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Inhibins, activins and follistatin: actions on the testis
Molecular and Cellular Endocrinology 180 (2001) 87 – 92 www.elsevier.com/locate/mce
Inhibins, activins and follistatin: actions on the testis D.M. de Kretser *, K.L. Loveland, T. Meehan, M.K. O’Bryan, D.J. Phillips, N.G. Wreford Monash Institute of Reproduction and De6elopment and Department of Anatomy and Cell Biology, Monash Uni6ersity, Monash Medical Centre, 246 Clayton Road, Victoria 3168, Australia Received 12 February 2001; accepted 18 March 2001
Abstract While the early studies of the inhibins, activins and follistatins concentrated on their role as endocrine regulators of FSH secretion, recent data has emphasized the local actions of the activins and follistatin. Inhibin, through its capacity to suppress FSH secretion can modulate numerous processes within the testis. However, to date, evidence to support a local role for inhibin is limited. In contrast, activin and its binding protein follistatin are produced by a large number of cell-types within the testis raising the possibility of a range of paracrine and autocrine actions. These include the modulation of androgen production, influence on the proliferation of Sertoli cells and germ cells as well as the capacity to influence the structural and functional features of mitochondria within germ cells. Some of these actions are carefully controlled in a temporal relationship during the development of testicular function in the rat in which there is no separation in time between birth and the onset of spermatogenesis. Given the range of actions of activin in different cell-types, recognition of systems that are designed to modulate its actions are crucial in enhancing our understanding of how these many roles can be compartmentalized. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Activin; Follistatin; Inhibin; Testis; Sertoli cell; Spermatogenesis
1. Introduction The inhibins, activins and follistatin were isolated on the basis of their ability to modify the secretion of FSH by the pituitary (Ling et al., 1985; Robertson et al., 1985; Ling et al., 1986; Vale et al., 1986; Robertson et al., 1987; Ueno et al., 1987). These actions were initially presumed to result from the endocrine action of these proteins produced at peripheral sites. While this has been shown for inhibin, where following castration, the circulating levels of this hormone decline to almost undectable levels, the same conclusions cannot be reached from the data concerning activin and follistatin (Robertson et al., 1988; Ishida et al., 1990). Following castration, activin A levels in adult male rats did not change over a 7-day period, indicating that sources * Corresponding author. E-mail address: [email protected] (D.M. de Kretser).
other than the testis contributed to circulating levels (McFarlane et al., 1996). Similarly, when rams were castrated, follistatin levels actually increased, a phenomenon which has subsequently been shown to result from the cytokine stimulation of follistatin as part of the acute phase response (Phillips et al., 1996). Numerous studies have shown that, apart from the action of inhibin on FSH levels, the modulation of FSH by activin and follistatin results from paracrine or autocrine actions at the pituitary (Bilezikjian et al., 1993, 1996). The bB and bA subunits are produced by the pituitary gland and act locally to exert a stimulatory action on FSH secretion which is negatively modulated by locally produced follistatin through its ability to bind and neutralize the action of the activins (Nakamura et al., 1990). Since FSH exerts numerous effects on the testis, the modulation of its secretion by inhibin, activin and follistatin can influence testicular function. This review however, is focused on the testicular production of these proteins and their local effects.
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2. Tissue and cellular distribution The distribution of the bA and bB subunits and follistatin at many tissue sites has raised the possibility that these substances exert paracrine and autocrine actions at those sites (Roberts et al., 1989; Michel et al., 1990). While the testis is not a major contributor to circulating levels of activin A and follistatin, both mRNA and protein for follistatin and the activin subunits are present in the testis indicating the capacity for local production (Roberts et al., 1989; Michel et al., 1990; Andersson et al., 1998). To fully understand the possible testicular actions of the activins and inhibins it is essential to recognize the sites of production. There is general agreement that inhibin B is the major protein regulator of FSH produced by the testis, largely secreted by the Sertoli cells (Anawalt et al., 1996). There is a positive relationship between inhibin B levels and Sertoli cell number in normal and pathophysiological states in vivo, supporting the concept that the Sertoli cell is the principal site of inhibin B production (Ramaswamy et al., 1999; Sharpe et al., 1999). These studies support earlier reports which established that the immature Sertoli cell in culture is the site of inhibin secretion and showed that FSH could stimulate its production (Le Gac and de Kretser, 1982). However there is also evidence that the Leydig cells have the capacity to produce bioactive inhibin (McLachlan et al., 1988; Risbridger et al., 1989). Recent studies have suggested that the Leydig cells can modulate the expression of the a subunit gene in the seminiferous tubules (Tena-Sempere et al., 1999). Data concerning the production of activin A and B are sparse due to the limited availability of specific assays. Consequently our understanding of the sites of production in the testis is limited to the mRNA and immunocytochemical localization of the bA and bB subunits. The bA and bB subunits are present in the Sertoli cells (Toboesch et al., 1988) and there is bioassay data and our unpublished data using a specific ELISA to indicate that activin A is produced by the Sertoli cells (Grootenhuis et al., 1989; de Winter et al., 1993). In addition, there is data which supports the view that the Leydig cells and the peritubular cells can secrete activin A (Lee et al., 1989; de Winter et al., 1994). Surprisingly immunocytochemical data indicate that the spermatogonia, primary spermatocytes and round spermatids in the human testis are potential sites of activin B production (Andersson et al., 1998). Our unpublished data show that in the rat testis, the identical stages of spermatogenesis have the capacity to produce activin B since they contain bB subunit mRNA and immunocytochemically detected protein. To define the site of paracrine and autocrine actions it is essential to know which cell types contain receptors for the activins. Several studies have shown that the
