Hormonal regulation of spermatogenesis and spermiogenesis

Journal of Steroid Biochemistry & Molecular Biology 109 (2008) 323–330

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Hormonal regulation of spermatogenesis and spermiogenesis夽 Nikolaos Sofikitis ∗ , Nikolaos Giotitsas, Panagiota Tsounapi, Dimitrios Baltogiannis, Dimitrios Giannakis, Nikolaos Pardalidis Department of Urology, Ioannina University School of Medicine, Panepistimioupolis, Metavatiko Building, Ioannina 45110, Greece

a r t i c l e Keywords: Hormones Spermatogenesis Spermiogenesis Growth factors

i n f o

a b s t r a c t Normal testicular function is dependent upon hormones acting through endocrine and paracrine pathways both in vivo and in vitro. Sertoli cells provide factors necessary for the successful progression of spermatogonia into spermatozoa. Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone which are the main hormonal regulators of spermatogenesis. Hormones such as testosterone, FSH and luteinizing hormone (LH) are known to influence the germ cell fate. Their removal induces germ cell apoptosis. Proteins of the Bcl-2 family provide one signaling pathway which appears to be essential for male germ cell homeostasis. In addition to paracrine signals, germ cells also depend upon signals derived from Sertoli by direct membrane contact. Somatostatin is a regulatory peptide playing a role in the regulation of the proliferation of the male gametes. Activin A, follistatin and FSH play a role in germ cell maturation during the period when gonocytes resume mitosis to form the spermatogonial stem cells and differentiating germ cell populations. In vitro cultures systems have provided evidence that spermatogonia in advance stage of differentiation have specific regulatory mechanisms that control their fate. This review article provides an overview of the literature concerning the hormonal pathways regulating spermatogenesis. © 2008 Elsevier Ltd. All rights reserved.

1. The role of the Sertoli cell in the regulation of germ cell life and death Spermatogenesis is a cyclic process, which can be divided into 12 stages (I–XII) in mice [1]. In stage VIII, As, Apr and a few Aal spermatogonia are present. From stage X onwards, these cells start to proliferate in such a way that the numbers of As and Apr spermatogonia remain relatively constant and more Aal spermatogonia are formed. At about stages II–III (stage XII is followed by stage I), proliferation stops and the cells become arrested in G1–G0 phase. Subsequently, in stages VII–VIII, without division, nearly all Aal spermatogonia formed during the period of active proliferation differentiate into A1 spermatogonia. The A1 spermatogonia enter S phase and in stage IX, divide into A2 spermatogonia, after which there are five subsequent divisions into A3, A4, ln and B spermatogonia and primary spermatocytes, respectively. In total, there are 9–11 mitotic divisions during spermatogonial development. When the numbers of A4, ln and B spermatogonia are low, the proliferation period is extended to stage VII. There appears to be a feedback mechanism between A4, ln and B spermatogonia and

夽 Presented at the ‘12th International Congress on Hormonal Steroids and Hormones & Cancer’ (Athens, Greece, 13–16 September 2006). ∗ Corresponding author. E-mail address: [email protected] (N. Sofikitis). 0960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2008.03.004

the As, Apr and Aal spermatogonia lying in between these cells. When the numbers of A4–B spermatogonia are about 50% lower than in the normal testis, the proliferation activity of the As, Apr and Aal spermatogonia continues beyond the stage II. Whether the spermatogonial stem cell (SSC) divisions are symmetrical or asymmetrical is a subject of debate. Symmetrical divisions imply divisions by either producing two stem cells or two interconnected cells destined to differentiate (Apr). Another possibility is that SSCs divide asymmetrically into a stem cell and a cell destined to produce Apr spermatogonia and, therefore, not all As spermatogonia are true stem cells. Two differentiation steps appear to occur in the developmental path of spermatogonia. First, there is the step from the As spermatogonia to the Apr spermatogonia. From then on, the germ cells consist of clones of interconnected cells of increasing size, as from Apr onwards all divisions are such that the daughter cells remain connected by bridges. The second differentiation step is that from Aal to A1 spermatogonia and this step brings about a marked change in cell behaviour. As mentioned above, several studies have assumed that SSC divisions are symmetrical with divisions by either producing two new stem cells or two interconnected cells destined to differentiate (Apr). Another probability is that stem cells divide asymmetrically into a stem cell and a cell destined to produce Apr spermatogonia. No definite answer can be given yet to the question of whether rodent stem cell divisions are symmetrical or asymmetrical. In Drosophila testes, germ line stem cells normally divide

