Human Reproduction Update Advance Access originally published online on April 7, 2005
Human Reproduction Update 2005 11(3):229-259; doi:10.1093/humupd/dmi007
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Efforts to create an artificial testis: culture systems of male germ cells under biochemical conditions resembling the seminiferous tubular biochemical environment
1 Laboratory for Molecular Urology and Genetics of Human Reproduction, Department of Urology, Ioannina University School of Medicine, Ioannina, Greece, 2 Department of Urology, Tottori University School of Medicine, Yonago, Japan, 3 Department of Obstetrics and Gynecology, Athens University School of Medicine, Athens, Greece, 4 MFC Clinic, Yonago, Japan, 5 Department of Obstetrics and Gynecology, Aristotle University School of Medicine, Thessaloniki and 6 Department of Urology, Thessalia University School of Medicine, Larissa, Greece
7 To whom correspondence should be addressed at: Department of Urology, Tottori University School of Medicine, 36-1 Nishimachi, 683-8504, Yonago, Japan. Email: akrosnin{at}hotmail.com
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Abstract |
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 TOP Abstract Regulation of spermatogenesis:...Technical prerequisites and...Methods to achieve meiotic...Scientific and clinical...Genetic and epigenetic risks...References |
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Induction of meiotic and post-meiotic alterations of male germ cells in vitro has been the target of several research efforts since 1960. However, to date, the establishment of an ideal culture system in which spermatogonial stem cells can be maintained and directed to proliferate and undergo meiosis and complete spermiogenesis does not exist. This is attributed to the difficulties concerning the isolation and purification of defined subpopulations of germ cells and the establishment of male germ cell lines. In addition, there is no adequate knowledge regarding the optimal biochemical conditions that promote the survival and differentiation of germ cells in long-term cultures. This review focuses on the methodologies that have been proved sufficient to achieve differentiation of cultured male germ cells. Furthermore, the factors regulating spermatogenesis and the technical prerequisites to achieve differentiation of cultured male germ cells are described. Finally, the role of in vitro cultures of immature diploid germ cells in the therapeutic management of men negative for haploid cells in their testes and the subsequent potential genetic and epigenetic risks are discussed.
Key words: artificial testis / in vitro culture system / meiotic maturation / spermatogonial stem cell
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Regulation of spermatogenesis: what we have learned from in vitro cultures of spermatogenic cells |
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TOPAbstractRegulation of spermatogenesis:...Technical prerequisites and...Methods to achieve meiotic...Scientific and clinical...Genetic and epigenetic risks...References |
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The role of the Sertoli cell in the regulation of male germ cell proliferation/differentiation and the apoptotic mechanisms of germ cells
In mammalian embryos, gonads develop in the bilateral gonadal ridges from the interaction of primordial germ cells (PGCs) with local somatic cells represented by two different populations: mesenchymal cells of the mesonephric region and epithelial cells of the overlying coelomic epithelium (Kierszenbaum, 1994
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In mouse, about eight PGCs depart from their site of origin, the yolk sac, and translocate to the epithelium of the hindgut adjacent to the yolk sac. PGCs leave the hindgut by active mechanism to enter the dorsal mesentery and finally settle in the gonadal ridge. Soon after reaching the gonadal ridges, PGCs increase in number and begin their cell–cell interaction with coelomic epithelial and mesenchymal cells to organize testicular cords. Male PGCs become gonocytes, the cell precursor of spermatogonia and enter a mitotic arrest stage (Kierszenbaum, 1994).
Spermatogenesis is a cyclic process, which can be divided into 12 stages (I–XII) in mice (Kierszenbaum, 1994). 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 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.
It should be noted that in rat, type A1–A4 spermatogonia are the renewing stem cells and mitosis of these cells constitutes the major phase of germ cell proliferation. The last division of the renewing stem cells (A4) in rat results in the first of two generations of differentiated spermatogonia [intermediate (1N) and B]. In monkey, Ap spermatogonia are the renewing stem cells and the major phase of germ cell proliferation results from mitosis of differentiated type B1–B4 spermatogonia (Plant and Marshall, 2001). The reserve stem cells in primates and rats rarely divide. There appear to be fundamental differences between monkey and rat in the mechanism used for stem cell renewal and germ cell proliferation. Stem cell renewal in rat involves sequential divisions to produce four generations of undifferentiated spermatogonia (A1, A2, A3 and A4) and therefore concomitantly contributes to germ cell proliferation (Kierszenbaum, 1994). On the other hand, in monkey stem cell renewal follows a simpler pattern involving only one type of undifferentiated spermatogonia (type Ap) and thus contributes to proliferation only indirectly by the production of the first of the four generations of differentiated B spermatogonia (Kierszenbaum, 1994).
