J. Endocrinol. Invest. 15: 67-75,1991
REVIEW ARTICLE
Growth factors and testis G. Giordano*, P. Del Monte*, and F. Minuto** *Cattedra di Endocrinologia and **Cattedra di Fisiopatologia Endocrina, DiSEM Universita di Genova, Genova, Italy
INTRODUCTION
direct and that other factors may play a role in the paracrine communication between Leydig cells and seminiferous tubules. Moreover, it has been shown that peritubular myoid cells are androgen responsive and synthesize proteins that modulate the functions of Sertoli cells (P-mod-S) suggesting that some actions by testosterone on Sertoli cells can be mediated by peritubular cells (15-16). These complex local regulations seem to be accomplished mainly through paracrine communication between testicular cells and therefore particular interest has been addressed to the growth factors and regulatory peptides identified within the testis (17) (Table 1). It must be underlined that many growth factors exhert pleiotypic effects in gonadal cells which are not simply related to their mitogenic action. On the other hand, many intragonadal regulatory peptides have been described opioid peptides (18, 19), inhibin-activins (20,21), immunoregulatory peptides (22, 23) neuropeptides (24-26) - and they too can influence cell replication. The distinction between the two groups therefore can be somehow artifactual. In the present review, however, we focus mainly on the paracrine regulation by classical growth factors.
It is well known that FSH and LH play a major role in the regulation of testicular function, but there is increasing evidence that their action can be modulated intratesticularly, according to the local requirements. The existence of active interactions among the various testicular cell types has been demonstrated (1-4) and recently extensively reviewed (5). Sertoli cells form the framework of the seminiferous epithelium and exert nursing functions on the germ cells, providing them with functional and structural support (6). Germ cells, in turn, can regulate Sertoli cells, which vary their activity depending on the stage of spermatogenesis (5). Several studies both in vivo and in vitro support the concept that Sertoli cells might influence Leydig cell function (3). FSH treatment of immature hypophysectomized rats can induce Leydig cells hypertrophy and hyperplasia (7), increase testicular LH receptors and enhance testicular steroidogenesis in response to hCG (8, 9). Moreover, co-culture of Leydig cells with Sertoli cells in presence of FSH shows an increase in both LH receptor number and in the steroidogenic response to hCG, effects that are not observed if FSH is added to the Leydig cells cultured alone (3, 10, 11). Furthermore, conditioned media from FSH-treated Sertoli cells potentiate Leydig cells steroidogenesis in response to LH (11-13). Although testosterone is the best known Leydig cell-derived factor influencing spermatogenesis, androgen receptors have been demonstrated in Sertoli cells, while they have not been detected in germ cells (14), thus it is thought that the actions of testosterone on spermatogenesis are mainly in-
INSULIN-LIKE GROWTH FACTORS (IGF) Several evidences point to the IGFs as an important paracrine regulator of testicular function. It is known that isolated GH deficiency is often associated with delayed puberty and with a reduced testosterone response to hCG administration, which usually normalize under GH therapy (27-30). Moreover, experimental studies evidenced that treatment of hypophysectomized animals with GH partially restores hCG-stimulated testosterone secretion, and that the continuous administration of GH immediately following hypophysectomy partially prevents the decrease of LH receptor level (8). These findings suggest that GH is involved, either directly or indirectly, in the maintenance of gonadotrophin responsiveness of Leydig cells. It has
IThis work was supported by research grants from Consiglio Nazionale delle Rlccrche n. 88.01970.04, 89.04176.04, 89.00188.70. Key-words: Growth factors, testis, insulin-like growth factors, epidermal growth factor, transforming growth factors, nerve growth factor, fibroblast growth factor. Correspondence.' Cattedra di Endocrinologia, ISMI. Viale Benedetto XV 6. 1-16132 Genova, Italy
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characterized (38). Several studies evidenced that rat (39) and pig Sertoli cells (40) release in the culture medium immunoreactive IGF-I, bound to a carrier protein to form a complex of 25000 d. After gel chromatography in acid conditions a smaller peptide of 8000 d is released. More recently, mRNA for IGF-I has been detected in Leydig cells (41) and both IGF-I and IGF binding protein have been identified in the conditioned media of both Leydig and peritubular cells (42). In Sertoli cells FSH and other agonists which increase cAMP stimulate IGF-I secretion, but inhibit IGF-BP release (42, 43). In Leydig cells, LH increases IGF-I production and a stimulatory effect has been demonstrated for FGF on both the cell types. The production of IGF-I by LeydigSertoli cells cocultures seems to exceed that expected from the monocultures, suggesting that cellcell interactions may also play a role in the control of testicular IGF production (44). Interestingly, thyroid hormones increase IGF secretion by rat Sertoli cells in vitro (45). No clear effect by GH has been demonstrated on cell cultures in vitro, while in testicular tissues of rats treated with gonadotrophins or GH an increase in IGF-I mRNA was detected (36). In another study, however, it has been shown that, while hypophysectomy induces a reduction of IGF-I testicular mRNA, GH treatment alone was unable to normalize IGF-I expression (37). No correlation could be detected between the ontogeny of testicular GHRH RNA, which increase to adult levels by day 30 (37), and either IGF-I or IGF-II mRNA levels. In particular testicular IGF-I mRNA was found to be elevated in neonatal rats, fell to an intermediate levels by day 20 and showed a further decrease after day 60. Evaluation by immunohistochemistry of IGF-I distribution in the rat testis showed that IGF-I immunoreactivity is present in spermatogenic, Sertoli and Leydig cells in the young rat, while only spermatogenic cells are positive in the mature animal (46). IGF-II mRNA can be detected in abundant quantities in the testes of 2 days old rats, but it falls rapidly by day 10 and it is expressed in extremely low levels in the adult rat testis (35-37). Furthermore, its expression appears to be independent from hormonal control (36). These observations are in keeping with the view that this factor is mainly involved in fetal development. The presence of IGF-I in human testis has been confirmed by immunohistochemical studies and IGF-I has been preferentially localized in Sertoli cells, while less evident positivity has been found associated with primary spermatocytes and with some Leydig cells (47). Both IGF-I and IGF-I binding protein are present in human seminal plasma (48-50).
