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Inhibin and related peptides: Mechanisms of action and regulation of secretion
J. steroid Biochem. Vol. 32, No. 18, pp. 193-197, Printedin Great Britain. All rights reserved
1989 Copyright 0
0022-4731/89 $3.00 + 0.00 1989 PergamonPress plc
INHIBIN AND RELATED PEPTIDES: MECHANISMS OF ACTION AND REGULATION OF SECRETION PAUL FRANCHIMONT, MARIE-T&R&E
HAZEE-HAGEISTEIN,
CHANTAL CHARLET-RENARD
Radioimmunoassay
and
JEAN-MARIE JASPAR,
ANDRE: DEMOULIN
Laboratory B23, University of Liege, B-4000 Liege, Belgium
Summary-The structure of inhibin is known; it consists of a heterodimer composed of one alpha and one beta subunit. The homodimer of beta A (beta A-beta A) and the heterodimer beta A-beta B, called activin A and B, respectively, stimulate the release and synthesis of FSH by gonadotrophs. Inhibin exerts effects at the hypophyseal, hypothalamic, and gonadal levels. Produced by granulosa cells in the female and by Sertoli cells in the male, inhibin synthesis is stimulated by FSH and reduced by hypophysectomy and progesterone. At present, there is no evidence for a signal from germinal cells to modify inhibin production. Inhibin secretion evolves in parallel with follicular maturation and aromatase activity, whereas luteinization arrests its production. Nevertheless, important differences in the regulation of inhibin secretion seem to exist from one species to another. Sperm inhibin levels can be correlated with spermatozoa number. Administration of inhibin to sheep induces either anovulation or an increase in the rate of ovulation depending on the scheme of treatment.
INTRODWTION
Since 1972, numerous studies have been directed towards the elucidation of the nature and bioactivity of inhibin, a gonadal peptide which specifically or preferentially decreases the secretion of follicle stimulating hormone (FSH). The physiological role of this hormone, its actions on the pituitary, hypothalamus, and gonads, its possible synergism with the sex steroids, and the mechanisms controlling its secretion have all been the object of extensive investigation [ 1,2]. This article reviews findings on the nature of inhibin, its action at the hypothalamic, pituitary and gonadal levels, and the mechanisms regulating its production by granulosa and Sertoli cells. NATURE OF INHIBIN AND RELATED PEWIDES
The structure of this gonadal hormone was not conclusively demonstrated until 1985 [3,4] and 1985 [5], when several investigators succeeded in isolating and characterizing a pure preparation of inhibin from porcine follicular fluid (FF). The protein was shown to be a heterodimer having a mol. wt of 32 kDa and consisting of one alpha (18 kDa) and one beta (14 kDa) subunit bound together by disulfide bonds. The polymorphic character of the molecule has been demonstrated by bioactivity of porcine FF inhibin molecules in the form of either major Proceedings of the XIII Meeting of the International Study Group for Steroid Hormones (Rome, Italy, 30 November-2
December 1987).
(32 kDa) or minor (100,80 and 55 kDa) constituents. Inhibin extracted from bovine FF has also been identified in three forms possessing biological inhibin activity at mol. wt of 95, 55 and 32 kDa, respectively [6]. This contrasts with the isolated alpha and beta subunits which are devoid of biological effects on FSH secretion by pituitary cells. In human and bovine FF, two forms of inhibin with a mol. wt of 32 kDa have been found to exist [7]. Although both inhibins possess the same alpha subunit, they differ in their beta subunits which either consist of type beta A (116 aa) or type beta B (115 aa), forming inhibin A and inhibin B, respectively. The beta A subunit is identical in human and bovine species; its NH,-terminal amino acid sequence is also similar to that of erythroid differentiation factor [8]. Porcine and human beta B subunits differ by a single amino acid. Their alpha subunits show 85% homology. Beta subunits may also be linked by disulfide bridges to form the homodimer beta A-beta A [9] termed FSH-releasing protein (FRP), also known as activin A, or as a beta A-beta B heterodimer [lo] called activin B. Activin A and B are capable of stimulating FSH secretion by pituitary cells in monolayer cell culture. Both activins have mol. wts of 28 kDa. The FSH stimulatory effect of activins distinguishes them from the action of gonadotrophinreleasing hormone (GnRH): Activins do not modify luteinizing hormone (LH) secretion, require a latency period of several hours before stimulating FSH secretion, act on both release and synthesis of FSH, and bind to different membrane receptors from those of GnRH.
