Proc. Natl. Acad. Sci. USAVol. 84, pp. 5082-5086, July 1987Physiological Sciences

Heterodimers and homodimers of inhibin subunits have differentparacrine action in the modulation of luteinizinghormone-stimulated androgen biosynthesis

(ovary/testis/gonadotropins/Leydig cells/theca cells)

AARON J. W. HSUEH*, KRISTINE D. DAHL*, JOAN VAUGHAN+, ERMELINDA TUCKER*, JEAN RIVIERt,C. WAYNE BARDIN*, AND WYLIE VALEt*Department of Reproductive Medicine, M-025, University of California, San Diego, La Jolla, CA 92093; tClayton Foundation Laboratory for Peptide Biology,Salk Institute for Biological Studies, La Jolla, CA 92037; and *The Population Council, Center for Biomedical Research, New York, NY 10021

Communicated by Ernst Knobil, April 2, 1987 (received for review December 1, 1986)

ABSTRACT Inhibin, a gonadal hormone capable of pref-erential suppression of pituitary follicle-stimulating hormone(FSH) secretion, has recently been purified. The major form ofthis protein is an a48 heterodimer encoded by two separategenes. In contrast to the FSH-suppressing action of the a,3heterodimer, the 1831 homodimer stimulates FSH secretion.Luteinizing hormone (LH)-secreting pituitary cells and gonad-al androgen-producing cells have long been shown to form aclosed-loop feedback axis. Based on recent studies demonstrat-ing the FSH stimulation of inhibin biosynthesis by ovariangranulosa and testis Sertoli cells, an additional closed-loopfeedback axis exists between pituitary FSH- and gonadalinhibin-producing cells. Because uncharacterized Sertoli cellfactors have been suggested to either stimulate or inhibitandrogen production by testicular Leydig cells, we have testedthe intragonadal paracrine actions of heterodimers andhomodimers of inhibin subunits. In primary cultures of testiscells, the a48 heterodimer of inhibin enhances Leydig cellandrogen biosynthesis stimulated by LH, whereas the (3(3homodimer suppresses androgen production. Furthermore,similar modulatory actions of inhibin-related proteins werefound in cultured ovarian theca-interstitial cells and thecaexplants treated with LH. In contrast, treatment with theinhibin-related proteins alone did not affect gonadal steroido-genesis. Our data indicate that the inhibin-related gene prod-ucts synthesized by Sertoli and granulosa cells may formheterodimers or homodimers to serve as intragonadalparacrine signals in the modulation of LH-stimulated androgenbiosynthesis and allow cross-communication between the twofeedback loops.

Earlier investigations on testicular (1, 2) and ovarian (3-6)proteins capable of differential suppression of follicle-stim-ulating hormone (FSH), but not luteinizing hormone (LH),secretion by the anterior pituitary gland have been followedby the recent isolation of inhibin from porcine (7-9) andbovine (10, 11) follicular fluid. Based on the amino acidsequence of these proteins, porcine (12), human (13), andbovine (14) inhibin genes have been cloned. These proteinsare composed of dissimilar a and 8 subunits that are derivedfrom separate genes. Although the af heterodimer of inhibinsuppresses FSH secretion, the p,8 homodimer stimulatesFSH release (15, 16). Furthermore, the gene for the inhibinp8 subunit is highly conserved and is homologous to the genesfor transforming growth factor type , (p-TGF) and Mullerianduct inhibiting substance (17).

In primary cultures of ovarian granulosa cells, FSH stim-ulates the secretion of both bioactive and immunoreactive

inhibin (18, 19), whereas the testicular inhibin is secreted bycultured Sertoli cells (20, 21). Because uncharacterizedSertoli cell factors have been shown to either enhance orsuppress Leydig cell functions (22-26), we tested the hypoth-esis that inhibin and related proteins may exert paracrineactions by modulating Leydig cell androgen production. Wefurther tested the hypothesis that inhibin and related proteinsregulate androgen production by ovarian theca interna cells.

MATERIALS AND METHODSAnimals. Male and female Sprague-Dawley rats were

obtained from Johnson Laboratories (Bridgeview, IL). Neo-natal male rats (7-day-old) were maintained at 8 to 10 perlactating mother, whereas adult male rats were hypophysec-tomized at 60-70 days of age and then used at 7-10 days afteroperation. For theca-interstitial cell cultures, immature fe-male rats were hypophysectomized at 21 days of age and usedat 4 days after operation. In addition, theca explants wereobtained from 30-day-old intact rats. Animals were housed inair-conditioned quarters under a 14-hr light, 10-hr dark cycle.