Table 1 - Growth factors involved in the local regulation of testicular function. Factor
Origin
IGF-I
Leyding c. Sertoli c. peritubular c. Leydig c.
EGF-TGF
Target
Actions Potentiation of LH-Induced steroidogenesis
Sertoli c.
Proliferation Metabolic effects Induction of differentiated function
germinal c.
Regulation of spermatogenesis (?)
a-TGF Sertoli c. germinal c. EGFa-TGF peritubular c. Leydig c.
Stimulation of meiosis Modulation of steroidogenesis
peritubular c. Proliferation
Beta-TGF Sertoli c.
Sertol i c.
(7)
Leydig c.
Inhibition of steroidogenesis
Sertoli c.
metabolic effects
perituberal c. differentiation NGF
germinal c.
germinal c.
seminiferous tubules
Sertoli c.
structure
pentubular c. bFGF
Sertoli c.
SGF
Semiferous t. Sertoli c.
Sertoli c. Leydig c.
Prol iferation Induction differentiated functions Mitogenic activity
been shown that in vivo administration of GH to hypophysectomized rats increases the number of type I IGF receptors on Leydig cells (31). Recently, a GHRH-like peptide has been identified within the testis (32), and localized, by immunohystochemistry, mainly in germ cells (33). Origin of /GFs in the testis
Ritzen first reported that a substance with somatomedin activity accumulated in the incubation medium of isolated rat seminiferous tubules (1), and immunoreactive IGF-I has been found in rat testicular extracts (34). Moreover, IGF-I mRNA has been detected in testicular tissue (35, 36, 37) and testicular cDNA encoding IGF-I precursors have been
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Growth factors and testis
Binding sites for IGFs in the testis High affinity binding sites for IGFs have been identified in testicular membrane preparations (51), in Leydig (52, 53) and in Sertoli cells (54, 55). Purified Leydig cells present type I IGF receptors, and Scatchard analysis reveals a linear binding. In rats treated with hCG, IGF-I receptor number on Leydig cells is increased, without affinity modifications (31, 56) and a similar effect has been shown on pig Leydig cells (53). Both type I and type II receptors have been identified in Sertoli cells in culture. IGFI binding is linear and fifty percent of displacement of the tracer is observed with a 2 nM concentration of IGF-I, while insulin is less effective. Using immunofluorescence techniques in rat Sertoli-germ cells co-cultures, immunoreactive IGF-I was found associated with pachitene spermatocytes but not with spermatogonia (57). In human testis the major positivity for the IGF-I receptor was found in secondary spermatocytes and in early spermatides (47), and, therefore, a possible role for this growth factor in the regulation of the meiotic process has been suggested. Sertoli cells were found less positive, while an intense positivity has been observed in some Leydig cells (47).
IGFs (54, 65), and FSH and IGF-I together exhert a synergistic effect on DNA synthesis in Sertoli cells obtained from neonatal rats (66). Tubular lumen
Interstitial tissue
Tubular lumen
IGF-I actions on testicular cells
IGF-I increases the number of LH receptors on Leydig cells in culture and hCG-stimulated testosterone secretion. Insulin is ten fold less potent than IGF-I in inducing these effects (58-60). IGF-I increases also hCG-stimulated cAMP production and 8 bromo cAMP-induced testosterone secretion. Therefore, it has effect at both the LH receptor sites and the steps beyond adenilate cyclase. An increase of 3 beta hydroxysteroid dehydrogenase activity has also been described (60). Cocultures of Leydig and Sertoli cells produced an increase in both hCG receptors and hCG-induced testosterone production (61-63). Addition to the medium of FSH, insulin or IGF-I produced a marked increase in these two parameters of Leydig cell function. Moreover, conditioned media from Sertoli cells cultured in the absence of insulin and FSH inhibited hCG responsiveness. Immunoneutralization experiments show that IGF-I is an important permissive factor to maintain steroidogenesis in Leydig cells and in cocultures, but an IGF-I antiserum could not completely neutralize the stimulatory effect of coculture (64). Other factors therefore seem to be involved. Sertoli cells prepared from immature rats and pig showed an increased incorporation of 3H-thymidine into DNA in response to micromolar concentrations of insulin or nanomolar concentrations of
FSH
Interstitial tissue
Vascular lumen
Fig. 1 - Schematic representation of the various growth factors and hormones involved in the paracrine regulation of testicular function. (TH = Thyroid hormones).