type I and type II activin receptors are present in Sertoli cells, primary spermatocytes and round spermatids (de Winter et al., 1992; Kaipia et al., 1993). Additionally, other investigators showed that iodinated activin A bound to primary spermatocytes and round spermatids (Krummen et al., 1994). These cells therefore represent sites at which the activins may exert their actions. Modulation of these actions can occur through the actions of follistatin which has been shown to be produced by the Sertoli cells, spermatogonia, primary spermatocytes and round spermatids (Michel et al., 1990; Kogawa et al., 1991; Meinhardt et al., 1998). It is important to recognize that in addition to cellular production, the sites at which follistatin is detected on cell surfaces may represent the binding and tissue storage of follistatin 288 which has a strong affinity to heparin sulphate proteoglycans (Sugino et al., 1993). Additionally since activin binds strongly to follistatin, the binding to proteoglycans represent tissue stores of activin. Evidence that this type of binding results in the accumulation of significant amounts of follistatin and activin, can be seen by the release of both proteins following an intravenous injection of heparin (Phillips et al., 2000). The amounts released can substantially raise the circulating levels of these proteins in men and women (Phillips et al., 2000). A new kinase deficient type 1 receptor has been identified in Xenopus (BAMBI) which can inhibit the actions of the activins, the bone morphogenetic proteins and TGFb. This is yet another protein with the capacity to modulate the actions of the activins (Onichtchouk et al., 1999). Using a cDNA probe complimentary to the human orthologue, hnma, the mRNA for this protein has been identified in the adult rat, mouse and human testis in spermatogonia, primary spermatocytes, round and elongating spermatids and in Sertoli cells, all sites that have the capacity to produce activin (Loveland et al., unpublished data). Further studies of the role of this protein are awaited with interest.
3. Actions on the testis
3.1. Physiological actions emerging from genetic manipulation Targeted disruption of the inhibin a subunit in mice resulted in the development of testicular tumours which became apparent around 21–28 days of age and resulted in death (Matzuk et al., 1992). These tumours probably arise from Sertoli cells but have the appearance of granulosa cells. The mice become severely cachetic, an action that has been shown to result from the high levels of activin that are present in these mice (Matzuk et al., 1994; Coerver et al., 1996). These studies also confirmed that the tumourigenic action
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resulted specifically from the absence of a functional a subunit gene, confirming the proposal that the a subunit is a tumour suppressor (Matzuk et al., 1992). Since the tumours in these mice disrupt the architecture of the testis, it has not been possible to identify any specific defects in spermatogenesis resulting from the targeted disruption. Targeted disruption of the bA subunit gene resulted in perinatal death of mice due to palatal developmental defects that prevented successful suckling (Matzuk et al., 1995a). Consequently, analysis of the local actions within the testis has not been possible. In contrast, the functional disruption of the bB subunit gene showed no obvious disturbance of spermatogenesis (Vassalli et al., 1994). It is possible that the absence of an effect on spermatogenesis resulted from functional compensation by the bA subunit. In an attempt to interrupt the action of the activins, Matzuk and colleagues disrupted the function of the activin type IIA gene and showed that the mice were fertile despite the presence of testes that were smaller than normal (Matzuk et al., 1995b). Since the FSH levels in these mice were markedly decreased, the smaller testicular size was attributed to decreased Sertoli cell numbers resulting from the inadequate stimulation of proliferation in the fetal and neonatal period during which these cells have the capacity to divide. Quantitative studies of the testes of these mice have confirmed that the Sertoli cells are decreased by 30% leading to a corresponding decline in sperm production, a finding comparable to the decrease in the number of these cells in mice with targeted disruption of the FSH b subunit gene (Wreford et al., 2001). It was not possible to specifically delineate a defect that could be attributed to the absence of activin action. Despite the failure to demonstrate a specific defect by the approach using targeted disruption of the relevant genes, over-expression of the bA subunit gene in mice resulted in spermatogenic disruption (Tanimoto et al., 1999). In this case, the promoter led to bA synthesis in spermatocytes. The pattern of disruption varied with the level of expression in different lines. Profiles of vacuolated tubules suggested the total absence of germ cells but in other areas, spermatids were more severely depleted than spermatogonia and primary spermatocytes. Over expression of the follistatin gene also resulted in infertility and spermatogenic defects that were related to the degree of follistatin over-expression (Guo et al., 1998). In mice with the highest level of expression, testis weights were decreased and spermatogenesis was disrupted and some tubules in some mice showed only Sertoli cells. These observations suggest that the excess follistatin can impair the local actions of the activins and the spermatogenic defect may result from this action. Unfortunately, targeted disruption of the follistatin gene resulted in perinatal death due to respiratory difficulties and hence it has not been possible
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to evaluate the effects on the adult testis (Matzuk et al., 1995c).
