- Email: [email protected]
Regulation of Leydig cell function by inhibins and activins
ANIMAL REPRODUCTION SCIENCE Animal Reproduction Science 42 (19%) 343-349
Regulation of Leydig cell function by inhibins and activins Gail P. Risbridger
’
Mortash University, Institute of Reproduction and Development, Monash Medical Centre, Level 3, Block E, 246 Clayton Road, Clayton, Vie., Australia
Abstract The role of inhibin related proteins is not confined to the regulation of pituitary FSH. These proteins are now recognised as growth and differentiation factors and in the testis they regulate both epithelial and interstitial cell functions. In species such as rodents and pigs, inhibin and activin are considered to he paracrine regulators of steroidogenesis whereby they modulate the endocrine LH signal. Porcine and rodent Leydig cells have been shown in vitro to respond to exogenously added activin A. We have previously proposed that activins have a significant role in Leydig cell development and in the fetal testis and at puberty, i.e. activin (like TGF /3) holds Leydig cell growth in abeyance until differentiation or puberty. The roles of activins, inhibins and binding proteins for these ligands, such as follistatins, in the development of Leydig cells remains to he determined. Keywords: Inbibin; Activin; Leydig cells
1. Introduction In 1932 inhibin was described as the active principle in aqueous extracts of the testes which was able to prevent the appearance of castration cells in the male rat pituitary
(McCullagh, 1932). In 1985, the purification of inhibin, which suppressed pituitary follicle-stimulating hormone (FSH) but not luteinising hormone (LH), was achieved (Robertson et al., 1985); this was closely followed by the discovery of activins which stimulated, rather than suppressed, FSH (Ling et al., 1986; Vale et al., 1986). In the following decade the focus of the inhibin field developed from the concept that inhibins were simply reproductive endocrine hormones to a recognition of their roles as paracrine
’Tel.: (61-3) 9550 3571; fax: (61-3) 9550 3584; e-mail: [email protected]. 0378-4320/%/$15.00
0 1996 Elsevier Science B.V. All rights reserved.
PII SO378-4320(96)01490-X
344
G.P. Risbridger/Animol
Reproduction Science 42 (19%) 343-349
and autocrine growth factors. In the testis, inhibins and activins were shown to regulate seminiferous tubule functions, but they also have roles in the interstitial tissue and particularly the Leydig cells. The action of inhibin and related proteins on Leydig cells provides the focus of this report. The first inhibin protein was purified from bovine follicular fluid and was the 58 kDa form which was composed of two disulphide linked polypeptide chains of 44 kDa and 14 kDa; designated as the a and l3 subunits, respectively (Robertson et al., 1985). Two different p subunit forms of inhibin were described PA and Pa, which gave rise to two forms of inhibin, inhibin A and inhibin B (Ling et al., 1986; Vale et al., 1986). The two l3 subunits, encoded on separate genes, also combine to form three dimeric forms of activin, activin A (PAPA), activin B (&$a) and activin AB (pApa>. Activin A has been widely studied, but less is known about activin AB and B as the availability of these proteins is limited. More recently, a putative l3, subunit was cloned from human liver (Hotten et al., 1995) and &, subunit cDNA isolated from Xenopus liver (Oda et al., 1995). This raises the probability that other activin molecules exist and remain to be isolated. These ligands act through specific receptors and a large family of serine/threonine kinase receptors was revealed, through which activin, inhibin and TGFP related factors can signal. In addition, binding proteins for these ligands play an integral role in the regulation and mechanism of action of inhibins and activins. o,-Macroglobulin is a binding protein for many cytokines, including inhibin, activin and TGFP. It is one of the main proteins in serum and appears to be a major binding protein for inhibin/activin (Kntmmen et al., 1993). It is viewed as acting to prevent proteolytic degradation of activin/inhibins or to deliver these ligands to target tissues. Follistatins have no homology to the inhibin subunits but will bind strongly to activins and in doing so can suppress bioactivity, such as the release of FSH (Nakamura et al., 1990). Although inhibin binds follistatin (Shimonaka et al., 1991), the affinity is lower than that for activin, and therefore inhibin (and TGFP) do not compete for activin binding to follistatin. The consequences of the binding interaction between follistatin and activins are important if the affinity of activin for its receptors is similar to the affinity for follistatins. A single follistatin gene has been isolated which consists of five exons and a signal peptide (see review by Michel et al., 1993). As with the inhibin subunits, the amino acid sequences of follistatin are highly conserved and across rat, sheep, human and pig there is 97% homology (Tisdall et al., 1992).
2. Evidence that inhibin and activin regulate Leydig cells The gonads are the main source of inhibin and related proteins which contribute to the endocrine regulation of the reproductive system. However, local paracrine actions within the testis are predicated upon the presence of specific receptors for these ligands on Leydig cells (De Winter et al., 1992; Feng et al., 1993). The effects of inhibin and activin on Leydig cells have been described predominantly in terms of their role in the regulation of steroidogenic enzyme activities and cell proliferation.