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G. Giordano, P. De/ Monte, and F. Minuto
Both IGF-I and IGF-II are able to increase the production of lactate, the incorporation of 3H-leucine into proteins and the transport of glucose (54). Moreover, FSH and IGF-I synergistically increase cAMP production and plasminogen activator secretion (67) by Sertoli cells in culture, and a stimulatory effect of IGF-I on androgen binding protein production has been demonstrated (66). The idea that the IGF-I may play a role in the regulation of spermatogenesis is supported by the findings that this peptide is produced by Sertoli cells and that it has been found associated with spermatic cells. It must be underlined that because the entry of circulating factors into the tubular fluid is restricted, germ cells are exposed only to the locally produced peptides. Vitamin A withdrawal in the rat results in an arrest of spermatogenesis and in this condition a reduction in testicular IGF-I content, which is reversed by vitamin A replenishment, has been observed (68). In men, IGF-I content in seminal fluid of azoospermic subjects shows a tendency to the reduction (50). Further studies, however, are necessary to understand the functions of testicular IGF-I on spermatogenesis. The role of the somatomedin binding proteins identified in the testis is still unknown, it seems likely that they play a role in the modulation of IGFs actions.
and Sertoli cells. Alpha-TGF is a polypeptide that shares sequence omology with EGF and binds with high affinity to the EGF receptor. Isolated peritubular and Sertoli cells contain mRNA for alpha-TGF and this peptide accumulate in media conditioned by both cell types (72). EGF/alpha TGF receptors have been identified in peritubular cells and in Leydig cells (72-74). Recently, it has been shown that alpha TGF can stimulate peritubular cells proliferation and it has been suggested that it can influence Sertoli cell function indirectly, through action on peritubular cells (5, 72). The effects of EGF on steroidogenesis are still controversial. In a study performed on Leydig cells derived from immature hypophysectomized rats it was observed that EGF inhibits (75) hCG-stimulated production of testosterone, androstenedione and 17 OH-progesterone, and the conversion of exogenous 17 alpha OH-Pg to androstenedione. On the contrary, an increased production of pregnenolone and progesterone was evidenced. Therefore it has been suggested that EGF inhibits both 17 alpha hydroxylase and 17-20 lyase. Other authors, however, have shown a stimulatory effect of EGF on steroidogenesis in Leydig cells (76). Recently, it has been shown that EGF decreases DHEA accumulation in hCG-treated porcine Leydig cells and activates testosterone formation by stimulation of 3 beta-hydroxysteroid dehydrogenase enzyme activity (74). Studies performed with stage synchronization of seminiferous epithelium by withdrawal and subsequent replenishment of vitamin A in the rat show increased testicular concentration of EGF at stages IX-XIV of the cycle of the seminiferous epithelium, where the three mitotic division of type A spermatogonia occur in the rat (68), providing evidence of a correlation between EGF or an EGF-like factor and the regulation of spermatogonial cell division.
EPIDERMAL GROWTH FACTORfTRANSFORMING GROWTH FACTOR ALPHA(EGFm~haTG~
The possibility that EGF is involved in the regulation of testicular function was first suggested by the observation that sialoadenomectomy in the rat is accompanied with a reduction of the number of mature sperm in the epididymis (69). The number of spermatides in the testis is also decreased, while the spermatocytes are slightly increased. Administration of EGF restores a normal spermatogenesis. It seems, therefore, that EGF plays a role in male reproductive function by stimulating the meiotic phase of spermatogenesis. Epidermal growth factor-like activity has been detected in human seminal plasma and a specific EGF binding activity was observed in testicular membrane preparations (70). Sertoli cells conditioned media contain a mitogen, hormonally regulated by FSH, testosterone and retinol, that competes with 1251-EGF for the binding to the EGF receptors (71) It seems likely that this peptide is related to alpha-TGF, a mitogen produced by normal and transformed cells, that has been recently identified in both peritubular
TRANSFORMING GROWTH FACTOR BETA (TGF beta) TGF-beta peptides belong to the same family as inhi bins, activins and anti mullerian hormone. Both Sertoli cells and peritubular cells produce TGF-beta (77). It has been shown that this peptide inhibits several Leydig cell functions, and in particular it reduces the number of LH receptors and the cAMP response to hCG. Moreover, a dose-dependent inhibition of hCG-stimulated testosterone production was observed when cells were pretreated with beta-TGF (78). This peptide also reduced forskolin and (Bu) cAMP-induced testosterone production, indicating that it can inhibit steroidogenesis distal to the formation of cAMP (78). The conversion of ex-
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Growth factors and testis
ogenously added testosterone precursors (progesterone and 17 -OH progesterone) to testosterone by hCG stimulated cells was also suppressed by the addition of beta-TGF, suggesting that this peptide affects the activity of the 17 alpha hydroxylase enzyme. Endogenous progesterone accumulation did not increase when hCG-stimulated testosterone production was inhibited by TGFbeta indicating that steps before the formation of progesterone are also inhibited (78). On the other hand, it has been shown that this peptide increases the activity of 3 beta-hydroxysteroid dehydrogenase, a key enzyme in the maturation of Leydig cells (79). TGF-beta receptors are present in Sertoli cells and this factor is able to stimulate lactate production and glucose uptake (80). In peritubular cells, TGF-beta can inhibit TGF-alpha/EGF stimulated growth, while it promotes cell differentiation and chemiotaxis (5, 77) and can increase the production of specific proteins, such as plasminogen activator inhibitor (81).
cells. NGF mRNA and NGF immunoreactivity have been identified mainly in spermatocytes and early spermatids (90, 91). Recently, NGF receptor gene expression has been observed in Sertoli cells, where it is down-regulated by androgens (92). These findings suggest that NGF may mediate regulatory interactions between germ cells and Sertoli cells. Moreover, NGF affects the morphology of the seminiferous tubules, and maintains their normal structure in vitro (93). By immunohistochemical methods, target cells for this peptide were identified within the lamina propria of the tubules (94).
SEMINIFEROUS GROWTH FACTOR This was the first growth factor isolated from the seminiferous tubules, and it has been partially characterized as a protein of 15750 m.w. with a pi of 5.2, immunologically different from both acid and basic FGF (17,95). It is a broad-spectrum mitogenic factor and is endowed with pleiotypic action in testicular cell lines (96, 97). In conclusion, there are now increasing evidences that a large number of growth factors and of other specific substances are involved in the physiological regulation of the testis. It should be underlined, however, that the actions of most growth factors are interdependent on the presence of other growth factors and regulatory peptides, each working like a player in an orchestra. This fascinating chapter is open to further development, particularly as regard the interrelationships between classical endocrine hormones and local paracrine factors.