3.2. Actions on androgen production In vitro incubation of Leydig cells from 21-day old rats with inhibin stimulated testosterone production but activin inhibited this production (Hsueh et al., 1987). However Risbridger and colleagues were unable to confirm the observation that inhibin stimulated testosterone secretion using highly purified Leydig cells from adult rats raising the possibility that this function may be age specific (Risbridger et al., 1989). Other studies have also failed to confirm the action of inhibin on testosterone secretion but have been able to show the inhibition by activin A in vitro (Lin et al., 1989). The in vivo physiological significance of these variable results remains unknown but no significant changes have been noted in testosterone levels in mice with targeted disruption of the inhibin/activin subunit genes.
3.3. Action on the maintenance of mitochondrial morphology in primary spermatocytes Our recent studies have identified the capacity for activin A to specifically maintain the ‘condensed’ mitochondrial morphology found in germ cells beyond the leptotene stage of the first meiotic prophase (Meinhardt et al., 2000). The ‘condensed’ mitochondrial appearance results from the dilatation of the intra-cristal spaces and the peripheral margination of the cristae leaving a central space within the mitochondrion. This action of activin A is dose-dependent and can be inhibited by an antiserum specific to activin A. These morphological changes reflect functional changes such as the loss of heat shock protein 60 (HSP-60) which is thought to act in concert with hsp 10 as a chaperone to fold newly formed mitochondrial proteins (Meinhardt et al., 1995; Paranko et al., 1996). Further, localization of the Lonprotease in mitochondria was lost after the zygotene stage raising the possibility of limited proteolytic degradation of mitochondrial proteins in the early meiotic stages but not thereafter (Seitz et al., 1995). Since there is now evidence that the mRNA for the bB subunit and its protein can be found in primary spermatocytes which also contain specific activin type I and II receptors, the action on mitochondria represents an example of an autocrine role. As the morphological changes in the mitochondria have functional correlates, the action of activin A is likely to result in functional changes, the nature of which must remain speculative. Given that activin can influence the occurrence of apoptosis in other cell types, it is possible that the mitochondrial changes may represent an action related to apoptosis.
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3.4. Modulation of spermatogonial proliferation Although the studies are limited in number, there is data to suggest that inhibin can inhibit spermatogonial proliferation (Van Dissel-Emiliani et al., 1989). Unfortunately the study was performed using partially purified native inhibin from follicular fluid and did not rule out possible contamination of the preparation with activin and follistatin. In one study with purified activin A, spermatogonial proliferation was stimulated when this protein was added to co-cultures of Sertoli cells and spermatogonia (Mather et al., 1990) suggesting that inhibin and activin may have opposing effects on spermatogonial proliferation. In contrast, in a study using fragment cultures of day 9 testes, activin A inhibited the FSH-stimulated proliferation of differentiated spermatogonia (Boitani et al., 1995).
3.5. Action during the de6elopment of the rat testis There is increasing evidence that the actions of activin and follistatin may exert discrete, age-specific actions during the early post-natal development of the rat testis. This concept arises from several lines of evidence. First, the localization of activin A protein and bA subunit mRNA appear in gonocytes but are not present in early spermatogonia (Meehan et al., 2000). In contrast, follistatin is absent in gonocytes at birth, and appears around day 3 persisting in spermatogonia. Localization of activin A to fetal gonocytes has also been found in the sheep testis (Jarred et al., 1999). In keeping with this changing localization, Meehan et al. (2000) showed that in fragment cultures of the rat testis, activin A stimulated gonocyte numbers and did not alter spermatogonial numbers. In contrast, follistatin in combination with FSH, stimulated spermatogonial numbers. Additionally, activin A inhibited proliferation of Sertoli cells in fragment cultures from day 3 rat testes. Further evidence of an age specificity emerged from the results of Boitani et al. (1995) who showed that in fragment cultures from day 3 rat testes, activin A had no effect on Sertoli cell proliferation, with or without FSH. However, at day 9, activin A and FSH in combination, but not separately, stimulated Sertoli cell proliferation. These data highlight the changing responsiveness of the testis to activin which characterizes the onset of spermatogenesis. It is possible that activin and follistatin may play an important role in the transformation of gonocytes to spermatogonia, perhaps acting in concert with other growth factors such as leukaemia inhibitory factor, transforming growth factor b, oncostatin M and fibroblast growth factor, all of which are known to exert effects on early testicular development (de Miguel et al., 1996; Van Dissel-Emiliani et al., 1996; de Miguel et al., 1997; Richards et al., 1999). Further studies of the
testes in knock-out models of the genes encoding the activin subunits and follistatin may shed further information of the physiological relevance of these in vitro effects. Since the events in the immediate post-natal development of the seminiferous epithelieum are temporally precise, the controlled expression of activin and follistatin may be crucial control mechanisms which determine the onset of the spermatogenic process.
