The germline of multicellular organisms, which passes

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1 Journal of Andrology, Vol. 21, No. 4, July/August 2000 Copyright American Society of Andrology Genetic and Cellular Analysis of Male Germ Cell Development Review ROBERT OLASO AND RENÉ HABERT From the Université Paris and INSERM U418, Paris, France. ABSTRACT: The evolution of the germline has been studied for many decades. Although the pathway of germ cell primordial migration and the kinetic evolution of the gonocytes are well known, their genetic and cellular controls are poorly understood. Recently, a genetic approach using gene knockout and a cellular investigation using several germ cell culture models has allowed a better understanding of the involvement of several genes and factors in the development of germ cells during fetal and neonatal life. Because of the obvious importance of the development of primordial germ cells and gonocytes in adult fertility and its eventual alteration by toxins or xenobiotics, a better knowledge of these interactions appears as an important research area. Key words: Primordial germ cell, gonocyte, testis, growth factor, proliferation, apoptosis. J Androl 2000;21: The germline of multicellular organisms, which passes genetic information from generation to generation, is fundamentally important in reproduction, heredity, and evolution. It has long been known that the primordial germ cells (PGCs) of mammals are extragonadal in origin (for a review see McLaren, 1999). These cells, which are derived from the epiblast/primitive ectoderm (Lawson and Hage, 1994; McLaren, 1999), are first recognizable in the extraembryonic mesoderm, and proliferate during their migration to the gonadal anlage. Once they have reached it, they are generally referred to as gonocytes (the term we will use in this review), but some authors use the word prespermatogonia or postmigratory PGC. The terms PGC and gonocytes reflect their different potentialities (Donovan et al, 1986). In the gonadal ridge, the gonocytes become enclosed by Sertoli cells in the emerging cords and continue to proliferate before entering a quiescent period (Table 1). Mitosis resumes just before or shortly after birth, and many of the gonocytes differentiate into spermatogonia, while others degenerate at this time. The development of gonocytes is characterized by 1 or 2 phases of degeneration that have been demonstrated to be caused by apoptosis (Coucouvanis et al, 1993; Mori et al, 1997; Wang et al, 1998; Boulogne et al, 1999a). These events are seen in all mammal species stud- Supported by Université Paris 7, INSERM and INRA. Correspondence to: Pr R. Habert, INSERM-INRA U 418 Université Paris 7, Tour 33/43, 2 Place Jussieu, Paris Cedex 05, France ( habert@paris7.jussieu.fr). Received for publication November 9, 1999; accepted for publication December 8, ied, except for the lamb (Hochereau-de Reviers et al, 1995) in which a quiescent phase has not been observed (Table 1). In this review, we will not be considering the origin of the germline and the migration of PGC, topics that have been extensively covered in previous reviews (Eddy et al, 1981; De Felici and Pesce, 1994; Lawson and Hage, 1994; McLaren, 1995; Buehr, 1997; McLaren, 1999; Wylie, 1999), but we will examine the proliferation and survival of both PGC and gonocytes, since there is no review dealing with gonocytes and new data imply to actualize previous reviews dealing with PGC (De Felici and Pesce, 1994; Buehr, 1997; Donovan et al, 1998). Furthermore, this topic has gained more interest since linkage between gonocyte development and testicular cancer is more and more evident, and there has been a dramatic increase in testicular cancer during these last decades (Skakkebaek et al, 1998). In early stages of testicular cancer (carcinoma in situ), cells have several histological and growth characteristics of gonocytes. This suggest that tumor could originate from fetal and neonatal periods (De Rooij, 1998). During the last years, 2 experimental strategies have been developed to study the control of development of PGC and gonocytes: a genetic analysis using several gene knockouts and a cellular investigation using germ cell culture models. Here we present the information brought by each one. In the second part of this paper we analyze the recent findings dealing with apoptosis, which specifically affects the germ cells in the developing testis.

2 498 Journal of Andrology July/August 2000 Table 1. Male germ cell evolution during fetal and neonatal life* Genocyte Degeneration References Mitosis Resumption Mitotic Arrest Gonocyte Proliferation Period Testicular Differentiation Genital Ridge Colonization First Detection of Primordial Germ Cells Species, Days of Gestation Gondos, 1980; Mackay et al, 1989; Miething, dpc 13 dpc dpc 11 dpc 9 10 dpc 8 dpc Hamster, 16 Gondos, 1980; Eddy et al, 1981; Vergouwen et al, 1991; Coucouvanis et al, 1993 Beaumont and Mandl, 1963; Jost, 1972; Gondos, 1980; Orth, dpc 9 dpp dpc 7 dpp dpc 1 dpp 16.5 dpc dpc 12.5 dpc 11.5 dpc 7.5 dpc Mice, dpp 18 dpc dpc 13.5 dpc 13.5 dpc 8 dpc Rat, 21 Gondos and Conner, 1973; Gondos, 1980; Gondos and Byskov, 1981 Black, 1971; Pelliniemi, 1975; Gondos, 1980 Zamboni and Upadhyay, 1982; Hochereau-de Reviers et al, dpc 14 dpp 5 dpp 22dpc 5dpp dpc dpc 25 dpc 29 dpc dpc Rabbit, 31 NG puberty dpc 23 dpc dpc dpc Pig, 68 Lamb, 150 Human, 270 Fujimoto et al, 1997; Gondos, 1980; Müller and Skakkebaek, 1984; Franscavilla et al, 1990 Matschke and Erickson, 1969; Wrobel and Süb, to 5 years dpc dpc 18 dpc Calf, 280 * dpc indicates days postconception; dpp, days postpartum;, phenomenon observed without precise dating; and NG, phenomenon not observed during gestation. Proliferation and Survival of PGC and Gonocytes Genetic Analysis One strategy used for the study of the development of germ cells is genetic analysis, as numerous mutations and gene knockouts affect the proliferation and/or survival of PGC and gonocytes. In the mouse, 5 