193
194
PAUL
FRANCHlMONT
Some analogies exist between inhibins and transforming growth factor-/I (TGF+). The latter is a homodimer consisting of two identical 12.5 kDa subunits bound by disuhide bonds. Inhibin and TGF-fi subunits possess 9 cysteine and 33 similar amino acid residues located at the same positions in their molecules [ 111. These similarities have led to speculation that both subunits are derived from a common ancestral gene. It appears that TGF-fl stimulates FSH release from pituitary cells and significantly increases aromatization of androgens and oestrogens by granulosa cells [ll, 121. SITES AND MECHANISMS OF ACTION OF INHIBINS AND RELATED PEPTIDES
The discovery of inhibin was prompted by its ability to impair FSH secretion as much in vivo as in vitro. Moreover, inhibin, activins and TGF-B also seem to act as paracrine signals regulating reproduction intragonadally. Finally, evidence for a hypothalamic site of action is also suggested by several experiments. These biological activities of inhibin occur regardless of the species from which it originates. Pituitary action
Inhibin’s certain action on the pituitary has been shown by its effect on basal FSH levels and on the FSH response to GnRH in isolated pituitary cell cultures. It is also true that an effect can be observed on the LH response to GnRH, but considerably larger doses of the inhibin preparation are consistently required and the inhibition curve differs from that for FSH. The degree of inhibitory activity is quantitatively and temporally cumulative, since an increase both in the dose and length of exposure results in an increased degree of inhibition of FSHpituitary release. Convincing evidence for the action of inhibin on FSH synthesis has been provided by the experiments of Chowdhury et a/.[131 who studied the incorporation of [‘Hlleucine into FSH and LH produced by organ cultures of rat anterior pituitaries in a spent medium previously used for 2-5 days to culture Sertoli cells. The Sertoli Cell Factor present in the culture medium selectively reduced the incorporation of labeled leucine into immunoprecipitable FSH without decreasing its incorporation into LH. At the gonadotrophic level, inhibin acts by binding to specific membrane receptors different from those for GnRH [14]. Under both basal and stimulated conditions, inhibin induces an increase in cyclic GMP release and production which is dose-dependent and appears to occur simultaneously with the inhibition of FSH and LH release. By contrast, inhibin causes a reduction of cyclic AMP release and production which is not dose-dependent and appears to be delayed in time with respect to FSH and LH modifications [15].
et al.
Therefore, these studies suggest that cyclic GMP acts as the second messenger in the inhibition of FSH and LH secretion by inhibin. The increase in cyclic GMP could, in turn, lead to a secondary reduction of cyclic AMP since it was demonstrated that cyclic GMP stimulated CAMP hydrolysis [ 161. Hypothalamic action
A study by Lugaro et al. suggests an action by inhibin at the hypothalamic level [17]. After injection of a IOO-ng native extract prepared from bull spermatozoa into the third ventricle, the investigators observed a reduction in FSH levels, in contrast to the absence of effect on LH. In vitro, we have demonstrated that inhibin preparations extracted from rete testis fluid and human seminal plasma decrease the endogenous LH-RH content of isolated rat hypothalami after short-term incubation with varying inhibin concentration [ 181. Lumpkin et al. also showed that inhibin preparations purified from rat rete testis fluid preferentially inhibit FSH secretion in the adult male rat by a hypothalamic mechanism [19]. Thus, the injection of inhibin into the third ventricle of castrated rats resulted in a decrease in plasma FSH levels during the 24-h post-injection period, whereas FSH levels increased steadily in controls. When compared with controls, plasma LH levels were not significantly different for any given time period. In contrast, FSH and LH releases induced by a LH-RH challenge administered 6 h post-injection were similar in inhibin and control groups, thereby arguing against a pituitary site of action of the inhibin injected into the third ventricle. Gonadal actions
Certain imperfectly purified preparations of inhibin have been shown to exert a direct inhibitory effect upon the synthesis of DNA by actively dividing spermatogonia in normal pubertal rats[20]. However, the lack of absolutely pure inhibin preparations used earlier makes it impossible to conclude whether these direct effects on gonadal cells were due to inhibin itself or to contamination of the preparations which have been tested with biologically active protein factors. Several paracrine actions on steroidogenesis in the ovary and testis have been discovered for inhibin, activin, and TGF-/I. Recently, it was demonstrated that recombinant porcine inhibin directly inhibits FSH-induced aromatization by rat granulosa cells [12]. Moreover, in primary cultures of testis cells, the alpha-beta heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the beta A-beta A activin homodimer suppresses androgen production [21]. Similar modulatory actions of inhibin-related proteins were shown to be present in LH-treated cultured ovarian thecainterstitial cells [21]. Finally, we have demonstrated that activin A (beta A-beta A homodimer) inhibits aromatase activity in rat granulosa cells [22].
Inhibin and related peptides The N-terminal sequence of the inhibin alpha subunit (alpha 1-26 Gly-27-Tyr 28-OH) has been tested on granulosa cells from immature female rat ovaries cultured with FSH and testosterone[23]. While an inhibitory effect by this sequence has been described on progesterone, CAMP, and 17/loestradiol production [23], we were unable to confirm such results in a similar biological model [22]. TGF-B is produced by ovarian thecal cells which can regulate granulosa cell growth and differentiation [24]. In addition, it has been shown to be a powerful agent capable of stimulating the aromatization of androgens into oestrogens in granulosa cells [11, 121.
