Recent Progress in Hormone Research: Proceedings of the 1987 Laurentian Hormone Conference

Zajac

PREFACE

Many new and exciting things are happening in endocrinology. The analysis of hormone action at the molecular level is opening new vistas to our understanding of hormonal regulation of physiological and developmental functions. This volume of Recent Progress in Hormone Research contains many examples of the molecular biological approach to unraveling the mysteries of hormone action. The topics covered range from the control of gene expression in differentiated systems to the control of differentiation itself. The structure and function of receptors and their role in the control of cell function via signal transduction and intracellular second messengers is examined by several authors. The surprising interactions and functions of the inhibin peptides and their role in the control of gonadotropin secretion is discussed vigorously. The role of growth factors and lymphokines in autocrine, paracrine, and endocrine physiology is becoming increasingly more important and is covered in lucid presentations. A topic that stimulates many questions, comments, and controversy is the concept of complementary peptides and their use as tools for examining hormonal interactions and regulation. The latest concepts and ideas concerning luteal regulation and fetal endocrinology complete the book and make us realize that cellular and molecular biological studies of hormone action must be integrated into the higher order scheme of physiological functions.

As usual, the discussion–question periods at the conference were lively and dynamic. These sessions were conducted in an admirable fashion guided by Gordon Ringold, Daryl Granner, Robert Fellows, Edward Rall, Marian Walters, Gerald Aurbach, Henry Friesen, and Neena Schwartz. On behalf of the Board of Directors I would like to thank each of them for a job done well. We also thank Robert Lacroix who recorded the sessions and Lucy Felicissimo and Linda Carsagnini who transcribed them. The final corrections of the discussion sessions were made by Georgietta Brown who deserves special thanks.

Financial support for the Laurentian Hormone Conference from pharmaceutical companies is especially important, and their contributions are greatly appreciated. This marked the first year of the Syntex Lecture which was made possible by a generous gift from Syntex. Other contributions include those from Serono Laboratories, Sterling-Winthrop Research, Wyeth Laboratories, Merck Sharp and Dohme Research Laboratories, Merrel Dow Research Institute, and Eli Lilly Laboratories.

James H. Clark

Chemical and Biological Characterization of the Inhibin Family of Protein Hormones¹

WYLIE VALE*, CATHERINE RIVIER*, AARON HSUEH†, CAROLYN CAMPEN*, HELENE MEUNIER*, THOMAS BICSAK†, JOAN VAUGHAN*, ANNE CORRIGAN*, WAYNE BARDIN‡, PAUL SAWCHENKO§, FELICE PETRAGLIA**, JOHN YU††, PAUL PLOTSKY*, JOACHIM SPIESS* and JEAN RIVIER*,     *The Clayton Foundation Laboratories for Peptide Biology; §Developmental Neurobiology Laboratory, The Salk Institute, La Jolla, California 92037; †Department of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093; ‡The Population Council, Center for Biomedical Research, New York, New York 10021; **Department Obstetrics and Gynecology, University of Modena, School of Medicine, Modena, Italy; ††Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, California 92037

Publisher Summary

This chapter discusses the chemical and biological characterization of the inhibin family of protein hormones, which is a family of peptides isolated from the follicular fluid or rete testis fluid on the basis of their ability to inhibit the secretion of the follicle-stimulating hormone (FSH) by cultured rat anterior pituitary cells. It also reviews the possible roles of inhibin and fibre-reinforced plastic (FRP)/activin in placenta, brain, and bone marrow. Inhibin-related dimers are broadly distributed anatomically and have powerful activities in several biological systems where inhibin and FRP/activin often exhibit opposite effects. While the physiologic roles of inhibin to regulate FSH secretion in the female rat and immature male rat are strongly supported, the significance of these hormones within the gonad, brain, placenta, and bone marrow have yet to be placed in in vivo context. Although the panoply of functions of inhibin and FRP/activin are certainly incompletely understood at this time, this family has already demonstrated a powerful mechanism for the generation of signal diversity whereby differential subunit association can result in the generation of dimers with opposing biological actions in multiple tissues.

I Introduction

The functions of the pituitary gonadotrophs are regulated by the dynamic interplay of neural, peripheral, and local signals. Although hypothalamic gonadotropin-releasing hormone (GnRH) stimulates the production of both leutinizing hormone (LH) and follicle-stimulating hormone (FSH) and they are often cosecreted, there are a variety of circumstances, some involving gonadal damage or extirpation with or without steroid replacement, in which the secretion of the two gonadotropins can be dissociated from one another. Mottram and Cramer (1923) reported that radiation-induced destruction of the seminiferous tubules of male rats caused hypertrophy of the pituitary gland. The administration of an aqueous testicular extract was observed by McCullough (1932) to prevent the appearance of castration cells in gonadectomized rats. The water-soluble testicular principle responsible for this activity was termed inhibin and was considered to be distinct from androtin, the factor soluble in organic solvents that was later shown to be testosterone. With concepts and tools provided by the identification, characterization, and development of assays for the two gonadotropins (Sairam and Papkoff, 1974), the effects of gonadal injury (Klinefelter et al., 1942) and of crude inhibin preparations on LH and FSH secretion could be studied. The demonstration that fluids and extracts of male and female gonads of many species selectively inhibit FSH production in a variety of in vivo and in vitro assays (Schwartz and Channing, 1977; Channing et al., 1985; Baker et al., 1976; Erickson and Hsueh, 1978; DeJong et al., 1979; Franchimont et al., 1979) supported the concept of inhibin as a negative regulator of FSH secretion. The levels of inhibin-like activity in plasma or gonadal fluids measured by bioassays were shown to vary with gonadectomy, gonadotropin administration, stages of the estrous cycle, maturation, and other conditions.

This article will deal exclusively with a family of peptides isolated from follicular fluid or rete testis fluid on the basis of its ability to inhibit the secretion of FSH by cultured rat anterior pituitary cells. This assay method (Vale et al., 1972) has been extensively validated and was employed for the purification of ovine somatostatin (Vale et al., 1975), ovine and rat corticotropin-releasing factor (CRF) (Vale et al., 1981; Spiess et al., 1983), and human and rat growth-releasing factor (GRF) (Rivier et al., 1982; Guillemin et al., 1982; Spiess et al., 1983). Erickson and Hsueh (1978) and subsequently most groups in the field groups used the pituitary cell culture method for the routine assay of inhibin. The peptides, "α-inhibin-92" and its derivatives such as α-inhibin-31 and "β-inhibin-94," have been purified from seminal fluids based upon other bioassays (Li et al., 1985; Seidah et al., 1984), but we and others find them to be inactive in the pituitary cell culture assay (Li and Ramasharma, 1987; Ling et al., 1985; Vale and Rivier, unpublished results) and will not be discussed here.