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 asymmetrically, giving rise to one stem cell and one gonialblast which initiates differentiation starting with the spermatogonial transient-amplifying divisions; Yamashita et al., 2003). 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.
Yamashita et al. (2003) have shown that the dividing Drosophila male germ line stem cells use intracellular mechanisms involving centrosome function and cortically localized adenomatous polyposis coli tumour suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation. According to Yamashita et al. (2003), the orientation of stem cells towards the niche appears to play a critical role in the mechanism that ensures a reliably asymmetric outcome of Drosophila male germ line stem cellular divisions consistently placing one daughter within the reach of short-range signals from the hub and positioning the other away from the niche. Oriented stem cell division may be a general feature of other stem cell systems, helping to maintain the correct balance between stem cell self-renewal and initiation of differentiation throughout the adult life.
The development of differential display RT–PCR procedures has led to the identification of many genes that are differentially regulated in various cell and tissue types. The above technique to identify genes that are expressed in isolated mouse testicular type A spermatogonia and in more advanced germ cells. The authors identified cDNA fragments for mDEAH9, RanBP5, GC3, GC12 and GC14 genes in the testis and type A spermatogonia from wild-type mice, but not in samples from mutant W/Wv mouse testis. RT–PCR analyses of isolated spermatogonia, pachytene spermatocytes and round spermatids revealed that mDEAH9, RanBP5, GC3, GC12 and GC14 genes were expressed in all three cellular populations. RanBP5 expression appeared to be regulated during the cycle of the seminiferous epithelium with the highest expression in stages III–VII. Expression of GC14 was greatest in the meiotic germ cellular subpopulations. In addition, it was identified that a murine testis cDNA encoding a homologue to human A-kinase anchoring protein-associated sperm protein (ASP). Northern blot and RT–PCR analyses did not detect ASP mRNA in mouse spleen, brain, liver, lung, heart, kidney, skeletal muscle, ovary or Sertoli cells. On the other hand, the above techniques localized ASP mRNA to the germ cell compartment of the seminiferous tubules in the testis.
Mouse models with reproductive defects as a major phenotype have been created and now number over 200 (Matzuk and Lamb, 2002). These models are helping to define mechanisms of reproductive function, as well as to identify potential new genes involved in the pathophysiology of reproductive disorders. Mouse models for the study of reproductive defects have been produced by spontaneous mutations, transgene integrations, retroviral infection of embryonic stem cells, ethylnitrosurea mutagenesis and gene targeting technology. Several genes required for vertebrate fertility are highly conserved in evolution with orthologues in Drosophila melanogaster (i.e. D DX4), fat facets (DFFRY) and boule (DAZ). Defects in sexual differentiation pathways can cause infertility in mice and humans of both sexes. It has been pointed out by Matzuk and Lamb (2002) that several gene defects or gene-related pathophysiologies leading to defects in sex determination or development (i.e. pseudohermatidism, sex reversal, Denys–Drash syndrome, pseudovaginal perineoscrotal hypospadias, cryptorchidism or congenital bilateral absence of vas deferens), defects in sperm production and function (i.e. myotonic dystrophy, Nooman syndrome, sickle cell anaemia, b-thalassaemia, Kartagener syndrome, primary ciliary dyskinesia, Fanconi anaemia or ataxia telangiectasia), endocrinopathies and numerical/structural chromosomal abnormalities result in human male infertility. Knockout animal models have provided strong evidence supporting the genetic basis of human male infertility in subpopulations of infertile men.
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 (Griswold, 1998). 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 (Meachem et al., 2001). 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 (Orth et al., 1988; Hess et al., 1993; Simorangkir et al., 1995).
Sertoli cells provide critical factors necessary for the successful progression of spermatogonia into spermatozoa. According to Griswold (1998), 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 (Debeljuk et al., 2003). 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 (Debeljuk et al., 2003).
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 (Allard et al., 1996). At the beginning of spermatogenesis there is a dramatic shift in the production (by Sertoli cells) of soluble SCF to membrane-bound SCF (Blanchard et al., 1998), 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 membrane-bound SCF are unable to bind spermatocytes (Marziali et al., 1993). 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 (Wu et al., 1993).