ORIGIN
OF INHIBIN AND REGULATION OF ITS SECRETION
Inhibin is produced by Sertoli cells in the male and by granulosa cells in the female [ 1,2]. Effect
ofhypophysectomy on inhibin production
In adult rats, hypophysectomy leads to a marked and significant reduction of inhibin testicular content from the seventh day following the surgery. Treatment with FSH (2 x 22 IU/day for 3 days) and with FSH + testosterone (5 mg/day for 3 days) increases inhibin testicular content. By contrast, sole treatment with testosterone does not lead to any significant modification of inhibin testicular levels [25]. Thus, hypophyseal function seems to be an essential element for inhibin production. Effect of gonadotrophins on inhibin production
In female rats, administration of FSH and pregnant mare serum gonadotrophin results in increased plasma inhibin concentrations and inhibin ovary content [26,27]; increased ovarian inhibin concentrations in humans are also found after FSH treatment [28,29]. Furthermore, the addition of FSH to healthy granulosa cells in culture leads to increases in inhibin production in the culture medium [30]. In contrast, bovine granulosa cells from atretic follicles do not respond to FSH by an increase in inhibin production [30]. In human [31] and bovine [30] species, 178oestradiol and inhibin production evolve in parallel with follicular maturation. When bovine granulosa cells luteinize, inhibin production decreases, whereas that of progesterone increases[30]. Recently, an inhibin radioimmunoassay was proposed [32] based on an antiserum against a synthetic replicate of the inhibin alpha chain N-terminal fragment (l-26) labelled at position 27 (Tyr-OH). When using such an assay, an increased production of inhibin in cultured granulosa cells from immature hypophysectomized, oestrogen-treated rats is observed in the presence of androstenedione (lo-’ M) and FSH. No effect is observed with LH or with androstenedione alone [33]. These experiments provide RIA
195
confirmation of the existence of an FSH/inhibin/FSH feedback loop. Similar results have been found for immunoreactive inhibin production by Sertoli cells from normal rats stimulated by FSH + androstenedione and by FSH alone, but not by LH, androstenedione, or 17B-oestradiol alone [33]. In contrast with the results observed using rat granulosa cells, Tsonis et a1.[34] have investigated the capacity of granulosa-lutein cells from human preovulatory ovarian follicles to produce inhibin in vitro. They found that LH was able to stimulate inhibin production in a dose-dependent manner, whereas administration of FSH at the same dose range had no effect. Addition of testosterone to the culture medium increased oestrogen formation and inhibin production. These findings seem to indicate that, in human beings, LH and testosterone stimulate inhibin production by granulosa-lutein cells in vitro, and suggest that inhibin production occurs under hormonal control in the corpus luteum as well as in the preovulatory follicle in the human ovary. This possibly suggests important differences in the regulation of inhibin secretion from one species to another. Using radioimmunoassay, it has also been demonstrated that FSH induces a dose-dependent increase in Sertoli cell inhibin production [35]. This increase in FSH action by the phosphodiesterase inhibitor, and the ability of both adenyl cyclase activators and the CAMP analogue dibutyryl CAMP to induce inhibin production by Sertoli cells, all point to a CAMPdependent mechanism involving FSH stimulation of inhibin production [35]. Interaction between gametogenesis and inhibin production
It is not evident that germinal cells can modulate inhibin secretion by Sertoli cells in vitro. Thus, coculture of Sertoli and germinal cells (spermatocytes and spermatids) do not modify inhibin production when compared with cultures of Sertoli cells alone [36]. There even exists a significant reduction of inhibin secretion by co-culture of Sertoli and germinal cells in the presence of FSH [37]. Effect of other substances
Somatomedin C alone or in association with FSH stimulates inhibin production in granulosa cell culture medium [38]. By contrast, epidermal growth factor inhibits FSH-induced inhibin production by rat granulosa cells [39]. Similarly, GnRH reduces FSH-induced inhibin secretion by granulosa cells [40]. lnhibin and placenta
Human placenta extracts exhibit inhibin bioactivity and immunoreactivity [41]. This inhibin-like immunoreactivity (inhibin-L.I.) is present in cells of the cytotrophoblast layer of human placenta at term and in primary cultures of human trophoblasts. Human chorionic gonadotrophin (hCG) stimulates
inhibin-L.I. Placental inhibin seems to exert a local tonic inhibitory action on hCG and GnRN-L.I. release [42]: Inhibin-L.I. L G&H--L.I. t--____-_____-____---I
&
hCG
Aeknowiedgement~-be
studies in this chapter carried out by this laboratory were supported by Grant No. 3.452786 of the Belgian Scientific Foundation for Medical Research (FRSM). We thank Mr and Mrs F. Shapiro (SYNTAXISBELGIWM) for their editorial and technical assistance with the manuscript. REFERENCES
J., Hazee1. Franchimont P., Verstraelen-Proyard Hagelstein M-T., Renard Ch., Demoulin A., Bourguignon J-P. and Hustin J.: Inhibin: from concept to reality. Y&m. Norm. 37 (1979) 243-302. 2. Franchimont P., Henderson K., Verhoeven G., HazeeHagelstein M-T., ~harlet-Renard Ch., Demoulin A., Bour~i~on J-P. and L~orn~-Yerna M-J.: Inhibin: mechanisms of action and secretion. In Intrugona~l Re~~atio# of Reprod~~tjon (Edited by P. Fran~~mont and C. P. Chartnina). Academic Press. New York 11981) . , pp. 167-192. _ 3. Ling N., Ying S-Y., Ueno N., Esch F., Denoroy L. and Guillemin R.! Isolation and partial characterization of a M. 32.000 orotein with inhibin activitv from noroine folli&lar thud. Proc. nut1 Acad. Sci. U.%.A. 8i (1985) 1217-7221. 4. Mivamoto K.. Hasenawa Y., Fukuda M.. Nomura M..