Reagents and Hormones. Deoxyribonuclease (DNase; bo-vine pancrease; 2100 units/mg), McCoy's 5A medium (mod-ified), fetal calf serum, and trypan blue stain were obtainedfrom GIBCO. Collagenase (144 units/mg) was obtained fromWorthington. Ovine LH (National Institute of Arthritis,Diabetes, and Digestive and Kidney Diseases oLH 23;contains 2.3 units of LH per mg, <0.5% FSH by weight and<0.1% growth hormone or prolactin by weight) was obtainedfrom the National Pituitary and Hormone Distribution Pro-gram, National Institute of Arthritis, Diabetes, and Digestiveand Kidney Diseases.Ovine inhibin (inhibin a,3 heterodimer) was isolated from

rete testis fluid by immunoaffinity chromatography (J.V. andW.V., unpublished work) using an antiserum to the NH2-terminal portion of the porcine inhibin a subunit. The activezone from immunoaffinity chromatography was further pu-rified by gel permeation on fast protein liquid chromatogra-phy (FPLC) (15) and reversed-phase HPLC. On NaDodSO4/PAGE, purified nonreduced ovine inhibin shows a singleband of Mr 32,000; following reduction, two bands of Mr21,000 and Mr 14,000 are observed. Porcine FSH releasingprotein, a homodimer of inhibin-/3A13A, was purified tohomogeneity from porcine follicular fluid as described byVale et al. (15).

Cell Cultures. Neonatal testis cell cultures have beenreported (27). In brief, testes of neonatal rats were dissectedfree of fat, decapsulated, and dispersed in a solution con-taining 0.1% collagenase, 10 jig of DNase per ml, and 10 mg

Abbreviations: FSH, follicle-stimulating hormone; LH, luteinizinghormone; hCG, human chorionic gonadotropin.

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Proc. Natl. Acad. Sci. USA 84 (1987) 5083

of bovine serum albumin per ml. The testes were incubatedat 370C for 45 min, during which time they were dissociatedinto a cell suspension by repeated pipetting at 15-min inter-vals. At the end of the incubation, cells were collected bycentrifugation and washed three times with medium. For thepreparation of adult testis cell cultures, testes of hypophy-sectomized adult rats were obtained and collagenase-dis-persed cells were cultured as described (28). The adult testiscells were cultured without treatment for 8 days with mediumchange every 2 days to allow the recovery and stabilizationof the responsiveness of the Leydig cells to LH/humanchorionic gonadotropin (hCG). The cell viability (75-85%)was determined by trypan blue exclusion testing.

Ovarian theca-interstitial cells were prepared as describedearlier (29) with modification. Ovaries from hypophysecto-mized rats were decapsulated and punctured to express thegranulosa cells. The remaining ovaries containing theca-interstitial tissues were then cut into 6-8 pieces and incu-bated in 2 ml of a solution containing 0.4% collagenase, 10 ,Agof DNase per ml, and 10 mg of bovine serum albumin per mlin McCoy's 5A medium for 1 hr at 370C. The pieces of ovarieswere flushed through pipettes with successively smallerdiameters at 20-min intervals. The dispersed cells were thencentrifuged and washed twice with McCoy's 5A medium, andthe final pellet was resuspended in a known volume of thismedium. Samples (25 ,Al) of the cell suspension were dilutedwith 25 ,ul of trypan blue, and aliquots were counted in ahemacytometer. The dispersion procedure yielded =4 x 106cells per ovary of which 85-90% were viable.