3.6. Importance of factors controlling the actions of acti6in This brief review of the actions of activins in the testis emphasizes the importance of control systems that have the capacity to modulate the action of these proteins, particularly to localize actions to a particular cell type. There are a number of mechanisms that can exert this modulation. First, the relative expression of the a and b subunits can alter the amounts of activin produced. Increasing a-subunit expression drives the Sertoli cell to produce inhibin and free a-subunit rather than activin (Hancock et al., 1992). Secondly, the production of follistatin within the same or adjacent cells can neutralize the action of activin although it failed to inhibit the activin-stimulated proliferation of germ cellSertoli cell cultures (Mather et al., 1990). Thirdly, the level and identity of the type I and type II activin receptors will influence the ability of activin to exert its actions. Fourth, the expression of heparin sulphate proteoglycans on the surface of cells can, through the intermediary of follistatin, assist the degradation of activin through lysosomal mechanisms (Hashimoto et al., 1997). Alternatively, the binding of activins, through follistatin to heparin sulphate proteoglycans, can result in a tissue store of activin which could be released through the action of heparin released by heparinases. Finally, both activin and follistatin can bind to the fast form of a2 macroglobulin, an action that may modulate the degradation of these proteins (Niemuller et al., 1995; Phillips et al., 1997). Such a mechanism may be important since a2 macroglobulin is produced by the Sertoli cells in the testis (Cheng et al., 1990). In conclusion, while inhibin probably exerts its action on the testis through a long loop system involving the modulation of FSH secretion, the effects of activin and follistatin are exerted through the local production of these proteins. Considerable further work is required to define these local actions and to define their physiological roles.
Acknowledgements Many of these studies were supported by a Program Grant from the National Health and Medical Research
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Council of Australia. The excellent research assistance of Anne O’Connor, Lynda Foulds and Elizabeth Christy is gratefully acknowledged.
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McFarlane, J.R., Foulds, L.M., Pisciotta, A., Robertson, D.M., de Kretser, D.M., 1996. Measurement of activin in biological fluids by radioimmunoassay, utilizing dissociating agents to remove the interference of follistatin. Eur. J. Endocrinol. 134, 481 – 489. McLachlan, R.I., Matsumoto, A.M., de Kretser, D.M., Burger, H.G., Bremner, W.J., 1988. The relative roles of follicle stimulating hormone and luteinizing hormone in the control of inhibin secretion in normal men. J. Clin. Invest. 82, 1 –5. Meehan, T., Schlatt, S., O’Bryan, M.K., de Kretser, D.M., Loveland, K.L., 2000. Regulation of germ cell and Sertoli cell development by activin, follistatin and FSH. Developmental Biol. 220, 225 – 257. Meinhardt, A., Parvinen, M., Bacher, M., Aumu¨ ller, G., Hakovirta, H., Yagi, A., Seitz, J., 1995. Expression of the mitochondrial heat shock protein 60 in distinct cell types and defined stages of rat seminiferous epithelium. Biol. Reprod. 52, 798 –807. Meinhardt, A., O’Bryan, M.K., McFarlane, J.R., Loveland, K.L., Mallidis, C., Foulds, L.M., Phillips, D.J., de Kretser, D.M., 1998. Localization of follistatin in the testis. J. Reprod. Fertil. 112, 233 – 241. Meinhardt, A., McFarlane, J.R., Seitz, J., de Kretser, D.M., 2000. Activin maintains the condensed type of mitochondria in germ cells. Mol. Cell. Endocrinol. 186, 111 – 117. Michel, U., Albiston, A., Findlay, J.K., 1990. Rat follistatin: gonadal and extragonadal expression and evidence for alternative splicing. Biochem. Biophys. Res. Commun. 173, 401 –407. Nakamura, T., Takio, K., Eto, Y., Shibai, K., Sugino, H., 1990. Activin-binding protein from rat ovary is follistatin. Science 247, 836 – 838. Niemuller, C.A., Randall, K.J., Webb, D.J., Gonias, S.L., Lamarre, J., 1995. a2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A a2macroglobulin complex. Endocrinology 136, 5343 –5349. Onichtchouk, D., Chen, Y.G., Dosch, R., Gawantka, V., Delius, H., Massague´ , J., Niehrs, C., 1999. Silencing of TGF-b signaling by the pseudoreceptor BAMBI. Nature 401, 480 –485. Paranko, J., Seitz, J., Meinhardt, A., 1996. Developmental expression of heat shock protein 60 (hsp60) in the rat testis and ovary. Differentiation 60, 159 – 167. Phillips, D.J., Hedger, M.P., McFarlane, J.R., Klein, R., Clarke, I.J., Tilbrook, A., Nash, A.D., de Kretser, D.M., 1996. Follistatin concentrations in male sheep increase following sham castration/ castration or injection of interleukin-1b. J. Endocrinol. 151, 119 – 124. Phillips, D.J., McFarlane, J.R., Hearn, M.T.W., de Kretser, D.M., 1997. Inhibin, activin and follistatin bind preferentially to the transformed species of a2-macroglobulin. J. Endocrinol. 155, 65 – 71. Phillips, D.J., Jones, K.L., McGaw, D.J., Groome, N.P., Smolich, J.J., Pa¨ rsson, H., de Kretser, D.M., 2000. Release of activin and follistatin during cardiovascular procedures is largely due to heparin adminstration. J. Clin. Endocrinol. Metab. 85, 2411 –2415. Ramaswamy, S., Marshall, G.R., McNeilly, A.S., Plant, T.M., 1999. Evidence that in a physiological setting Sertoli cell number is the major determinant of circulating concentrations of inhibin B in the adult male rhesus monkey (Macaca mulatta). J. Androl. 20, 430 –434. Richards, A.J., Enders, G.C., Resnick, J.L., 1999. Activin and TGFb limit murine primordial germ cell proliferation. Dev. Biol. 207, 470 –475. Risbridger, G.P., Clements, J., Robertson, D.M., Drummond, A.E., Muir, J., Burger, H.G., de Kretser, D.M., 1989. Immuno and bioactive inhibin and inhibin a subunit expression in rat Leydig cell cultures. Mol. Cell. Endocrinol. 66, 119 –122. Roberts, V., Meunier, H., Sawchenko, P.E., Vale, W., 1989. Differential production and regulation of inhibin subunits in rat testicular cell types. Endocrinology 125, 2350 –2359. .