FIBROBLAST GROWTH FACTOR (FGF) Basic FGF has been purified from bovine testis (82, 83). The purified protein is a truncated form of bFGF (aa 16-146), with full mitogenic activity on fibroblast and endothelial cells. Recently bFGF has been demonstrated in Sertoli cell conditioned media (84). Furthermore, it has been shown that bFGF exerts a mitogenic effect on immature pig Sertoli cells and that the simultaneous addition of FGF and IGF-I led to a synergistic response (65). Moreover, in presence of insulin, FGF increased FSH binding, cAMP production and plasminogen activator activity of pig Sertoli cells (65). FGF effect on transferrin secretion by Sertoli cells appears to be biphasic, in fact acute treatment stimulates the release of transferrin by some Sertoli cells, while chronic treatment decreases the fraction of Sertoli cells secreting this factor (85). Moreover, it has been shown that FGF stimulates c-fos and B-jun gene expression in Sertoli cells (84). Recently, FGF actions on Leydig cells have been described. It inhibits LH-stimulated androgen production (86), 5-alpha reductase activity (87) and 5-3 beta hydroxysteroidodehydrogenase isomerase activity (88), but it stimulates aromarase activity (89). Therefore, it can be involved in the local regulation of androgen production.
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67. Saez J.M, Chatelain P.G., Perrard-Saporl M.H., Jaillard C, Naville D. Differentiating effects of somatomedln-C/insulln-like growth factor I and insulin on Leydig and Sertoli cells functions. Reprod. Nutr. Dev. 28.' 989,1988.
56. Lin T., Blaisdell J, Haskell J.F. Type I IGF receptors of Leydig cells are upregulated by human chorionic gonadotropin. Biochem. Biophys. Res. Commun. 149: 852, 1987.
68. Eartlett JM.S, Spiteri-Grech J, Nieschlag E. Regulation of Insulin-like growth factor I and stagespecific levels of epidermal growth factor in stage synchronized rat testes. Endocrinology 127747,1990.
57. Tres L, Smith E.P, Van Wyk J.J, Kierszenbaum AL Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell cocultures. Exp. Cell Res. 162: 33,1986.
69. Tsutsumi 0, Kurachi H., Oka 1. A physiological role of epidermal growth factor in male reproductive function. Science 233. 975, 1986.
58. De Mellow J.S. M, Handelsman D.J., Baxter RC. Short term exposure to insulin-like growth factors stimulates testosterone production by testicular interstitial cells. Acta Endocrinol. (Copenh.) 115. 483,1987.
70. Elson S.D., Browne CA, Thorburn G.D. Identification of epidermal growth factor-like activity in human male reproductive tissues and fluids J. Clin. Endocrlnol. Metab. 58. 589, 1984. 71. Holmes SO, Spotts G , Smith RG. Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF)binding to its receptor. J. Bioi. Chem. 261.4076,1986. 72. Skinner M.K., Takacs K, Coffey RJ. Transforming growth factor alpha gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Endocrinology 124. 845, 1989.
59. Kasson B. G, Hsueh AJ,W. Insulin-like growth factor-I augments gonadotropinstimulated androgen biosynthesis by cultured rat testicular cells. Mol. Cell. Endocrinol. 52 27,1987. 60. Lin T., Vinson N, Haskell J., Murono E.P. Induction of 3beta-hydroxysteroid dehydrogenase activity by insulin-like growth factor-I in primary culture of purified Leydig cells. Adv. Exp. Med. Bioi. 219. 603,1987. 61. Perrard Sapori MH, Chatelain P.C, Rogemond N, Saez J.M. Modulation of Leydig cell functions by culture with Sertoli cells or with Sertoli cell conditioned medium: effect of insulin, somatomedin-C and FSH. Mol. Cell. Endocrinol. 50: 193,1987.
73. Teerds KJ, Rommerts F.F., Dorrington JH Immunohistochemical detection of transforming growth factor-alpha in Leydig cells during the development of the rat testis. Mol. Cell. Endocrinol. 69. R1-6, 1990.
62. Morera AM., Chauvin MA, de Peretti E., Binoux M, Benhamed M. Somatomedin-C/lnsulin-like growth factor-I: an intratesticular differentiative factor of Leydig cells? Horm. Res. 28. 50.1987.
74. Sordoillet C., Chauvin MA, De Peretti E., Morera AM., Benhemed M. Epidermal growth factor directly stimulates steroidogenesis in primary cultures of porcine Leydig cells: actions and sites of action. Endocrinology 128. 2160, 1991.
63. Benhamed M., Morera AM., Chauvin M.C., de Peretti Somatomedin-C/lnsulin-like growth factor I as a pos-
75. Welsh T.H, Hsueh AJ.W. Mechanism of the inhibitory action of epidermal growth
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76.
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factor on testicular androgen biosynthesis in vitro. Endocrinology 110. 1498, 1982. Verhoeven G, Cailleau J Stimulatory effects of epidermal growth factor on steroidogenesis in Leydig cells. Mol. Cell. Endocrinol. 49.' 99,1986. Skinner M.K., Moses HL Transforming growth factor beta gene expression and actions in the seminiferous tubule: peritubular cell-Sertoli cell interactions. Mol. Endocrinol. 3. 625, 1989. Bart C., Fauser J. M., Hsueh J.w. Effect of transforming growth factor beta on human chorionic gonadotropin induced testosterone production by cultured rat testicular cells. Life Sci. 43.1363,1988. Benhamed M., Esposito G, Sordoillet C, de Peretti E, Chauvin MA, Ghiglieri C., Revol A, Morera AM. Transforming growth factor beta and its related peptides in the testis: an intragonadal polypeptide control system. In: Serio M. (Ed.), Perspective in andrology. Raven Press, New York, Serono Symp. Pub!. 53: 191, 1989. Esposito G., Keramidas M., Mauduit, Feige J.J., Morera AM, Benahmed M. Direct regulating effects of transforming growth factor-beta 1 on lactate production in cultured porcine Sertoli cells. Endocrinology 128. 1441, 1991.