Igarashi M., KangaGa K. and Matsuo H:: Isolation of porcine follicular fluid inhibin of 32 K dalton. B~ochem. biopkys. Res. Common. I29 (1985) 396-403. 5. Rivier J., Spiess J., McCfintock R., Vaughan J. and Vale W.: ~rification and partial characte~~tion of inhibin from porcine follicular fluid. ~~oc~ern, biophys, Res.
activity of cultured rat granulosa cells. Biockem. biophys. Res. Commun. 136 (1986) 969-975.
13. Chowdhury M., Steinberger A. and Steinberger E.: Inhibition of de nouo synthesis of FSH by the Sertoli Cell Factor (SCF). Endocrinology X03 (1978) 644-647. 14. Sairam M. R.. Ranganatham M. R. and Lamothe I!.: vocation and ch~racter~tio~ of a Fo~itropjn suppressing prinoipte from bull ejaculate. i(iol. Reprod. IS (1978) Abstr. no. 58. Franchimont P., Craze F., Hende~on K., HazeeHagelstein M-T. and ~omte-Yema M-J.: Re~lation of inhlbin by granulosa cells in vitro. In Gona& Protein a~d~pt~des and Their Bio~ogicai Sfg@cance (Edited by M. R. Sairam and L. E. Atkinson). World Scientific Publ. (1983) pp. 127-139. Erneux Ch., Van Sande J., Dumont J. and Boeymaens J-M.: Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3’-S’-monophosphate and guanosine 3’-S-monophosphate. Eur. J. Biothem. 72 (1977) 137-147. 17. Luaaro ci.. Cassellato M. M.. Mazola G.. Fachini G. and Carrea G.: Evidence for the existence in spermatozoa of a factor inhibiting hormone synthesis. _ Ne~r~~dorr~no~ogy 15 (1974) 62-68. 18 Demoulin A.. Bourauianon J-P. and Fr~chimont P.: In vitro inhibition of hypothalamic LH-RH by human inhibin. Acta endocr. Copenh. 2% Suppl. (1979) Abstr. 227. 19, Lumpkin M., Negro-Vilar A., Franchimont P. and McCann S.: Evidence for a hypothalamic site of action of inhibin to suppress FSH release. Endocrinology 108 (1981) 1101-l 104. 20. Franchimont P., Croze F., Demoulin A., Bologne R. and Hustin J.: Effect of inhibin on rat testicular desoxyribonucleic acid (DNA) synthesis in v&u and in aitrb. Actn endoe&.
Cooenh. 98 lt9811 312-320.
21. Hsueh A. J. W., i)iahl K. D., V&zghan J_, Tucker E., Rivier J., Bardin C. W. and Vale W.: Heterodimers and hom~imers of inhibin subunits have different paracrine action in the modulation of luteini~ng hormoneCommu#. 133 (1985) 120-127. stimulated androgen biosynthesis. Prot. nail Acad. Sci. 6. Fukuda M., Mivamoto K. Hasenawa Y.. Nomura M.. U.S.A. 84 (1987) 5082-5086, Igarashi M.1 Kangawa K. and M>tsuo Hz Isolation or 22. Franchimont P., Jaspar J-M., Hazee-Hagetstein M-T., bovine foSlicular fluid inhibin of about 32 kRa. a%#olec. Charlet-Renard Ch., Van Heuverswijn H., De Waele P., Cell. Endocr. 44 (1986) 5560. Marmenout A., Bosman F. and Van de Voorde A.: 7. Mason A. J., Niall H. D. and Seeburg P. II.: Structure Effect of inhibin alpha and beta subunit and their polymers on aromatase activity of rat granulosa cells. of two human ovarian inhibin. Biochem. biophys. Res. Commun. 135 (1986) 957-964. Submitted far publication. 8. Eto Y., Tsuji T., Takezawa M., Takano S., Yokogawa 23. Hillier S. G., Tsonis C. G. and Eidne K. A.: Inhibition Y. and Shibai S.: Purification and characterization of of FSH-stimulated granulosa cell function by a synerythroid differentiation Factor (EDF) isolated from thetic fragment of the porcine inhibin alpha subunit: evidence for involvement of GnRH receptors. 3. Endorr. human leukemia cell line THP-1. Biochem. biophys. Res, Commrm. 142 (1987) iO95-1103. It3 (1987) R2-R9. 9. Vale W., Rivier J., Vaughan J., M~~lintock R., Cor24. Skinner M. K., Keski-Oja J.. &teen K. G. and Moses rigan A., Woo W., Karr D. and Spiess J.: ~fication H. L.: Ovarian thecal ceils produce transforming growth and characterization of an FSH releasina urotein from factor bets which can regulate granulosa cell growth. E~dorri~o~ogy 121 ( 1987) 786-792. porcine ovarian follicular fluid. Na&e’ 321 (1986) 776-779. 