Neonatal testis (1 x 105 viable cells per well) or ovariantheca-interstitial (5 x 104 viable cells per well) cells wereincubated in culture wells (16-mm diameter, 24-well culturedishes; Corning) containing 0.5 ml of McCoy's 5A mediumsupplemented with L-glutamine (2 mM), penicillin (100units/ml), and streptomycin sulfate (100 u.g/ml). Cell cul-tures were maintained at 370C under a water-saturatedatmosphere of 5% C02/95% air. After 2 or 3 days ofincubation, the medium was changed, and cells were incu-bated with or without various concentrations of LH, retetestis fluid inhibin, or ,A homodimer of inhibin for 2 or 3more days as indicated. Adult testis cells (106 viable cells per35-mm culture well containing 1 ml of medium) were culturedin the same medium and treated with various hormonesbetween days 8 to 10 of culture (28). At the end of theincubation period, the medium was frozen (-20°C) untilassayed for testosterone and androstenedione content fortestis and ovarian cultures, respectively.To test the effect of inhibin-related proteins on androgen

production by oyarian theca cells, follicle shells containingtheca interna cells were prepared and cultured as describedby Richards et al. (30). In brief, small antral follicles wereisolated from ovaries of 30-day-old female rats. Theca shellswere separated from granulosa cells by piercing the follicularwall to release loosely attached granulosa cells, followed byscraping of the mural granulosa cells from basal lamina.Theca shells were cultured individually in 0.5 ml of McCoy's5A medium containing penicillin, streptomycin, and 1% fetalcalf serum. After 3 days of culture, the medium was changed,and the explants were cultured in serum-free McCoy's 5Amedium in the absence or presence of hCG (5 ng/ml) with orwithout ca and 8,3f dimers of inhibin for 3 more days.

Steroid Radioimmunoassay and Statistical Analysis. Thecontent of androgens in the culture medium was analyzedusing specific radioimmunoassay. The testosterone and an-drostenedione antisera were raised in rabbits againsttestosterone-3-oxime-conjugated bovine serum albumin and4-androstene-3, 17-dione-6,B-ol-hemisuccinate-conjugatedbovine serum albumin, respectively. The specificity of theseantisera has been described (31). Statistical analysis was doneusing Student's t test. All experiments were repeated at least

three times with comparable results; only one representativeexperiment is presented.

RESULTS

LH stimulated a dose-dependent increase in testosteroneproduction by primary cultures of neonatal testicular cells(Fig. 1). Although ineffective by itself, a purified inhibin (a/3heterodimer at 7 ng/ml) preparation obtained from ovine retetestis fluid enhanced LH-stimulated androgen production atall doses of LH tested (P < 0.01). At 3 ng of LH per ml, thea,3 heterodimer of inhibin increased LH action 1.8-fold. Incontrast to the augmenting action of the inhibin a,(3 hetero-dimer, the 3,8( homodimer of inhibin (5 ng/ml) purified fromporcine follicular fluid suppressed Leydig cell androgenproduction at all doses of LH tested. Likewise, treatmentwith the homodimer alone did not affect testosterone bio-synthesis. When both heterodimer and homodimer wereincluded, an intermediate level of androgen biosynthesis wasinduced by LH. Similar to studies using the neonatal testiscell cultures, treatment with LH stimulated a dose-dependentincrease in testosterone production by cultured testis cellsobtained from adult rats (Table 1). Likewise, cotreatmentwith the inhibin a/3 heterodimer augmented LH action at alldoses of the gonadotropin tested.We further tested the modulatory effects of dimers of

inhibin subunits on ovarian androgen biosynthesis. Fig. 2shows that treatment with LH resulted in a dose-dependentstimulation Qf androstenedione production by cultured theca-interstitial cells. Although treatment with ca,3 and ,83 dimersof inhibin subunits did not affect androgen biosynthesis bythemselves (P > 0.05), concomitant treatment with LH andthese gonadal proteins modulated LH action. As found forthe testis cells, the a/3 heterodimer further enhanced LH-stimulated androgen production by ovarian cells, whereas thep,8 homodimer suppressed LH action. These proteins also

40

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20

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00.3 iLH (ng/ml)

FIG. 1. Modulatory effects of inhibin dimers on LH-stimulatedandrogen production by cultured neonatal testis cells. Primarycultures of testis cells from intact neonatal rats were prepared asdescribed (27). Cells were cultured for 3 days, and the medium waschanged before treatment for 3 more days with increasing doses ofLH with or without the inhibin heterodimer (7 ng/ml) purified fromovine rete testis fluid. Some cells were also treated with LH plus 8Ahomodimer of inhibin (5 ng/ml) purified from porcine follicular fluid,or LH plus both dimers of inhibin subunits. Medium content oftestosterone was measured by radioimmunoassay. Results depictmean ± SEM of triplicate cultures. Similar results were obtained inthree separate experiments. C, control.