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asymmetrically, giving rise to one stem cell and one gonialblast which initiates differentiation starting with the spermatogonial transient-amplifying divisions [2]. The hub, a cluster of somatic cells at the testis apical tip, functions as a stem cell niche: apical hub cells express the signalling ligand unpaired, which activates the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway within germ line stem cells to maintain stem cell identity. 2. Factors secreted by Sertoli cells Sertoli cells are the only somatic cells in the seminiferous tubuli. The essential role of Sertoli cells in testicular function is stressed by the fact that (a) ‘germ cell-only testes’ have never been observed, (b) the great majority of in vitro culture systems for successful differentiation of germ cells requires the presence of Sertoli cells and (c) the number of germ cells sustained by the testes is directly related to the population of Sertoli cells [3]. Sertoli cells limit the expansion of the spermatogonial population, with each Sertoli cell supporting a defined number of germ cells. Sertoli cells form niches for germ cells; these niches allow a certain number of germ cells to reside in or repopulate the seminiferous tubules [4]. Moreover, in experiments using animal models in which the size of the testis and/or the spermatogenic output was manipulated by changing the number of Sertoli cells, there was a relatively constant ratio between Sertoli cells and spermatids before and after the manipulation [5–7]. Sertoli cells provide critical factors necessary for the successful progression of spermatogonia into spermatozoa. According to Griswold [3], glycoproteins secreted by Sertoli cells important for spermatogenesis could be divided into three categories: (a) those that facilitate the transport of ions and hormones or provide bioprotective functions, such as androgen-binding protein (ABP), transferrin and ceruloplasmin, (b) proteases and proteases inhibitors (these proteins have a role in tissue remodelling processes that occur, for instance, during spermiation or movement of preleptotene spermatocytes into the adluminal compartment of the seminiferous tubule) and (c) structural components of the basement membrane between the Sertoli cells and the peritubular cells. A specific transferrin system has evolved to ensure that the tight Sertoli cell junctions are circumvented so that the germ cells can receive a supply of ferritin by Sertoli cells. Recently, vasoactive peptides and tachykinins have been localized in Sertoli cells [8]. Tachykinins have been shown to stimulate the release of lactate and transferrin by Sertoli cells in vitro and also to stimulate aromatase activity by Sertoli cells [8]. Furthermore, the Sertoli cells secrete other glycoproteins that function as growth factors or paracrine factors, such as the Mullerian duct inhibiting substance, c-kit ligand (stem cell factor; SCF), inhibin and glial cell line-derived neurotrophic factor (GDNF). Both survival and proliferation of spermatogonia were found to be stimulated by the administration of SCF [9]. At the beginning of spermatogenesis there is a dramatic shift in the production (by Sertoli cells) of soluble SCF to membrane-bound SCF [10], suggesting an important role for the SCF–c-kit system in spermatogonial differentiation. C-kit-receptor and SCF also mediate Sertoli cell–spermatocytes cellular adhesions. This is confirmed by studies indicating that Sertoli cells from mice mutant for the membranebound SCF are unable to bind spermatocytes [11]. Other molecules, the cadherins, secreted by Sertoli cells also have an important role in the maintenance of the structure of testicular tissue and cell architecture and identity. Expression of mRNA coding for the three ‘classical’ cadherins, E-, P- and N-cadherin, has been demonstrated in the developing rat testis [12]. Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone, which are the main hormonal regulators of