Robertson, D.M., Foulds, L.M., Leversha, L., Morgan, F.J., Hearn, M.T.W., Burger, H.G., Wettenhall, R.E.H., de Kretser, D.M., 1985. Isolation of inhibin from bovine follicular fluid. Biochem. Biophys. Res. Commun. 126, 220 – 226. Robertson, D.M., Klein, R., de Vos, F.L., McLachlan, R.I., Wettenhall, R.E.H., Hearn, M.T.W., Burger, H.G., de Kretser, D.M., 1987. The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin. Biochem. Biophys. Res. Comm. 149, 744 – 749. Robertson, D.M., Hayward, S., Irby, D.C., Jacobsen, J.V., Clarke, L.J., McLachlan, R.I., de Kretser, D.M., 1988. Radioimmunoassay of rat serum inhibin: changes after PMSG-stimulation and gonadectomy. Mol. Cell. Endocrinol. 58, 1 – 8. Seitz, J., Mobius, J., Bergmann, M., Meinhardt, A., 1995. Mitochondrial differentiation during meiosis of male germ cells. Int. J. Androl. 18, 7 – 11. Sharpe, R.M., Turner, K.J., McKinnell, C., Groome, N.P., Atanassova, N., Millar, M.R., Buchanan, D.L., Cooke, P.S., 1999. Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number. J. Androl. 20, 94 – 101. Sugino, K., Kurosawa, N., Nakamura, T., Takio, K., Shimasaki, S., Ling, N., Titani, K., Sugino, H., 1993. Molecular heterogeneity of follistatin, an activin binding protein. J. Biol. Chem. 68, 15 579 – 15 587. Tanimoto, Y., Tanimoto, K., Sugiyama, F., Horiguchi, H., Murakami, K., Yagami, K., Fukamizu, A., 1999. Male sterility in transgenic mice expressing activin bA subunit gene in testis. Biochem. Biophys. Res. Commun. 259, 699 – 705. Tena-Sempere, M., Kero, J., Rannikko, A., Yan, W., Huhtaniemi, I., 1999. The pattern of inhibin/activin a- and bB-subunit messenger ribonucleic acid expression in rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate. Endocrinology 140, 5761 – 5770. Toboesch, A.M.W., Robertson, D.M., Trapman, J., Klaasen, P., de Paus, R.A., de Jong, F.H., Grootegoed, J.A., 1988. Effects of FSH and IGF 1 on immature rat Sertoli cells: inhibin a and b-subunit mRNA levels and inhibin secretion. Mol. Cell. Endocrinol. 55, 101 – 105. Ueno, N., Ling, N., Ying, S., Esch, F., Shimasaki, S., Guillemin, R., 1987. Isolation and partial characterization of follistatin: a singlechain Mr 35 000 monomeric protein that inhibits the release of follicle stimulating hormone. Proc. Natl. Acad. Sci. USA 84, 8282 – 8286. Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W., Karr, D., Spiers, J., 1986. Purification and characterization of an FSH releasing protein from porcine follicular fluid. Nature 321, 776 – 779. Van Dissel-Emiliani, F.M., Grootenhuis, A.J., de Jong, F.H., de Rooij, D.G., 1989. Inhibin reduces spermatogonial numbers in testes of adult mice and Chinese hamsters. Endocrinology 125, 1898 – 1903. Van Dissel-Emiliani, F.M., de Boer-Brouwer, M., de Rooij, D.G., 1996. Effect of fibroblast growth factor-2 on Sertoli cells and gonocytes in coculture during the perinatal period. Endocrinology 137, 647 – 654. Vassalli, A., Matzuk, M.M., Gardner, H.A.R., Lee, K., Jaenisch, R., 1994. Activin/inhibin bB subunit gene disruption leads to defects in eyelid development and female reproduction. Genes Dev. 8, 414 – 427. Wreford, N.G., Kumar, T.R., Matzuk, M.M., de Kretser, D.M., 2001. Analysis of the testicular phenotype of the follicle stimulating hormone (FSH) b subunit knock-out and the activin type II receptor knock-out mice by stereological analysis. Endocrinology, In Press.