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81. Nargolwalla C., McCabe D, Fritz I.B. Modulation of levels of messenger RNA for tissuetype plasminogen activator in rat Sertoli cells, and levels of messenger RNA for plasminogen activator inhibitor in testis peritubular cells. Mol. Cell. Endocrinol. 70.' 73,1990.
93.
Fibroblasts growth factor inhibits luteinizing hormone stimulated androgen production by cultured rat testicular cells. Endocrinology 123: 2935, 1990. Murono E.P., Washburn AL. Fibroblast growth factor inhibits of 5-alpha reductase activity in cultured immature Leydig cells. Mol. Cell. Endocrinol. 68.' R 19-23, 1990. Murono E.P, Washburn AL. Basic fibroblast growth factor inhibits delta 5-3 beta hydroxysteroid dehydrogenase-isomerase activity in cultured immature Leydig cells. Biochem. Biophys. Res. Commun. 168.' 248,1990. Raeside J.I., Berthelon M.C., Sanchez P., Saez J.M. Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF). Biochem. Biophys. Res. Commun. 151.163,1988. Olson L., Ayer-LeLievre C., Ebendal T., Seiger A Nerve growth factor-like immunoreactivities in rodent salivary glands and testes. Cell. Tissue Res. 248 275,1987. Ayer-LeLievre C, Olson L., Ebendal T, Hallbook F, Persson H. Nerve growth factor mRNA and protein in the testis and epididimis of the mouse and rat. Proc. Natl. Acad. Sci. U.SA 85.' 2628, 1988. Persson H., Ayer-LeLievre C., Soder 0, Villar M.J., Metsis M., Olson L, Ritzen M., Hokfelt T Expression of beta nerve growth factor receptor mRNA in Sertoli cells downregulated by testosterone. Science 247: 704, 1990. Seidl K., Holstein AF. Organ culture of human seminiferous tubules: a useful tool to study the role of nerve growth factor in the testis. Cell Tissue Res. 261. 539,1990.
82. Ueno N., Baird A, Esch F, Ling N., Guillemin R. Isolation and partial characterization of basic fibroblast growth factor from bovine testis. Mol. Cell. Endocrinol. 49: 189,1987. 83. Story MT, Sas se J., Kakuska D, Jacobs SC., Lawson RK A growth factor in bovine and human testes structurally related to basic fibroblast growth factor. J. Urol. 140.422,1988.
94. Seidl K, Holstein AF. Evidence for the presence of nerve growth factor (NGF) and NGF receptors in human testis. Cell Tissue Res. 261.' 549, 1990. 95. Feig LA, Klagsbrun M, Bellve AR. Mitogenic polypeptide of the mammalian seminiferous epithelium. Biochemical characterization and partial purification. J. Cell BioI. 97 1435, 1983.
84. Smith EP., Hall S, Monaco L., French F., Wilson E.M., Conti M. A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells. Mol. Endocrinol. 3. 958, 1989.
96. Braunhut S.J., Rufo GA, Ernisee B.J, Zheng B.J., Bellve AR. The seminiferous growth factor induces proliferation of TM4 cells in serum free medium. Bioi. Reprod. 42.' 639, 1990. 97. Zheng WX, Butwell TJ, Heckert L., Griswold MD, Bellve AR. Pleiotypic actions of the seminiferous growth factor on two testicular cell lines: comparison with acidic and basic fibroblasts growth factors. Growth factors 3: 73, 1990.
85. Boockfor FR, Schwarz L.K. Fibroblast growth factor modulates the release of transferrin from cultured Sertoli cells. Mol. Cell. Endocrinol. 73.' 187,1990. 86. Fauser B.C., Baird A, Husueh AJ.
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REVIEW ARTICLE
Growth factors and testis G. Giordano*, P. Del Monte*, and F. Minuto** *Cattedra di Endocrinologia and **Cattedra di Fisiopatologia Endocrina, DiSEM Universita di Genova, Genova, Italy
INTRODUCTION
direct and that other factors may play a role in the paracrine communication between Leydig cells and seminiferous tubules. Moreover, it has been shown that peritubular myoid cells are androgen responsive and synthesize proteins that modulate the functions of Sertoli cells (P-mod-S) suggesting that some actions by testosterone on Sertoli cells can be mediated by peritubular cells (15-16). These complex local regulations seem to be accomplished mainly through paracrine communication between testicular cells and therefore particular interest has been addressed to the growth factors and regulatory peptides identified within the testis (17) (Table 1). It must be underlined that many growth factors exhert pleiotypic effects in gonadal cells which are not simply related to their mitogenic action. On the other hand, many intragonadal regulatory peptides have been described opioid peptides (18, 19), inhibin-activins (20,21), immunoregulatory peptides (22, 23) neuropeptides (24-26) - and they too can influence cell replication. The distinction between the two groups therefore can be somehow artifactual. In the present review, however, we focus mainly on the paracrine regulation by classical growth factors.