spermatogenesis. Mutations of the FSH receptor have been associated with variably severe reduction in sperm count, but fertility has been maintained [13]. FSH alone or in synergy with testosterone has been shown to prevent germ cell loss or to restore spermatogenesis quantitatively in hypophysectomized animals [14]. In vitro studies in human testicular tissue material demonstrate the role of FSH and testosterone in the prevention of germ cell apoptosis [15,16], suggesting that both hormones act as germ cell survival factors. The role of Sertoli cells in the regulation of the apoptotic mechanisms of germ cells is confirmed by the fact that the expression of Fas ligand in the testis is mainly localized in Sertoli cells [17]. 3. Germ cell regeneration and death 3.1. Spontaneous apoptosis in the human testis The maintenance of normal architecture of the seminiferous tubuli is achieved by a dynamic balance of germ cellular regeneration and elimination. Sinha Hikim et al. [18] have provided strong evidence that germ cell death during normal spermatogenesis in men occurs via apoptosis and have indicated the presence of ethnic differences in the inherent susceptibility of germ cells to the apoptotic cell death. In addition, apoptosis has been reported to be a possible mechanism of spermatogonial death in pre-pubertal boys [18] or of 2-methoxy acetic acid-induced spermatocyte death in cultured seminiferous tubules of middle-aged human donors [18]. The above investigators provided strong evidence for the presence of germ cell apoptotic process in the human. The exact incidence of adult male germ cell apoptosis remains unclear, since not all degenerating germ cells display the classical morphology of apoptosis. Spermatogonia and round spermatids almost certainly die by apoptosis, since they demonstrate many of the apoptosis classical morphological and biochemical features, such as compaction of DNA at the nuclear margin and labelling of nuclei by terminal deoxynucleotidal transferase [19]. Apoptotic round spermatids often degenerate en masse as multinucleated symplasts. Apoptotic germ cells are either sloughed into the tubule lumen or phagocytosed by Sertoli cells. The extent of spermatocyte and elongated spermatid apoptosis is less clear; some can be labelled with terminal deoxynucleotidal transferase and annexing V, but they do not show the characteristic nuclear changes usually associated with apoptosis possibly due to the unusual morphology and DNA configuration of these cells [19]. Whether male germ cells survive or die is determined by a complex network of signals. These include paracrine signals such as the SCF, leukaemia inhibitory factor (LIF) and Desert Hedgehog (Dhh) [20], as well as endocrine signals such as pituitary gonadotrophins, estrogens and testosterone, among others [21,22]. In addition, male germ cells respond to external signals, and to their internal milieu, by activating intracellular signalling pathways that ultimately determine their fate. 3.2. The role of the Bcl-2 signalling pathway in regulating the mitochondria-dependent apoptotic pathway in human Proteins of the Bcl-2 family provide one signalling pathway which appears to be essential for male germ cell homeostasis. Some members of this family promote cell survival (i.e. Bcl-2, Bcl-xl and Bcl-w, among others) while others antagonize it (e.g. Bax, Bak and Bim, among others). Up-regulation of Bax expression is a feature of germ cell apoptosis in vitro. The pro-survival protein Bcl-xl may play an important role in determining germ cell fate. Bcl-xl potentially promotes germ cell survival during embryogenesis. Bcl-xl may also regulate germ cell survival during the first wave of spermatogenesis since it is expressed at high levels in testis at this time.