25 Au C. L., Irby D. C., Robertson D. M. and de Kretser IO. Ling N,, Ying S-Y., Ueno N., Shimasaki S., Esch F., D. M.: Eff~ts of testosterone on testicular inhibin and Hotta M. and Guitlemin R.: Pituitary FSH is released fluid production in intact and hypophysectomized adult by a heterodimer of the /3-subunits from the two forms rats. J. Reprod. Fert. 76 (1986) 257-266. of inhibin. Nature 321 (1986) 779-782. 26. Lee W. K., McMaster J., Quigg H., Findlay J. and 11. Yin8 S-Y., Becker A., Baird A., Ling N., Ueno N., Esch Leversha L.: Ovarian and peripheral blood inhibin F. and Guillemin R.: Tvoe beta transformina arowth concentrations increase with gonadotrophin treatment factor (TGF beta} is a potent stimulator of rhi basal in immature rat. Endocrinology 108 (1981) 2403-2405. secretion of follicle stimulating hormone (FSH) in a 27. Lee W. K., McMaster J., Quigg H. and Leversha L.: pituitary monolayer system. Biochem. btophys. R-es. Ovarian and circulating inhibin levels in immature CommurL 13s (1986) 950-956. female rats treated with gonadotrophin and after cas12. Ying S-Y., Becker A., Ling N., Ueno N. and Guillemin tration. Endocrinology Iii (1982) 1849-1854. R.: Inhibin and beta type transfo~ing growth factor 28. ~hanning C. P.: Folh&lar guid survey and possible role (TGF beta) have opposite modulating effects on the of follic~ar Ruid inhibin activity in the human. Inferfollicte stimulating hormone (FSH) induced aromatase rilii.v 6 (1983) 251-263.
Inhibin and I,elated peptides 29. Channing C. P., Chacon M., Tanabe K., Gagliano .P.
and Tildon T.: Follicular fluid inhibin activity and steroid levels in ovarian tissue obtained at autopsy for human infants from 18-200 days of age. Ferr. Sferil. 42 (1984) 861-869. 30. Henderson K. M., Franchimont P., Charlet-Renard Ch. and McNatty K. P.: Effect of follicular atresia in inhibin production by bovine granulosa cells in vitro and inhibin concentrations in the follicular fluid. J. Reprod. Fert. 72 (1984) 1-8. 31 McLachlan
R. I., Robertson D. M., Healy D. L., Findlay .I. K., de Kretser D. M. and Burger H. G.: The radioimmunoassay of human serum inhibin. In Inhibin: Non -steroidal Regulation of Follicle Stimulating Hormone Secretion (Edited bv H. G. Buraer. D. M. de Kretser, J. K. Fihdlay and M. Igarashi; Raven Press, New York, Serono Symposium (1987) Vol. 42, pp. 105-I 18. 32. Rivier C. and Vale W.: Inhibin: measurement and role in the immature female rat. Endocrinology 120 (1987) 1688-1690. 33. Ying S. Y., Czvik J., Becker A., Ling N., Ueno N. and Guillemin R.: Secretion of follicle-stimulating hormone and production of inhibin are reciprocally related. Med. Sci. 84 (1987) 4631-4635. 34. Tsonis C. G.. Hillier S. G. and Blaird D. T.: Production of inhibin bibactivity by human granulosa-lutein cells: stimulation by LH and testosterone in vitro. J. Endocr. 112 (1987) Rl I-R14. 35 Bicsak T. A., Vale W., Vaughan J., Tucker E. M., Cappel S. and Hsueh A. J. W.: Hormonal regulation of inhibin production by cultured sertoli cells. Molec. Cell Endocr. 49 (1987) 211-217.
197
36. Steinberger A.: Regulation of inhibin secretion in the testis. In Intragonadal Regulation of Reproduction (Edited by P. Franchimont and C. P. Charming). Academic Press: New York (1981) pp. 283-298. -’ 37. Ultee-van Gessel A. M.. Leembora F. G.. de Jone F. H. and van der Molen H. J.: I&vitro se&etion 07 inhibin-like activity by Sertoli cells from normal and prenatally irradiated immature rats. J. Endocr. 109 (1986) 411418. 38. Zhiwen Z., Carson R. S., Herington A. C., Lee V. W. K. and Burger H. G.: Follicle-stimulating hormone and somatomedin-C stimulate inhibin production by rat granulosa cells in vitro. Endocrinology 120 (1987) 1633-1638. 39. Franchimont P., Hazee-Hagelstein M-T., CharletRenard Ch. and Jaspar J-M.: Effect of mouse epidermal growth factor on DNA and protein synthesis, progesterone and inhibin production by bovine granulosa cells in culture. Acta endocr. Copenh. 111 (1986) 122-127. 40. Bicsak T. A., Tucker E. M., Cappel S., Vaughan J., Rivier J., Vale A. and Hsueh A. J. W.: Hormonal regulation of granulosa cell inhibin biosynthesis. Endocrinology 119 (1986) 271 I-2719.