G.P. Risbridger/Animal
Reproduction Science 42 (19%) 343-349
345
2.1. Steroidogenesis
The effects of inhibin/activin on steroidogenesis were studied in a number of types of Leydig cells. Dual, but opposing, actions of inhibin and activin A on steroidogenesis were first reported using interstitial cell cultures prepared from immature, 7-day-old animals or from adult hypophysectomised rats. The action of inhibin A was to increase LH-stimulated testosterone production further, whereas activin A inhibited production (Hseuh et al., 1987). Another study on highly purified adult rat Leydig cells also reported an inhibitory effect of activin on testosterone release, but in these cultures inhibin had no effect alone and only antagonised the stimulatory action of activin in vitro (Lin et al., 1989). A further variation in the action of inhibin related proteins was described on immature porcine Leydig cells (Maudit et al., 1991). Human chorionic gonadotropin (hCG) stimulated dihydroepiandrostenone (DHEA) accumulation was reduced in vitro, but the conversion of pregnenolone and DHEA was increased and no net effect on testosterone production was observed. Leydig cell tumour lines commonly synthesise progesterones rather than testosterone, and activin A inhibited progesterone synthesis by the R2C tumour cells; inhibin had no effect either alone or in combination with activin A and therefore did not antagonise the inhibitory effect of activin (Vale and Gonzalez-Manchon, 1989). Overall, the exact details of the effects of inhibin and activin on steroidogenesis in vitro differ, and could be explained by the differences between the culture conditions used or by the species or type of Leydig cell cultured. Further potential for the action of activin A on Leydig cell steroidogenesis is suggested by studies describing the stimulation of So-reductase activity in human genital skin fibroblasts (Antonapillar et al., 1995). An increase in the activity of this enzyme characterises the development of immature forms of Leydig cells and the production of reduced androgens; could this involve an action of activin A? 2.2. DNA synthesis Activin is a member of the TGFP family of proteins and these proteins have proliferative and antiproliferative effects on other cell types, both within the testis and in other tissues (Hedger et al., 1989). In the normal testis, most attention has focused on the role of activin and inhibin in the regulation of spermatogonial cell proliferation (Van Dissel-Emiliani et al., 1989; Mather et al,, 1990), yet during the development of Leydig cell populations, there are periods of intense proliferation (Hardy et al., 1989; Russell et al., 1995). Although it is not known if inhibins or activins can regulate Leydig cell proliferation, TGFP inhibits DNA synthesis by immature Leydig cells under LH stimulation (Kahn et al., 1992). These authors suggested that TGFP regulates the growth of immature Leydig cells and restricts their growth until puberty (Teerds et al., 1994). It is therefore possible that activin has the same, or a similar action, on Leydig cells. This has been tested on the rat Leydig turnour cell line, R2C. Minor inhibitory effects of activin A on DNA synthesis were reported but a prolonged period of 4 days of culture was required to observe such an effect (Vale and Gonzalez-Manchon, 1989). TGFP also
346
G.P. Risbridger/Animal Reproduction Science 42 (1996) 343-349
inhibited proliferation of the R2C tumour cells, and was more potent in its action than activin A.
3. Discussion arising from the observed actions of inhibin and activins on Leydig cells These data clearly demonstrate that in vitro activin/inhibin regulate Leydig cell steroidogenesis and DNA synthesis, but a number of questions are posed by this information. First, the reason for Leydig cell DNA synthesis and steroidogenesis to be regulated by both activin/inhibin and TGFP is not known. In general, the action of TGFP on Leydig cell steroidogenesis is more potent than that of activin A (Lin et al., 1987; Lin et al., 19891, and in R2C tumour cells TGFP is more potent in terms of the effects on DNA synthesis and steroidogenesis (Vale and Gonzalez-Manchon, 1989). The growth factors may act through separate pathways, and the relative concentrations of the two hormones may be important; or they may act through the same pathways via the same receptors as promiscuity of receptor systems does occur in vitro. Secondly, it cannot be assumed that the actions of inhibin B and activin B or activin AB are the same as those that have been reported for inhibin A or activin A. The results of PA and pa inhibin subunit gene disruption studies suggest that the functions of activin A and B do not overlap during embryonic development (Vassalli et al., 1994; Matzuk et al., 1995a; Matzuk et al., 1995b). Therefore, the effects of the specific activin/inhibin on the regulation of Leydig cell functions may differ; but this has to be tested and determined. Thirdly, although activin A antagonised the action of inhibin A on steroidogenesis by immature and adult rat interstitial cells (Hseuh et al., 1987; Lin et al., 19891, this was not the case for all effects on Leydig cell steroidogenesis or on DNA synthesis. The effects of activin were observed in the absence of any effect of inhibin alone or in combination with activin (Vale and Gonzalez-Manchon, 1989). Furthermore, the ability of binding proteins, e.g. follistatins, to modify some of the biological actions of activin needs to be considered. Immunostaining identified follistatin binding protein to Leydig cells (Kaipia et al., 19921, and suggests a significant role