II Characterization of Inhibin and FRP/Activin

In 1985, four groups reported the isolation of proteins from follicular fluid that could suppress the secretion of FSH by cultured rat anterior cells. Follicular inhibins were noted to be heterodimers, linked by disulfide bridges and some N-terminal sequences of both chains of inhibin were provided (Robertson et al., 1985; Miyamoto et al., 1985; Ling et al., 1985; Rivier et al., 1985). From porcine follicular fluid the latter three groups purified Mr ∼ 32,000 dimers consisting of Mr ∼ 18,000 and Mr ∼ 14,000 subunits which are now by convention referred to as the α and β chains, respectively. The Mr ∼ 56,000 dimer purified by Robertson et al. (1985) from bovine follicular fluid consisted of an Mr ∼ 44,000 α chain and an Mr ∼ 14,000 β chain. Subsequently, Robertson et al. (1986a) isolated a smaller dimer from bovine follicular fluid that was similar to inhibins purified from porcine follicular fluid.

Based upon N-terminal sequence information, synthetic oligonucleotide probes were designed and used to select clones encoding inhibin subunits from porcine and bovine ovarian cDNA libraries. The sequences of precursors for the porcine α subunit (Mason et al., 1985; Mayo et al., 1986) and two distinct β subunits, βA and βB (Mason et al., 1985), were deduced from cDNA sequences. Each subunit and its precursor are encoded by a separate mRNA. The dimer αβ A comprises inhibin A and αβB comprises inhibin B. Although bovine α and βA were identified (Forage et al., 1986) no bovine βB was described. Porcine probes were employed to select and sequence human α clones from ovarian (Mason et al., 1986) or placental (Mayo et al., 1986) libraries and βA and βB clones from ovarian (Mason et al., 1986) libraries. Most recently, α, βA, and βB subunits of rat inhibin have been cloned and their precursor sequences deduced (Esch et al., 1987; Woodruff et al., 1987).

The three prohormones (Fig. 1) contain several clusters of multiple basic residues which can serve as potential proteolytic processing sites as well as several potential Asn-linked glycosylation sites. The mature, most abundant forms of each subunit, Mr ∼ 18,000 α and Mr ∼ 14,000 βA and βB, are peptides of 134, 116, and 115 residues, respectively, and are derived from the C-terminal regions of their prohormones. As shown in Fig. 2, the Mr ∼ 18,000 α chains from all species characterized contain seven cysteine residues and at least one potential glycosylation site (the human α chain has two sites). The mature Mr ∼ 14,000 β subunits (Fig. 3) have nine cysteines and no consensus Asn-linked glycosylation sites. Although the location of intra- and intermolecular disulfide bridges have not been assigned, each subunit is presumably extensively folded. Amino acid sequences exhibit ∼80% similarities between porcine, bovine, human, and murine inhibin α subunits and >95% similarities within βA or βB subunits. The mature βA subunits are identical in all four species and the mature βB subunits differ by only a single residue. βA and βB subunits are 70% similar to one another and much less so to the α subunit. The β subunits and the α subunits to a lesser extent are structurally related, particularly with regards to the distribution of cysteine residues, to a family of growth factors that includes transforming growth factor β (TGF-β) (Derynck et al., 1985), Müllerian duct inhibiting substance (MIS, Cate et al., 1986) which causes regression of the Müllerian duct during development of the male, and the fly decapentaplegic gene complex which apparently plays a role in embryonic dorsal–ventral determination (Padgett et al., 1987).

FIG. 1 Human inhibin subunit precursors. Mature portions of each subunit are shaded. From Mason et al. (1986) and Mayo et al. (1986).

FIG. 2 Protein sequences of mature α subunits from several species. –, identity with porcine inhibin α; cross-hatched areas, potential Asn-linked glycosylation sites; stippled areas, cysteine residues. Modified region was synthesized for use as immunogen (Rivier et al., 1986; Vaughan et al., 1987).

FIG. 3 Protein sequences of mature β subunits from several species. Symbols as per Fig. 2 legend.

We have also isolated and partially characterized an ovine inhibin of Mr ∼ 32,000 from ram rete testis fluid (Bardin et al., 1987; Vaughan et al., 1987). Ovine male inhibin is a dimer whose subunits have N-terminal sequences similar to those of follicular inhibin (Figs. 2 and 3). The N-terminal sequences of the α subunits of ovine male inhibin and bovine female inhibin are identical whereas ovine βA differs from those of the other species by one residue. From these results we suggest that male and female inhibin subunits are identical although differences in processing as well as in ratios of inhibin A to inhibin B might exist.

Follicular fluids contain a variety of biologically active species including multiple inhibin-related dimers comprised of different sized α chains (as a result of varying glycosylation, incomplete precursor processing or degradation of the mature form) with either βA or βB subunits also of different lengths. For example, the Mr ∼ 54,000 bovine inhibin purified by Robertson et al. (1985) contained an N-terminally extended α subunit. In addition, Miyamoto et al. (1986), using immunoblotting procedures, have observed high-molecular-weight trimeric forms of Mr ∼ 120,000, 108,000, and 88,000 that appear to be composed of an Mr ∼ 62,000 β chain linked by disulfides to αβ dimers of different sizes (Mr ∼ 65,000, 55,000, and 32,000); unfortunately, the type of β chain (A or B) was not resolved in this study. The role, if any, played by such trimers in the assembly of αβ and ββ dimers (see below) is presently unknown. Furthermore, there are other FSH release inhibiting fractions (Rivier et al., 1985) that are not immunologically related to inhibin α or β chains and which upon purification contain only monomeric proteins (N. Ling, personal communication; and Rivier, Vaughan, Corrigan, McClintock, Spiess, and Vale, unpublished results). Finally, we have observed in our initial report of the isolation of inhibin (Rivier et al., 1985) fractions that could stimulate FSH secretion.