The bradykinin-B2-receptor (B2-r) has been demonstrated to be present on rat Sertoli cells (Monsees et al., 1996, 1999, 2002, 2003). In situ hybridization (ISH) and immunochemistry revealed that B2-r mRNA and protein are solely present on specific cells within the testis. B2-r is located on endothelial cells of blood vessels, Sertoli cells, pachytene spermatocytes and in round and elongated spermatids. Moreover, in the sexually mature rat the expression of B2-r mRNA and protein are dependent on the stage of the spermatogenic cycle (Monsees et al., 2003). This stage-dependent expression of the protease (tissue kallikrein) and the B2-r together with their locations on certain germ cells suggests a potential function of the tissue kallikrein system as a local factor with an effect on spermatogenesis. In organ cultures of immature rat testis a significant stimulation of prespermatogonial cell proliferation after exposure to bradykinin has been observed (Atanassova et al., 1998).
Sertoli cells have receptors for 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 (Tapanainen et al., 1997). FSH alone or in synergy with testosterone has been shown to prevent germ cell loss or to restore spermatogenesis quantitatively in hypophysectomized animals (Marshall et al., 1995). In vitro studies in human testicular tissue material demonstrate the role of FSH and testosterone in the prevention of germ cell apoptosis (Erkkila et al., 1997; Tesarik et al., 2001), 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 FasL in the testis is mainly localized in Sertoli cells (Lee et al., 1997).
Support of xenogeneic germ cell maturation in vivo
Experiments have demonstrated that donor spermatogonia transplanted into the seminiferous tubules of syngeneic animals form cell associations with the recipient Sertoli cells that are characteristic of the normal recipient testis (Brinster and Zimmermann, 1994; Russell et al., 1996; Sofikitis et al., 2003). A subpopulation of donor spermatogonia injected into the lumen of recipient seminiferous tubules moves towards the basal compartment of the testis, implying that the adluminal surface of the recipient Sertoli cells can recognize the donor spermatogonia and direct them to their normal location in the basal compartment. Moreover, successful experiments of xenogeneic germ cell transplantation (Clouthier et al., 1996; Russell and Brinster, 1996; Sofikitis et al., 2003) provide strong evidence that the information necessary for the differentiation of male germ cells (i.e. to complete meiosis and spermiogenesis) is inherent in the germ cells and the role of the Sertoli cells is to facilitate the progression of this process. The maintenance of primate germ cell clusters in mouse testes after the transplantation of primate cells into mouse seminiferous tubules (Nagano et al., 2001b) clearly demonstrates that antigens, growth factors and signalling molecules that participate in the interaction between the donor germ cells and the recipient Sertoli cells have been preserved for 100 million years in these widely divergent species (Nagano et al., 2001b).
Molecular and hormonal mechanisms regulating male germ cell differentiation
Regulation of spermatogonia kinetics and meiosis
Development of a clonogenic assay to evaluate the role of growth factors in gonocyte proliferation and differentiation
The complex process of spermatogenesis has been the target of intensive research efforts for many years. Nowadays, we have a relatively good understanding of the development, proliferation and differentiation of SSCs especially under in vitro culture conditions. In addition, the importance of interaction between somatic cells (Sertoli cells) and SSCs and the role of hormones and intratesticular biochemical factors (growth factors, endorphins) in the various events of the spermatogenic process have been recognized. In vivo, in a normal testis, all the events in SSC development occur at the same time in different regions of the seminiferous tubules and are, therefore, difficult to study. Thus, an in vitro culture system in which SSCs can be maintained, proliferate and proceed through meiosis to the formation of spermatids (in long-term cultures) could be ideal in order to study the complex events of mammalian spermatogenesis. However, no such system exists today because (a) only a limited number of SSCs can be isolated and processed for studies involving in vitro culture systems, (b) germ cells have a limited viability in culture conditions and (c) it is difficult to distinguish SSCs from the more differentiated type A spermatogonia in vitro due to the lack of specific stem cell markers.
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. (1999). 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 gonocyte-derived 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 (Hasthorpe et al., 2000). Nagano et al. (2003) 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 (Orth and Boehm, 1990; de Miguel et al., 1996).
The in vitro clonogenic method has been also used to determine the growth factors that regulate mouse gonocyte proliferation and differentiation (Hasthorpe et al., 2000). In the latter study, it was found that transforming growth factor-b (TGF-b) 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) Hasthorpe, 2003] are necessary for the growth of gonocytes in vitro. However, Nagano et al. (2003) 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 (Meng et al., 2000). Hasthorpe (2003) 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 15-day-old animals (Hasthorpe, 2003).