for follistatins in regulating the effect of activin on Leydig cells. If follistatin binds and neutralises the bioactivity of activins on Leydig cells, then changes to the relative levels of follistatin could provide another means of regulating activin. action, other than through changes to the levels of the receptor or activin ligands themselves. The regulatory role of follistatins alone or in combination with activin A in the regulation of steroidogenesis, or cell proliferation, has not been explored. CX~,-macroglobulinis also a binding protein for inhibin and activin (Vaughan and Vale, 19931, and the interplay between follistatin and a,-macroglobulin in the binding of inhibin related proteins is not known. In the testis, a,-macroglobulin content increases with age (Zhu et al., 1994), but the relative levels of follistatin and OL ,-macroglobulin and the distribution of the complexes with inhibins and activins in the testis remains to be recorded. Fourthly, and most importantly, there is a wide variation in the effect of inhibins and
G.P. Risbridger/Animal
Reproduction
Science 42 (1996) 343-349
347
activins as described herein, which may be due to the type and maturity of the Leydig cells that were studied. There is more than one population of Leydig cells and there are multiple stages in the development of Leydig cells from stem cell precursors to immature and mature forms. The two populations of Leydig cells (e.g. foetal and adult) respond differently to control by LH (Huhtaniemi, 19941, and it would not be surprising if there are variations in the response to regulation by inhibins/activins or indeed any other paracrine factors. The actions of inhibin/activins at particular phases of the development of Leydig cell populations has not been undertaken in a systematic manner. Differences in culture conditions and the species of Leydig cells (rat or pig) that were cultured, are other reasons why variations in the effects of inhibins and activins are observed. The predominant effect of activin A on steroidogenesis by immature rodent Leydig cells, measured as testosterone production, was inhibitory. In contrast, there was no net effect on immature porcine Leydig cell testosterone production (Maudit et al., 1991). Therefore, there appears to be a species-specific difference in the response to the ligand. Nothing is known about the regulation of ovine Leydig cell function by inhibin/activin but the ovine testis during foetal development represents an excellent model for the study of inhibins and activins in the regulation of Leydig cell development and differentiation in an alternate species. In the sheep foetus, testosterone is present 5 days before the onset of sexual differentiation (Anal, 1969). This dissociation between testicular morphogenesis and functional differentiation of the Leydig cells has been reported in other species such as lamb, rat and human. The onset of Leydig cell differentiation cannot be due to pituitary LH alone, as gonadal development occurs in the anencephalic foetus. The role of growth factors (such as inhibins and activins) is therefore worthy of examination. Although Thomas et al. (1995) have reported the local&ion of (Ybut not p inhibin subunits, in the ovine foetal testis, we have recently localised activin immunoreactivity to foetal Leydig cells in mid gestation (G.R. Risbridger, unpublished observations, 1996). Together with the detection of follistatin immunoreactivity in the ovine foetal testis (Wongprasartsuk et al., 1994), the role of these ligands and binding proteins in the development and regulation of foetal Leydig cell function is currently under investigation.
4. Summary This brief review demonstrates that inhibin and activin are paracrine regulators of Leydig cells. However, there are a number of questions that arise from the observations which demonstrate the complexity of the action of these growth factors in their role in growth and differentiation of cells. The questions raised specifically in this review are illustrated with the example of Leydig cells, but are not specific to these cells or tissue; indeed, the questions raised are relevant to any study of inhibins and activins. References Antonapillar, I., Wahe, M., Yamamoto, J. and Horton, R., 1995. Activin aad inhibii have opposite effects on steroid. Sa-rcductase activity in genital skin fibroblasts. Mol. Cell. Endocrinol., 107: 99- 104.
348
G.P. Risbridger / Animal Reproduction Science 42 (19%) 343-349
Attal, J., 1%9. Levels of testosterone, androstenedione, estrone and estradiol-17s in the testes of fetal sheep. Endocrinology, 85: 280-289. De Winter, J.P., Themman, A.P.N., Hoogerbrugge, J.W., Klaij, LA., Grootegoed, J.A. and de Jong, F.H., 1992. Activin receptor mRNA expression in rat testicular cell types. Mol. Cell. Endocrinol., 83: Rl-R8. Feng, A-M., Madigan, M.B. and Chen, C-LC., 1993. Expression of type II activin receptor genes in the male and female reproductive tissues of the rat. Endocrinology, 132: 2593-2600. Hardy, M.R., Zirkin, B.R. and Ewing, L.L., 1989. Kinetic studies on the development of the adult population of Leydig cells in testis of the pubertal rat. Endocrinology, 124: 762-770. Hedger, M.R., Drummond, A.E., Robertson, D.M., Risbridger, G.R. and de Kretser, D.M., 1989. Divergent actions of inhibin, activin and transforming growth factor B on [3H] thymidine incorporation by rat thymocytes and 3T3 fibroblasts in vitro. Mol. Cell. Endocrinol., 61: 133-138. Hotten, G., Neidhatdt, R., Schneider, C. and Pohl, J., 1995. Cloning of a new member of the TGF-B family:-a putative new activin B, chain. Biochem. Biophys. Res. Commun., 206(2): 608-613. Hseuh, A.J.W., Dahl, K.D., Vaughan, J., Tucker, E., Rivier, J., Bardm, C.W. and Vale, W.W., 1987. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of lutemizing hormone-stimulated androgen biosynthesis. Proc. Natl. Acad. Sci. USA, 84: 5082. Huhtaniemi, I., 1994. Fetal testis-a very special endocrine organ. Eur. J. Endocrinol., 130: 25-31. Kahn, S., Teems, K. and Donington, J., 1992. Growth factor requirements for DNA synthesis by Leydig cells from the immature rat. Biol. Reprod., 46 335-341. Kaipia, A., Penttila, T.L., Shimaski, S., Ling, N., Parvinen, M. and Toppari, J., 1992. Expression of :mhibm BA and Ba, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium. Endocrinology, 131: 2703-2710. Kmmmen. L.A., Woodruff, T.K., DeGuzman, G., Cox, E.T., Baly, D.L., Mann, E., Garg, S., Wong, W-L, Cossum, P.. Mather. J.P., 1993. Identification, and characterization of binding proteins for inhibin and a&in in human strum and follicular fluids. Endocrinology, 132: 431-443. Lm, T., Blaisdell, J. and Haskell, J.F., 1987. Transforming growth factor-B inhibits Leydig cell steroidogenesis in primary culture. Biochem. Biophys. Res. Commun.. 146: 387-394. Lin, T.. CaIkms, H., Morris, P.L., Vale, W.W. and Bardin, C.W., 1989. Regulation of Leydig cell function in primary culture by inhibin and activin. Endocrinology, 125: 2134-2140. Ling, N., Yng, S-Y, Ueno, N., Shimasaki, S., Esch, R., Hotta, M. and Guillemin, R., 1986. Pituitary FSH is released by the heterodimer of the B-subunits from the two forms of inhibin. Nature, 321: 779-782. Mather, J.R., Attie, K.M., Woodruff, T., Rice, G.C. and Phillips, D.M., 1990. Activin stimulates spermatogonial proliferation in germ-Sertoli cell co-cultures from immature rat. Endocrinology, 127: 3206-3214. Matzuk, M.M.. Kumar, T.R. and Bradley, A., 1995a. Different phenotypes for mice deficient in either activins or activin receptor type II. Nature, 374: 356-360. Matzuk, M.M.. Lu, N., Vogel, H., Sellheyer, K., Roop, D.R. and Bradley, A., 1995b. Multiple defects and perinatal death in mice deficient in follistatin. Nature, 374: 360-363. Maudit, C., Chativin, M.A., de Pemtti, E., Morera, A.M. and Benalimed, M., 1991. Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells. Biol. Reprod., 45: 101-109. McCullagh. D.R., 1932 Dual endocrine activity of the testes. Science, 76: 19-20. Michel, U., Famworth, P. and Findlay, J.K., 1993. Follistatins-more than follicle-stimulating hormone suppressing proteins. Mol. Cell. Endocrinol., 91: 11-11. Nakamura, T.. Takio, K., Eto, Y.. Shibai, H., Titani, K. and Sugino, H., 1990. Activin-binding from rat ovary is follistatin. Science, 247: 836-838. Gda, S., Nishimatsu. S-I., Murakami, K. and Ueno, N., 1995. Molecular cloning and functional analysis of a new activin B subunit: a dorsal mesoderm-inducing activity in Xenopus. Robertson, M.. Foulds, L.M., Leversha, L., Morgan, F.J., Hearn, M.T.W., Burger, H.G., Wettenhall, R.E.H. and de Kretser, D.M., 1985. Isolation of inhibin from bovine follicular fluid. B&hem. Biophys. Res. Commun., 126: 220-226. Russell, L.D., de Francat, L.R., Hess, R. and Cooke, P.. 1995. Characteristics of mitotic cells in developing and adult testes with observations on cell lineages. Tissue Cell, 27: 105- 128. Shimonaka, M., Inouye, S., Shimasaki, S. and Ling, R., 1991. Follistatin binds to both activin and inhibin through common beta-subunit. Endocrinology, 128: 3313-3315.
G.P. Risbridger/Animal
Reproduction
Science 42 (1996) 343-349
349
Teerds, K.J., Veldhuizen-Tsoerkan, M.B., Rommerts, F.F.G., de Rooij, D.G. and Dorrington, J.H., 1994. Proliferation and differentiation of testicular interstitial cells: aspect of Leydig cell development in the (pre) pubertal and adult testis. In: G. Verhoeven and U.-F. Habenicht (Editors), Molecular and Cellular Endocrinology of the Testis. Springer, Berlin, pp. 37-65. Thomas, G.B., Davidson, E.J., Engehmrdt, H., Baird, D.T., McNeilly, AS. and Brooks, A.N., 1995. Expression of mRNA and immunocytochemical localization of inhibin o- and inhibin B,-subunits in the fetal sheep testis. J. Endocrinol., 145: 35-42. Tisdall, D.J., Hill, D.F., Petersen, G.B. and Fleming, J.S., 1992. Ovine follistatin: characterization of cDNA and expression in sheep ovary during the luteal phase of the oestrous cycle. J. Mol. Endocrinol., 8: 258-264. Vale, W. and Gonzalez-Manchon, C., 1989. Activin-A, inhibin and transforming growth factor-p modulate growth of two gonadal cell lines. Endocrinology, 125: 1666-1672. Vale, W., Rivier, J., Vaughan, J.. McClintock, R., Corriagan, A., Woo, W., Karr, D. and Spiess, J., 1986. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature, 321: 776-779. Van Dissel-Emiliani, F.M.F., Grootenhuis, A.J., de Jong, F.H. and de Rooij, D.G., 1989. Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters. Endocrinology, 125: 1898-1903. Vassalli, A., Matzuk. M.M., Gardner, H.A.R., Lee, K-F. and Jaenisch, R., 1994. Activin/inhibin BB subunit gene disruption leads to defects in eyelid development and female reproduction. Genes Dev., 8: 414427. Vaughan, J.M. and Vale, W.W., 1993. cr.,-Macroglobulin is a binding protein of inhibin and activin. Endocrinology, 132: 2038-2050. Wongprasartsuk, S., Jenkin, G., McFarlane, J.R., Goodman, M. and de Kretser, D.M., 1994. Inhibin and follistatin concentrations in fetal tissues and fluids during gestation in sheep: evidence for activin in amniotic fluid. J. Endocrinol., 141: 219-229. Zhu, Li-Ji, Cheng, C.Y., Phillips, D.M., Bardin, C.W., 1994. The immunohistochemical localization of a ,-macroglobulin in rat testes is consistent with its role in germ cell movement and spermiation. J. Androl., 15: 575-581.