spontaneous mutations and 1 insertional mutation affect the germline during development. They are white spotting (W), steel (Sl), hertwig s anemia (an), teratoma (ter), germ cell deficient (gcd), and atrichosis (at) mutants. Mutations on loci of W and Sl, which encode a tyrosine kinase receptor (c-kit) and its ligand, known as c-kit ligand (KL) but also steel factor (SF), stem cell factor (SCF), or mast cell growth factor (MCF; Loveland and Schlatt, 1997), show adult sterility and a markedly reduced population of PGC in fetuses of 9 10 days postconception (dpc). These may be due to a defect in PGC migration (Buehr, 1997; Loveland and Schlatt, 1997) but seem also to be due to a failure of proliferation during PGC migration (although an excessive rate of cell death cannot be excluded; McCoshen and McCallion, 1975; Buehr et al, 1993). The role of W and Sl products has been confirmed by in vitro studies (see below). Mutants of an are often sterile as adults (Russell et al, 1985). During development, mutants undergo a reduction in germ cell number at 12 dpc. However, it has not been established whether this results from the impairment of migration, proliferation, and/or survival of PGC. Furthermore, between 12 and 15 dpc, when gonocytes proliferate in the wild type, the an mutant shows a total absence of proliferation, and considerable degeneration of the gonocytes. The ter mutation causes adult sterility resulting from PGC deficiency at around 8 dpc at the beginning of the migratory and proliferative stages (Sakurai et al, 1995). The number of germ cells remains low thereafter. The ter mutation affects the proliferation of PGCs, but not their migration. The authors suggest that the ter locus encodes an unknown factor that may induce PGC proliferation before the c-kit/steel system. The gcd mutation is an insertional one that reduces the PGC number in 11.5-dpc mouse fetuses and leads to adult sterility (Pellas et al, 1991). The authors do not report if gcd impairs migration, proliferation, and/or survival of germ cells. The insertion is near the leukemia inhibitory factor (LIF) and oncostatin M (OSM) genes (Duncan et al, 1995), 2 cytokines that affect PGC proliferation and survival. There is no rearrangement in these genes, but the insertion could act by modifying their expression. The last mutation, at, causes a depletion of germ cells

3 Olaso and Habert Male Germ Cell Development in the adult (Handel and Eppig, 1979). The total absence of spermatogenesis suggests an embryonic and/or postnatal failure in germline development, although no studies have been performed during this period. In contrast with W and Sl, the identity of the affected or disrupted genes in an, ter, gcd, and at mutations has not yet been established. Another type of mutation is that obtained after homologous recombination (knockout or gene invalidation) in mice. The results of several recent gene knockouts have permitted a better understanding of the proliferative/survival phase. As for other physiological functions, the knockout strategy can be used to evaluate the function of adefined gene in germ cell development, but this approach has some intrinsic limitations since it can cause death in early development or compensatory effects between the members of multigene families. Despite these limitations, the knockout strategy has revealed that several genes are involved in the control of proliferation and/ or survival of PGC and gonocytes. The knockout of zfx gene (Luoh et al, 1997), which encodes a zinc-finger protein, leads to a reduction by half of the number of spermatozoa in adults with fertility keeping. This defect in spermatogenesis results from a reduced PGC number at 11.5 dpc. The authors report no abnormal PGC migration. This result suggests that the zfx gene impairs the PGC proliferation and/or enhances PGC cell death. The disruption of TIAR, an RNA binding protein, results in adult sterility (Beck et al, 1998). Gonocytes are severely reduced in number at 11.5 dpc and completely absent at 13.5 dpc. This is not the result of an impairment of PGC migration. It is noteworthy that TIAR is highly expressed by gonocytes. The mechanism of action of TIAR protein is not established, but in an embryonic stem cell (ES) culture system, it has been suggested that TIAR exerts its action via the LIF system (Beck et al, 1998). The dazla gene also encodes an RNA binding protein. Mutant mice lacking dazla are infertile. They present a deficiency of germ cells at 19 dpc (Ruggiu et al, 1997), but germ cell number is normal at 15 dpc. This suggests that dazla gene is essential for the survival of gonocytes during a short critical phase corresponding to the quiescent period in mouse. The male null mutant for the desert hedgehog (Dhh), a vertebrate homologue of the Drosophila hedgehog (hh) gene, is sterile (Bitgood et al, 1996) and it shows a reduction in testis size from day 1 postconception (pc) to adulthood. Germ cells are totally absent in many seminiferous tubules in the adult. The evolution of germ cell number during development has not been reported, but the expression of Dhh in Sertoli cells from 11.5 dpc suggests that Dhh plays a role as early as this developmental stage. 