The role of hormones in the regulation of 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 (Hasthorpe, 2003). On the other hand, in a study by Nagano et al. (2003) the addition of activin to culture systems significantly reduced the number of SSCs. In the latter study, the authors (Nagano et al., 2003) 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 (2003) used the in vitro clonogenic method while Nagano et al. (2003) applied a two-step enzymatic digestion method on cryptorchid testes to collect spermatogonia as had been described by Ogawa et al. (1997). 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 (2003). Furthermore, the use of feeder layers in a study by Nagano et al. (2003) 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 (Meehan et al., 2000). Meehan et al. (2000) 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. (2000) 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. (1988) 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 (Mather et al., 1990; Boitani et al., 1995). The receptor of EGF is functional in spermatogonia and EGF has been proposed to inhibit testicular germ cell differentiation. Haneji et al. (1991) have proven that EGF blocks the proliferation of adult mouse type A spermatogonia stimulated by FSH. In contrast, Wahab-Wahlgren et al. (2003) 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.
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. (1999) 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 macrophage-colony stimulating factor also enhanced the survival of porcine type A spermatogonial cells. Nakayama et al. (1999) 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. (1995) who have demonstrated that IGF-I and transforming growth factor-a (TGF-a) stimulate the differentiation of mouse type A spermatogonia in 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 (Sousa et al., 2002).
Hormonal, molecular and genetic mechanisms regulating spermiogenesis
Hormones
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 (Martin du Pan and Campana, 1993). 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. (1996) and Sofikitis et al. (1999) 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 (Zirkin, 1998). Studies in our laboratory have demonstrated released step 8 round spermatids within the epididymal lumen of rats with low intratesticular testosterone profiles (Sofikitis et al., 1999). It has been proven that lowering of intratesticular testosterone concentration results in the apoptotic death of germ cells (Kim et al., 2001). 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 (Show et al., 2003). 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 (Puthalakath et al., 2001). 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 (Russell et al., 1986; Show et al., 2003). Show et al. (2003) 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 meiosis. However, testosterone only has been considered to sustain complete spermatid differentiation (McLachlan et al., 1995; Singh and Handelsman, 1996). On the other hand, Tesarik et al. (1998a,b) 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. Additional studies by Baccetti et al. (1997) showed that exogenous FSH administration in combination with or without human chorionic gonadotrophin in infertile men improved sperm counts. Furthermore, the administration of FSH had a positive role in sperm cytostructural parameters (Baccetti et al., 1997). Studies by Krishnamurthy et al. (2000) have shown that there is an increase in propidium iodide stainability of elongated spermatids and an increased sperm head size in FSH receptor knockout mice. These findings suggest a disturbance in the normal replacement of histones by protamines during spermiogenesis, leading to poor condensation of spermatid nuclei (Krishnamurthy et al., 2000) in FSH receptor knockout mice. FSH turns Sertoli cells competent to bind round spermatids. In addition, studies in humans showed that FSH stimulates meiosis II and round spermatid flagellum extrusion, whereas testosterone potentiates FSH action and stimulates late spermatid differentiation (Sousa et al., 2002).
Studies by Dinulovic and Rodonjic (1990) in diabetic patients tend to suggest a role of insulin in the spermiogenetic process. The identification of insulin gene family members in round spermatids of human and rat testes has provided additional evidence for a role of insulin and IGF in spermiogenesis (Lok et al., 2000). Another hormone having a potential role in spermiogenesis is prolactin. Prolactin receptor expression has been found in round and elongating spermatids of intact sheep testes (Jabbour and Lincoln, 1999). However, in rat early round spermatids Hondo et al. (1995) did not detect prolactin receptor mRNA expression. The latter findings probably indicate species-dependent differences in the hormones regulating spermiogenesis.
The presence of type I and type II activin receptors in round spermatids indicate that activins may have some actions on early haploid male germ cells (de Winter et al., 1992). Testicular inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells (including the stages from pachytene spermatocytes to early spermatids; Andersson et al., 1998). Marchetti et al. (2003) found that the inhibin bB subunit was immunolocalized in germ cells (pachytene spermatocytes to round spermatids) but not in Sertoli cells. Activin actions may be modulated by actions of follistatin, which has been shown to be produced by Sertoli cells, spermatogonia, primary spermatocytes and round spermatids (Meinhardt et al., 1998).
Molecular and genetic mechanisms
Round spermatids express the precursor forms of the nerve growth factor (NGF) gene product, but not the mature form of NGFb. NGFb moiety of the NGF precursor proteins exhibits trophic activity in the rescue of Sertoli cell viability, consistent with the paracrine regulation of spermatogenesis.