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In the adult testis, Bcl-xl is less abundant than in immature testis and appears to be restricted to spermatocytes and spermatids. The pro-survival protein Bcl-w plays an important role in the regulation of testicular germ cell number. The incidence of germ cell apoptosis in Bcl-w knockout mice becomes dramatically elevated between 2 and 4 weeks of age [23]. Other members of the Bcl-2 family are expressed in the testis, but their role in spermatogenesis has not been clarified (i.e. Bad, Bok, Bcm and Boo9, among others). Oldereid et al. [24] have shown that spontaneous apoptosis occurs in male germ cell subpopulations in the human and that Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments. They provided evidence for a specific role for these proteins in the processes of cellular differentiation and maturation during the human spermatogenesis process. Bcl-2 and Bak are preferentially expressed in the compartments of human spermatocytes and differentiating human spermatids [24]. Bcl-x is preferentially expressed in human spermatogonia. Bax demonstrates a preferential expression in the nuclei of human round spermatids. Bad can be detected by immunochemistry in the acrosome region of various stages of human spermatids. On the other hand, Mcl-1 staining does not demonstrate a particular pattern in the human testis. In the human testis, Bcl-w, p53 and p21 cannot be detected. Since Bax is an apoptotic promoter, the Bax preferential expression in human round spermatids may suggest that round spermatids may be particularly prone to apoptosis when DNA is damaged. The apoptotic rate in spermatogonia is significantly lower in aged men compared with controls [25]. However, in that study [25], the balance of spermatogonial proliferation and apoptosis showed no significant difference between the group of aged men and the control group. This is believed to be one of the reasons explaining why spermatogonial numbers in aged men are similar to that of controls. On the other hand, the apoptotic rate of primary spermatocytes in aged men is significantly elevated compared with younger controls resulting in a decrease of the number of human primary spermatocytes per Sertoli cell in aged men. Furthermore, Kimura et al. [25] have demonstrated that the expression of Bcl-xl is inversely related with the apoptotic rate in human primary spermatocytes, suggesting that Bcl-xl may contribute to the regulation of human primary spermatocyte apoptosis. 3.3. Growth factors and apoptotic pathways in the testis Grataroli et al. [26] demonstrated the tumour necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) and its receptors in different human testicular germ cell types. In addition, TRAIL, DR5/TRAIL-R2 (receptor) and DcR2/TRAIL-R4 (receptor) are localized in Leydig cells. DR4/TRAIL-R1 (receptor) is seen in human peritubular and Sertoli cells. It appears that the TRAIL pathway may have a role in the induction of apoptosis in the human testis [26]. Several paracrine signals are regulators of germ cell fate. LIF promotes the survival of primordial germ cells (PGC) in culture. Other factors that promote PGC survival in vitro include interleukin-4 (IL4), basic fibroblast growth factor (FGF), a soluble form of SCF and the bone morphogenetic protein (BMP)-4. In contrast, transforming growth factor-b (TGF-beta) has been reported to promote gonocyte apoptosis in vitro. The survival of gonocytes co-cultured with Sertoli cells is promoted by bFGF, LIF and ciliary neurotrophic factor [27–29]. In adults, members of the BMP family promote germ cell survival in vivo. BMP-8A and BMP-8P appear to provide survival signals to spermatocytes. Dhh secreted by Sertoli cells is another paracrine signal known to promote germ cell survival indirectly. GDNF, neurturin, persephin and artemin are related members of transforming growth factor-b (TGF-b) superfamily [30–33]. GDNF mRNA was found to be expressed in many tissues in addition to the brain and kidney, including intestine, stomach, muscle, carti-