41. McLachlan R. I., Healy D. L., Robertson D. M., Burger H. G. and de Kretser D. M.: The human placenta: a novel source of inhibin. Biochem. biophys. Res. Commun. 140 (1986) 485490. ._ 42. Petraglia F., Sawchenko P., Lim A. T. W., Rivier J. and Vale W.: Localization, secretion and action of inhibin in human placenta. Science 237 (1987) 187-189.
1989 Copyright 0
0022-4731/89 $3.00 + 0.00 1989 PergamonPress plc
INHIBIN AND RELATED PEPTIDES: MECHANISMS OF ACTION AND REGULATION OF SECRETION PAUL FRANCHIMONT, MARIE-T&R&E
HAZEE-HAGEISTEIN,
CHANTAL CHARLET-RENARD
Radioimmunoassay
and
JEAN-MARIE JASPAR,
ANDRE: DEMOULIN
Laboratory B23, University of Liege, B-4000 Liege, Belgium
Summary-The structure of inhibin is known; it consists of a heterodimer composed of one alpha and one beta subunit. The homodimer of beta A (beta A-beta A) and the heterodimer beta A-beta B, called activin A and B, respectively, stimulate the release and synthesis of FSH by gonadotrophs. Inhibin exerts effects at the hypophyseal, hypothalamic, and gonadal levels. Produced by granulosa cells in the female and by Sertoli cells in the male, inhibin synthesis is stimulated by FSH and reduced by hypophysectomy and progesterone. At present, there is no evidence for a signal from germinal cells to modify inhibin production. Inhibin secretion evolves in parallel with follicular maturation and aromatase activity, whereas luteinization arrests its production. Nevertheless, important differences in the regulation of inhibin secretion seem to exist from one species to another. Sperm inhibin levels can be correlated with spermatozoa number. Administration of inhibin to sheep induces either anovulation or an increase in the rate of ovulation depending on the scheme of treatment.
INTRODWTION
Since 1972, numerous studies have been directed towards the elucidation of the nature and bioactivity of inhibin, a gonadal peptide which specifically or preferentially decreases the secretion of follicle stimulating hormone (FSH). The physiological role of this hormone, its actions on the pituitary, hypothalamus, and gonads, its possible synergism with the sex steroids, and the mechanisms controlling its secretion have all been the object of extensive investigation [ 1,2]. This article reviews findings on the nature of inhibin, its action at the hypothalamic, pituitary and gonadal levels, and the mechanisms regulating its production by granulosa and Sertoli cells. NATURE OF INHIBIN AND RELATED PEWIDES
The structure of this gonadal hormone was not conclusively demonstrated until 1985 [3,4] and 1985 [5], when several investigators succeeded in isolating and characterizing a pure preparation of inhibin from porcine follicular fluid (FF). The protein was shown to be a heterodimer having a mol. wt of 32 kDa and consisting of one alpha (18 kDa) and one beta (14 kDa) subunit bound together by disulfide bonds. The polymorphic character of the molecule has been demonstrated by bioactivity of porcine FF inhibin molecules in the form of either major Proceedings of the XIII Meeting of the International Study Group for Steroid Hormones (Rome, Italy, 30 November-2
December 1987).
(32 kDa) or minor (100,80 and 55 kDa) constituents. Inhibin extracted from bovine FF has also been identified in three forms possessing biological inhibin activity at mol. wt of 95, 55 and 32 kDa, respectively [6]. This contrasts with the isolated alpha and beta subunits which are devoid of biological effects on FSH secretion by pituitary cells. In human and bovine FF, two forms of inhibin with a mol. wt of 32 kDa have been found to exist [7]. Although both inhibins possess the same alpha subunit, they differ in their beta subunits which either consist of type beta A (116 aa) or type beta B (115 aa), forming inhibin A and inhibin B, respectively. The beta A subunit is identical in human and bovine species; its NH,-terminal amino acid sequence is also similar to that of erythroid differentiation factor [8]. Porcine and human beta B subunits differ by a single amino acid. Their alpha subunits show 85% homology. Beta subunits may also be linked by disulfide bridges to form the homodimer beta A-beta A [9] termed FSH-releasing protein (FRP), also known as activin A, or as a beta A-beta B heterodimer [lo] called activin B. Activin A and B are capable of stimulating FSH secretion by pituitary cells in monolayer cell culture. Both activins have mol. wts of 28 kDa. The FSH stimulatory effect of activins distinguishes them from the action of gonadotrophinreleasing hormone (GnRH): Activins do not modify luteinizing hormone (LH) secretion, require a latency period of several hours before stimulating FSH secretion, act on both release and synthesis of FSH, and bind to different membrane receptors from those of GnRH.