499 Recently, it has been reported that the inactivation of connexin 43, a membrane protein that is part of gap junctions in the testis, causes a reduction of germ cell number in fetuses and in neonates (Juneja et al, 1999). This reduction is observed as early as 11.5 dpc. The gene responsible for Fanconi anemia (FA) in humans was target-disrupted. The resulting newborn and adult mutant mice have reduced numbers of germ cells, but adults are not sterile (Whitney et al, 1996). Adult males have heterogeneous seminiferous tubules: some are normal, others are totally devoid of germ cells. These data rule out a meiotic defect and suggest an impairment of the formation of some seminiferous cords during fetal life. Furthermore, this phenotype is similar to that observed after inactivation of c-kit/sf system and the authors suggest that the FA and c-kit pathways may interact with each other. The inactivation of the breast cancer susceptibility gene-2 (Brca-2) leads to an absence of all germ cells in the adult (Connor et al, 1997), but the possibility that the defect appears during development has not been studied. Lastly, the target disruption of the bone morphogenetic protein 8 (Bmp8) causes male infertility due to a reduction in germ cell proliferation followed by an increase in germ cell death (Zhao et al, 1996). These defects appear in pubertal animals, but a decrease in testis weight and germ cell number are already apparent in 1-week-old mice, the youngest so far studied. Whether Bmp8 inactivation affects the germline before birth remains to be investigated. In conclusion, the developmental control of PGC and gonocytes probably involves several genes. Because of the fundamental importance of this process, probably numerous redundancies compensate the defect in 1 of the components of the control system. Furthermore, this approach does not detect minor changes in the development of the germline throughout fetal and neonatal life, which can be compensated at adult stage. This makes the genetic approach difficult to interpret except when the mutation leads to sterility. It must also be emphasized that most of the mutations leading to sterility have been observed at the adult stage (Bhasin et al, 1998) and their effects throughout development have not yet been studied. It is clear that future identification of the genes that control proliferation and survival of germline will have important implication for our understanding of the normal evolution of germline and its alterations in human and animal reproductive disease. Cellular Analysis The past few years have seen a number of groups making use of feeder-dependent cultures to study the effect of

4 500 Journal of Andrology July/August 2000 various factors on PGC and gonocytes (Donovan et al, 1986; Godin et al, 1990; De Felici and Dolci, 1991; Matsui et al, 1991; Resnick et al, 1992). More recently, Sertoli cell/gonocyte coculture models (Van Dissel-Emiliani et al, 1993, 1996; Boulogne et al, 1999b) and purifiedgonocyte culture models (Li et al, 1997) have been developed. We and others (Kancheva et al, 1990; Boitani et al, 1993; Jannini et al, 1993; Prépin, 1993) have also used the organotypic culture system previously developed by Jost s group (Agelopoulou et al, 1984). These in vitro studies demontrate the fundamental role of somatic cells and show that numerous factors, hormones, and vitamins have the capacity to modulate the number, proliferation, and/or survival of PGC and gonocytes. However, these studies must be considered with caution because these factors may act directly on PGC and gonocytes, or indirectly through somatic cocultured cells. Role of Somatic Cells Numerous data reveal the fundamental role of somatic cells in the control of PGC and gonocyte development: 1. Conditioned medium from the gonadal ridge of dpc mouse fetuses increases the number of PGCs in cell culture (Godin et al, 1990). Similarly, conditioned medium from Sertoli cells of neonatal rat stimulates the mitotic activity of 3-dpp gonocytes in organotypic culture (Kancheva et al, 1990). 2. Gonocyte mitosis resumes at the same time both in vivo and in neonatal testis cultured in synthetic medium deprived of hormone and growth factor (Mc- Guinness and Orth, 1992), and this resumption is observed in neonatal Sertoli cell/gonocyte cocultures but not in purified gonocyte cultures (Li et al, 1997). In the same line, the PGCs and the gonocytes cannot survive in vitro for more than 2 days when they are cultured alone (De Felici and Dolci, 1991; Matsui et al, 1991; Van Dissel-Emiliani et al, 1993; Li et al, 1997). The addition of extracellular matrix components such as fibronectin, laminin, and type I collagen scarcely improve PGC survival (De Felici and Dolci 1989). 3. The number of germ cells in the adult rat depends on the number of the Sertoli cells that differentiate during fetal and neonatal life (Orth et al, 1988). These data suggest that somatic cells, and particularly Sertoli cells, are strictly required for the survival and multiplication of PGC and gonocytes. The action of these somatic cells may be exercised by the production of soluble factors that can act as paracrine regulators of the multiplication and/or survival of germ cells during fetal and neonatal life as happens in adults (Jégou, 1992, 1993). Furthermore, diffusible factors can pass through the gap junctions observed between Sertoli cells and gonocytes in vitro as well as in vivo (Orth and McGuinness, 1991; Van Dissel-Emiliani et al, 1993; Orth and Jester, 1995). Lastly, this control may also be exercised by membrane contact, but the unique membrane component evidenced as a potential candidate until now is the membrane form of SF, which is more efficient than the soluble one is. Role of Locally Produced Factors Cellular and/or organotypic culture systems have been used to show that numerous factors are able to modulate the number, proliferation, and/or survival of PGC and/or gonocytes by acting either directly or via the cocultured somatic cells (Tables 2 and 3). However, few of these molecules have yet fulfilled the 4 classical criteria needed to establish that a molecule found to have an in vitro effect might play a local regulatory effect (Bardin et al, 1990): 1) the molecule should be synthesized and secreted locally, 2) its secretion should be regulated by appropriate physiological signals, 3) its local concentration should be compatible with the kd of its receptor(s), and 4) its removal should influence the development and/or the function of the target cells. Role of TGFs and Related Factors Numerous experimental arguments suggest that a member of the transforming growth factor beta (TGF) family may control PGC and gonocyte development. We and others have detected the 3 isoforms TGF1, TGF2, and TGF3 in fetal and neonatal testis (Teerds and Dorrington, 1993; Gautier et al, 1994; Olaso et al, 1997; Cupp et al, 1999a; Olaso et al, 1999). In Table 4, our results are summarized and compared with the findings of previous (Teerds and Dorrington, 1993) and later (Cupp et al, 1999a) workers (Tables 5 and 6). We have shown that the immunolocalization of each isoform in each testicular cell type varies according to the developmental stage. This is confirmed by Cupp et al (1999a), although they find TGF2 and TGF3 appearing some days later than we do (Table 6). This discrepancy is probably linked to the difficulty of getting good histological pictures during early development because of the high water content of tissues. The presence of TGF2 in gonocytes at 21 dpc is clearly visible in the Figure, which differs from the data from Teerds and Dorrington (1993) and Cupp et al (1999a). We have also demonstrated that fetal testis in vitro secretes a TGF1-like bioactive material (Gautier et al, 1997). TGF1, TGF2, and TGF3 messenger ribonucleic acid (mrna) have also been detected in fetal testis (Gautier et al, 1997; Cupp et al, 1999a) and in neonatal testis (Mullaney and Skinner, 1993; Cupp et al, 1999a). Our group has also demonstrated that the level of TGF1 mrna and the secretion of a TGF1-like bioactive material in vitro are increased by Luteinizing hormone (LH) plus follicle-stimulating hormone (FSH) but not by LH or FSH alone (Gautier et al, 1997). This has been confirmed in

5 Olaso and Habert Male Germ Cell Development 501 Table 2. Effect of several factors on mice primordial germ cells/gonocytes in culture Factors* TGF1 /10.5/ /11.5 Activin /10.5/11.5 Steel factor /10.5/11.5 / LIF /10.5/11.5 OSM CNTF IL-3 IL-4 IL-11 sky/gas6 FGF-2 TNF camp PACAP Retinoic acid Age, Days Postconception Effect References / / / 10.5/11.5 / / 10.5/11.5/12.5 /10.5/ / /11.5/13.5 /11.5/13.5 Decreased number Decreased mitosis Decreased number Decreased mitosis Increased mitosis Decreased apoptosis Increased mitosis Decreased apoptosis Increased mitosis Increased mitosis Increased mitosis Increased mitosis Godin and Wylie, 1991; Richards et al, 1999 De Felici and Dolci, 1991 Richards et al, 1999 Richards et al, 1999 Richards et al, 1999 Dolci et al, 1991, 1993; Godin et al, 1991; Matsui et al, 1991 Godin et al, 1991; Dolci et al, 1993 Matsui et al, 1991 Pesce et al, 1993 Matsui et al, 1991 De Felici and Dolci, 1991; Resnick et al, 1992; Dolci et al, 1993; Cheng et al, 1994; Koshimizu et al, 1996 De Felici and Dolci, 1991; Dolci et al, 1993 Matsui et al, 1991 Dolci et al, 1993 Pesce et al, 1993 Hara et al, 1998 Hara et al, 1998 Koshimizu et al, 1996 Cheng et al, 1994 Cooke et al, 1996 Rich, 1995 Rich, 1995 Koshimizu et al, 1996 Koshimizu et al, 1996 Matsubara et al, 1996 Resnick et al, 1992; Cheng et al, 1994; Resnick et al, 1998 Kawase et al, 1994 Kawase et al, 1994 Kawase et al, 1994 De Felici et al, 1993; Dolci et al, 1993 De Felici et al, 1993; Dolci et al, 1993 Pesce et al, 1996 Pesce et al, 1996 Koshimizu et al, 1995 Koshimizu et al, 1995 * TGF1 indicates transforming growth factor beta-1; LIF, leukemia inhibitory factor; OSM, oncostatin M; CNTF, ciliary neurotropic factor; IL, interleukin; FGF-2, fibroblast growth factor 2; TNF, tumor necrosis factor alpha; camp, cyclic adenosine monophosphate; and PACAP, pituitary adenylate cyclase activating peptide. part by Cupp et al (1999a), who observed that FSH does not modify the level of TGF1, TGF2, or TGF3 mrna. A negative effect of TGF1 on PGC number in cell culture was first reported by Godin and Wylie (1991). However, such an effect has not been observed by De Felici and Dolci (1991). This discrepancy can be explained by the concentration of TGF1 used (25 ng/ml and 1 ng/ml, respectively). Our group has studied the effect of TGF on rat gonocytes development using the organotypic culture system (Olaso et al, 1998). In this system, TGF1 or TGF2 decreases the number of gonocytes in testis explanted at 13.5 dpc by increasing their apoptosis without modifying their mitotic activity. This effect is observed with doses as low as 2 ng/ml of TGF1. The negative effect of TGF is not observed with 17.5-dpc testis (during the quiescent period) but is again present in 3-days postpartum (dpp) testis (the stage of gonocyte mitosis and apoptosis resumption). Lastly, Richards et al (1999) have confirmed the negative effect of TGF1 on the number of mouse PGC and gonocytes in a cell culture system. They observed that TGF1 decreases the mitotic activity of PGC, but did not analyze the apoptotic activity of PGC. It is noteworthy that Morita et al (1999) have reported that TGF1 also increases apoptosis in the oogonia of 13.5-dpc cultured mouse ovary. All these data suggest that TGF reduces germ cell number by different processes: it inhibits the mitotic activity of PGC (and enhances PGC apoptosis?) and it enhances gonocyte apoptosis. The members of the TGF family have been shown to mediate their signals through a complex of heterodimeric

6 502 Journal of Andrology July/August 2000 Table 3. Effect of several factors on rat gonocytes in culture Factors* Age Effect References TGF1/TGF2 AMH Steel factor LIF OSM CNTF FGF-2 PDGF Thyroid hormone Estradiol Thymulin Retinoic acid 13.5 dpc/ 13.5 dpc 13.5 dpc 17.5 dpc 14.5 dpc 14.5 dpc 14.5 dpc 2 dpp 0/ 2 dpp 2 dpp 0 dpp 0 dpp 2 dpp 2 dpp 0/2 dpp 0 dpp 2 dpp 2 dpp 13.5 dpc 2 6 dpp 0/1/7 dpp 14.5 dpc 14.5 dpc/ 14.5 dpc 1 dpc Decrease in number Increase in apoptosis Decrease in number Increase in apoptosis Increase in mitosis Increase in number Increase in number Increase in mitosis Increase in number Increase in number Increase in mitosis Increase in mitosis Increase in number Decrease in apoptosis Increase in mitosis Increase in number Increase in mitosis Decrease in number Increase in mitosis Increase in apoptosis Increase in number No change in apoptosis Decrease in number Olaso et al, 1998 Olaso et al, 1998 Olaso et al, 1998 Olaso et al, 1998 Olaso et al, personal communication Olaso et al, personal communication