Regulation of Leydig cell function by inhibins and activins Gail P. Risbridger
’
Mortash University, Institute of Reproduction and Development, Monash Medical Centre, Level 3, Block E, 246 Clayton Road, Clayton, Vie., Australia
Abstract The role of inhibin related proteins is not confined to the regulation of pituitary FSH. These proteins are now recognised as growth and differentiation factors and in the testis they regulate both epithelial and interstitial cell functions. In species such as rodents and pigs, inhibin and activin are considered to he paracrine regulators of steroidogenesis whereby they modulate the endocrine LH signal. Porcine and rodent Leydig cells have been shown in vitro to respond to exogenously added activin A. We have previously proposed that activins have a significant role in Leydig cell development and in the fetal testis and at puberty, i.e. activin (like TGF /3) holds Leydig cell growth in abeyance until differentiation or puberty. The roles of activins, inhibins and binding proteins for these ligands, such as follistatins, in the development of Leydig cells remains to he determined. Keywords: Inbibin; Activin; Leydig cells
1. Introduction In 1932 inhibin was described as the active principle in aqueous extracts of the testes which was able to prevent the appearance of castration cells in the male rat pituitary
(McCullagh, 1932). In 1985, the purification of inhibin, which suppressed pituitary follicle-stimulating hormone (FSH) but not luteinising hormone (LH), was achieved (Robertson et al., 1985); this was closely followed by the discovery of activins which stimulated, rather than suppressed, FSH (Ling et al., 1986; Vale et al., 1986). In the following decade the focus of the inhibin field developed from the concept that inhibins were simply reproductive endocrine hormones to a recognition of their roles as paracrine
’Tel.: (61-3) 9550 3571; fax: (61-3) 9550 3584; e-mail: [email protected]. 0378-4320/%/$15.00
0 1996 Elsevier Science B.V. All rights reserved.
PII SO378-4320(96)01490-X
344
G.P. Risbridger/Animol
Reproduction Science 42 (19%) 343-349
and autocrine growth factors. In the testis, inhibins and activins were shown to regulate seminiferous tubule functions, but they also have roles in the interstitial tissue and particularly the Leydig cells. The action of inhibin and related proteins on Leydig cells provides the focus of this report. The first inhibin protein was purified from bovine follicular fluid and was the 58 kDa form which was composed of two disulphide linked polypeptide chains of 44 kDa and 14 kDa; designated as the a and l3 subunits, respectively (Robertson et al., 1985). Two different p subunit forms of inhibin were described PA and Pa, which gave rise to two forms of inhibin, inhibin A and inhibin B (Ling et al., 1986; Vale et al., 1986). The two l3 subunits, encoded on separate genes, also combine to form three dimeric forms of activin, activin A (PAPA), activin B (&$a) and activin AB (pApa>. Activin A has been widely studied, but less is known about activin AB and B as the availability of these proteins is limited. More recently, a putative l3, subunit was cloned from human liver (Hotten et al., 1995) and &, subunit cDNA isolated from Xenopus liver (Oda et al., 1995). This raises the probability that other activin molecules exist and remain to be isolated. These ligands act through specific receptors and a large family of serine/threonine kinase receptors was revealed, through which activin, inhibin and TGFP related factors can signal. In addition, binding proteins for these ligands play an integral role in the regulation and mechanism of action of inhibins and activins. o,-Macroglobulin is a binding protein for many cytokines, including inhibin, activin and TGFP. It is one of the main proteins in serum and appears to be a major binding protein for inhibin/activin (Kntmmen et al., 1993). It is viewed as acting to prevent proteolytic degradation of activin/inhibins or to deliver these ligands to target tissues. Follistatins have no homology to the inhibin subunits but will bind strongly to activins and in doing so can suppress bioactivity, such as the release of FSH (Nakamura et al., 1990). Although inhibin binds follistatin (Shimonaka et al., 1991), the affinity is lower than that for activin, and therefore inhibin (and TGFP) do not compete for activin binding to follistatin. The consequences of the binding interaction between follistatin and activins are important if the affinity of activin for its receptors is similar to the affinity for follistatins. A single follistatin gene has been isolated which consists of five exons and a signal peptide (see review by Michel et al., 1993). As with the inhibin subunits, the amino acid sequences of follistatin are highly conserved and across rat, sheep, human and pig there is 97% homology (Tisdall et al., 1992).
2. Evidence that inhibin and activin regulate Leydig cells The gonads are the main source of inhibin and related proteins which contribute to the endocrine regulation of the reproductive system. However, local paracrine actions within the testis are predicated upon the presence of specific receptors for these ligands on Leydig cells (De Winter et al., 1992; Feng et al., 1993). The effects of inhibin and activin on Leydig cells have been described predominantly in terms of their role in the regulation of steroidogenic enzyme activities and cell proliferation.