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Table 1. Modulatory effects of inhibin a,8 heterodimer onLH-stimulated androgen production by cultured testis cellsobtained from adult hypophysectomized rats

Testosterone production,Treatment ng/ml

Control 0.5 ± 0.2Inhibin ac3 (5 ng/ml) 0.5 ± 0.2LH

1 ng/ml 4.9 ± 0.43 ng/ml 13.6 ± 0.710 ng/ml 12.2 ± 0.4

LH1 ng/ml + inhibin ac3 7.7 ± 0.3*3 ng/ml + inhibin ac1 25.0 ± 0.7*10 ng/ml + inhibin a,c3 20.9 ± 1.2*

Adult male Sprague-Dawley rats (60-70 days of age) werehypophysectomized for 7-10 days. Testes were decapsulated, andcells were dissociated by collagenase as described (28). After 8 daysof culture, the medium was changed, and cells were treated for 2 dayswith hCG with or without the ac1 heterodimer of inhibin (5 ng/ml).Medium content of testosterone was measured by radioimmuno-assay. Mean ± SEM of triplicate cultures.*Significantly different from corresponding groups without treat-ment with the inhibin c1,8 heterodimer; P < 0.01.

antagonize each other's action when incubated together withLH.To further define the site of action of inhibin-related

proteins in the ovary, hormonal modulation of androgenproduction by theca explants was studied. Isolated follicleshells containing theca interna cells were treated with hCG (5ng/ml) with or without the inhibin ap heterodimer (7 ng/ml)

25

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FIG. 2. Modulatory effects of inhibin dimers on LH-stimulatedandrogen production by cultured ovarian theca-interstitial cells.Ovaries from immature hypophysectomized rats were puncturedwith a fine needle to eliminate granulosa cells, and the remainingtissues were dissociated by collagenase/DNase to obtain theca-interstitial cells. Ovarian cells were cultured for 2 days, and themedium was changed before treatment for 2 mQre days with orwithout increasing doses of LH in the absence or presence of theinhibin heterodimer (7 ng/ml) purified from ovine rete testis fluid.Some cells were also treated with LH plus the ,3A homodimer (5ng/ml) of inhibin purified from porcine follicular fluid or LH plusboth dimers of inhibin subunits. Medium content of androstenedionewas measured by radioimmunoassay. Results depict mean SEM oftriplicate cultures. Similar results were obtained in three separateexperiments with similar results.

Table 2. Modulatory effects of inhibin dimers on hCG-stimulatedandrogen production by ovarian theca explants in vitro

Androstenedione production,Treatment ng/ml

Control 0.08 ± 0.01Inhibin a,8 heterodimer

(7 ng/ml) 0.05 ± 0.01Inhibin ,38, homodimer

(5 ng/ml) 0.06 ± 0.01hCG (5 ng/ml) 1.05 ± 0.15hCG + inhibin a,1 heterodimer

(7 ng/ml) 1.86 ± 0.20*hCG + inhibin ,38, homodimer

(5 ng/ml) 0.62 ± 0.11*

Small antral follicles were obtained from 30-day-old female rats.Follicle shells containing theca interna cells were obtained bypiercing the follicles to release granulosa cells followed by scrapingthe follicle wall. Individual theca explants were cultured in McCoy's5A medium containing 10o fetal calf serum for 3 days before mediumchange. Theca explants were then cultured for 3 more days inserum-free medium in the absence or presence of hCG with orwithout inhibin-related proteins. Concentrations of androstenedionein the conditioned media were measured by radioimmunoassay.Mean ± SEM; n = 6.*Significantly different from the corresponding group without treat-ment with inhibin-related proteins; P < 0.01.

or the ,8P homodimer (5 ng/ml) for 3 days (Table 2). Althoughtreatment with the inhibin-related proteins by themselveswas ineffective, the a,8 heterodimer enhanced LH-stimulatedandrostenedione production by 80%, whereas the pp homo-dimer of inhibin subunits suppressed LH action by 41% (P <0.01).The dose-dependent action of these gonadal proteins was

further tested. Neonatal testis and ovarian theca-interstitialcells were treated with LH with or without increasing dosesof the inhibin-related proteins. As shown in Fig. 3, the a1heterodimer of inhibin enhanced LH action with an apparentED50 value of 1.8 ng/ml (0.56 x 10-10 M) at both testis andovarian cells. Conversely, the pp homodimer of inhibininhibited LH-stimulated androgen production by testis andovarian cells with ED50 values of 1.4 and 1.2 ng/ml (0.64 and0.55 x 10-10 M), respectively.