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lage, lung and testis [34]. Expression of GDNF mRNA in testis is related with the expansion of the Sertoli cell population. GDNF contributes to the paracrine regulation of spermatogonial self-renewal and differentiation [35]. The platelet-derived growth factor-A (PDGF-A) and PDGF-B genes encode A and B chains of the PDGF and are located on human chromosome 7p and 22q, respectively [36]. PDGF is produced by many cells and exerts its effects on cells by receptor phosphorylation, leading to cellular responses including migration, proliferation, contraction and alteration of cellular metabolic activities such as matrix synthesis, cytokine production and lipoprotein uptake [37]. PDGF-A gene, PDGF-B gene and the genes encoding the PDGF receptor alpha and beta subunits are expressed in the human fetal testis and this expression increases in the adult testis, suggesting a connection between the PDGF system and the initiation of spermatogenesis [38]. Human Leydig cells express both the ligands and receptors of the PDGF system. This allows us to hypothesize that the ontogeny of this cell type is profoundly influenced by PDGF [38]. 3.4. Effects of factors derived by Sertoli cells on the fate of testicular germ cells In addition to paracrine signals, germ cells also depend upon signals derived from Sertoli by direct membrane contact. Membrane-bound SCF is expressed on Sertoli cell precursors in the embryonic genital ridge and its receptor, the c-kit tyrosine kinase, is expressed on the surface of adjacent PGCs. SCF is also required during the first wave of spermatogenesis. In adults, membrane-bound SCF is expressed on the basal regions of Sertoli cells, while c-kit is expressed on the corresponding surface of spermatogonia. When SCF/c-kit interaction in adults is blocked in vivo, the incidence of apoptosis in spermatogonia and spermatocytes is increased. The ckit gene is the cellular homologue of the feline sarcoma oncogene v-kit [39]. It is located at the White spotting (W) locus in the mouse and on chromosome 4 in the human [40,41]. The ligand SCF has been identified as an analogue of the murine Steel (Sl) gene and is located on chromosome 12 in the human encoded by 9 exons [42]. In the post-natal and adult testis, the c-kit is detected in the proliferating spermatogonia A1–A4 and is also present in interstitial somatic Leydig cells [43,44]. In contrast, Sertoli cells are the unique source of SCF in the testis [45]. SCF/c-kit system is involved in different functions in the testis, including germ cell (GC) migration, cell adhesion, cellular proliferation and anti-apoptotic actions. W and Sl homozygous mutations, resulting in the absence of functional production of c-kit or SCF, respectively, are associated with the absence of germ cells in the post-natal testis. These alterations of spermatogenesis are related to defects in PGC migration and/or induction of apoptosis [46]. Therefore, the SCF/c-kit complex appears to represent one of the key regulators of spermatogenesis. Pentikainen et al. [47] have shown that the Fas–FasL system regulates germ cell apoptosis in the human testis. Expression of FasL has been observed in the human testis. Antagonistic antibodies to the FasL block human germ cell apoptosis in vitro [47]. Among the apoptotic receptors comprising the tumour necrosis factor receptor superfamily, CD95/APO-1 (Fas) is the best characterized. FasL, a cell surface molecule binds to its receptor Fas, thus inducing apoptosis of Fas-bearing cells [48]. Fas is abundantly expressed in various tissues, particularly in activated T and B cells, thymocytes, hepatocytes and the heart tissue [49]. Of particular interest is the observation that FasL is constitutively expressed by cells in immune privileged sites such as the testis [50] and the anterior chamber of the eye [51,52]. Fas is expressed at low levels in the mouse testis [17,53] and appears to be restricted to some germ cells [17]. In addition, Sertoli cells may