Spermiogenesis is a very sensitive process to alterations in molecular and genetic factors. Generation of animal models by genetic engineering offers the opportunity to discover genetically regulated molecular factors that are implicated in the spermiogenetic process. Alterations in the expression of molecular agents in the testicular tissue due to defects in gene expression (null mutations, gene overexpression, exogenous gene expression and gene misexpression) could lead to deficiency in the completion of different steps of spermiogenesis. Histone replacement by transition proteins (TP) and protamines during spermiogenesis may be affected by disruption of the Tarbp2 gene resulting in infertility and oligospermia (Zhong et al., 1999). A partial or complete failure to synthesize the protamines results in delayed replacement of TP and the spermatids show abnormal nuclear morphogenesis, developmental arrest and degeneration (Zhong et al., 1999). Premature translation of pre-existing protamine-1 (Prm1) mRNA causes precocious condensation of spermatid nuclear DNA and abnormal head morphogenesis (Lee et al., 1995). Successful interaction of mature protamine-2 with chromatin is required for the displacement of TP2 (Wu et al., 2000). Step 15 spermatids in Camk4–/– mice demonstrate a loss of protamine-2. These animals are characterized by prolonged retention of TP2. Mice lacking the major TP1 have been obtained following targeted deletion of the Tnp1 gene. Tnp1–/– mice demonstrate a normal sperm production quantitatively, but only 23% of the spermatozoa show any movement, and most of these do not show forward progression (Yu et al., 2000). In these animals, sperm heads with a blunted or bent tip are seen in 16% of epididymal spermatozoa possibly generated by the abnormal chromatin condensation that could reduce the rigidity of the fine apex of the spermatozoon (Yu et al., 2000). Tnp1 contains a cAMP-responsive element (CRE) that serves as a binding site for the CRE modulator (CREM). CREM is involved in the regulation of the Tnp1 gene expression, and human CREM protein is synthesized in steps 1–3 round spermatids. This may explain why a reduction in Crem expression and a lack of both CREM and TP1 have been demonstrated in human arrested spermatids at step 3 (Steger et al., 1999). Mice with deletion in Crem presented a spermatogenesis arrest at the round spermatid step (De Cesare et al., 1999). CREM is involved in regulating gene expression in round spermatids. Transcriptional activity of the CREM protein is thought to be regulated by the activator of CREM in the testis (ACT). Steger et al. (2004) applying RT–PCR and ISH demonstrated cell-specific gene expression of ACT in the man, cynomolgus monkey and mouse. Steger et al. (2004) have suggested that there is a conserved function of ACT during the evolution of mammalian spermatogenesis. They also suggested that there is a role for CREM in the ACT transcriptional regulation.
Deficiencies in intratesticular molecular factors due to genetic defects affect the organization and reorganization of the cytoskeleton during spermiogenesis. Thus, homozygous c-ros knockout mice are sterile and the epididymal spermatozoa exhibit bent tails and compromised flagellar vigour within the uterus (Yeung et al., 2000). Testicular haploid expression gene (THEG) is expressed in round and elongated spermatids. The molecular products of this gene appear to play a role in the spermiogenesis since abnormal or absent flagella in mice with THEG disruption have been demonstrated and may be due to impairment of the assembly of cytoskeletal proteins such as the tubulins (Yanaka et al., 2000). A specific block in spermiogenesis was observed in homozygous JunD–/– mice. JunD is one of the three mammalian Jun proteins that contribute to the AP-1 transcription factor complex. Jun proteins can form either homodimers or heterodimers with members of the related Fos family or with the ATF family to create the AP-1 transcription factor. Embryonic JunD expression is initially detected in the developing heart and cardiovascular system. JunD(–/–) males exhibit multiple age-dependent defects in reproduction, hormone imbalance and impaired spermatogenesis with abnormalities in head and flagellum sperm structures (Thepot et al., 2000). Lack of molecular factors encoded by the latter gene results in an absence of flagella in spermatids in the lumen of the seminiferous tubules (Thepot et al., 2000; Escalier, 2001). The absence of JunD led to sperm flagellar growth impairment. Additional defects in sperm nuclear and cytoskeletal morphology and in mitochondrial localization can be observed in nectin-null mutant mice. Nectin-2 is a component of cell–cell anchoring junctions playing a role in the connection of the cytoskeletal elements of neighbouring cells. Thus, this molecular system participates in the regulation of cell shape and differentiation through signalling pathways (Bouchard et al., 2000). Further interesting observations on the male gamete cytoskeleton are demonstrated in the null mutant for the zinc-finger transcription factor Egr4. In the latter animals, the flagella is often fragmented, sharply kinked or have tightly coiled distal ends. Spermatozoa with heads that are either separated entirely or bent sharply back on the flagella are observed (Tourtellotte et al., 1999; Escalier, 2001).