We hypothesized that this FSH releasing activity was due to an FSH releasing protein (FRP) and purified it by several FPLC and HPLC steps. Purified FRP showed a major band on SDS–PAGE of Mr ∼ 28,000 which following reduction converted to a single species of Mr ∼ 15,000. Edman degradation of this preparation yielded a single peptide sequence that was identical to the N-terminal 38 amino acids of inhibin βA. Following specific proteolytic cleavage and purification of fragments, additional peptides corresponding to residues 70 −85, 88 −102, and 103 −116 of inhibin βA were identified by sequence analysis. In all we established that 83 of 116 residues including the amino and carboxy termini were the same in FRP and inhibin βA. These results led us to propose that FRP is a homodimer of inhibin βA (Vale et al., 1986). At the same time as our characterization of FRP, Ling et al. (1986a) identified another FSH-releasing protein, activin, as a heterodimer, inhibin βAβB, and subsequently confirmed our identification of the βAβA dimer (Ling et al., 1986b). Both groups raised the possibility of the existence of a βBβB homodimer that would also release FSH. Conventionally, FRP or activin A, AB, and B would refer to inhibin βAβA, βAβB, and βBβB, respectively, and the ββ dimers would be referred to generically as FRPs or activins.

The finding of a homodimeric form of an inhibin β subunit is noteworthy in view of the fact that both TGF-β and MIS are also homodimers. Although Ying et al. (1986a) reported that TGF-β is a potent FSH secretagogue in vitro, we have found no such stimulatory effects of TFG-β on FSH release under a wide range of conditions. It is likely that as we begin to elucidate the full spectrum of actions of FRP/activin and inhibin we will begin to consider these proteins as full functional members of the TGF-β family of growth/developmental factors.

III In Vitro Hypophysiotropic Actions of Inhibin and FRP/Activin

Inhibins purified from either porcine follicular fluid or ram rete testis fluid are potent inhibitors of the basal release of FSH by cultured anterior pituitary cells. Inhibin acts in vitro with a several hour latency and maximal effects on FSH secretory rates are attained between 8 and 24 hours following initiation of treatment. Inhibin bioassays are, therefore, typically conducted over a 48- or 72-hour period. The effects of inhibin on FSH release are dose related exhibiting EC50 values of between 3 and 60 pM depending upon the source of inhibin and circumstances such as length of time, serum concentration, and presence of other substances. Under nonstimulated conditions, inhibin has little or no effect on the secretion of LH, although decreases in cellular LH as well as FSH content can be detected in our studies. In cells stimulated by GnRH, or other means such as phorbol myristate acetate, highly purified inhibin suppresses the release of both LH and FSH (Fig. 4, from Campen and Vale, 1988). Effects of crude inhibin preparations on LH secretion had been noted previously (Massicotte et al., 1984a; Scott and Burger, 1981; DeJong, 1979; Franchimont et al., 1979) and are in agreement with results of some (Fukuda et al., 1987) but not all groups (Robertson et al., 1986b) working with highly purified inhibin. The ability of inhibin to lower total amounts (medium + cells) of LH and FSH suggests that the peptide suppresses biosynthesis of both gonadotropins. It is possible that the selectivity of inhibin to block FSH and not LH secretion observed under basal conditions is a reflection of the higher tonic secretory rate of FSH with its correspondingly greater dependence on newly synthesized hormone levels. When LH secretory rates are elevated, then the consequences of partial biosynthetic inhibition could be revealed as an inhibition of release. It should be pointed out that in vivo, inhibin preparations exhibit high selectivity to lower FSH and not LH levels. When supplies permit, it will be interesting to determine whether longer term administration of inhibin in vivo will reduce LH stores and ultimately suppress LH secretion.

FIG. 4 The effect of purified ovine inhibin (Vaughan et al., 1987) on basal (left panel) and GnRH-stimulated (right panel) gonadotropin release from primary rat pituitary cultures (Vale et al., 1986). Dissociated rat pituitary cells were established in primary culture for 3 days at 37°C in a 5% CO2/95% air incubator. On day 4, cultures were washed, fresh media added, and purified inhibin added at the concentrations shown. Cells were then preincubated for 72 hours. Media were then collected from each culture, and stored until assayed by RIA for determination of basal release of LH and FSH. The same cultures were then washed four times with fresh media, and fresh ovine inhibin was added back at the same concentration as used for the preincubation period. GnRH (10 nM) was then added, and the cultures were incubated for 4 hours. Media was collected and stored until assayed by RIA for GnRH-stimulated FSH and LH release.

Steroid hormones are well known to modulate gonadotropin release acting at both pituitary and brain levels. Past studies have suggested that inhibin and steroids may interact to control gonadotropin secretion. For example, Massicotte et al. (1984a, b) found that granulosa cell culture medium (GCM) reversed the stimulatory effect of estradiol-17β on GnRH-mediated FSH and LH secretion in anterior pituitary cultures derived from adult female rats. Furthermore, they reported that the combination of GCM and the androgen dihydrotestosterone (DHT) led to a greater inhibition of GnRH-mediated LH release than by DHT alone. DHT was found to stimulate basal FSH secretion and to exert little, if any, effect on GnRH-mediated FSH secretion (Massicotte et al., 1984b).

The direct interaction between inhibin and steroids has been tested in our laboratory using highly purified ovine inhibin, along with testosterone, dihydrotestosterone, androstenedione, and the estrogen estradiol-17β. The addition of androgens increased the basal release of FSH by 40 −50%. Increasing concentrations of purified inhibin suppressed the secretion of FSH to the same level as nonsteroid treated cells (IC50 = 3 pM). The addition of 10 nM estradiol-17β did not affect the release of FSH and did not alter the ability of inhibin to suppress the release of FSH in a dose-dependent manner. Furthermore, neither androgen, estrogen, nor inhibin affected the basal release of LH.

In order to determine the effects of inhibin and steroids on GnRH-stimulated gonadotropin secretion, cultures were treated with increasing doses of inhibin in the absence or presence of steroids and then exposed to GnRH. Inhibin suppressed GnRH-stimulated release of both FSH and LH in a dose-dependent fashion (IC50 = 8.8 and 18.4 pM, respectively). The addition of androgens alone also suppressed GnRH-stimulated FSH and LH release, although estradiol had no effect. The addition of androgens and inhibin resulted in a greater total inhibition of both LH and FSH release than that caused by either inhibin or androgen alone. The calculated IC50 value for the suppression of GnRH-stimulated gonadotropin release by inhibin in the presence of androgen is 2- to 3-fold lower than that for inhibin alone, suggesting that the pathways by which these two substances alter gonadotropin secretion may overlap.