Olaso et al, personal communication De Miguel et al, 1996 De Miguel et al, 1996; Nikolova et al, 1997 De Miguel et al, 1996 De Miguel et al, 1996 De Miguel et al, 1997 De Miguel et al, 1997 De Miguel et al, 1996 De Miguel et al, 1996 Van Dissel-Emiliani et al, 1996 Van Dissel-Emiliani et al, 1996 Li et al, 1997 Li et al, 1997 Li et al, 1997 Jannini et al, 1993 Jannini et al, 1993 Li et al, 1997 Li et al, 1997 Prépin, 1993 Prépin et al, 1994 Prépin et al, 1994 Livera et al, 2000 Livera et al, 2000 Livera et al, 2000 Livera et al, 2000 Livera et al, 2000 Livera et al, 2000 Boulogne et al, 1999 Boulogne et al, 1999 * TGF1/TGF2 indicates transforming growth factors beta-1 and -2; AMH, anti-müllerian hormone; LIF, leukemia inhibitory factor; OSM, oncostatin M; CNTF, ciliary neurotropic factor; FGF-2, fibroblast growth factor 2; and PDGF, platelet-derived growth factor. dpc indicates days postconception; dpp, days postpartum. serine/threonine kinase receptors (type I and type II; Derynck and Feng, 1997). To elucidate the mechanism of action of TGF in the testis, we have identified the locations of the TGF receptors and observed TGF type I (TRI) and type II (TRII) receptors in gonocytes throughout fetal and neonatal development (Olaso et al, 1998). This is in accordance with the detection of mrna of TRI and TRII in gonocytes (Richards et al, 1999). Taken together, these data suggest that TGF directly exerts its effect on the germ cells, which agree with the negative effect of TGF on purified gonocyte cultures recently observed (Richards et al, 1999). It is interesting to note that a mutation of punt, the homologue of the mammalian TGF type II receptor (TRII) in Drosophila melanogaster, leads to an overproliferation of germ cells followed by an impairment of germ cell differentiation and a degeneration of these cells (Matunis et al, 1997). The same phenotype has been observed for the schnurri mutation (Matunis et al, 1997). Schnurri encodes for a transcription factor involved in the pathway of decapentaplegic (dpp), which is the Drosophila homologue of TGF (Pei-chih Hu et al, 1998). These data support and extend the observations made in rat and mouse culture models. There is no spontaneous mutation of the TGF system in mammals, and the inactivations of the TGF1, TGF2 or TGF3 genes do not contribute to an understanding of the role of TGF in the development of the germline (Shull et al, 1992; Kulkarni et al, 1993; Kaartinen et al, 1995; Proetzel et al, 1995; Sanford et al, 1997). Null mutant mice for 1 of these genes die in the perinatal stage, and no effect has been reported in the germline. On the contrary, 1 group (Shull and Doetschman, 1994) succeed in getting an adult fertile male mouse TGF1 /. Unfortunately, the lack of effect of this null mutation on the development of the testis is of limited significance be-

7 Olaso and Habert Male Germ Cell Development 503 Table 4. Localization of transforming growth factors beta-1, -2, and -3 (TGF1, TGF2, and TGF3) in the developing rat testis Results from our group s studies (Gautier et al, 1994; Olaso et al, 1997; Olaso et al, 1999) Cells Sertoli cells TGF1 TGF2 TGF3 Leydig cells TGF1 TGF2 TGF3 Germ cells TGF1 TGF2 TGF3 Fetal Age, Days Postconception Stage* * indicates staining not detectable;, faint or undetectable staining;, moderate staining; and, marked staining. Postnatal Age, Days Postpartum cause of the redundancy in the system. To answer this problem, 1 group has inactivated the gene TRII (Oshima et al, 1996), which encodes an obligatory step in the TGF pathway. However, the early death of mutant mice in utero before the formation of gonads prevents any analysis of germ cell development. One way of removing this obstacle will be the use of conditional gene targeting (Sauer, 1998), which allows the creation of spatially and temporally controlled somatic mutations. Activin, a member of the TGF family, can also act on PGC and gonocytes development. Activin and its type II receptor are expressed in fetal and neonatal rat testis (Roberts et al, 1991; Kaipia et al, 1994; Roberts and Barth, 1994), and type I and II receptors are expressed by PGC (Richards et al, 1999). Activin inhibits thymidine incorporation in 14.5-dpc testes in vitro (Kaipia et al, 1994). Activin also reduces the number of PGC and fetal gonocytes in cell culture; the effect on PGC results from an inhibition of their mitotic activity (Richards et al, Table 5. Results from Teerds and Dorrington s (1993) study* Cells Sertoli cells TGF1 TGF2 Leydig cells TGF1 TGF2 Germ cells TGF1 TGF Days Postconception Stage 7 Days Postpartum * TGF1 and TGF2 indicate transforming growth factors beta-1 and -2, respectively;, staining not detectable;, faint or undetectable staining;, moderate staining; and, marked staining. 1999). No data are available on the effect of activin on neonatal gonocytes development. The anti-müllerian hormone (AMH), another member of the TGF family, is expressed by Sertoli cells in fetal testis (Josso et al, 1993). Although AMH receptor type I (AMHRI) has not yet been precisely identified, the AMH receptor type II (AMHR II) is well known (Josso and di Clemente, 1997; Lane and Donahoe, 1998). AMHR II is present in fetal and neonatal male gonads (Teixeira et al, 1996) and AMH can act as a local regulator of somatic cell development in fetal testis (Rouiller-Fabre et al, 1998). Mutants overexpressing AMH at high levels show a male germ cell deficiency (Behringer et al, 1990), whereas null mutant adult mice AMH / have apparently normal testes but their spermatogenic activity has not been precisely studied (Behringer et al, 1994). In the same Table 6. Results from the study of Cupp et al (1999a)* Cells Sertoli cells TGF1 TGF2 TGF3 Leydig cells TGF1 TGF2 TGF3 Germ cells TGF1 TGF2 TGF Days Postconception Stage 0 Days Postpartum * TGF1, TGF2, and TGF3 indicate transforming growth factors beta-1, -2, and -3, respectively;, staining not detectable;, faint or undetectable staining;, moderate staining; and, marked staining.