In null mice for Sla12a2 gene (normally expressing the Na+–K+–2Cl– co-transporter) few spermatids are present, but defects are striking when the male gametes gradually acquire the features of spermatozoa (Escalier, 2001). Defects in the molecular system of Na+–K+–2Cl– co-transporter result in morphological abnormalities of spermatids. Spermatids show abnormalities in acrosomal vesicle during the cap phase and abnormalities in the nuclear shape (Pace et al., 2000). Other morphological abnormalities of the male gamete accompany the lack of factors that are normally expressed by the CsnK2a2 gene. CsnK2a2 could be a candidate globozoospermia gene. Mice deficient for CsnK2a2 show abnormalities of spermatid nuclear morphogenesis. Further abnormalities are observed in the nuclear and acrosomal shape (Xu et al., 1999).
Robertson et al. (1999) have demonstrated that deficiency in the production of aromatase enzyme cyp19 due to targeted disruption of the cyp19 gene in ArKO mice results in maturation arrest at early stages of spermiogenesis. Round spermatids do not complete elongation and spermiation. Furthermore, morphological defects in round spermatids are seen in tubules exhibiting spermiogenic arrest. Moreover, abnormalities of spermatid cap phase, acrosomal vesicle and nuclear shape are observed. These findings may suggest that estrogens have a role in spermatid differentiation (Robertson et al., 1999).
Production of polyploid spermatids and male gamete DNA fragmentation are demonstrated after the disruption of the protein phosphatase catalytic subunit Pp1c
(Varmuza et al., 1999; Jurisikova et al., 1999; Escalier, 2001).
Deficiency in the production of an epithelial, microtubule-associated protein due to defects in the expression of the E-MAP-115 gene results in abnormal shape and progressive degeneration in all condensed spermatids. Abnormalities of the microtubular manchette and nuclear shape are also observed (Komada et al., 2000; Escalier, 2001). Subnormal expression of the molecular products of the gene Tg737, which encodes one of the components of the raft protein complex, designated Polaris in the mouse and IFT88 in both Chlamydomonas and mouse, results in defective ciliogenesis and abnormalities in flagellar development in spermatids as well as asymmetry in left–right axis determination (Kierszenbaum, 2002). Polaris/IFT88 is detected in the manchette of mouse and rat spermatids. Intramanchette transport has the features of intraflagellar transport machinery but, in addition, facilitates nucleocytoplasmic exchange activities during spermiogenesis (Kierszenbaum, 2002).
During spermiogenesis, histone-to-protamine exchange causes chromatin condensation. Spermatozoa from infertile men are known to exhibit an increased protamine-1 (PRM1) to protamine-2 (PRM2) protein ratio. Patients undergoing testicular sperm extraction followed by ICSI procedures reveal low fertilization rates. Steger et al. (2001) investigated whether the outcome of ICSI could be related to the presence of round spermatids expressing PRM1 mRNA and PRM2 mRNA. The above investigators showed that the PRM1 mRNA to PRM2 mRNA ratio in round spermatids may serve as a possible predictive factor for the outcome of ICSI (Steger et al., 2001). In another study Steger et al. (2002) have shown that PRM1 mRNA and PRM2 mRNA in round spermatids are associated with RNA-binding proteins. In addition, Steger et al. (2003) have demonstrated that there is a decreased PRM1 transcript level in the testes from infertile men. The result is an aberrant Prm1/Prm2 mRNA ratio that plays an important role for the development of male infertility and may serve as a possible predictive factor for the outcome of ICSI (Steger et al., 2003).
A dynamic balance of germ cell regeneration and death
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. (1998) 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 (Sinha Hikim et al., 1998) or of 2-methoxy acetic acid-induced spermatocyte death in cultured seminiferous tubules of middle-aged human donors (Sinha Hikim et al., 1998). 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 (Print and Loveland, 2000). 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 (Print and Loveland, 2000).
Whether male germ cells survive or die is determined by a complex network of signals. These include paracrine signals such as SCF, LIF and Desert Hedgehog (Dhh) (Gnessi et al., 1997), as well as endocrine signals such as pituitary gonadotrophins, estrogens and testosterone, among others (Schlatt et al., 1997; O'Donnell et al., 2001). In addition, male germ cells respond to external signals, and to their internal milieu, by activating intracellular signalling pathways that ultimately determine their fate.