Inhibins, activins and follistatin: actions on the testis D.M. de Kretser *, K.L. Loveland, T. Meehan, M.K. O’Bryan, D.J. Phillips, N.G. Wreford Monash Institute of Reproduction and De6elopment and Department of Anatomy and Cell Biology, Monash Uni6ersity, Monash Medical Centre, 246 Clayton Road, Victoria 3168, Australia Received 12 February 2001; accepted 18 March 2001
Abstract While the early studies of the inhibins, activins and follistatins concentrated on their role as endocrine regulators of FSH secretion, recent data has emphasized the local actions of the activins and follistatin. Inhibin, through its capacity to suppress FSH secretion can modulate numerous processes within the testis. However, to date, evidence to support a local role for inhibin is limited. In contrast, activin and its binding protein follistatin are produced by a large number of cell-types within the testis raising the possibility of a range of paracrine and autocrine actions. These include the modulation of androgen production, influence on the proliferation of Sertoli cells and germ cells as well as the capacity to influence the structural and functional features of mitochondria within germ cells. Some of these actions are carefully controlled in a temporal relationship during the development of testicular function in the rat in which there is no separation in time between birth and the onset of spermatogenesis. Given the range of actions of activin in different cell-types, recognition of systems that are designed to modulate its actions are crucial in enhancing our understanding of how these many roles can be compartmentalized. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Activin; Follistatin; Inhibin; Testis; Sertoli cell; Spermatogenesis
1. Introduction The inhibins, activins and follistatin were isolated on the basis of their ability to modify the secretion of FSH by the pituitary (Ling et al., 1985; Robertson et al., 1985; Ling et al., 1986; Vale et al., 1986; Robertson et al., 1987; Ueno et al., 1987). These actions were initially presumed to result from the endocrine action of these proteins produced at peripheral sites. While this has been shown for inhibin, where following castration, the circulating levels of this hormone decline to almost undectable levels, the same conclusions cannot be reached from the data concerning activin and follistatin (Robertson et al., 1988; Ishida et al., 1990). Following castration, activin A levels in adult male rats did not change over a 7-day period, indicating that sources * Corresponding author. E-mail address: [email protected] (D.M. de Kretser).
other than the testis contributed to circulating levels (McFarlane et al., 1996). Similarly, when rams were castrated, follistatin levels actually increased, a phenomenon which has subsequently been shown to result from the cytokine stimulation of follistatin as part of the acute phase response (Phillips et al., 1996). Numerous studies have shown that, apart from the action of inhibin on FSH levels, the modulation of FSH by activin and follistatin results from paracrine or autocrine actions at the pituitary (Bilezikjian et al., 1993, 1996). The bB and bA subunits are produced by the pituitary gland and act locally to exert a stimulatory action on FSH secretion which is negatively modulated by locally produced follistatin through its ability to bind and neutralize the action of the activins (Nakamura et al., 1990). Since FSH exerts numerous effects on the testis, the modulation of its secretion by inhibin, activin and follistatin can influence testicular function. This review however, is focused on the testicular production of these proteins and their local effects.
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2. Tissue and cellular distribution The distribution of the bA and bB subunits and follistatin at many tissue sites has raised the possibility that these substances exert paracrine and autocrine actions at those sites (Roberts et al., 1989; Michel et al., 1990). While the testis is not a major contributor to circulating levels of activin A and follistatin, both mRNA and protein for follistatin and the activin subunits are present in the testis indicating the capacity for local production (Roberts et al., 1989; Michel et al., 1990; Andersson et al., 1998). To fully understand the possible testicular actions of the activins and inhibins it is essential to recognize the sites of production. There is general agreement that inhibin B is the major protein regulator of FSH produced by the testis, largely secreted by the Sertoli cells (Anawalt et al., 1996). There is a positive relationship between inhibin B levels and Sertoli cell number in normal and pathophysiological states in vivo, supporting the concept that the Sertoli cell is the principal site of inhibin B production (Ramaswamy et al., 1999; Sharpe et al., 1999). These studies support earlier reports which established that the immature Sertoli cell in culture is the site of inhibin secretion and showed that FSH could stimulate its production (Le Gac and de Kretser, 1982). However there is also evidence that the Leydig cells have the capacity to produce bioactive inhibin (McLachlan et al., 1988; Risbridger et al., 1989). Recent studies have suggested that the Leydig cells can modulate the expression of the a subunit gene in the seminiferous tubules (Tena-Sempere et al., 1999). Data concerning the production of activin A and B are sparse due to the limited availability of specific assays. Consequently our understanding of the sites of production in the testis is limited to the mRNA and immunocytochemical localization of the bA and bB subunits. The bA and bB subunits are present in the Sertoli cells (Toboesch et al., 1988) and there is bioassay data and our unpublished data using a specific ELISA to indicate that activin A is produced by the Sertoli cells (Grootenhuis et al., 1989; de Winter et al., 1993). In addition, there is data which supports the view that the Leydig cells and the peritubular cells can secrete activin A (Lee et al., 1989; de Winter et al., 1994). Surprisingly immunocytochemical data indicate that the spermatogonia, primary spermatocytes and round spermatids in the human testis are potential sites of activin B production (Andersson et al., 1998). Our unpublished data show that in the rat testis, the identical stages of spermatogenesis have the capacity to produce activin B since they contain bB subunit mRNA and immunocytochemically detected protein. To define the site of paracrine and autocrine actions it is essential to know which cell types contain receptors for the activins. Several studies have shown that the
type I and type II activin receptors are present in Sertoli cells, primary spermatocytes and round spermatids (de Winter et al., 1992; Kaipia et al., 1993). Additionally, other investigators showed that iodinated activin A bound to primary spermatocytes and round spermatids (Krummen et al., 1994). These cells therefore represent sites at which the activins may exert their actions. Modulation of these actions can occur through the actions of follistatin which has been shown to be produced by the Sertoli cells, spermatogonia, primary spermatocytes and round spermatids (Michel et al., 1990; Kogawa et al., 1991; Meinhardt et al., 1998). It is important to recognize that in addition to cellular production, the sites at which follistatin is detected on cell surfaces may represent the binding and tissue storage of follistatin 288 which has a strong affinity to heparin sulphate proteoglycans (Sugino et al., 1993). Additionally since activin binds strongly to follistatin, the binding to proteoglycans represent tissue stores of activin. Evidence that this type of binding results in the accumulation of significant amounts of follistatin and activin, can be seen by the release of both proteins following an intravenous injection of heparin (Phillips et al., 2000). The amounts released can substantially raise the circulating levels of these proteins in men and women (Phillips et al., 2000). A new kinase deficient type 1 receptor has been identified in Xenopus (BAMBI) which can inhibit the actions of the activins, the bone morphogenetic proteins and TGFb. This is yet another protein with the capacity to modulate the actions of the activins (Onichtchouk et al., 1999). Using a cDNA probe complimentary to the human orthologue, hnma, the mRNA for this protein has been identified in the adult rat, mouse and human testis in spermatogonia, primary spermatocytes, round and elongating spermatids and in Sertoli cells, all sites that have the capacity to produce activin (Loveland et al., unpublished data). Further studies of the role of this protein are awaited with interest.