It is well known that FSH and LH play a major role in the regulation of testicular function, but there is increasing evidence that their action can be modulated intratesticularly, according to the local requirements. The existence of active interactions among the various testicular cell types has been demonstrated (1-4) and recently extensively reviewed (5). Sertoli cells form the framework of the seminiferous epithelium and exert nursing functions on the germ cells, providing them with functional and structural support (6). Germ cells, in turn, can regulate Sertoli cells, which vary their activity depending on the stage of spermatogenesis (5). Several studies both in vivo and in vitro support the concept that Sertoli cells might influence Leydig cell function (3). FSH treatment of immature hypophysectomized rats can induce Leydig cells hypertrophy and hyperplasia (7), increase testicular LH receptors and enhance testicular steroidogenesis in response to hCG (8, 9). Moreover, co-culture of Leydig cells with Sertoli cells in presence of FSH shows an increase in both LH receptor number and in the steroidogenic response to hCG, effects that are not observed if FSH is added to the Leydig cells cultured alone (3, 10, 11). Furthermore, conditioned media from FSH-treated Sertoli cells potentiate Leydig cells steroidogenesis in response to LH (11-13). Although testosterone is the best known Leydig cell-derived factor influencing spermatogenesis, androgen receptors have been demonstrated in Sertoli cells, while they have not been detected in germ cells (14), thus it is thought that the actions of testosterone on spermatogenesis are mainly in-
INSULIN-LIKE GROWTH FACTORS (IGF) Several evidences point to the IGFs as an important paracrine regulator of testicular function. It is known that isolated GH deficiency is often associated with delayed puberty and with a reduced testosterone response to hCG administration, which usually normalize under GH therapy (27-30). Moreover, experimental studies evidenced that treatment of hypophysectomized animals with GH partially restores hCG-stimulated testosterone secretion, and that the continuous administration of GH immediately following hypophysectomy partially prevents the decrease of LH receptor level (8). These findings suggest that GH is involved, either directly or indirectly, in the maintenance of gonadotrophin responsiveness of Leydig cells. It has
IThis work was supported by research grants from Consiglio Nazionale delle Rlccrche n. 88.01970.04, 89.04176.04, 89.00188.70. Key-words: Growth factors, testis, insulin-like growth factors, epidermal growth factor, transforming growth factors, nerve growth factor, fibroblast growth factor. Correspondence.' Cattedra di Endocrinologia, ISMI. Viale Benedetto XV 6. 1-16132 Genova, Italy
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G. Giordano, P. Del Monte, and F. Minuto
characterized (38). Several studies evidenced that rat (39) and pig Sertoli cells (40) release in the culture medium immunoreactive IGF-I, bound to a carrier protein to form a complex of 25000 d. After gel chromatography in acid conditions a smaller peptide of 8000 d is released. More recently, mRNA for IGF-I has been detected in Leydig cells (41) and both IGF-I and IGF binding protein have been identified in the conditioned media of both Leydig and peritubular cells (42). In Sertoli cells FSH and other agonists which increase cAMP stimulate IGF-I secretion, but inhibit IGF-BP release (42, 43). In Leydig cells, LH increases IGF-I production and a stimulatory effect has been demonstrated for FGF on both the cell types. The production of IGF-I by LeydigSertoli cells cocultures seems to exceed that expected from the monocultures, suggesting that cellcell interactions may also play a role in the control of testicular IGF production (44). Interestingly, thyroid hormones increase IGF secretion by rat Sertoli cells in vitro (45). No clear effect by GH has been demonstrated on cell cultures in vitro, while in testicular tissues of rats treated with gonadotrophins or GH an increase in IGF-I mRNA was detected (36). In another study, however, it has been shown that, while hypophysectomy induces a reduction of IGF-I testicular mRNA, GH treatment alone was unable to normalize IGF-I expression (37). No correlation could be detected between the ontogeny of testicular GHRH RNA, which increase to adult levels by day 30 (37), and either IGF-I or IGF-II mRNA levels. In particular testicular IGF-I mRNA was found to be elevated in neonatal rats, fell to an intermediate levels by day 20 and showed a further decrease after day 60. Evaluation by immunohistochemistry of IGF-I distribution in the rat testis showed that IGF-I immunoreactivity is present in spermatogenic, Sertoli and Leydig cells in the young rat, while only spermatogenic cells are positive in the mature animal (46). IGF-II mRNA can be detected in abundant quantities in the testes of 2 days old rats, but it falls rapidly by day 10 and it is expressed in extremely low levels in the adult rat testis (35-37). Furthermore, its expression appears to be independent from hormonal control (36). These observations are in keeping with the view that this factor is mainly involved in fetal development. The presence of IGF-I in human testis has been confirmed by immunohistochemical studies and IGF-I has been preferentially localized in Sertoli cells, while less evident positivity has been found associated with primary spermatocytes and with some Leydig cells (47). Both IGF-I and IGF-I binding protein are present in human seminal plasma (48-50).
Table 1 - Growth factors involved in the local regulation of testicular function. Factor
Origin
IGF-I
Leyding c. Sertoli c. peritubular c. Leydig c.
EGF-TGF
Target
Actions Potentiation of LH-Induced steroidogenesis
Sertoli c.
Proliferation Metabolic effects Induction of differentiated function
germinal c.
Regulation of spermatogenesis (?)
a-TGF Sertoli c. germinal c. EGFa-TGF peritubular c. Leydig c.
Stimulation of meiosis Modulation of steroidogenesis
peritubular c. Proliferation
Beta-TGF Sertoli c.
Sertol i c.
(7)
Leydig c.
Inhibition of steroidogenesis
Sertoli c.
metabolic effects
perituberal c. differentiation NGF
germinal c.
germinal c.
seminiferous tubules
Sertoli c.
structure
pentubular c. bFGF
Sertoli c.
SGF
Semiferous t. Sertoli c.
Sertoli c. Leydig c.