The role of the Bcl-2 signalling pathway in governing 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. 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 (Ross et al., 1998). 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. (2001) 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 (Oldereid et al., 2001). 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 (Kimura et al., 2003). However, in that study (Kimura et al., 2003), 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. (2003) 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. In another study, Erkkila et al. (2002) showed that human germ cell death in vitro is inhibited effectively and dose-dependently by lactate, indicating that lactate has an important effect on the regulation of cellular death in human male germ cells. Thus, Erkkila et al. (2002) have demonstrated that human testicular germ cell death is effectively regulated by lactate, which may be regarded as a potential compound for optimizing in vitro methods for the maintenance of the function of male germ cells for assisted reproduction technique (ART) purposes. Regarding the mechanisms of the anti-apoptotic role of lactate in the human, it appears that the death suppressing mechanism of lactate is not related with changes in intracellular AMP, ADP and ATP levels. Erkkila et al. (2002) have hypothesized that the action of lactate is downstream along the cell-death pathway activated by the Fas receptor of the germ cells.
Contribution of transcription factors to the mechanism responsible for the elimination of human damaged meiotic germ cells
Transcription factors may provide additional fate-determining signals; c-myc may regulate the apoptosis of pre-meiotic germ cells. The E2f family of transcription factors can induce both apoptosis and proliferation in somatic tissues (Holmberg et al., 1998). Mechanisms responsible for the detection and elimination of damaged meiotic germ cells are present in the human male as evidenced by the high incidence of spermatocyte apoptosis and round spermatid apoptosis described in infertile men. In addition, men with ataxia telangiectasia and mice lacking the ATM gene are infertile as a result of extensive spermatocyte apoptosis (Burgoyne and Baker, 1984). The CREM is a transcription factor required for the expression of post-meiotic germ cell-specific genes. Spermatids of mice lacking the CREM are arrested in the first step of spermiogenesis and appear to be subsequently removed by apoptosis. Several mechanisms may mediate the selective apoptosis of damaged germ cells. For example, the cell cycle regulator p53 appears to be necessary for the radiation-induced apoptosis of spermatogonia (Hasegawa et al., 1998). There are also p53-independent mechanisms. Failed synapsis appears to induce spermatocyte apoptosis through a p53-independent pathway.
Paracrine mechanisms and growth factors in apoptotic pathways in the testis
Grataroli et al. (2004) 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 (Grataroli et al., 2004).
Several paracrine signals are regulators of germ cell fate. LIF promotes the survival of PGC in culture. Other factors that promote PGC survival in vitro include interleukin-4 (IL-4), basic FGF (bFGF), a soluble form of SCF and the bone morphogenetic protein (BMP)-4. In contrast, 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 (Dolci et al., 1991; Matsui et al., 1992; Cooke et al., 1996). 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 TGF-b superfamily (Lin et al., 1993; Kotzbauer et al., 1996; Baloh et al., 1998; Milbrandt et al., 1998). GDNF mRNA was found to be expressed in many tissues in addition to the brain and kidney, including intestine, stomach, muscle, cartilage, lung and testis (Trupp et al., 1995). 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 (Meng et al., 2000).
The PDGF-A and PDGF-B genes encode A and B chains of the PDGF and are located on human chromosome 7p and 22q, respectively (Antoniades and Hunkapiller, 1983). 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 (Heldin and Westermark, 1999). 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 (Basciani et al., 2002). 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 (Basciani et al., 2002).
Regulation of the fate of testicular germ cells by factors derived from Sertoli cells by direct membrane contact
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 c-kit gene is the cellular homologue of the feline sarcoma oncogene v-kit (Besmer et al., 1986). It is located at the White spotting (W) locus in the mouse and on chromosome 4 in the human (Giebel et al., 1992; Vandenbark et al., 1992). 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 nine exons (Flanagan et al., 1991). 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 (Manova et al., 1990; Orth et al., 1996). In contrast, Sertoli cells are the unique source of SCF in the testis (Rossi et al., 1991). 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 (Mauduit et al., 1999). Therefore, the SCF/c-kit complex appears to represent one of the key regulators of spermatogenesis.
Pentikainen et al. (1999) 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 (Pentikainen et al., 1999). 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 (Nagata and Golstein, 1995).