DISCUSSIONIn addition to the well-known paracrine actions of gonadalsteroids at their site of production (32), the present findingthat inhibin enhances LH-mediated Leydig and theca cellandrogen production provides evidence of a paracrine actionof this gonadal protein (Fig. 4). Although inhibin was orig-inally isolated from the follicular fluid and believed to be agranulosa cell product, the ovine inhibin used here is isolatedfrom the rete testis fluid and believed to be of Sertoli cellorigin. In addition, our recent data have demonstrated theFSH stimulation of inhibin production by cultured rat Sertolicells (21). Thus, inhibin may prove to be one of the putativeSertoli cell- or seminiferous tubule-derived nonsteroidalfactors capable of enhancing LH-mediated Leydig cellsteroidogenesis (23-25). The present observation may alsoexplain paradoxical findings showing that FSH enhancesLeydig cell LH responsiveness and receptor content (22).

In addition to the two long-loop feedback axes betweenpituitary (LH- and FSH-secreting) gonadotrophs and testic-ular (androgen- and inhibin-producing) cells, inhibin mayprovide the cross-communication between the two loops.Thus, testis inhibin not only suppresses pituitary FSH re-lease, but also enhances LH-regulated Leydig cell productionof androgens, which exert negative feedback at the pituitaryLH-producing cells (Fig. 4). An analogous situation may also

LH+a ,

ILH+a13+0ILH

LH+005 F

Proc. Natl. Acad Sci. USA 84 (1987)

Proc. Natl. Acad. Sci. USA 84 (1987) 5085

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INHIBIN DIMERS (ng/ml)

INHIBIN OIMERS (ng/ml)

FIG. 3. Dose-dependent modulation of testicular and ovarian cellandrogen production by dimers of inhibin subunits. Testis (A) orovarian (B) cells were cultured as described in Figs. 1 and 2,respectively. Cells were treated with LH (1 ng/ml for testis cells and10 ng/ml for ovarian cells) with or without increasing doses ofheterodimer and homodimer of inhibin. Medium content of arndro-gens was measured by radioimmunoassay. Mean ± SEM of triplicatecultures.

exist in the female because treatment with the inhibinheterodimer enhances androgen production by cultured ovar-ian theca interna cells. Both FSHI and LH have been shownto stimulate cultured granulosa cells to produce inhibin (18),which suppresses pituitary FSH release as well as enhancestheca cell steroidogenesis.Although not shown in Fig. 4, androgens produced by

testis Leydig and ovarian theca cells also exert importantparacrine actions by enhancing steroidogenesis and otherfunctions of Sertoli (33) and granulosa (32) cells. Takentogether, gonadal steroids and inhibin-related proteins mayplay a role in the feedback regulation of the hypothalamic-pituitary-gonadal axis. In addition to the potential role ofinhibin as an intragonadal paracrine hormone, a recent reportsuggests a possible autocrine action of inhibin in the sup-pression of estrogen production by cultured granulosa cells(34). Although few studies deal with ovarian androgen-

Hypothalamus

Pituitary

(-)

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l--Testis

(-) s

Hypothalamus

Pi tui tar3

Ovary

y

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I-- Theca

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FIG. 4. Diagram ofthe hypothalamic-pituitary-gonadal feedbackmechanisms. Based on findings ofparacrine and endocrine actions ofheterodimer and homodimer of inhibin subunits, the cross-commu-nication between two hypothalamic-pituitary-gonadal feedback ax-es can be postulated. In males (Upper), LH acts exclusively on testisLeydig cells to stimulate the production of androgens that exertnegative feedback on LH secretion. Likewise, FSH stimulates theproduction of Sertoli cell inhibin that suppresses FSH release butenhances the LH stimulation of androgen biosynthesis at the Leydigcell level. In addition, the ,/,8 homodimer may exert pituitary andintratesticular actions opposite to those induced by the inhibinheterodimer. In females (Lower), LH stimulates thecal cell produc-tion of androgens, which exert negative feedback on LH release,whereas ESH stimulates granulosa cell production of inhibin, whichsuppresses FSH release but enhances the LH stimulation of thecalcell androgen biosynthesis. In addition, LH may also stimulategranulosa cell inhibin production in mature follicles (18). Likewise,the /3,8 homodimer of inhibin may antagonize the action of the a,/heterodimer of inhibin.

producing cells, several uncharacterized follicular fluidnonsteroidal factors have been shown to modulate granulosacell steroidogenesis (35).