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employ the Fas system to regulate the germ cell fate. Furthermore, with regard to the human testis, the expression and the function of Fas and FasL are a matter of debate. Besides the immunoregulative role of FasL in the testis, the Fas system has also been proposed as a key regulator of physiological germ cell apoptosis [17,47,54,55]. Caspase inhibitors inhibit programmed human germ cell death, suggesting that Fas-associated human germ cell apoptosis is mediated via the caspase pathway. It appears that human germ cell death can be inhibited [47] by blocking the interaction between Fas and FasL. The FasL is constitutively expressed by the human Sertoli cells and is suggested to bind to the Fas molecule of the germ cells. Thus, it causes death of these Fas-bearing germ cells. In rat testis, upregulation of Fas was observed in germ cells undergoing apoptosis after in vivo administration of Sertoli cell toxicants [17]. However, in human testis the expression of Fas does not seem to be up-regulated during an enhanced apoptotic process after the withdrawal of survival factors. Therefore, additional pathways leading to increased apoptosis in the human testis may occur. Alternatively, Pentikainen et al. [47] hypothesized that Fas activation may be more effective in unfavourable conditions, thus enhancing the ability of the Fas–FasL system to mediate apoptotic human germ cell death. 3.5. A role of hormones in the regulation of germ cell apoptosis Hormones such as testosterone, FSH and luteinizing hormone (LH) are known to influence the germ cell fate. Their removal induces germ cell apoptosis. Estrogen treatment, which is thought to mimic a gradual withdrawal of gonadotrophins, also induces apoptosis of all germ cells including elongated spermatids [56]. In addition, Pentikainen et al. [57] have demonstrated that estradiol acts as a germ cell survival factor in the human testis in vitro [57]. In human seminiferous tubuli, apoptosis is induced under serum-free conditions in vitro [15]. The fact that this apoptosis is suppressed by testosterone indicates that testosterone in the human male is a critical germ cell survival factor. The mechanism by which androgen withdrawal induces germ cell death remains unclear. It is tempting to speculate that androgen withdrawal alters the expression of the Bcl family proteins in germ cells, since Bcl-xl and Bcl-2 in the testis are altered following long-term anti-androgen treatment for prostate cancer. Somatostatin (SRIF) is a regulatory peptide hormone playing a role in the regulation of the proliferation of the male gametes. Its biological actions are mediated by five receptors (sst1–sst5) [58]. The injection of an SRIF analogue (SMS201995) in healthy adult males is followed by a rapid (2 h after the injection) rise in serum testosterone level. Such an increase in testosterone secretion occurs without a simultaneous increase in LH secretion, suggesting that SRIF can modulate testosterone secretion at the testicular level [59]. The presence of SRIF and its receptors in human testes [60] supports the existence of auto/paracrine loops controlling local testosterone secretion. Indeed, sst3, sst4 and sst5 are expressed in human normal testicular tissue, while sst1 and sst2 are usually not detected. Goddard and co-workers have provided evidence for an inhibitory role of SRIF in the control of spermatogonial proliferation. In the perinatal porcine testis, SRIF might exert its actions both directly on spermatogonia by preventing SCF-induced proliferation and indirectly by inhibiting SCF mRNA expression by Sertoli cells [61]. 4. The role of growth factors in gonocyte proliferation and differentiation The cascade of events that mediate the transition of gonocytes to type A spermatogonia is one of the least understood processes that occur during spermatogenesis. Successful research efforts in germ cell cultures have resulted in the development of a clonogenic

method to assay the capacity of gonocytes to proliferate in vitro as described by Hasthorpe et al. [62]. In the latter study a mouse gonocyte was selected taking into consideration its relative large cell size using micromanipulation techniques and placed in a collagen IV-coated microtitre well containing Iscove’s modified Dulbecco’s medium and 20% fetal calf serum (FCS) for 4–5 days. The gonocytederived colonies consisted of between four and more than 256 cells per colony which enabled certain spermatogonial subtypes to be identified and collected. In that in vitro culture system, Sertoli cells had an inhibitory activity on gonocyte-derived colony formation when added to the cultures of gonocytes. A Sertoli cell line had an even more pronounced inhibitory effect on the proliferation of gonocytes in vitro [63]. Nagano et al. [64] found that Sertoli cell lines resulted in a great reduction of SSCs after 7 days of culture. These findings suggested that Sertoli cellular exocrine or paracrine factors, which are known to support spermatogonial differentiation, cause a significant reduction in the number of SSCs cultured. It appears that the maintenance of SSCs in culture can be achieved by the suppression of germ cell differentiation. These findings are consistent with other studies demonstrating no stimulatory role of Sertoli cells in the proliferation of gonocytes in co-cultures of Sertoli cells with gonocytes [65,66]. The in vitro clonogenic method has been also used to determine the growth factors that regulate mouse gonocyte proliferation and differentiation [63]. In the latter study, it was found that TGFb and epidermal growth factor (EGF) did not exert any inhibitory effect on gonocyte-derived colony formation. Furthermore, Mullerian inhibitory factor and leukaemia inhibitory factor (LIF) had no effects on the proliferation of gonocytes. The authors demonstrated that the growth of gonocytes in vitro was optimal in the presence of FCS. Thus, it has been concluded that no specific growth factors [with a probable exception of platelet-derived growth factor (PDGF) [67]] are necessary for the growth of gonocytes in vitro. However, Nagano et al. [64] found that the addition of GDNF to a culture of mouse SSCs had a positive role in SSC maintenance. Forced expression of GDNF in transgenic mouse testes resulted in the accumulation of undifferentiated spermatogonia in vivo without a change in SSC proliferation kinetics. Thus, it was suggested that GDNF inhibits spermatogonial differentiation [35]. Hasthorpe [67] evaluated the role of SCF in the differentiation of spermatogonia. In that study SCF did not exert any effect on mouse type A spermatogonia colony-forming cells, indicating that more highly differentiated spermatogonia represent the target-cellular population for the SCF. The majority of gonocytes express c-kit mRNA, but fail to respond to SCF, indicating that the receptor is not functional. The PDGF appears to stimulate the proliferation of gonocytes, but not the proliferation of type A spermatogonia recovered from 15day-old animals [67]. 5. Effects of hormones on gonocyte proliferation and differentiation The addition of activin to the gonocyte culture system overrides the antagonistic effect of somatic testicular cells (which produce inhibin bA subunit) on gonocyte proliferation. The addition of activin increased gonocyte colony formation; however, in that study, a very little effect on spermatogonia cells was demonstrated [67]. On the other hand, in a study by Nagano et al. [64] the addition of activin to culture systems significantly reduced the number of SSCs. In the latter study, the authors [64] suggested that the stimulation of spermatogonial proliferation by activin may be exerted on more advanced spermatogonia rather than on SSCs. These inconsistent findings may be attributed to the different methods used for the selection of a spermatogonial population for culture. Hasthorpe [67] used the in vitro clonogenic method while Nagano et al. [64]