8 504 Journal of Andrology July/August 2000 Distribution of TGF2 in 21.5-dpc testis. Arrow indicates gonocyte; arrowhead, Leydig cell. Scale bar 20 m. way, we have recently observed that AMH reduces the number of gonocytes in organotypic culture of fetal rat testis by increasing apoptosis (Olaso et al, unpublished data). Role of c-kit and Steel Factor We earlier remarked that the W and Sl mutants have their number of PGC largely lowered (McCoshen and McCallion, 1975; Buehr et al, 1993), suggesting that the couple tyrosine kinase receptor/ligand, c-kit/sf, is necessary for the proliferation (and/or survival) of PGC in vivo. In vitro studies suggest that SF is necessary for the survival of PGC (Dolci et al, 1991; Godin et al, 1991; Dolci et al, 1993). Moreover, the membrane form is more effective in culture that the soluble form (Godin et al, 1991; Matsui et al, 1991; Dolci et al, 1993), which is in agreement with the sterility observed in Steel-dickies mice (Sld) that produce only the soluble form (Loveland and Schlatt, 1997). On the other hand, Matsui et al (1991) reported that SF can act as a mitogenic factor for PGC. Concerning the gonocytes development, SF also increases their number in culture at 11.5 dpc by decreasing their rate of apoptosis (Pesce et al, 1993) but SF has no effect on the number of gonocytes from older fetal stages or from neonates (Matsui et al, 1991; De Miguel et al, 1996), although c-kit is expressed by neonatal gonocytes (Orth et al, 1996). In vitro, SF therefore acts mainly as a survival factor. However, when SF is associated with other factors such as LIF or fibroblast growth factor 2 (FGF-2), it can act on PGC as a mitogenic factor (Matsui et al, 1991, 1992; Resnick et al, 1992; Hara et al, 1998). It can therefore be hypothesized that it is this mitogenic effect that is significant in vivo. Role of LIF and Related Factors LIF also has a positive effect on germ cell development. It increases the number and the mitotic activity of PGC (De Felici and Dolci, 1991; Matsui et al, 1991; Resnick et al, 1992; Dolci et al, 1993; Koshimizu et al, 1996) and enhances the survival of fetal gonocytes (Cheng et al, 1994) by decreasing apoptosis (Pesce et al, 1993). LIF is also able to increase mitosis and survival of postnatal gonocytes (De Miguel et al, 1996; Nikolova et al, 1997). LIF can be detected in neonatal testis (De Miguel et al, 1996) but it has not yet been established whether LIF is expressed in PGC, in somatic cells along the route of migratory PGC, and in the fetal testis. The LIF signal is mediated by the LIF-receptor (LIF-R) and the subunit gp130 (Cheng et al, 1994), which are both expressed in the gonadal ridges (Koshimizu et al, 1996). Interestingly, addition of anti- LIF-R or anti-gp130 antibodies to culture medium decreases the PGC number (Cheng et al, 1994; Koshimizu et al, 1996). However, the inactivation of LIF (Stewart et al, 1992) or LIF-R (Ware et al, 1995) does not modify the PGC number. This discrepancy can probably be explained by a redundancy in the system. Indeed, other ligands of the LIF family, present in vivo but not in vitro, can bind to the subunit gp130 and may not require the presence of the LIF-R. This hypothesis is confirmed by the fact that inactivation of gp130 reduces the PGC number (Hara et al, 1998). Among gp130 ligands, OSM can be involved since it increases gonocyte numbers (De Miguel et al, 1997; Hara et al, 1998) and stimulates the mitotic activity of neonatal gonocytes (De Miguel et al, 1997), whereas it has no effect on PGC development (Hara et al, 1998). OSM has been detected in fetal and neonatal testis (De Miguel et al, 1997), but no data are available on the expression of the OSM receptor (OSMR) in the fetal and neonatal testis. Previous studies have reported that OSM increases the number of mouse PGC and gonocytes in vitro (Cheng et al, 1994; Koshimizu et al, 1996), but these results were obtained using human recombinant human OSM, which acts via murine LIFR-gp130 and not via murine OSMRgp130 (Ichihara et al, 1997), and then mimics the effect of LIF on murine PGC. Ciliary neurotropic factor (CNTF), another gp130 ligand, has no effect on PGC number but weakly increases the number of fetal gonocytes in vitro (Cheng et al, 1994; Koshimizu et al, 1996) and seems to act like a survival factor for postnatal gonocytes (De Miguel et al, 1996). CNTF is expressed in neonatal testis, but there is no report of its expression prior to this stage. Koshimizu et al (1996) found no CNTF receptor in gonadal ridges, and the inactivation of CNTF has no effect on germline development (Masu et al, 1993). In the adult, interleukins (ILs) play an important role in the control of germ cell differentiation throughout spermatogenesis (Jégou, 1993). In the fetus, IL6, another ligand of gp130, has no effect on PGC and gonocyte numbers in vitro (De Felici and Dolci, 1991; De Miguel et al, 1996; Koshimizu et al, 1996). By contrast, IL3, IL4, and IL11 increase PGC number and IL4 seems to act like a survival factor (Cooke et al, 1996), but none of these factors has an effect on fetal gonocyte number (Rich, 1995; Cooke et al, 1996; Koshimizu et al, 1996). There

9 Olaso and Habert Male Germ Cell Development is no report of the expression of ILs within fetal or neonatal testis. Role of Other Local Factors Among the tyrosine kinase receptor/ligand couples, sky/gas 6, expressed in germ cells and Sertoli cells, potentiates the effect of the SF on PGC proliferation and/or survival (Matsubara et al, 1996). The FGF-2 is present in fetal and neonatal testis (Mullaney and Skinner, 1992; Koike and Noumura, 1994) and is observed in gonocytes between 18 dpc and 5 dpp (Koike and Noumura, 1994). FGF-2 can bind to the 4 FGF receptors (FGFR1 to FGFR4; Klint and Claesson-Welsh, 1999). PGC are able to bind FGF-2 in culture, and FGFR1 and FGFR2 but not FGFR3 and FGFR4 have been detected in gonadal ridges of 11.5-dpc mice (Resnick et al, 1998). During late fetal life, gonocytes express FGFR1 and FGFR3, whereas Sertoli cells and Leydig cells express only FGFR1 and FGFR2 and FGFR4 have not been detected in the testis (Cancilla and Risbridger, 1998). In coculture, FGF-2 increases the number of PGC and gonocytes (Resnick et al, 1992; Cheng et al, 1994; Resnick et al, 1998) and has a mitogenic effect on postnatal gonocytes (Van Dissel-Emiliani et al, 1996). This proliferative effect is not observed in purified culture of postnatal gonocytes (Li et al, 1997). Taken together, these data suggest that FGF-2 may act directly or indirectly on PGC, and seems to act indirectly on postnatal gonocytes via Sertoli cells. It is noteworthy that no germline defect is reported in FGFR1 or FGFR3 knockout mice, and that FGFR2 knockout mice die before gonad formation (Klint and Claesson-Welsh, 1999). Platelet-derived growth factor (PDGF) does not act on PGC number in vitro (De Felici and Dolci, 1991) nor on proliferation of purified gonocytes of 2 dpp, but it stimulates the multiplication of