Highly purified FRP (inhibin βAβA) is a potent and selective FSH secretogogue in vitro, stimulating release of FSH by cultured rat pituitary cells with an EC50 of ∼25 pM (Fig. 5). The effects of FRP can be distinguished from those of GnRH in several ways: GnRH acts immediately to increase FSH release whereas FRP has a long latency in vitro, requiring ≥24 hours to reach maximal effectiveness. GnRH releases LH while FRP, by itself, has no effect on LH secretion. The action of GnRH is completely blocked by a GnRH antagonist which has no influence on the response to FRP, indicating that its effects are not mediated by the GnRH receptor. Pituitary cells treated with GnRH become rapidly desensitized within hours whereas FRP continues to stimulate FSH production for days with no attenuation. Prolonged exposure of pituitary cells to GnRH results in depletion of cellular FSH stores, whereas FRP, while stimulating secretion to the same extent, increases amounts of stored FSH (Fig. 6). Thus the continuous exposure of cells to FRP appears to stimulate FSH biosynthesis.

FIG. 5 Effects of two concentrations of purified ovine inhibin (αβA) on the release of FSH in response to purified porcine FRP (inhibin βAβA, activin A) by cultured anterior pituitary cells. Inhibin and FRP added simultaneously to 3-day-old cultures of male rat anterior pituitary cells. Media removed after 72 hours for RIA.

FIG. 6 Comparison of effects of GnRH and FRP (inhibin αβA) on amounts of secreted and intracellular FSH in rat anterior pituitary cell cultures over a 48-hour period.

Purified inhibin lowers basal production of FSH and obscures the effects of lower concentrations of FRP. Higher concentrations of FRP overcome the effects of inhibin resulting in a net stimulation of FSH secretion. In the few experiments our limited supplies have permitted, it appears that highest levels of FRP can completely abolish any effects of inhibin. Although the latter observation suggests that the inhibition could involve competition at a common site, perhaps a plasma membrane receptor, we would still have to explain the effects of inhibin in the absence of added FRP as well as the converse. Our finding of inhibin α and β subunit mRNAs in the pituitary (see below) suggests that pituitary cells could make ββ or αβ dimers constitutively and raises the possibility that effects seen in response to the addition of one dimer could plausibly reflect antagonism of the other. As will be developed in this article, FRP and inhibin can functionally antagonize one another in every system that we have explored thoroughly.

Crude follicular fluid has a net inhibitory effect on FSH secretion. The existence of gonadal FRPs/activins had probably been overlooked in the past because of the presence of a relative abundance of αβ vs ββ dimers which are not separated by simple gel permeation methods. Previous discussions of an FSH releasing factor (FRF) have assumed it to be present in the hypothalamus-median eminence and partially purified preparations of FRF have been smaller and more rapidly acting than FRP (Mizunuma et al., 1983; Igarashi and McCann, 1964; Schally et al., 1966). Whether ββ dimers circulate in blood is unknown at this time as is their physiological significance in the control of FSH secretion.

Although there are no effects of the βAβA dimer alone on the secretion of LH we decided to explore whether FRP could interact with inhibin under GnRH-stimulated conditions where inhibin influences LH release. We observed that FRP does indeed antagonize the inhibitory effects of inhibin on GnRH-stimulated LH secretion (Fig. 7).

FIG. 7 Effects of 72-hour pretreatment with ovine inhibin (αβA) and/or porcine FRP (inhibin βAβA) on GnRH-induced LH and FSH secretion over a 4-hour period by cultured anterior pituitary cells.

In our original report of the structure and biological activities of FRP, we noted its inhibitory effects on basal ACTH and growth hormone (GH) secretion by cultured rat anterior pituitary cells. FRP is a very potent inhibitor of both spontaneous as well as GRF-mediated GH secretion (Fig. 8) and biosynthesis (Billestrup et al., 1988). The effects of cotreatment of cells with both inhibin and FRP results in a shift to the right of the FRP dose–response curve. Whether FRP and/or inhibin are involved in GH regulation in vivo has yet to be evaluated.

FIG. 8 Effects of 72-hour pretreatment with porcine FRP in presence and absence of ovine inhibin on GRF-mediated release of GH by cultured anterior pituitary cells.

IV Development of Antisera toward Inhibin Subunits

We have generated antibodies directed against inhibin by the use of conjugated synthetic subunit fragments as immunogens. This approach, reviewed by Lerner (1982), has been used successfully to develop antisera to a variety of larger proteins. The lack of information about the position of intra- and intermolecular disulfide bonds complicates the design of synthetic peptides for immunization. To avoid generation of antibodies against epitopes that do not exist in the intact hormone we have chosen to make fragments corresponding mainly to regions lacking cysteine residues and which have a tyrosine or histidine (for coupling and iodinating) at one terminus. When the region of interest lacked a tyrosine at the termini, a tyrosine connected by a spacer glycine was added. For the cysteine-rich β subunit, we have made cyclic peptides that we hoped would mimic a loop in the native protein. We have obtained useful antibodies towards the cysteine-free N-terminal region of porcine α inhibin using porcine inhibin α (1-25)-Gly-Tyr conjugated to human α-globulins. Cuevas et al. (1987) have also raised antisera toward the N-terminal region of the inhibition α subunit. The best antibodies toward β subunits were raised in rabbits immunized with conjugated cyclic Ac-inhibin βA (83-113)-NH2 and cyclic Ac-inhibin βB (80-112)-NH2 (Vaughan et al., 1988). These antisera recognize proteins in bovine, human, ovine, rat, rhesus monkey, and human gonadal fluids and have been used for radioimmunoassays (RIAs), immunoblots, immunoprecipitation, affinity chromatography, and passive immunization. Other groups have obtained inhibin antisera by use of native inhibin or its subunits as immunogens (McLachlan et al., 1986; Miyamoto et al., 1986).

V Gonadal Production and Actions of Inhibin and FRP/Activins

The gonadal origins of inhibin had been assumed since the recognition of its existence (McCullough, 1932). Bioassays and more recently immunocytochemical and in situ hybridization methods have indicated that inhibin and its subunits are produced in granulosa cells of the ovary and Sertoli cells of the testis in vivo and in vitro (Steinberger, 1979; Erickson and Hsueh, 1978; Cuevas et al., 1987; Bardin et al., 1987; Bicsak et al., 1986; Channing et al., 1985, review).