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applied a two-step enzymatic digestion method on cryptorchid testes to collect spermatogonia as had been described by Ogawa et al. [68]. The latter method results in the recovery of a heterogeneous population of germ cells and allows the inclusion of testicular somatic cells within the population of the recovered cells. These somatic cells might have negatively affected the proliferation of SSCs in a study by Nagano et al. [64]. Furthermore, the use of feeder layers in a study by Nagano et al. [64] might also have negatively influenced the proliferation of the SSC population. In general, in vitro attempts to improve the viability and differentiation of cultured gonocytes by adding several growth factors to the basic medium did not yield any clear results till date. It appears that activin A, follistatin and FSH play a role in germ cell maturation during the period when gonocytes resume mitosis to form the SSCs and differentiating germ cell populations [69]. Meehan et al. [69] have proposed that germ cells have the potential to regulate their own maturation initially through the production of endogenous activin A. Sertoli cells were observed to produce the activin/inhibin bA subunit, the inhibin a subunit and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. The authors used 1- and 3-day-long cultures of 3-day-old rat testicular fragments and observed that treatment with activin A produced a significantly higher ratio of germ cells to Sertoli cells, whereas treatment with follistatin and FSH increased the number of spermatogonia. Meehan et al. [69] suggested that locally produced activin can stimulate gonocyte proliferation immediately after birth in the rat testis. Therefore, it is possible that activin and follistatin may play a vital role in the transition of gonocytes to spermatogonia. Toebosch et al. [70] demonstrated that FSH acts indirectly on the gonocytes by inducing Sertoli cell expression of follistatin and inhibin. It appears that the maturation of gonocytes to form spermatogonia could result from the combined effects of follistatin and inhibin as activin antagonists, with FSH as the stimulus for inhibin production, thus producing effects on both germ cells and Sertoli cells that enable germ cell maturation. In vitro culture systems presented evidence that FSH and activin stimulate Sertoli cell proliferation during early post-natal testis development [71,72]. The receptor of EGF is functional in spermatogonia and EGF has been proposed to inhibit testicular germ cell differentiation. Haneji et al. [73] have proven that EGF blocks the proliferation of adult mouse type A spermatogonia stimulated by FSH. In contrast, Wahab-Wahlgren et al. [74] have shown that EGF stimulates spermatogonial proliferation in adult rat seminiferous tubules in vitro and might have an important role in the paracrine regulation of spermatogenesis. 6. Late spermatogonial development In vitro culture systems have provided evidence that spermatogonia in advance stage of differentiation have different regulatory mechanisms (comparatively with the mechanisms regulating the gonocyte proliferation) that control their fate. Thus, SCF and its receptor c-kit play an important role in relatively late spermatogonial development. Dirami et al. [75] showed that the SCF acts as a mitogen and survival factor for spermatogonia type A cultured in a potassium-rich medium Potassium Simplex Optimized Medium (KSOM). In the same study, granulocyte macrophagecolony stimulating factor also enhanced the survival of porcine type A spermatogonial cells. Nakayama et al. [76] demonstrated that insulin-like growth factor-I and -II (IGF-I and IGF-II, respectively) as well as insulin promote spermatogonial differentiation into primary spermatocytes. These findings are consistent with the findings in a previous study by Tajima et al. [77] who have demonstrated that IGF-I and transforming growth factor-a (TGFa) stimulate the differentiation of mouse type A spermatogonia in