Prol iferation Induction differentiated functions Mitogenic activity
been shown that in vivo administration of GH to hypophysectomized rats increases the number of type I IGF receptors on Leydig cells (31). Recently, a GHRH-like peptide has been identified within the testis (32), and localized, by immunohystochemistry, mainly in germ cells (33). Origin of /GFs in the testis
Ritzen first reported that a substance with somatomedin activity accumulated in the incubation medium of isolated rat seminiferous tubules (1), and immunoreactive IGF-I has been found in rat testicular extracts (34). Moreover, IGF-I mRNA has been detected in testicular tissue (35, 36, 37) and testicular cDNA encoding IGF-I precursors have been
68
Growth factors and testis
Binding sites for IGFs in the testis High affinity binding sites for IGFs have been identified in testicular membrane preparations (51), in Leydig (52, 53) and in Sertoli cells (54, 55). Purified Leydig cells present type I IGF receptors, and Scatchard analysis reveals a linear binding. In rats treated with hCG, IGF-I receptor number on Leydig cells is increased, without affinity modifications (31, 56) and a similar effect has been shown on pig Leydig cells (53). Both type I and type II receptors have been identified in Sertoli cells in culture. IGFI binding is linear and fifty percent of displacement of the tracer is observed with a 2 nM concentration of IGF-I, while insulin is less effective. Using immunofluorescence techniques in rat Sertoli-germ cells co-cultures, immunoreactive IGF-I was found associated with pachitene spermatocytes but not with spermatogonia (57). In human testis the major positivity for the IGF-I receptor was found in secondary spermatocytes and in early spermatides (47), and, therefore, a possible role for this growth factor in the regulation of the meiotic process has been suggested. Sertoli cells were found less positive, while an intense positivity has been observed in some Leydig cells (47).
IGFs (54, 65), and FSH and IGF-I together exhert a synergistic effect on DNA synthesis in Sertoli cells obtained from neonatal rats (66). Tubular lumen
Interstitial tissue
Tubular lumen
IGF-I actions on testicular cells
IGF-I increases the number of LH receptors on Leydig cells in culture and hCG-stimulated testosterone secretion. Insulin is ten fold less potent than IGF-I in inducing these effects (58-60). IGF-I increases also hCG-stimulated cAMP production and 8 bromo cAMP-induced testosterone secretion. Therefore, it has effect at both the LH receptor sites and the steps beyond adenilate cyclase. An increase of 3 beta hydroxysteroid dehydrogenase activity has also been described (60). Cocultures of Leydig and Sertoli cells produced an increase in both hCG receptors and hCG-induced testosterone production (61-63). Addition to the medium of FSH, insulin or IGF-I produced a marked increase in these two parameters of Leydig cell function. Moreover, conditioned media from Sertoli cells cultured in the absence of insulin and FSH inhibited hCG responsiveness. Immunoneutralization experiments show that IGF-I is an important permissive factor to maintain steroidogenesis in Leydig cells and in cocultures, but an IGF-I antiserum could not completely neutralize the stimulatory effect of coculture (64). Other factors therefore seem to be involved. Sertoli cells prepared from immature rats and pig showed an increased incorporation of 3H-thymidine into DNA in response to micromolar concentrations of insulin or nanomolar concentrations of
FSH
Interstitial tissue
Vascular lumen
Fig. 1 - Schematic representation of the various growth factors and hormones involved in the paracrine regulation of testicular function. (TH = Thyroid hormones).
69
G. Giordano, P. De/ Monte, and F. Minuto
Both IGF-I and IGF-II are able to increase the production of lactate, the incorporation of 3H-leucine into proteins and the transport of glucose (54). Moreover, FSH and IGF-I synergistically increase cAMP production and plasminogen activator secretion (67) by Sertoli cells in culture, and a stimulatory effect of IGF-I on androgen binding protein production has been demonstrated (66). The idea that the IGF-I may play a role in the regulation of spermatogenesis is supported by the findings that this peptide is produced by Sertoli cells and that it has been found associated with spermatic cells. It must be underlined that because the entry of circulating factors into the tubular fluid is restricted, germ cells are exposed only to the locally produced peptides. Vitamin A withdrawal in the rat results in an arrest of spermatogenesis and in this condition a reduction in testicular IGF-I content, which is reversed by vitamin A replenishment, has been observed (68). In men, IGF-I content in seminal fluid of azoospermic subjects shows a tendency to the reduction (50). Further studies, however, are necessary to understand the functions of testicular IGF-I on spermatogenesis. The role of the somatomedin binding proteins identified in the testis is still unknown, it seems likely that they play a role in the modulation of IGFs actions.
and Sertoli cells. Alpha-TGF is a polypeptide that shares sequence omology with EGF and binds with high affinity to the EGF receptor. Isolated peritubular and Sertoli cells contain mRNA for alpha-TGF and this peptide accumulate in media conditioned by both cell types (72). EGF/alpha TGF receptors have been identified in peritubular cells and in Leydig cells (72-74). Recently, it has been shown that alpha TGF can stimulate peritubular cells proliferation and it has been suggested that it can influence Sertoli cell function indirectly, through action on peritubular cells (5, 72). The effects of EGF on steroidogenesis are still controversial. In a study performed on Leydig cells derived from immature hypophysectomized rats it was observed that EGF inhibits (75) hCG-stimulated production of testosterone, androstenedione and 17 OH-progesterone, and the conversion of exogenous 17 alpha OH-Pg to androstenedione. On the contrary, an increased production of pregnenolone and progesterone was evidenced. Therefore it has been suggested that EGF inhibits both 17 alpha hydroxylase and 17-20 lyase. Other authors, however, have shown a stimulatory effect of EGF on steroidogenesis in Leydig cells (76). Recently, it has been shown that EGF decreases DHEA accumulation in hCG-treated porcine Leydig cells and activates testosterone formation by stimulation of 3 beta-hydroxysteroid dehydrogenase enzyme activity (74). Studies performed with stage synchronization of seminiferous epithelium by withdrawal and subsequent replenishment of vitamin A in the rat show increased testicular concentration of EGF at stages IX-XIV of the cycle of the seminiferous epithelium, where the three mitotic division of type A spermatogonia occur in the rat (68), providing evidence of a correlation between EGF or an EGF-like factor and the regulation of spermatogonial cell division.