A REGULATION OF INHIBIN PRODUCED BY GRANULOSA CELLS

Recent studies from our laboratories have probed the regulation of inhibin production by rat granulosa cells (Bicsak et al., 1986), the source of inhibin in the ovary (Erickson and Hsueh, 1978). By using a radioimmunoassay designed to detect the N-terminal portion of the porcine inhibin α subunit, we were able to demonstrate that FSH stimulates secretion of immunoreactive inhibin. Gel permeation chromatography combined with an in vitro anterior pituitary cell inhibin bioassay revealed that the cells secrete bioactive inhibin with Mr ∼ 32,000, as well as an Mr ∼ 50,000 protein which is immunochemically related to inhibin α but biologically inactive.

Subsequent in vitro labeling with [³²S]cysteine followed by immunoprecipitation with antiinhibin α antibody showed subunits, while the 50,000 Mr species is probably an unprocessed monomeric precursor of the inhibin α chain (Bicsak et al., 1988). The presence of unprocessed inhibin subunits is consistent with the findings from cDNA sequence studies suggesting that both inhibin α and β subunits are synthesized as precursor proteins (Esch et al., 1987; Forage et al., 1986; Mason et al., 1985, 1986; Mayo et al., 1986; Stewart et al., 1986; Woodruff et al., 1987). The observation of free high Mr inhibin α chain is also consistent with the fact that levels of α subunit mRNA in the ovary are always much larger than those of β subunit mRNA (Davis et al., 1986; Mason et al., 1986; Mayo et al., 1986). We were unable to detect any high Mr dimeric inhibin, composed of an unprocessed α and fully processed β subunit, as is found in bovine follicular fluid (Robertson et al., 1985). This may be due to species differences.

In addition to FSH, it appears that a number of hormones are able to modulate inhibin production by granulosa cells. After induction of LH (Erickson et al., 1979) and prolactin (Wang et al., 1979) receptors by treatment of the cells with FSH (Hsueh et al., 1984), we found that LH, but not prolactin, augmented inhibin production (Bicsak, 1987). The inability of Ying et al (1986b) to stimulate granulosa cell inhibin with LH is probably due to the fact that their cells were not primed with FSH prior to LH treatment, and therefore did not have any LH receptors. Our unpublished observations also show that LH has no effect on inhibin production by immature granulosa cells.

Also of particular interest was our finding that IGF-I and vasoactive intestinal peptide (VIP) both stimulated inhibin production by cultured rat granulosa cells (Bicsak et al., 1986). Both IGF-I (Davoran and Hsueh, 1986) and VIP (Ahmed et al., 1986) have been shown to be present in the ovary, and may therefore play a paracrine role in the regulation of inhibin production. Our observation that IGF-I increases granulosa cells inhibin production was confirmed by a subsequent study by Zhiwen et al. (1987) in which bioactive inhibin was measured in the conditioned medium from granulosa cells incubated with either FSH or IGF-I.

In contrast to gonadotropins, IGF-I, and VIP, treatment of the cells with either GnRH or EGF inhibited FSH-stimulated inhibin production. Both EGF (Hsueh et al., 1981) and GnRH (Hsueh and Erickson, 1979) have been shown to inhibit granulosa cell aromatase activity and LH receptor induction. While the presence of EGF-like molecules in the ovary has not yet been demonstrated, recent evidence suggests that GnRH-like peptides may exist in the ovary (Aten et al., 1986) and could therefore exert an intraovarian effect on inhibin production.

B REGULATION OF INHIBIN PRODUCTION BY SERTOLI CELLS

Earlier studies have suggested that Sertoli cells are the site of testis inhibin production (Steinberger and Steinberger, 1976a). The hormonal regulation of inhibin production by cultured rat Sertoli cells was also examined using the specific RIA to porcine inhibin α (Morris et al., 1988). FSH, but not human chorionic gonadotropin (hCG) or prolactin, caused a dose-dependent increase in inhibin production (EC50 for FSH = 2.4 ng/ml) (Bicsak et al., 1986); both secreted and intracellular levels of inhibin were increased, but the secreted form represented one-half to two-thirds of the total. The FSH-stimulated production of inhibin was augmented by addition of a phosphodiesterase inhibitor, and could be mimicked by cholera toxin, forskolin, or dibutyryl cAMP, all of which are known to increase intracellular cAMP levels. Examination of the conditioned media from forskolin-treated Sertoli cells by gel filtration chromatography revealed a single peak of bioactive and immunoreactive inhibin, at a molecular weight of approximately 32,000, similar to that observed for the porcine and bovine follicular fluid inhibins. Thus, FSH activated the cAMP pathways to stimulate Sertoli cell production of inhibin which in turn suppresses pituitary FSH release to form a closed-loop feedback system (Bicsak et al., 1986).

Inclusion of either dihydrotestosterone or estradiol in the cultures had no effect on inhibin production, both in the presence and absence of FSH. The lack of any effect on androgens on either basal or FSH-stimulated levels of Sertoli cell inhibin is somewhat surprising in light of the observed augmenting action of androgens on granulosa cell inhibin production (Bicsak et al., 1986). Some studies have suggested that androgens stimulate testicular inhibin production (Steinberger, 1981; Verhoeven and Franchimont, 1983). However, our data do corroborate the observations of Au et al. (1985) which suggested that FSH is the sole in vivo modulator of testicular inhibin measured by the pituitary bioassay. Also, increased bioactive inhibin levels during pubertal development in the rat are correlated with elevation of serum FSH, but not testosterone (Au et al., 1986).

C INTRAGONADAL FUNCTIONS OF INHIBIN AND FRP

In addition to its primary role at the pituitary level, it appears that inhibin and proteins related to inhibin may also function within the gonads as local hormones. Inhibin and FRP exhibit slight autocrine-like effects to modulate FSH-induced aromatase activity in granulosa cells where FRP potentiates and inhibin inhibits FSH induced production of estrogen (Ying et al., 1986b; Hsueh, Bicsak, and Vale, unpublished results). A study from our laboratory (Hsueh et al., 1987) has revealed a more important paracrine role for inhibin and FRP in the ovary, namely, in the regulation of theca cell androgen production. Using both enzymatically dispersed theca-interstitial cell preparations and intact theca-shell cultures (Carson et al., 1981), it was shown that inhibin stimulates LH-stimulated androstenedione production, presumably by the theca interna cells. In addition, FRP was observed to have the opposite effect, being inhibitory with respect to LH-stimulated androgen production. Neither inhibin nor FRP had any effect on androgen levels in the absence of LH. Similar effects of inhibin and FRP were observed with LH-stimulated Leydig cell testosterone production, suggesting that inhibin and FRP may also have a paracrine role in the testis. In primary cultures of testis cells, the αβ heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the ββ homodimer suppresses androgen production. As is the case for effects of GH secretion, inhibin is a stimulatory factor while FRP/activin is an inhibitor. Such results are ironically inconsistent with the inhibin/activin nomenclature for this hormone family.