G.P. Risbridger/Animal
Reproduction Science 42 (19%) 343-349
345
2.1. Steroidogenesis
The effects of inhibin/activin on steroidogenesis were studied in a number of types of Leydig cells. Dual, but opposing, actions of inhibin and activin A on steroidogenesis were first reported using interstitial cell cultures prepared from immature, 7-day-old animals or from adult hypophysectomised rats. The action of inhibin A was to increase LH-stimulated testosterone production further, whereas activin A inhibited production (Hseuh et al., 1987). Another study on highly purified adult rat Leydig cells also reported an inhibitory effect of activin on testosterone release, but in these cultures inhibin had no effect alone and only antagonised the stimulatory action of activin in vitro (Lin et al., 1989). A further variation in the action of inhibin related proteins was described on immature porcine Leydig cells (Maudit et al., 1991). Human chorionic gonadotropin (hCG) stimulated dihydroepiandrostenone (DHEA) accumulation was reduced in vitro, but the conversion of pregnenolone and DHEA was increased and no net effect on testosterone production was observed. Leydig cell tumour lines commonly synthesise progesterones rather than testosterone, and activin A inhibited progesterone synthesis by the R2C tumour cells; inhibin had no effect either alone or in combination with activin A and therefore did not antagonise the inhibitory effect of activin (Vale and Gonzalez-Manchon, 1989). Overall, the exact details of the effects of inhibin and activin on steroidogenesis in vitro differ, and could be explained by the differences between the culture conditions used or by the species or type of Leydig cell cultured. Further potential for the action of activin A on Leydig cell steroidogenesis is suggested by studies describing the stimulation of So-reductase activity in human genital skin fibroblasts (Antonapillar et al., 1995). An increase in the activity of this enzyme characterises the development of immature forms of Leydig cells and the production of reduced androgens; could this involve an action of activin A? 2.2. DNA synthesis Activin is a member of the TGFP family of proteins and these proteins have proliferative and antiproliferative effects on other cell types, both within the testis and in other tissues (Hedger et al., 1989). In the normal testis, most attention has focused on the role of activin and inhibin in the regulation of spermatogonial cell proliferation (Van Dissel-Emiliani et al., 1989; Mather et al,, 1990), yet during the development of Leydig cell populations, there are periods of intense proliferation (Hardy et al., 1989; Russell et al., 1995). Although it is not known if inhibins or activins can regulate Leydig cell proliferation, TGFP inhibits DNA synthesis by immature Leydig cells under LH stimulation (Kahn et al., 1992). These authors suggested that TGFP regulates the growth of immature Leydig cells and restricts their growth until puberty (Teerds et al., 1994). It is therefore possible that activin has the same, or a similar action, on Leydig cells. This has been tested on the rat Leydig turnour cell line, R2C. Minor inhibitory effects of activin A on DNA synthesis were reported but a prolonged period of 4 days of culture was required to observe such an effect (Vale and Gonzalez-Manchon, 1989). TGFP also
346
G.P. Risbridger/Animal Reproduction Science 42 (1996) 343-349
inhibited proliferation of the R2C tumour cells, and was more potent in its action than activin A.
3. Discussion arising from the observed actions of inhibin and activins on Leydig cells These data clearly demonstrate that in vitro activin/inhibin regulate Leydig cell steroidogenesis and DNA synthesis, but a number of questions are posed by this information. First, the reason for Leydig cell DNA synthesis and steroidogenesis to be regulated by both activin/inhibin and TGFP is not known. In general, the action of TGFP on Leydig cell steroidogenesis is more potent than that of activin A (Lin et al., 1987; Lin et al., 19891, and in R2C tumour cells TGFP is more potent in terms of the effects on DNA synthesis and steroidogenesis (Vale and Gonzalez-Manchon, 1989). The growth factors may act through separate pathways, and the relative concentrations of the two hormones may be important; or they may act through the same pathways via the same receptors as promiscuity of receptor systems does occur in vitro. Secondly, it cannot be assumed that the actions of inhibin B and activin B or activin AB are the same as those that have been reported for inhibin A or activin A. The results of PA and pa inhibin subunit gene disruption studies suggest that the functions of activin A and B do not overlap during embryonic development (Vassalli et al., 1994; Matzuk et al., 1995a; Matzuk et al., 1995b). Therefore, the effects of the specific activin/inhibin on the regulation of Leydig cell functions may differ; but this has to be tested and determined. Thirdly, although activin A antagonised the action of inhibin A on steroidogenesis by immature and adult rat interstitial cells (Hseuh et al., 1987; Lin et al., 19891, this was not the case for all effects on Leydig cell steroidogenesis or on DNA synthesis. The effects of activin were observed in the absence of any effect of inhibin alone or in combination with activin (Vale and Gonzalez-Manchon, 1989). Furthermore, the ability of binding proteins, e.g. follistatins, to modify some of the biological actions of activin needs to be considered. Immunostaining identified follistatin binding protein to Leydig cells (Kaipia et al., 19921, and suggests a significant role for follistatins