In direct contrast to the FSH-suppressing action of the a,3heterodimer of inhibin, the /3,8 homodimer stimulates FSHrelease by the pituitary (15, 16). Interestingly, the /,33homodimer inhibits Leydig and theca cell LH-regulatedandrogen production as compared with the enhancing actionof the af heterodimer. Indeed, the heterodimer and homo-dimer antagonize each other's action. Although the /,3/homodimer was purified from porcine follicular fluid, itscellular origin is unknown. Assuming that it is also producedby inhibin-secreting granulosa and Sertoli cells, this gonadalprotein may also serve important intragonadal paracrinefunctions. The potential importance of this protein is under-

A

LH+ Po

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5086 Physiological Sciences: Hsueh et al.

scored by the extreme conservation of the inhibin gene inall the mammalian species studies (12-14). In addition to itshomology with 8-TGF and Mullerian duct inhibiting sub-stance (17), the 1a homodimer of porcine inhibin wasrecently shown to have identical sequence as an erythroiddifferentiation factor isolated from a human leukemia cell line(36). Because the ,3,3 homodimer may exert both pituitary andgonadal actions opposite to those induced by inhibin, thebiosynthesis of the heterodimer and homodimer of inhibin isbelieved to be under stringent and dynamic control. It is ofinterest to study the mechanisms involved in the dimerizationofthe inhibin gene products. The independent genes for a and,3 chains of inhibin are probably under differential hormonalregulation.Both the heterodimer and homodimer of inhibin subunits

modulate gonadal androgen production. ED50 values are of<10- 10 M. Due to the observed high concentrations of inhibinin ovarian follicular and testicular rete testes fluid, thesefindings are believed to be of physiological significance.Additional studies on the gonadal receptors for the inhibin-related proteins should further substantiate their role ingonadal functions.

Testicular and ovarian androgens are produced exclusivelyby Leydig and theca interna cells, respectively. Although thehomodimer and heterodimer of inhibin subunits probably acton the androgen-producing Leydig cells in the testis cellcultures, one cannot exclude the involvement of other tes-ticular cell types due to the impurity of the cell preparationsused. However, the use of ovarian theca explants in vitrostrongly suggests that the ovarian theca interna cells are thetarget cells for these inhibin-related proteins. Conclusivedemonstration of the physiological significance of the presentfindings awaits future confirmation using in vivo experi-mental approaches. Nevertheless, the findings of similarmodulatory actions of inhibin-related proteins in four gonadalandrogen-producing cell models underscore the significanceof these results.

In addition to the well-established primary regulatory roleof LH in gonadal androgen biosynthesis, the present studiessuggest intragonadal paracrine actions of inhibin subunithetero/homodimers on testis Leydig and ovarian thecal cellsand suggest a mechanism for cross-communication betweenthe FSH-inhibin feedback axis and the classical LH-andro-gen feedback axis. Studies on the intricate regulation of theformation of inhibin heterodimfer and homodimer and theirinteraction with gonadal receptors during different physio-logical states would be of substantial interest.

We thank National Hormone and Pituitary Distribution Programfor ovine LH and hCG. This work was supported by NationalInstitutes of Health Program Project Grants HD-13527 and AM-26741, and conducted in part by the Clayton Foundation, CaliforniaDivision. W.V. is a Senior Investigator of the Clayton Foundationand K.D.D. is the recipient of National Institutes of Health Post-doctoral Fellowship HD 06875. Inhibins and related proteins werepurified under Contract N01-HD-32826 from the ContraceptiveDevelopment Branch of National Institute of Child Health andHuman Development and B85-28A/ICCR from the Population Coun-cil and the U.S. Agency for International Development.1. McCullagh, G. R. (1932) Science 76, 19-21.2. Franchimont, P., Verstraelen-Proyard, J., Hazee-Hagelstein,

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Proc. Natl. Acad. Sci. USA 84 (1987)