3. Actions on the testis
3.1. Physiological actions emerging from genetic manipulation Targeted disruption of the inhibin a subunit in mice resulted in the development of testicular tumours which became apparent around 21–28 days of age and resulted in death (Matzuk et al., 1992). These tumours probably arise from Sertoli cells but have the appearance of granulosa cells. The mice become severely cachetic, an action that has been shown to result from the high levels of activin that are present in these mice (Matzuk et al., 1994; Coerver et al., 1996). These studies also confirmed that the tumourigenic action
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resulted specifically from the absence of a functional a subunit gene, confirming the proposal that the a subunit is a tumour suppressor (Matzuk et al., 1992). Since the tumours in these mice disrupt the architecture of the testis, it has not been possible to identify any specific defects in spermatogenesis resulting from the targeted disruption. Targeted disruption of the bA subunit gene resulted in perinatal death of mice due to palatal developmental defects that prevented successful suckling (Matzuk et al., 1995a). Consequently, analysis of the local actions within the testis has not been possible. In contrast, the functional disruption of the bB subunit gene showed no obvious disturbance of spermatogenesis (Vassalli et al., 1994). It is possible that the absence of an effect on spermatogenesis resulted from functional compensation by the bA subunit. In an attempt to interrupt the action of the activins, Matzuk and colleagues disrupted the function of the activin type IIA gene and showed that the mice were fertile despite the presence of testes that were smaller than normal (Matzuk et al., 1995b). Since the FSH levels in these mice were markedly decreased, the smaller testicular size was attributed to decreased Sertoli cell numbers resulting from the inadequate stimulation of proliferation in the fetal and neonatal period during which these cells have the capacity to divide. Quantitative studies of the testes of these mice have confirmed that the Sertoli cells are decreased by 30% leading to a corresponding decline in sperm production, a finding comparable to the decrease in the number of these cells in mice with targeted disruption of the FSH b subunit gene (Wreford et al., 2001). It was not possible to specifically delineate a defect that could be attributed to the absence of activin action. Despite the failure to demonstrate a specific defect by the approach using targeted disruption of the relevant genes, over-expression of the bA subunit gene in mice resulted in spermatogenic disruption (Tanimoto et al., 1999). In this case, the promoter led to bA synthesis in spermatocytes. The pattern of disruption varied with the level of expression in different lines. Profiles of vacuolated tubules suggested the total absence of germ cells but in other areas, spermatids were more severely depleted than spermatogonia and primary spermatocytes. Over expression of the follistatin gene also resulted in infertility and spermatogenic defects that were related to the degree of follistatin over-expression (Guo et al., 1998). In mice with the highest level of expression, testis weights were decreased and spermatogenesis was disrupted and some tubules in some mice showed only Sertoli cells. These observations suggest that the excess follistatin can impair the local actions of the activins and the spermatogenic defect may result from this action. Unfortunately, targeted disruption of the follistatin gene resulted in perinatal death due to respiratory difficulties and hence it has not been possible
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to evaluate the effects on the adult testis (Matzuk et al., 1995c).
3.2. Actions on androgen production In vitro incubation of Leydig cells from 21-day old rats with inhibin stimulated testosterone production but activin inhibited this production (Hsueh et al., 1987). However Risbridger and colleagues were unable to confirm the observation that inhibin stimulated testosterone secretion using highly purified Leydig cells from adult rats raising the possibility that this function may be age specific (Risbridger et al., 1989). Other studies have also failed to confirm the action of inhibin on testosterone secretion but have been able to show the inhibition by activin A in vitro (Lin et al., 1989). The in vivo physiological significance of these variable results remains unknown but no significant changes have been noted in testosterone levels in mice with targeted disruption of the inhibin/activin subunit genes.