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organ culture in vitro. In contrast, neither the PDGF nor the fibroblast growth factor (FGF) has the above stimulatory effect. Studies employing a Vero cell conditioned medium rich in growth factors and interleukins showed that in humans FSH inhibits spermatogonia degeneration and stimulates meiosis entry, being further potentiated by testosterone [78]. Tesarik et al. [79,80] demonstrated that high concentrations of FSH represent a prerequisite for the completion of meiosis and spermiogenesis in vitro. In the latter studies, cultured round spermatids underwent nuclear changes similar to those occurring during the normal spermiogenesis process, characterized by nuclear condensation, peripheral migration and protrusion after the addition of high concentrations of FSH into the culture medium. It should be emphasized that the high concentration of FSH (25 IU/l) is necessary to obtain alterations in spermatid morphology in an in vitro culture system. 7. Hormonal mechanisms regulating spermiogenesis Spermiogenesis is a metamorphosis process involving the maturation and differentiation of the early haploid male gamete to a mature spermatozoon. During spermiogenesis, alterations occur in the male gamete nuclear proteins, cellular size, cellular shape, the position and size of pro-acrosomal granules and the localization of the centrioles. This fascinating process that converts a round immotile haploid gamete to an elongated cell with potential for movement is regulated by a complex of factors/mechanisms. The presence of immunoreactive ABP in Sertoli cell processes that surround the elongated spermatids has suggested a role of ABP in spermiogenesis [81]. ABP has a high affinity for androgens probably contributing to the generation of high androgen concentrations in the vicinity of certain meiotic germ cells. O’Donnell et al. [82] and Sofikitis et al. [83] have suggested that following withdrawal of intratesticular testosterone in a rat animal model, round spermatids are unable to proceed through the transition between steps 7 and 8 of spermiogenesis and therefore cannot complete the elongation process. This effect may be mediated by the loss of the adhesions of the spermatids with the sustentacular Sertoli cells [84]. Studies in our laboratory have demonstrated released step 8 round spermatids within the epididymal lumen of rats with low intratesticular testosterone profiles [83]. It has been proven that lowering of intratesticular testosterone concentration results in the apoptotic death of germ cells [85]. In addition, a consequence of decrease in intratesticular testosterone is that round spermatids lose their adhesion to the Sertoli cells, slough into the lumen of the seminiferous tubules and are occasionally phagocytized by Sertoli cells. A recent study has suggested that the Bcl-2-modifying factor (Bmf) is likely to play an important role in germ cell death in response to reduced intratesticular testosterone profiles [86]. Bmf was found to reside in the subacrosomal space of spermatids of steps 4–16 of the spermiogenetic process. The localization of Bmf to this region is not surprising because Bmf is normally sequestered to the actin cytoskeleton via its conserved dynein light chain-binding domain [87]. The actin polymers in the subacrosomal space have been shown to disappear at the late steps of spermiogenesis (step 19) just before the release of mature spermatozoa from the Sertoli cells [86,88]. Show et al. [86] have provided strong evidence supporting the absence of the Bmf protein from the subacrosomal space near the end of spermiogenesis (step 16), suggesting that Bmf is degraded just before the spermatid is released from the seminiferous epithelium. The role of FSH in the regulation of spermiogenesis has been a controversial issue. In the international literature, there are studies supporting the notion that FSH may stimulate early events in spermatogenesis including spermatogonial proliferation and meio-

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