Our data indicate that the inhibin-related gene products synthesized by Sertoli and granulosa cells may form heterodimers or homodimers to serve as intragonadal paracrine signals in the modulation of LH-stimulated androgen biosynthesis and allow cross-communication between the two feedback loops. Although inhibin was originally isolated from the follicular fluid and believed to be a granulosa cell product, the ovine inhibin used by us is isolated from the rete testis fluid and believed to be of Sertoli cell- or seminiferous tubule-derived nonsteroidal factors capable of enhancing LH-mediated Leydig cell steroidogenesis (Gospodarowicz et al., 1987; Greep et al., 1942; Hernandez et al., 1987). The present observation may also explain paradoxical findings showing that FSH enhances Leydig cell LH responsiveness and receptor content (Fukuda et al., 1986).

In addition to the two long-loop feedback axes between pituitary (LH-and FSH-secreting) gonadotrophs and testicular (androgen- and inhibin-producing) cells, inhibin may provide the cross-communication between the two loops. Thus, testis inhibin not only suppresses pituitary FSH release, but also enhances LH-regulated Leydig cell production of androgens, which exert negative feedback at the pituitary LH-producing cells (Fig. 9). An analogous situation may also exist in the female because treatment with the inhibin heterodimer enhances androgen production by cultured ovarian theca internal cells. Both FSH and LH have been shown to stimulate cultured granulosa cells to produce inhibin (Falck, 1959), which suppresses pituitary FSH release as well as enhances theca cell steroidogenesis. Inhibin and related proteins may prove to be important modulators of gonadal steroidogenesis and differentiation.

FIG. 9 Summary of potential interactions within the hypothalamic-pituitary–gonadal axis.

VI Role of Inhibin in the Regulation of FSH Secretion in Vivo

In the past, the role played by inhibin in modulating FSH secretion has been studied by injecting charcoal-extracted follicular fluid (at the time presumed to contain only inhibin) under various circumstances of elevated FSH release (such as following castration or during the secondary FSH surge of estrus), and measuring plasma gonadotropin levels [reviews in (Channing et al., 1985; Franchimont et al., 1979; Grady et al., 1982; Setchell and Main, 1974)]. These studies have indicated that follicular fluid could selectively suppress FSH, but not LH secretion (Campbell and Schwartz, 1979; Jones et al., 1985; Koiter et al., 1983; Lorenzen and Schwartz, 1979; Lumpkin et al., 1984; Marder et al., 1977; Piacsek and Goodspeed, 1978; Pomerantz, 1978; Thomas and Nikitovitch-Winer, 1984; Williams and Lipner, 1981). However, the exact physiological function of inhibin is still controversial: in the female rat, a role for inhibin in modulating the estrus surge of FSH has experimental support (DePaolo et al., 1979; Hoffmann et al., 1979; Ruch et al., 1981; Schwartz and Channing, 1977), but its importance during sexual maturation is unclear. In the male rat, there seems to be little consensus, as some investigators proposed evidence for, as well as against, a modulating function of inhibin during and following sexual maturation [review in (deJong, 1979; Franchimont et al., 1979; Steinberger and Chowdhury, 1974)]. However, the overall conclusion appeared to be that if inhibin did play a role in modulating FSH secretion in male rats, it was probably restricted to either the infantile/juvenile period (Hermans et al., 1980; Massicotte et al., 1984a), or periods of maximally elevated FSH secretion (Summerville and Schwartz, 1981).

Because there is now evidence that follicular fluid contains not only inhibin, but also FRP/activin (Vale et al., 1986; Ling et al., 1986a, b), it is difficult to evaluate the significance of earlier studies based on only partially purified starting material. Similarly, experiments designed to investigate the factors modulating inhibin secretion had relied on bioassays for inhibin [reviews in (Channing et al., 1985; Franchimont et al., 1979; Grady et al., 1982)], which measured not only inhibin, but the net FSH release-modifying effects of the biological fluids studied.

With the availability of specific antisera directed against the N-terminal segment of the α chain of inhibin (Rivier et al., 1985; Vaughan et al., 1988), we have reinvestigated the physiological role of inhibin, as well as the mechanisms which regulate its secretion, in both male and female rats.

A FEMALE RATS

Earlier studies using bioassays to measure inhibin levels had reported an increase in plasma inhibin levels until the time of first ovulation (Sander et al., 1985, 1986). Using our RIA for inhibin, we measured very low plasma inhibin levels in 10-day-old female rats, followed by steady increases until day 18 (Rivier and Vale, 1987b). These changes were accompanied by a concomitant increase in plasma FSH levels, a rise believed to be essential for normal follicular development (Uilenbroeck et al., 1976). Because inhibin is mainly secreted by granulosa cells (Bicsak et al., 1986), our observation of increased levels of circulating inhibin during this stage of sexual development is in good agreement with the presence of an increasing number of follicles in the maturing rat (Ojeda et al., 1980; Uilenbroeck et al., 1976). Studies designed to evaluate the role of inhibin indicated that the immunoneutralization of endogenous inhibin, achieved by the intravenous injection of antiinhibin serum (Rivier et al., 1985), caused no measurable changes in the plasma FSH levels of 10-day-old female rats, but consistently increased FSH release in older animals (Rivier and Vale, 1987). Taken all together, these results suggest that endogenous inhibin plays a physiological role in modulating FSH secretion by female rats after the second week of life.

In sexually mature female rats, the fluctuations of circulating FSH levels which are observed during the estrous cycle (Schwartz, 1969) are believed to be dependent upon the interaction of modulating signals including GnRH, sex steroids, and inhibin. In particular, there is evidence that the primary surge of FSH, which occurs in the afternoon of proestrus, is dependent upon GnRH (Condon et al., 1984; Schwartz et al., 1985), while the secondary (estrous) surge is mainly controlled by changes in inhibin secretion (DePaolo et al., 1979; Hasegawa et al., 1981; Hoffmann et al., 1979; Ruch et al., 1981; Schwartz and Channing, 1977). We have observed that in adult cycling female rats, the administration of antiinhibin serum elevated plasma FSH levels at all stages of the cycle (Rivier et al., 1987a), including the afternoon of proestrus and the morning of estrus (Rivier et al., 1986). Thus it appears that endogenous inhibin does modulate the release of FSH at all times of the estrous cycle.