purified gonocytes of (Li et al, 1997), suggesting that it may act directly on neonatal gonocytes after 2 dpp. PDGF receptor (PDGFR) and PDGF are both expressed in gonocytes and Sertoli cells in the late embryonic stage and after birth (Gnessi et al, 1995; Li et al, 1997). Disruption of PDGF signaling leads to embryonic or perinatal death and the development of germ cells has not been studied in these mutants (Ataliotis and Mercola, 1997). Tumor necrosis factor alpha (TNF) and its receptors TNF-RI and TNF-RII are expressed in most embryonic tissues (Kohchi et al, 1994). Addition of TNF to PGC in cell culture stimulates their multiplication in the earliest stages but has no effect at a later stage (Kawase et al, 1994). The action of the TNF is amplified by the presence of SF and/or LIF (Kawase et al, 1994). Factors such as IGF1, NGF, and EGF have no effect on PGC and/or gonocyte numbers in vitro (De Felici and Dolci, 1991; Li et al, 1997). Lastly, various studies from De Felici s group indicate 505 a proliferative effect of cyclic adenosine monophosphate (camp) in PGC and gonocyte cultures (De Felici et al, 1993; Dolci et al, 1993; Pesce et al, 1996). Furthermore, these workers have shown that the pituitary adenylate cyclase activating peptide (PACAP) increases the numbers of PGC and gonocytes, and stimulates the multiplication of the gonocytes via the activation of adenylate cyclase (Pesce et al, 1996). Role of Hormones The proliferation and survival of PGC and gonocytes seem to be independent of gonadotrophin since the decapitation of fetuses does not seem to modify germline evolution in rats (Creasy and Jost, 1966; and our observations), pigs (Van Vorstenbosch et al, 1987), or lambs (Hochereau-de Reviers et al, 1995). Futhermore, the addition of LH and FSH to fetal and neonatal testis in culture does not change the apoptotic and mitotic activities of the gonocytes (Boulogne et al, 1999a). Thyroid hormone (TH) acts via 2 types of receptors (THR and THR). THR is absent from fetal and neonatal testis, whereas THR is expressed by fetal and neonatal testis and particularly in seminiferous cords (Jannini et al, 1994). TH increases the number of 3-dpp gonocytes in organotypic culture and in vivo by stimulating their survival (Jannini et al, 1993). Estradiol may originate from either plasma or testis. Fetal Sertoli cells express aromatase, and this expression is stimulated by FSH and inhibited by AMH (Rouiller- Fabre et al, 1998). Estradiol is present in the fetal testis although its concentration is minute (1 pg/testis; Habert and Picon, 1984). Furthermore, xenobiotics and toxins with estrogenic activity can be expected to modify the gonocyte development since they modify adult spermatogenesis (Toppari et al, 1996). The effects of estradiol are mediated by two types of receptors, ER and ERß. These receptors are observed in fetal and neonatal testis. ER is confined to the Leydig cells throughout development (Saunders et al, 1998), whereas ER is expressed in gonocytes, Sertoli cells, and Leydig cells during the same period (Saunders et al, 1998; Van Pelt et al, 1999). As suggested by these distributions, estradiol can affect gonocyte both directly and indirectly. It has been reported that estradiol stimulates the proliferation of purified gonocytes at but not at 2 dpp (Li et al, 1997). Lastly, in organotypic culture, thymulin, which is secreted by the thymus, increases the number of 13.5-dpc gonocytes and stimulates the mitosis of 2- to 6-dpp gonocytes (Prépin, 1993; Prépin et al, 1994). Role of Vitamins Vitamin A and its bioactive product, retinoic acid (RA), have long been known to be involved in the reproductive process (Thompson et al, 1964). The action of RA is me-

10 506 Journal of Andrology July/August 2000 diated by 2 classes of nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are each expressed in 3 isoforms:,,. Weand others have reported the presence, in fetal and neonatal testis, of all these isoforms which change throughout the development, and according to the testicular cell type (Boulogne et al, 1999b; Cupp et al, 1999b). RAR, RAR (Boulogne et al, 1999b; Cupp et al, 1999b) and all RXRs (Boulogne et al, 1999b) are expressed in gonocytes, suggesting that RA can act directly on the germline. Koshimizu et al (1995) report that RA increases the number and mitotic activity of mouse PGC and fetal gonocytes cultured on a feeder layer. By contrast, our group has recently observed that RA causes a dramatic reduction in the number of 14.5-dpc rat gonocytes in an organotypic culture system (Livera et al, 2000). This results from an increase in apoptosis and mitosis, with cell death being dominant. Moreover, in the same system, RA causes no change in rat gonocyte number during the quiescent period (1 dpc), whereas it increases the number of 3-dpp gonocytes (when they resume mitosis and apoptosis) by stimulating their mitotic activity. On the contrary, in a 3- dpp testicular cell culture system, RA decreases gonocyte number without affecting their mitotic index. In this system, RA seems to act as an apoptotic factor toward gonocytes (Boulogne et al, 1999b). This discrepancy (as a function of experimental conditions) is probably linked to the importance of culture system on the effects of RA on mitosis or apoptosis. Indeed, the organotypic model allows the preservation of the structure, the organization, and the paracrine/autocrine relationship in the testis, while they are destroyed in germ cell culture. In conclusion, all these data suggest a complex combination of positive and negative factors acting in concert to regulate the proliferation and survival of PGC and gonocytes. Although several factors listed earlier are candidates for controlling germ cell development, it must be emphasized that, in most cases, the potential physiological significance remains to be established since evidence from in vivo experiments supporting the presumed action of a given factor are lacking except for the c-kit/steel factor system. Apoptosis of PGC and Gonocytes The degeneration of developmental germ cells has been known for many years (for a review, see Bartke, 1995; De Felici, 1997). This degeneration has been recently characterized as apoptosis, and the developmental pattern of germ cell apoptosis has been reported in mice (Coucouvanis et al, 1993; Mori et al, 1997; Wang et al, 1998), hamsters (Miething, 1992), and rats (Boulogne et al, 1999a). In the fetal testis, gonocytes are the only cell type to undergo apoptosis (Boulogne et al, 1999a). The pattern of germ cell apoptosis seems to parallel that of their mitotic activity. A first phase of apoptosis is seen in mouse, hamster, and rat fetal gonocytes, and a second surge is seen during postnatal life in hamsters and rats but not in mice, in which the mechanism of postnatal degeneration seems to be necrosis (Miething, 1992; Wang et al, 1998; Boulogne et al, 1999a). The physiological significance of germ cell apoptosis is not clear. Some authors