193
194
PAUL
FRANCHlMONT
Some analogies exist between inhibins and transforming growth factor-/I (TGF+). The latter is a homodimer consisting of two identical 12.5 kDa subunits bound by disuhide bonds. Inhibin and TGF-fi subunits possess 9 cysteine and 33 similar amino acid residues located at the same positions in their molecules [ 111. These similarities have led to speculation that both subunits are derived from a common ancestral gene. It appears that TGF-fl stimulates FSH release from pituitary cells and significantly increases aromatization of androgens and oestrogens by granulosa cells [ll, 121. SITES AND MECHANISMS OF ACTION OF INHIBINS AND RELATED PEPTIDES
The discovery of inhibin was prompted by its ability to impair FSH secretion as much in vivo as in vitro. Moreover, inhibin, activins and TGF-B also seem to act as paracrine signals regulating reproduction intragonadally. Finally, evidence for a hypothalamic site of action is also suggested by several experiments. These biological activities of inhibin occur regardless of the species from which it originates. Pituitary action
Inhibin’s certain action on the pituitary has been shown by its effect on basal FSH levels and on the FSH response to GnRH in isolated pituitary cell cultures. It is also true that an effect can be observed on the LH response to GnRH, but considerably larger doses of the inhibin preparation are consistently required and the inhibition curve differs from that for FSH. The degree of inhibitory activity is quantitatively and temporally cumulative, since an increase both in the dose and length of exposure results in an increased degree of inhibition of FSHpituitary release. Convincing evidence for the action of inhibin on FSH synthesis has been provided by the experiments of Chowdhury et a/.[131 who studied the incorporation of [‘Hlleucine into FSH and LH produced by organ cultures of rat anterior pituitaries in a spent medium previously used for 2-5 days to culture Sertoli cells. The Sertoli Cell Factor present in the culture medium selectively reduced the incorporation of labeled leucine into immunoprecipitable FSH without decreasing its incorporation into LH. At the gonadotrophic level, inhibin acts by binding to specific membrane receptors different from those for GnRH [14]. Under both basal and stimulated conditions, inhibin induces an increase in cyclic GMP release and production which is dose-dependent and appears to occur simultaneously with the inhibition of FSH and LH release. By contrast, inhibin causes a reduction of cyclic AMP release and production which is not dose-dependent and appears to be delayed in time with respect to FSH and LH modifications [15].
et al.
Therefore, these studies suggest that cyclic GMP acts as the second messenger in the inhibition of FSH and LH secretion by inhibin. The increase in cyclic GMP could, in turn, lead to a secondary reduction of cyclic AMP since it was demonstrated that cyclic GMP stimulated CAMP hydrolysis [ 161. Hypothalamic action
A study by Lugaro et al. suggests an action by inhibin at the hypothalamic level [17]. After injection of a IOO-ng native extract prepared from bull spermatozoa into the third ventricle, the investigators observed a reduction in FSH levels, in contrast to the absence of effect on LH. In vitro, we have demonstrated that inhibin preparations extracted from rete testis fluid and human seminal plasma decrease the endogenous LH-RH content of isolated rat hypothalami after short-term incubation with varying inhibin concentration [ 181. Lumpkin et al. also showed that inhibin preparations purified from rat rete testis fluid preferentially inhibit FSH secretion in the adult male rat by a hypothalamic mechanism [19]. Thus, the injection of inhibin into the third ventricle of castrated rats resulted in a decrease in plasma FSH levels during the 24-h post-injection period, whereas FSH levels increased steadily in controls. When compared with controls, plasma LH levels were not significantly different for any given time period. In contrast, FSH and LH releases induced by a LH-RH challenge administered 6 h post-injection were similar in inhibin and control groups, thereby arguing against a pituitary site of action of the inhibin injected into the third ventricle. Gonadal actions
Certain imperfectly purified preparations of inhibin have been shown to exert a direct inhibitory effect upon the synthesis of DNA by actively dividing spermatogonia in normal pubertal rats[20]. However, the lack of absolutely pure inhibin preparations used earlier makes it impossible to conclude whether these direct effects on gonadal cells were due to inhibin itself or to contamination of the preparations which have been tested with biologically active protein factors. Several paracrine actions on steroidogenesis in the ovary and testis have been discovered for inhibin, activin, and TGF-/I. Recently, it was demonstrated that recombinant porcine inhibin directly inhibits FSH-induced aromatization by rat granulosa cells [12]. Moreover, in primary cultures of testis cells, the alpha-beta heterodimer of inhibin enhances Leydig cell androgen biosynthesis stimulated by LH, whereas the beta A-beta A activin homodimer suppresses androgen production [21]. Similar modulatory actions of inhibin-related proteins were shown to be present in LH-treated cultured ovarian thecainterstitial cells [21]. Finally, we have demonstrated that activin A (beta A-beta A homodimer) inhibits aromatase activity in rat granulosa cells [22].
Inhibin and related peptides The N-terminal sequence of the inhibin alpha subunit (alpha 1-26 Gly-27-Tyr 28-OH) has been tested on granulosa cells from immature female rat ovaries cultured with FSH and testosterone[23]. While an inhibitory effect by this sequence has been described on progesterone, CAMP, and 17/loestradiol production [23], we were unable to confirm such results in a similar biological model [22]. TGF-B is produced by ovarian thecal cells which can regulate granulosa cell growth and differentiation [24]. In addition, it has been shown to be a powerful agent capable of stimulating the aromatization of androgens into oestrogens in granulosa cells [11, 121.