Earlier in vivo and in vitro studies had suggested that gonadotropins exerted a stimulatory action on the secretion of inhibin (Kimura et al., 1983; Lee et al., 1981, 1982; Sander et al., 1985; Savoy-More et al., 1986). We injected pregnant mare serum gonadotropin (PMSG), which has both LH- and FSH-like activity (Gospodarowicz, 1972; Papkoff, 1974), and observed that it markedly increased plasma in inhibin levels in both immature (Rivier et al., 1986) and mature (Rivier et al., 1986) female rats. The inability of hCG to alter inhibin release (C. Rivier, unpublished) suggests that FSH represents the primary stimulator of inhibin release in the intact rat. It should be noted, however, that Bicsak et al. (1986) have reported that hCG increased inhibin secretion by cultured granulosa cells (Bicsak et al., 1986). At the present time, we do not know the reason for this discrepancy.

B MALE RATS

As mentioned earlier, the importance of inhibin in modulating reproductive functions in the male rat, as well as the pattern of secretion of inhibin by Sertoli cells, have not yielded any unifying theory (Odell and Swerdloff, 1976; Piacsek and Goodspeed, 1978). Using bioassays to follow inhibin secretion, various investigators have reported that inhibin secretion by Sertoli cells increased with age in the male rat (Au et al., 1986), decreased (Ultee-van and deJong, 1987; Ultee-van et al., 1986) or remained constant (Steinberger, 1979; Steinberger and Steinberger, 1976a). We have observed that radioimmunoassayable inhibin showed a consistent decrease from day 8 through day 90 of age (Rivier et al., 1988). These changes were accompanied by an age-related lowering of inhibin levels in testicular extracts and decrease in alpha-chain immunocytochemical staining of testicular slices. These changes also paralleled the marked decline in alpha-chain expression seen in the maturing testes (Meunier et al., 1987). Thus, all our results point to an age-related decrease in the level of inhibin expression and presence in the testes, as well as in the levels of circulating inhibin.

The role played by inhibin had been suggested, in particular, by experiments showing that the increases in FSH secretory following acute castration were significantly larger in younger than in older male rats, and could not be suppressed by exogenous sex steroids in the infantile/juvenile animal (Hermans et al., 1980). Other investigators, however, obtained evidence that testosterone prevented castration-induced FSH release at all ages (Odell and Swerdloff, 1976; Summerville and Schwartz, 1981). On the other hand, the injection of partially purified follicular fluid has been reported to reliably suppress secretion in adult-castrated male rats (deJong et al., 1979; Jones et al., 1985; Lorenzen et al., 1981; Massicotte et al., 1984a).

As in the case of the female rat, we have addressed the question of the physiological role played by inhibin, by injecting antiinhibin serum to male rats of various ages, and measuring plasma FSH 12 hours later. Because the immunoneutralization of endogenous inhibin increased plasma FSH levels of male rats aged 10 through 24 days, but not in older animals, our results suggest that endogenous inhibin may only exert a physiological role in modulating FSH secretion by infantile male rats. In these animals, the changes in plasma FSH levels which are measured during sexual maturation (Dohler and Wuttke, 1975; Lee et al., 1975; Negro-Vilar et al., 1973a; Odell and Swerdloff, 1976; Piacsek and Goodspeed, 1978) are presently believed to reflect, at least in part, changes in the brain and pituitary sensitivity to testosterone negative feedback (Negro-Vilar et al., 1973b; Odell and Swerdloff, 1976; Piacsek and Goodspeed, 1978). Our results suggest that during the first 3 weeks of life, inhibin may also represent a physiologic regulator of FSH release.

VII Tissue Expression of Inhibin Subunits

The anatomic distribution of proteins can be explored by a variety of immunologic and molecular techniques. We have employed a very specific and sensitive S1-nuclease analysis method to measure the levels of inhibin subunit mRNA levels (Meunier et al., 1987). This technique is based upon the ability of sample RNAs to protect small, single-stranded cDNA probes of predetermined sizes for α, βA, and βB subunits from degradation by S1-nuclease enzyme. The amounts of inhibin-RNAs were estimated by measuring the amount of each RNA-protected cDNA probe on acrylamide gel after a 2-hour digestion with Sl-nuclease.

The relative amounts of inhibin subunits in gonads and other tissues are shown in Fig. 10. In the ovary, inhibin α mRNA is much more abundant than either βA or βB subunit mRNA. This is in agreement with observations of Mason et al. (1985) using Northern analysis. The adult testis also expresses much more α than β messages. Although Esch et al. (1987) have reported that the βA subunit is missing in the male rat, we have shown that the levels of message encoding this protein are easily detectable in the testes of immature rats (Meunier et al., 1987). With maturation, concentrations of all messages decline but those of βA fall more rapidly than those of α or βB. A decline in the production of inhibin with age in the male rat is consistent with our observation of decreased immunologic evidence for inhibin subunits in the testis as well as the failure of passive immunization to elevate plasma FSH levels in the adult male rat (Rivier et al., 1987c).

FIG. 10 Levels of inhibin α, βA, and βB RNAs in various tissues. Total RNA was prepared from mature male and female animals. The following female tissues were used: ovary, placenta, pituitary, adrenal, spleen, kidney, brain, spinal cord (and vertebrae) thymus, and mammary glands. The following male tissues were used: testis, bone marrow, liver, and pancreas. Forty micrograms of total RNA from the indicated tissues was hybridized in liquid solution with an excess of single-stranded ³²P-labeled cDNA probe complementary to inhibin α, βA, and βB subunits. Samples were then incubated with S1-nuclease for 2 hours. The RNA/cDNA hybrids were precipitated with ethanol, denatured, and loaded on 5% acrylamide/8 M gel to separate all three inhibin cDNA probes of predetermined sizes. Autoradiograms of several exposures were scanned by densitometer, and the integration values for inhibin α, βA, and βB subunits were corrected according to the specific activity of each S1 probe. The corrected integration values are shown as arbitrary densitometric units. Liver, pancreas, thymus, and mammary glands did not contribute signals above background and, therefore, are not included in this figure. The results shown are representative of at least three independent experiments.