Fas is abundantly expressed in various tissues, particularly in activated T and B cells, thymocytes, hepatocytes and the heart tissue (Watanabe-Fukunaga et al., 1992). Of particular interest is the observation that FasL is constitutively expressed by cells in immune privileged sites such as the testis (Suda et al., 1993) and the anterior chamber of the eye (Bellgrau et al., 1995; Sofikitis et al., 2003). Fas is expressed at low levels in the mouse testis (French et al., 1996; Lee et al., 1997) and appears to be restricted to some germ cells (Lee et al., 1997). In addition, Sertoli cells may 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 (Korbutt et al., 1997; Lee et al., 1997; Pentikainen et al., 1999; Riccioli et al., 2003).
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 (Pentikainen et al., 1999) 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, up-regulation of Fas was observed in germ cells undergoing apoptosis after in vivo administration of Sertoli cell toxicants (Lee et al., 1997). 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. (1999) 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.
A role of hormones in the regulation of apoptosis in human germ cells
Hormones such as testosterone, FSH and 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 (Blanco-Rodriguez and Martinez-Garcia, 1996). In addition, Pentikainen et al. (2000) have demonstrated that estradiol acts as a germ cell survival factor in the human testis in vitro (Pentikainen et al., 2000).
In human seminiferous tubuli, apoptosis is induced under serum-free conditions in vitro (Erkkila et al., 1997). 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 (MacGregor et al., 1999).
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) (Reubi, 1997). 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 (Vasankari et al., 1995). The presence of SRIF and its receptors in human testes (Baou et al., 2000) 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 (Goddard et al., 2001).
Apoptosis in spermatozoa
In human, the presence of nuclear DNA damage in ejaculated spermatozoa has pointed to a possible role of apoptosis during spermatogenesis. Sakkas et al. (1999) have suggested that apoptosis is a major mechanism in regulating spermatogenesis in the human and that there are clear differences in molecular markers of apoptosis between males with normal and abnormal sperm parameters. Sakkas et al. (1999) have proposed that the presence of Fas-labelled spermatozoa in the ejaculate of men with abnormal semen parameters is indicative of ‘abortive apoptosis’ having taken place, whereby the normal apoptotic mechanisms have misfunctioned (Sakkas et al., 2003), have been overridden or have not been completed.
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Technical prerequisites and outcome of successful in vitro culture systems |
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TOPAbstractRegulation of spermatogenesis:...Technical prerequisites and...Methods to achieve meiotic...Scientific and clinical...Genetic and epigenetic risks...References |
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Recovery and purification of spermatogonia cells prior to culture
Methods to generate animal models with relatively large proportions of undifferentiated spermatogonia and SSCs
Rapid and effective preparation of pure populations of spermatogonia is the basis to achieve induction of in vitro spermatogenesis. However, obtaining a pure population of spermatogonia or SSCs is considered difficult due to the problems associated with the limited number of spermatogonia in the testis (Meachem et al., 2001) and the technical difficulties concerning the identification of specific biochemical or surface antigen markers characterizing the spermatogonia cells and their subpopulations (de Rooij and Grootegoed, 1998). Until now, the most common way to prepare a relatively pure population of SSCs for culture is to collect SSCs from neonatal or pre-pubertal testes. In the latter testes spermatogenesis is arrested at an early stage and thus fewer contaminating differentiating/differentiated germ cells are present and a larger number of SSCs is present (Creemers et al., 2002).
In experimental animals, testicular pathologies can be induced in which undifferentiated spermatogonia A are virtually the single germ cell present (Table I). Induction of vitamin A deficiency in rodents results in deterioration of spermatogenesis until virtually only type A spermatogonia remain present (Mitranond et al., 1979). Most, if not all, of these remaining spermatogonia A (in vitamin A deficiency models) are quiescent and unable to differentiate (Van Pelt et al., 1995). Van Pelt et al. (1996) used a Percoll gradient system in order to isolate spermatogonia cells from vitamin A-deficient 10-week-old rats. The administration of the Sertoli cell toxicant 2,5-hexanedione (Boekelheide, 1988; Allard et al., 1995) or exposure to X-irradiation (Kangasniemi et al., 1996; Table I) in animal models results in testicular histologies characterized by actively proliferating but not differentiating type A spermatogonia. The latter animal models can serve as a source of SSCs without contaminating differentiating germ cells. The cryptorchid model provides a rich alternative source of SSCs that can be further processed for in vitro cultures; one cell in 200 testicular cells in prepared testicular cellular suspensions in cryptorchid animals is an SSC (Shinohara et al.,