ORIGIN
OF INHIBIN AND REGULATION OF ITS SECRETION
Inhibin is produced by Sertoli cells in the male and by granulosa cells in the female [ 1,2]. Effect
ofhypophysectomy on inhibin production
In adult rats, hypophysectomy leads to a marked and significant reduction of inhibin testicular content from the seventh day following the surgery. Treatment with FSH (2 x 22 IU/day for 3 days) and with FSH + testosterone (5 mg/day for 3 days) increases inhibin testicular content. By contrast, sole treatment with testosterone does not lead to any significant modification of inhibin testicular levels [25]. Thus, hypophyseal function seems to be an essential element for inhibin production. Effect of gonadotrophins on inhibin production
In female rats, administration of FSH and pregnant mare serum gonadotrophin results in increased plasma inhibin concentrations and inhibin ovary content [26,27]; increased ovarian inhibin concentrations in humans are also found after FSH treatment [28,29]. Furthermore, the addition of FSH to healthy granulosa cells in culture leads to increases in inhibin production in the culture medium [30]. In contrast, bovine granulosa cells from atretic follicles do not respond to FSH by an increase in inhibin production [30]. In human [31] and bovine [30] species, 178oestradiol and inhibin production evolve in parallel with follicular maturation. When bovine granulosa cells luteinize, inhibin production decreases, whereas that of progesterone increases[30]. Recently, an inhibin radioimmunoassay was proposed [32] based on an antiserum against a synthetic replicate of the inhibin alpha chain N-terminal fragment (l-26) labelled at position 27 (Tyr-OH). When using such an assay, an increased production of inhibin in cultured granulosa cells from immature hypophysectomized, oestrogen-treated rats is observed in the presence of androstenedione (lo-’ M) and FSH. No effect is observed with LH or with androstenedione alone [33]. These experiments provide RIA
195
confirmation of the existence of an FSH/inhibin/FSH feedback loop. Similar results have been found for immunoreactive inhibin production by Sertoli cells from normal rats stimulated by FSH + androstenedione and by FSH alone, but not by LH, androstenedione, or 17B-oestradiol alone [33]. In contrast with the results observed using rat granulosa cells, Tsonis et a1.[34] have investigated the capacity of granulosa-lutein cells from human preovulatory ovarian follicles to produce inhibin in vitro. They found that LH was able to stimulate inhibin production in a dose-dependent manner, whereas administration of FSH at the same dose range had no effect. Addition of testosterone to the culture medium increased oestrogen formation and inhibin production. These findings seem to indicate that, in human beings, LH and testosterone stimulate inhibin production by granulosa-lutein cells in vitro, and suggest that inhibin production occurs under hormonal control in the corpus luteum as well as in the preovulatory follicle in the human ovary. This possibly suggests important differences in the regulation of inhibin secretion from one species to another. Using radioimmunoassay, it has also been demonstrated that FSH induces a dose-dependent increase in Sertoli cell inhibin production [35]. This increase in FSH action by the phosphodiesterase inhibitor, and the ability of both adenyl cyclase activators and the CAMP analogue dibutyryl CAMP to induce inhibin production by Sertoli cells, all point to a CAMPdependent mechanism involving FSH stimulation of inhibin production [35]. Interaction between gametogenesis and inhibin production
It is not evident that germinal cells can modulate inhibin secretion by Sertoli cells in vitro. Thus, coculture of Sertoli and germinal cells (spermatocytes and spermatids) do not modify inhibin production when compared with cultures of Sertoli cells alone [36]. There even exists a significant reduction of inhibin secretion by co-culture of Sertoli and germinal cells in the presence of FSH [37]. Effect of other substances
Somatomedin C alone or in association with FSH stimulates inhibin production in granulosa cell culture medium [38]. By contrast, epidermal growth factor inhibits FSH-induced inhibin production by rat granulosa cells [39]. Similarly, GnRH reduces FSH-induced inhibin secretion by granulosa cells [40]. lnhibin and placenta
Human placenta extracts exhibit inhibin bioactivity and immunoreactivity [41]. This inhibin-like immunoreactivity (inhibin-L.I.) is present in cells of the cytotrophoblast layer of human placenta at term and in primary cultures of human trophoblasts. Human chorionic gonadotrophin (hCG) stimulates
inhibin-L.I. Placental inhibin seems to exert a local tonic inhibitory action on hCG and GnRN-L.I. release [42]: Inhibin-L.I. L G&H--L.I. t--____-_____-____---I
&
hCG
Aeknowiedgement~-be
studies in this chapter carried out by this laboratory were supported by Grant No. 3.452786 of the Belgian Scientific Foundation for Medical Research (FRSM). We thank Mr and Mrs F. Shapiro (SYNTAXISBELGIWM) for their editorial and technical assistance with the manuscript. REFERENCES
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