Furthermore, gene expression of inhibin alpha- and beta-subunits have been demonstrated in bone marrow and thymus [62] and adrenal cortex [63]. Very recently, we have observed that these gonadal proteins also play a role in immune system Becker and Ying, unpublished data. These findings suggest that these molecules may be part of a more general system that makes up one kind of defense against degenerative disease or the aging processes. On the other hand, novel neurotropic action of activin has been demonstrated recently. Using regiondefined activin specific, polyclonal antibodies, Sawchenko et a1.[64,65] have identified the molecule in the neurons in magnocellular compartments of the paraventricular (PVN) and supraoptic (SON) nuclei ascending from a group of cells centered in the nucleus of the solitary tract. These are the same neurons that also found to be immunoreactive positive when identified with somatostatin-28 antiserum. Furthermore, experimental evidence strongly suggests that these activin-containing projections represent a physiologically significant afferent system mediating the milk ejection reflex [66]. First, microinfusion of activin into the PVN produced a two-fold elevation of immunoreactive oxytocin in the systemic circulation. Second, activation of endogenous activin-containing pathways via electrical stimulation results in the elevation of systemic immunoreactive oxytocin levels. Last, micro-infusion of antiserum to activin attenuates immunoreactive oxytocin secretion. These findings indicate that the novel protein, activin, other than the endocrine, paracrine actions, plays an important role in the physiological functions of the PVN and SON of the hypothalamus. Activin was also a novel molecule produced by human monocytes. Eto et a/.[471 have isolated a protein on the basis of its ability to induce the differentiation of Friend cells from a line of human monocytes (THP-I), originally derived from a oneyear-old boy with leukemia. This protein, named erythroid differentiation factor (EDF), was found to be a homodimer of the betaA-subunit of porcine

Inhibins, activins and follistatins follicular inhibin, and therefore identical to activin A. EDF was shown to have FSH-enhancing activity in pituitary monolayer culture system [49,67], demonstrating that the same molecule possesses different biological activity in two different systems (reproduction vs erythroid differentiation). This potential role in erythropoiesis was further extended to the induction of hemoglobin production in the differentiation of human erythroid cell line IL563 by Yu et n1.[68]. They found that activin and inhibin, acting antagonistically, modulate the induction of hemoglobin production in this cell line. We have examined the expression of follistatin and inhibin subunits in rat overies and placentae during different states of gestation. Tissue extracts prepared from day 10 of pregnant rat ovaries and placentae express a specific kilobase follistatin mRNA species whereas lower levels of follistatin mRNA are detected on ovaries and placentae on days 14 and 20 of pregnancy. The follistatin mRNA expressed in the placenta is higher than that in the ovary. These findings suggest that follistatin may play a role in the early development or cell differentiation. The biological activities of these three gonadal proteins can be summarized as follows:

(4 Inhibin

specifically suppresses the secretion and cell content of FSH in a delayed action. The production of inhibin is regulated mainly by FSH; therefore, a reciprocal relation between FSH and inhibin is established. (b) Activin has opposite endocrine function to inhibin, but in the presence of inhibin, the activity of inhibin overrode that of activin. (4 Follistatin specifically suppresses the release of FSH, approximately one third potency of inhibin. This probably is due to the fact that inhibin suppresses both FSH secretion and cell content of FSH whereas follistatin primarily inhibit the release of FSH. (4 In addition to the endocrine function, these three gonadal proteins also influence gonadotropin-mediated steroidogenesis. Outside the reproductive system, inhibin and 03 activin also play a role in hemoglobin production, erythroid cell differentiation; all three proteins together with TGFb, are involved in immunosuppression. (0 Follistatin may have specific effect on the embryo development during the early stage of pregnancy. Acknowledgemenrs-This

grants HD-22867

work was

supported

by

NIH

and HD-24648. REFERENCES

Mottram J. C. and Cramer T. V.: On the general effects of exposures to radium on metabolism and tumor growth in the rat and the special effects on testis and pituitary. Q. JI exp. Physiol. 13 (1923) 209-228.

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