3.3. Action on the maintenance of mitochondrial morphology in primary spermatocytes Our recent studies have identified the capacity for activin A to specifically maintain the ‘condensed’ mitochondrial morphology found in germ cells beyond the leptotene stage of the first meiotic prophase (Meinhardt et al., 2000). The ‘condensed’ mitochondrial appearance results from the dilatation of the intra-cristal spaces and the peripheral margination of the cristae leaving a central space within the mitochondrion. This action of activin A is dose-dependent and can be inhibited by an antiserum specific to activin A. These morphological changes reflect functional changes such as the loss of heat shock protein 60 (HSP-60) which is thought to act in concert with hsp 10 as a chaperone to fold newly formed mitochondrial proteins (Meinhardt et al., 1995; Paranko et al., 1996). Further, localization of the Lonprotease in mitochondria was lost after the zygotene stage raising the possibility of limited proteolytic degradation of mitochondrial proteins in the early meiotic stages but not thereafter (Seitz et al., 1995). Since there is now evidence that the mRNA for the bB subunit and its protein can be found in primary spermatocytes which also contain specific activin type I and II receptors, the action on mitochondria represents an example of an autocrine role. As the morphological changes in the mitochondria have functional correlates, the action of activin A is likely to result in functional changes, the nature of which must remain speculative. Given that activin can influence the occurrence of apoptosis in other cell types, it is possible that the mitochondrial changes may represent an action related to apoptosis.
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3.4. Modulation of spermatogonial proliferation Although the studies are limited in number, there is data to suggest that inhibin can inhibit spermatogonial proliferation (Van Dissel-Emiliani et al., 1989). Unfortunately the study was performed using partially purified native inhibin from follicular fluid and did not rule out possible contamination of the preparation with activin and follistatin. In one study with purified activin A, spermatogonial proliferation was stimulated when this protein was added to co-cultures of Sertoli cells and spermatogonia (Mather et al., 1990) suggesting that inhibin and activin may have opposing effects on spermatogonial proliferation. In contrast, in a study using fragment cultures of day 9 testes, activin A inhibited the FSH-stimulated proliferation of differentiated spermatogonia (Boitani et al., 1995).
3.5. Action during the de6elopment of the rat testis There is increasing evidence that the actions of activin and follistatin may exert discrete, age-specific actions during the early post-natal development of the rat testis. This concept arises from several lines of evidence. First, the localization of activin A protein and bA subunit mRNA appear in gonocytes but are not present in early spermatogonia (Meehan et al., 2000). In contrast, follistatin is absent in gonocytes at birth, and appears around day 3 persisting in spermatogonia. Localization of activin A to fetal gonocytes has also been found in the sheep testis (Jarred et al., 1999). In keeping with this changing localization, Meehan et al. (2000) showed that in fragment cultures of the rat testis, activin A stimulated gonocyte numbers and did not alter spermatogonial numbers. In contrast, follistatin in combination with FSH, stimulated spermatogonial numbers. Additionally, activin A inhibited proliferation of Sertoli cells in fragment cultures from day 3 rat testes. Further evidence of an age specificity emerged from the results of Boitani et al. (1995) who showed that in fragment cultures from day 3 rat testes, activin A had no effect on Sertoli cell proliferation, with or without FSH. However, at day 9, activin A and FSH in combination, but not separately, stimulated Sertoli cell proliferation. These data highlight the changing responsiveness of the testis to activin which characterizes the onset of spermatogenesis. It is possible that activin and follistatin may play an important role in the transformation of gonocytes to spermatogonia, perhaps acting in concert with other growth factors such as leukaemia inhibitory factor, transforming growth factor b, oncostatin M and fibroblast growth factor, all of which are known to exert effects on early testicular development (de Miguel et al., 1996; Van Dissel-Emiliani et al., 1996; de Miguel et al., 1997; Richards et al., 1999). Further studies of the
testes in knock-out models of the genes encoding the activin subunits and follistatin may shed further information of the physiological relevance of these in vitro effects. Since the events in the immediate post-natal development of the seminiferous epithelieum are temporally precise, the controlled expression of activin and follistatin may be crucial control mechanisms which determine the onset of the spermatogenic process.
3.6. Importance of factors controlling the actions of acti6in This brief review of the actions of activins in the testis emphasizes the importance of control systems that have the capacity to modulate the action of these proteins, particularly to localize actions to a particular cell type. There are a number of mechanisms that can exert this modulation. First, the relative expression of the a and b subunits can alter the amounts of activin produced. Increasing a-subunit expression drives the Sertoli cell to produce inhibin and free a-subunit rather than activin (Hancock et al., 1992). Secondly, the production of follistatin within the same or adjacent cells can neutralize the action of activin although it failed to inhibit the activin-stimulated proliferation of germ cellSertoli cell cultures (Mather et al., 1990). Thirdly, the level and identity of the type I and type II activin receptors will influence the ability of activin to exert its actions. Fourth, the expression of heparin sulphate proteoglycans on the surface of cells can, through the intermediary of follistatin, assist the degradation of activin through lysosomal mechanisms (Hashimoto et al., 1997). Alternatively, the binding of activins, through follistatin to heparin sulphate proteoglycans, can result in a tissue store of activin which could be released through the action of heparin released by heparinases. Finally, both activin and follistatin can bind to the fast form of a2 macroglobulin, an action that may modulate the degradation of these proteins (Niemuller et al., 1995; Phillips et al., 1997). Such a mechanism may be important since a2 macroglobulin is produced by the Sertoli cells in the testis (Cheng et al., 1990). In conclusion, while inhibin probably exerts its action on the testis through a long loop system involving the modulation of FSH secretion, the effects of activin and follistatin are exerted through the local production of these proteins. Considerable further work is required to define these local actions and to define their physiological roles.
Acknowledgements Many of these studies were supported by a Program Grant from the National Health and Medical Research
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Council of Australia. The excellent research assistance of Anne O’Connor, Lynda Foulds and Elizabeth Christy is gratefully acknowledged.
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