have postulated that germ cell apoptosis could be one of the means of maintaining an optimal germ cell/sertoli cell ratio (Huckins, 1978; Orth et al, 1988). Alternatively, this could be a negative selection of chromosomally abnormal germ cells, which are the consequence of high proliferative rate (Clermont, 1962; Dym, 1994). These hypotheses remain to be tested. Numerous survival factors have been shown to act on PGC and gonocytes. One of the intracellular effectors activated by growth factor receptors can be phosphatidylinositol-3-kinase (PI3K; Datta et al, 1997; Del Peso et al, 1997). PI3K is strongly expressed by neonatal gonocytes (Li et al, 1997) and, recently, Morita et al (1999) have demonstrated that PI3K is required for the germ cell survival of fetal ovary in culture. TGF, which is known to induce apoptosis in different cell types (Grande, 1997), and AMH have been shown to increase the rate of fetal gonocyte apoptosis in organotypic culture systems (Olaso et al, 1998; Olaso et al, unpublished data). The presence of both TGF receptors in gonocytes (Olaso et al, 1998) strongly suggests a direct apoptotic effect of TGF on germ cells, whereas a direct effect of AMH remains to be investigated. The TNF/TNFR system is known to induce apoptosis in several cell types (Kidd, 1998). In PGC, by contrast, TNF acts like a mitogenic factor. No study has analyzed the effect of TNF in gonocytes. Among the receptors of the TNFR family, Fas, a transmembrane receptor, is also known to be implicated in the apoptotic signal (Kidd, 1998). Fas is present in gonocytes (Wang et al, 1998) and its ligand, FasL, is secreted by the Sertoli cells of immature rats (Lee et al, 1997). This system seems to be functional because in germ cell/sertoli cell coculture, the activation of Fas increases germ cell apoptosis, and treatment with the antisense oligonucleotide of Fas reduces germ cell apoptosis (Lee et al, 1997). The action of Fas/FasL in fetal and neonatal testis in vitro and in vivo remains to be demonstrated. Another gene, c-myc, which encodes a nuclear transcription factor, is involved in the induction of apoptosis, although its mechanism of action is not exactly certain (Dang, 1999). C-myc is expressed in different types of germ cells, including PGC (Coucouvanis and Jones, 1993), but the inactivation of c-myc results in embryonic death and prevents analysis of the germline. Moreover,

11 Olaso and Habert Male Germ Cell Development the overexpression of c-myc in testis leads to adult male sterility with an absence of germ cells (Suzuki et al, 1996). These authors report a first alteration of spermatogenesis at 4 dpp, an effect which, thereafter, grows more pronounced. This alteration seems to be due to an increase in the rate of germ cell apoptosis. The molecular mechanism of apoptosis in PGC and gonocytes is still unclear, although in the testis, some antiapoptotic and apoptotic factors have been detected. The role of Bcl-2, which is an antiapoptotic factor, in spermatogenesis is still unclear. Its role in spermatogenesis is debatable, since its presence in germ cells has not been firmly established (Matsui, 1998; Woolveridge et al, 1998). The overexpression of Bcl-2 in mouse inhibits apoptosis and the differentiation of germ cells in immature mice but, paradoxically, induces a massive apoptosis in germ cells of more aged animals and leads to male sterility (Furuchi et al, 1996; Rodriguez et al, 1997). By contrast, the inactivation of Bcl-2 does not seem to disrupt spermatogenesis (Veis et al, 1993). This suggests that either Bcl-2 is not required for the germ cell apoptotic process, or there is redundancy in the system. The overexpression of Bcl-xL, another death-protecting gene, induces disruptions similar to those observed with Bcl-2 (Rodriguez et al, 1997). Furthermore, the transfection of the Bcl-xL gene into cultured PGC decreases apoptosis (Watanabe et al, 1997). It seems, therefore, that Bcl-xL or a related gene could be involved in the regulation of germ cell apoptosis during development. This related gene is not Bcl-w, which is required only after 18 dpp in mouse testis (Ross et al, 1998). Lastly, the inactivation of Bax (Knudson et al, 1995), a proapoptotic factor, leads to an accumulation of anomalous premeiotic cells and gigantic multinuclear cells accompanied by a massive apoptosis, as seen in mice carrying the transgene Bcl-2. This same phenotype is observed in mice expressing a weak level of p53 (Rotter et al, 1993). Taken together, all these data indicate that the disruption of the normal apoptotic process during the development of the germline leads to an accumulation of undifferentiated germ cells which, after their anomalous development, commit suicide. Of the genes involved in the apoptosis distal pathway, some have been disrupted. The inactivation of caspase-1 (Kuida et al, 1995; Li et al, 1995) does not seem to disturb spermatogenesis. The inactivation of caspase-3 or caspase-9 (Kuida et al, 1996; Hakem et al, 1998) leads to an early death in utero that prevents germline analysis. On the other hand, the inactivation of caspase-2 (Bergeron et al, 1998) leads to an accumulation of ovary germ cells, which are resistant to apoptotic agents, suggesting that the apoptotic process in female germ cells requires the presence of caspase-2, but the authors report no observations on the male germ cells. In conclusion, although the knowledge of the genes and 507 factors controlling apoptosis and proliferation of germ cells are likely to be important for understanding both normal and abnormal germ cell development, such as tumorigenesis, further studies are needed to understand the molecular mechanism of germ cell apoptosis. Conclusions In the past few years, our knowledge on the biology of germ cells during development has been considerably extended and we are just beginning to understand some of the molecular mechanisms and to characterize some factors involved in the PGC/gonocytes development. This progress results from a combination of genetic and cellular investigations that have made possible the discovery of many genes and factors that are candidates for affecting the number, proliferation, and survival of PGC and gonocytes. All these processes seem to depend on a complex network of positive and negative factors involved in evoluting relationships between germ cells and somatic cells. Because of the obvious importance of the development of PGC and gonocytes for adult fertility and its eventual alteration by toxins or xenobiotics, a better knowledge of these interactions appears as a priority research axis. Acknowledgments We thank Catherine Pairault for her constructive comments, Christine Olaso for her help in typing the manuscript, and Owen Parkes for English revision. References Agelopoulou R, Magre S, Patsavoudi E, Jost A. Initial phases of the rat differentiation in vitro. J Embryol Exp Morph. 1984;83: Ataliotis P, Mercola M. 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