EPIDERMAL GROWTH FACTORfTRANSFORMING GROWTH FACTOR ALPHA(EGFm~haTG~
The possibility that EGF is involved in the regulation of testicular function was first suggested by the observation that sialoadenomectomy in the rat is accompanied with a reduction of the number of mature sperm in the epididymis (69). The number of spermatides in the testis is also decreased, while the spermatocytes are slightly increased. Administration of EGF restores a normal spermatogenesis. It seems, therefore, that EGF plays a role in male reproductive function by stimulating the meiotic phase of spermatogenesis. Epidermal growth factor-like activity has been detected in human seminal plasma and a specific EGF binding activity was observed in testicular membrane preparations (70). Sertoli cells conditioned media contain a mitogen, hormonally regulated by FSH, testosterone and retinol, that competes with 1251-EGF for the binding to the EGF receptors (71) It seems likely that this peptide is related to alpha-TGF, a mitogen produced by normal and transformed cells, that has been recently identified in both peritubular
TRANSFORMING GROWTH FACTOR BETA (TGF beta) TGF-beta peptides belong to the same family as inhi bins, activins and anti mullerian hormone. Both Sertoli cells and peritubular cells produce TGF-beta (77). It has been shown that this peptide inhibits several Leydig cell functions, and in particular it reduces the number of LH receptors and the cAMP response to hCG. Moreover, a dose-dependent inhibition of hCG-stimulated testosterone production was observed when cells were pretreated with beta-TGF (78). This peptide also reduced forskolin and (Bu) cAMP-induced testosterone production, indicating that it can inhibit steroidogenesis distal to the formation of cAMP (78). The conversion of ex-
70
Growth factors and testis
ogenously added testosterone precursors (progesterone and 17 -OH progesterone) to testosterone by hCG stimulated cells was also suppressed by the addition of beta-TGF, suggesting that this peptide affects the activity of the 17 alpha hydroxylase enzyme. Endogenous progesterone accumulation did not increase when hCG-stimulated testosterone production was inhibited by TGFbeta indicating that steps before the formation of progesterone are also inhibited (78). On the other hand, it has been shown that this peptide increases the activity of 3 beta-hydroxysteroid dehydrogenase, a key enzyme in the maturation of Leydig cells (79). TGF-beta receptors are present in Sertoli cells and this factor is able to stimulate lactate production and glucose uptake (80). In peritubular cells, TGF-beta can inhibit TGF-alpha/EGF stimulated growth, while it promotes cell differentiation and chemiotaxis (5, 77) and can increase the production of specific proteins, such as plasminogen activator inhibitor (81).
cells. NGF mRNA and NGF immunoreactivity have been identified mainly in spermatocytes and early spermatids (90, 91). Recently, NGF receptor gene expression has been observed in Sertoli cells, where it is down-regulated by androgens (92). These findings suggest that NGF may mediate regulatory interactions between germ cells and Sertoli cells. Moreover, NGF affects the morphology of the seminiferous tubules, and maintains their normal structure in vitro (93). By immunohistochemical methods, target cells for this peptide were identified within the lamina propria of the tubules (94).
SEMINIFEROUS GROWTH FACTOR This was the first growth factor isolated from the seminiferous tubules, and it has been partially characterized as a protein of 15750 m.w. with a pi of 5.2, immunologically different from both acid and basic FGF (17,95). It is a broad-spectrum mitogenic factor and is endowed with pleiotypic action in testicular cell lines (96, 97). In conclusion, there are now increasing evidences that a large number of growth factors and of other specific substances are involved in the physiological regulation of the testis. It should be underlined, however, that the actions of most growth factors are interdependent on the presence of other growth factors and regulatory peptides, each working like a player in an orchestra. This fascinating chapter is open to further development, particularly as regard the interrelationships between classical endocrine hormones and local paracrine factors.
FIBROBLAST GROWTH FACTOR (FGF) Basic FGF has been purified from bovine testis (82, 83). The purified protein is a truncated form of bFGF (aa 16-146), with full mitogenic activity on fibroblast and endothelial cells. Recently bFGF has been demonstrated in Sertoli cell conditioned media (84). Furthermore, it has been shown that bFGF exerts a mitogenic effect on immature pig Sertoli cells and that the simultaneous addition of FGF and IGF-I led to a synergistic response (65). Moreover, in presence of insulin, FGF increased FSH binding, cAMP production and plasminogen activator activity of pig Sertoli cells (65). FGF effect on transferrin secretion by Sertoli cells appears to be biphasic, in fact acute treatment stimulates the release of transferrin by some Sertoli cells, while chronic treatment decreases the fraction of Sertoli cells secreting this factor (85). Moreover, it has been shown that FGF stimulates c-fos and B-jun gene expression in Sertoli cells (84). Recently, FGF actions on Leydig cells have been described. It inhibits LH-stimulated androgen production (86), 5-alpha reductase activity (87) and 5-3 beta hydroxysteroidodehydrogenase isomerase activity (88), but it stimulates aromarase activity (89). Therefore, it can be involved in the local regulation of androgen production.
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