Inhibin subunits are also expressed in nongonadal tissues including brain, spinal cord, pituitary, adrenal, spleen, and kidney where α subunit mRNAs predominate. In placenta and bone marrow, levels of β subunits mRNAs are in excess. Our working hypothesis is that the ratios of α to β subunits expressed determine the amounts of inhibin relative to FRP/ activin produced. Accordingly, taken as a whole the gonads would produce predominantly αβ dimers whereas placenta and bone marrow would make mainly ββ dimers. In the remainder of this article, possible roles of inhibin and FRP/activin in placenta, brain, and bone marrow will be discussed.

VIII Inhibin-Related Proteins in the Placenta

The demonstration of the presence of mRNAs for inhibin subunits (Mayo et al., 1986; Meunier et al., 1987) in the human and rat placenta is consistent with results of bioassays and radioimmunoassays (McLachlan et al., 1986). Immunocytochemical studies with antiinhibin α serum revealed numerous immunoreactive cells in the placental villi localized in the central, cytotrophoblast, layer (Petraglia et al., 1987).

We have studied the production and actions of inhibin-related proteins in primary cultures of human trophoblasts. The secretion of immunoreactive (ir)-inhibin (with antiinhibin α subunit) by cultured trophoblasts could be stimulated by the addition of hCG presumably acting through adenylate cyclase. 8-Br-cAMP and agents such as forskolin and cholera toxin that elevate intracellular cAMP levels also release ir-inhibin (Petraglia et al., 1987). In exploring activities of inhibin and FRP on production of various substances made by the placenta, we have found that FRP (ββ) stimulates the secretion of GnRH, hCG, and progesterone by trophoblast cultures (Petraglia et al., 1988). Inhibin (αβ) can prevent the effects of FRP on the production of all of these substances. In Fig. 11 we summarize some of the interactions that we have observed in the trophoblast cultures, which reveal an array of potential paracrine/autocrine regulatory possibilities. The roles of the ββ and αβ dimers in the physiology of the placenta and pregnancy will be an area of intense investigation in the future.

FIG. 11 Summary of interactions within human placental cell culture.

IX FRP and the Control of Oxytocin Secretion

With the availability of affinity purified antibodies toward all inhibin subunits, we have begun to map their distributions in the colchicine-treated rat brain (Sawchenko et al., 1988). Antisera directed toward two different regions of the inhibin βA subunit (66 −79 and 81 −113) both stained a cluster of neurons centered in the caudal part of the nucleus of the solitary tract (NTS) near the spinal–medullary transition zone and a smaller group of cells in the ventrolateral medullary reticular formation. Axons and terminals exhibiting inhibin β immunoreactivity were visualized in untreated rats and displayed a distribution consistent with the established projections of the NTS and/or ventrolateral medulla. The most prominent terminal fields were detected in the paraventricular (PVN), supraoptic (SON), and dorsomedial nuclei of the hypothalamus, the parventricular nucleus of the thalamus, and the bed nucleus of the stria terminalis. In view of the reproductive roles of inhibin-related proteins, it is noteworthy that the inhibin β-stained terminals were in the regions of the septal and preoptic areas where neurons containing GnRH are found, and more prominently in the portions of the PVN and SON in which the oxytocin-expressing magnocellular neurosecretory neurons are concentrated.

In order to evaluate the possible functional significance of the distribution of inhibin β fibers, we have infused purified FRP bilaterally into the PVN of anesthetized rats (Plotsky, Sawchenko, and Vale, unpublished results). The administration of only 70 fmol of FRP was sufficient to elevate plasma oxytocin levels within 10 minutes while having no effect on the secretion of vasopressin in the same animals. This is certainly the most rapid effect that we have been able to observe for inhibin-related peptides and may indicate that a different cellular mode of action is involved.

The distribution of inhibin β cell bodies and fibers and the effects of FRP are consistent with a role for this or a related protein in the central regulation of oxytocin secretion. The importance of these pathways in the regulation of milk ejection and parturition remains to be defined.

X FRP/Inhibin and Erythrodifferentiation

Recently, Eto et al. (1987) reported the isolation of an erythroid differentiation factor (EDF) from a human leukemic cell line that could stimulate erythropoiesis in a mouse Friend cell line. Based upon homodimeric structure and some N-terminal sequence analysis they proposed that EDF is identical to FRP (inhibin βAβA). We have supported this hypothesis by showing that highly purified FRP induces hemoglobin synthesis in the K562 human erythroleukemic cell line as well as proliferation of erythroid progenitor cells in human bone marrow culture (Yu et al., 1987). In the latter system, which reflects the early stages of erythrodifferentiation, FRP markedly potentiated the effects of erythropoietin on stem cells to induce colony-forming units and hence acts as a mitogen. Whereas in the K562 cells which is a model proerythroblast line and not erythropoietin sensitive, FRP stimulates differentiation on its own and inhibits cell division. The addition of inhibin alone had no effect on erythrodifferentiation in either bone marrow cultures or K562 cells, however, the αβ dimer could antagonize all actions of FRP in those systems.

The presence of substantial inhibin βA subunit mRNA in the bone marrow as well as the isolation of the inhibin βAβA homodimer (EDF, FRP) from a human leukemic cell line suggest that this hormone might be available as a paracrine regulator of erythrodifferentiation. Although, the α subunit mRNA is not presently detectable in the marrow and presumably, therefore, inhibin αβ is produced locally in negligible amounts, it is plausible that inhibin could reach the bone marrow from the blood and function in a hormonal mode.

XI Conclusions

Inhibin-related dimers are broadly distributed anatomically and have powerful activities in several biological systems where inhibin and FRP/activin often exhibit opposite effects (Fig. 12). While the physiologic roles of inhibin to regulate FSH secretion in the female rat and immature male rat are strongly supported, the significance of these hormones within the gonad, brain, placenta, and bone marrow have yet to be placed in in vivo context. Although the panoply of functions of inhibin and FRP/activin are certainly incompletely understood at this time, this family has already demonstrated a powerful mechanism for the generation of signal diversity whereby differential subunit association can result in the generation of dimers with opposing biological actions in multiple tissues.

FIG. 12 Summary of biological activities of inhibin (αβ) and FRP (inhibin βAβA, activin A).

ACKNOWLEDGMENTS

Research in the authors’ laboratories was supported in part by grants and contracts HD-13527, N01-HD32826, NO1-HD62943, HL-35137, and AM-26741 from the NIH, and B85 −28A/ICCR from the Population Council and the USAID and conducted in part by The Clayton Foundation for Research, California Division. W.V., C.R., P.S., J.S., and P.P. are Clayton Foundation Investigators. The authors thank B. Connor and S. McCall for manuscript