in regulating the effect of activin on Leydig cells. If follistatin binds and neutralises the bioactivity of activins on Leydig cells, then changes to the relative levels of follistatin could provide another means of regulating activin. action, other than through changes to the levels of the receptor or activin ligands themselves. The regulatory role of follistatins alone or in combination with activin A in the regulation of steroidogenesis, or cell proliferation, has not been explored. CX~,-macroglobulinis also a binding protein for inhibin and activin (Vaughan and Vale, 19931, and the interplay between follistatin and a,-macroglobulin in the binding of inhibin related proteins is not known. In the testis, a,-macroglobulin content increases with age (Zhu et al., 1994), but the relative levels of follistatin and OL ,-macroglobulin and the distribution of the complexes with inhibins and activins in the testis remains to be recorded. Fourthly, and most importantly, there is a wide variation in the effect of inhibins and
G.P. Risbridger/Animal
Reproduction
Science 42 (1996) 343-349
347
activins as described herein, which may be due to the type and maturity of the Leydig cells that were studied. There is more than one population of Leydig cells and there are multiple stages in the development of Leydig cells from stem cell precursors to immature and mature forms. The two populations of Leydig cells (e.g. foetal and adult) respond differently to control by LH (Huhtaniemi, 19941, and it would not be surprising if there are variations in the response to regulation by inhibins/activins or indeed any other paracrine factors. The actions of inhibin/activins at particular phases of the development of Leydig cell populations has not been undertaken in a systematic manner. Differences in culture conditions and the species of Leydig cells (rat or pig) that were cultured, are other reasons why variations in the effects of inhibins and activins are observed. The predominant effect of activin A on steroidogenesis by immature rodent Leydig cells, measured as testosterone production, was inhibitory. In contrast, there was no net effect on immature porcine Leydig cell testosterone production (Maudit et al., 1991). Therefore, there appears to be a species-specific difference in the response to the ligand. Nothing is known about the regulation of ovine Leydig cell function by inhibin/activin but the ovine testis during foetal development represents an excellent model for the study of inhibins and activins in the regulation of Leydig cell development and differentiation in an alternate species. In the sheep foetus, testosterone is present 5 days before the onset of sexual differentiation (Anal, 1969). This dissociation between testicular morphogenesis and functional differentiation of the Leydig cells has been reported in other species such as lamb, rat and human. The onset of Leydig cell differentiation cannot be due to pituitary LH alone, as gonadal development occurs in the anencephalic foetus. The role of growth factors (such as inhibins and activins) is therefore worthy of examination. Although Thomas et al. (1995) have reported the local&ion of (Ybut not p inhibin subunits, in the ovine foetal testis, we have recently localised activin immunoreactivity to foetal Leydig cells in mid gestation (G.R. Risbridger, unpublished observations, 1996). Together with the detection of follistatin immunoreactivity in the ovine foetal testis (Wongprasartsuk et al., 1994), the role of these ligands and binding proteins in the development and regulation of foetal Leydig cell function is currently under investigation.
4. Summary This brief review demonstrates that inhibin and activin are paracrine regulators of Leydig cells. However, there are a number of questions that arise from the observations which demonstrate the complexity of the action of these growth factors in their role in growth and differentiation of cells. The questions raised specifically in this review are illustrated with the example of Leydig cells, but are not specific to these cells or tissue; indeed, the questions raised are relevant to any study of inhibins and activins. References Antonapillar, I., Wahe, M., Yamamoto, J. and Horton, R., 1995. Activin aad inhibii have opposite effects on steroid. Sa-rcductase activity in genital skin fibroblasts. Mol. Cell. Endocrinol., 107: 99- 104.
348
G.P. Risbridger / Animal Reproduction Science 42 (19%) 343-349
Attal, J., 1%9. Levels of testosterone, androstenedione, estrone and estradiol-17s in the testes of fetal sheep. Endocrinology, 85: 280-289. De Winter, J.P., Themman, A.P.N., Hoogerbrugge, J.W., Klaij, LA., Grootegoed, J.A. and de Jong, F.H., 1992. Activin receptor mRNA expression in rat testicular cell types. Mol. Cell. Endocrinol., 83: Rl-R8. Feng, A-M., Madigan, M.B. and Chen, C-LC., 1993. Expression of type II activin receptor genes in the male and female reproductive tissues of the rat. Endocrinology, 132: 2593-2600. Hardy, M.R., Zirkin, B.R. and Ewing, L.L., 1989. Kinetic studies on the development of the adult population of Leydig cells in testis of the pubertal rat. Endocrinology, 124: 762-770. Hedger, M.R., Drummond, A.E., Robertson, D.M., Risbridger, G.R. and de Kretser, D.M., 1989. Divergent actions of inhibin, activin and transforming growth factor B on [3H] thymidine incorporation by rat thymocytes and 3T3 fibroblasts in vitro. Mol. Cell. Endocrinol., 61: 133-138. Hotten, G., Neidhatdt, R., Schneider, C. and Pohl, J., 1995. Cloning of a new member of the TGF-B family:-a putative new activin B, chain. Biochem. Biophys. Res. Commun., 206(2): 608-613. Hseuh, A.J.W., Dahl, K.D., Vaughan, J., Tucker, E., Rivier, J., Bardm, C.W. and Vale, W.W., 1987. Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of lutemizing hormone-stimulated androgen biosynthesis. Proc. Natl. Acad. Sci. USA, 84: 5082. Huhtaniemi, I., 1994. Fetal testis-a very special endocrine organ. Eur. J. Endocrinol., 130: 25-31. Kahn, S., Teems, K. and Donington, J., 1992. Growth factor requirements for DNA synthesis by Leydig cells from the immature rat. Biol. Reprod., 46 335-341. Kaipia, A., Penttila, T.L., Shimaski, S., Ling, N., Parvinen, M. and Toppari, J., 1992. Expression of :mhibm BA and Ba, follistatin and activin-A receptor messenger ribonucleic acids in the rat seminiferous epithelium. Endocrinology, 131: 2703-2710. Kmmmen. L.A., Woodruff, T.K., DeGuzman, G., Cox, E.T., Baly, D.L., Mann, E., Garg, S., Wong, W-L, Cossum, P.. Mather. J.P., 1993. Identification, and characterization of binding proteins for inhibin and a&in in human strum and follicular fluids. Endocrinology, 132: 431-443. Lm, T., Blaisdell, J. and Haskell, J.F., 1987. Transforming growth factor-B inhibits Leydig cell steroidogenesis in primary culture. Biochem. Biophys. Res. Commun.. 146: 387-394. Lin, T.. CaIkms, H., Morris, P.L., Vale, W.W. and Bardin, C.W., 1989. Regulation of Leydig cell function in primary culture by inhibin and activin. Endocrinology, 125: 2134-2140. Ling, N., Yng, S-Y, Ueno, N., Shimasaki, S., Esch, R., Hotta, M. and Guillemin, R., 1986. Pituitary FSH is released by the heterodimer of the B-subunits from the two forms of inhibin. Nature, 321: 779-782. Mather, J.R., Attie, K.M., Woodruff, T., Rice, G.C. and Phillips, D.M., 1990. Activin stimulates spermatogonial proliferation in germ-Sertoli cell co-cultures from immature rat. Endocrinology, 127: 3206-3214. Matzuk, M.M.. Kumar, T.R. and Bradley, A., 1995a. Different phenotypes for mice deficient in either activins or activin receptor type II. Nature, 374: 356-360. Matzuk, M.M.. Lu, N., Vogel, H., Sellheyer, K., Roop, D.R. and Bradley, A., 1995b. Multiple defects and perinatal death in mice deficient in follistatin. Nature, 374: 360-363. Maudit, C., Chativin, M.A., de Pemtti, E., Morera, A.M. and Benalimed, M., 1991. Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells. Biol. Reprod., 45: 101-109. McCullagh. D.R., 1932 Dual endocrine activity of the testes. Science, 76: 19-20. Michel, U., Famworth, P. and Findlay, J.K., 1993. Follistatins-more than follicle-stimulating hormone suppressing proteins. Mol. Cell. Endocrinol., 91: 11-11. Nakamura, T.. Takio, K., Eto, Y.. Shibai, H., Titani, K. and Sugino, H., 1990. Activin-binding from rat ovary is follistatin. Science, 247: 836-838. Gda, S., Nishimatsu. S-I., Murakami, K. and Ueno, N., 1995. Molecular cloning and functional analysis of a new activin B subunit: a dorsal mesoderm-inducing activity in Xenopus. Robertson, M.. Foulds, L.M., Leversha, L., Morgan, F.J., Hearn, M.T.W., Burger, H.G., Wettenhall, R.E.H. and de Kretser, D.M., 1985. Isolation of inhibin from bovine follicular fluid. B&hem. Biophys. Res. Commun., 126: 220-226. Russell, L.D., de Francat, L.R., Hess, R. and Cooke, P.. 1995. Characteristics of mitotic cells in developing and adult testes with observations on cell lineages. Tissue Cell, 27: 105- 128. Shimonaka, M., Inouye, S., Shimasaki, S. and Ling, R., 1991. Follistatin binds to both activin and inhibin through common beta-subunit. Endocrinology, 128: 3313-3315.
G.P. Risbridger/Animal
Reproduction
Science 42 (1996) 343-349
349
Teerds, K.J., Veldhuizen-Tsoerkan, M.B., Rommerts, F.F.G., de Rooij, D.G. and Dorrington, J.H., 1994. Proliferation and differentiation of testicular interstitial cells: aspect of Leydig cell development in the (pre) pubertal and adult testis. In: G. Verhoeven and U.-F. Habenicht (Editors), Molecular and Cellular Endocrinology of the Testis. Springer, Berlin, pp. 37-65. Thomas, G.B., Davidson, E.J., Engehmrdt, H., Baird, D.T., McNeilly, AS. and Brooks, A.N., 1995. Expression of mRNA and immunocytochemical localization of inhibin o- and inhibin B,-subunits in the fetal sheep testis. J. Endocrinol., 145: 35-42. Tisdall, D.J., Hill, D.F., Petersen, G.B. and Fleming, J.S., 1992. Ovine follistatin: characterization of cDNA and expression in sheep ovary during the luteal phase of the oestrous cycle. J. Mol. Endocrinol., 8: 258-264. Vale, W. and Gonzalez-Manchon, C., 1989. Activin-A, inhibin and transforming growth factor-p modulate growth of two gonadal cell lines. Endocrinology, 125: 1666-1672. Vale, W., Rivier, J., Vaughan, J.. McClintock, R., Corriagan, A., Woo, W., Karr, D. and Spiess, J., 1986. Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature, 321: 776-779. Van Dissel-Emiliani, F.M.F., Grootenhuis, A.J., de Jong, F.H. and de Rooij, D.G., 1989. Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters. Endocrinology, 125: 1898-1903. Vassalli, A., Matzuk. M.M., Gardner, H.A.R., Lee, K-F. and Jaenisch, R., 1994. Activin/inhibin BB subunit gene disruption leads to defects in eyelid development and female reproduction. Genes Dev., 8: 414427. Vaughan, J.M. and Vale, W.W., 1993. cr.,-Macroglobulin is a binding protein of inhibin and activin. Endocrinology, 132: 2038-2050. Wongprasartsuk, S., Jenkin, G., McFarlane, J.R., Goodman, M. and de Kretser, D.M., 1994. Inhibin and follistatin concentrations in fetal tissues and fluids during gestation in sheep: evidence for activin in amniotic fluid. J. Endocrinol., 141: 219-229. Zhu, Li-Ji, Cheng, C.Y., Phillips, D.M., Bardin, C.W., 1994. The immunohistochemical localization of a ,-macroglobulin in rat testes is consistent with its role in germ cell movement and spermiation. J. Androl., 15: 575-581.