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Human Reproduction Update, Vol.7, No.2 pp. 191±210, 2001 Impact of genetic engineering on the understanding of spermatogenesis Denise Escalier INSERM U25, HoÃpital Necker, Paris Cedex 15, France. Email:[email protected] Address for correspondence: INSERM U25, HoÃpital Necker, 149 Rue de SeÁvres, F-75743 Paris Cedex 15, France. Tel: 33 1 44 49 53 67; Fax: 33 1 43 06 23 88; E-mail: [email protected] To date, about 100 genes have been found, by genetic engineering, to be implicated in spermatogenesis. Primordial germ cells, spermatogonia, spermatocytes I and elongating spermatids are particularly sensitive. Transgenic and knockout mice permit an approach to be made to the question of genetic factors involved in DNA damage repair, thermal injury, sperm chromatin compaction and sex-speci®c recombination. Knockout mice reveal unexpected functional redundancies of testis-speci®c genes. This review considers how functional divergences can exist among homologous genes from different species, and to what extent the phenotypes of knockout mice can be similar to those from spontaneous mutations. Additional anomalies in reproductive function have frequently been found in these mice, as were found factors leading to tumour susceptibility and/or various diseases. Finally, knockout mice remind us that, in nearly all cases, hemizygous individuals retain a fertility and a wild-type sperm phenotype, although half of the spermatozoa share a genetic defect. The ®ndings strongly emphasize the importance of understanding epidemiology in male infertility, to identify hereditary forms of impaired spermatogenesis, and to create DNA and pathological germ cell banks. Key words: Germ cell/meiosis/spermatozoa/spermatogenesis/testis TABLE OF CONTENTS Genetic engineering strategies Introduction Genetic engineering strategies Spermatogenesis revisited by means of knockout (KO) mice Other data relative to reproduction Diseases and male infertility An animal model with some restraints Conclusions References Data on mouse spermatogenesis have been obtained by several genetic engineering strategies, such as null mutations in the genome (so-called knock-out), gene overexpression, exogenous gene expression and gene misexpression (Table I). Multiple mutants consist either in the inactivation of several genes or gene expression by transgenesis in a null mutant for a functional rescue. Various information has been obtained from multiple mutants. The method allowed mimicry of human deletions of the Y chromosome RBM and DAZ genes (Vogel et al., 1999), induction of the rescue of male fertility in Kit receptor-de®cient mice by loss of p53 (Jordan et al., 1999), and the survival of Brca1-de®cient mice by p53 deletion (Cressman et al., 1999). Double mutants for p53 and the zinc-®nger transcription factor Egr4 have allowed the demonstration that germ cell apoptosis occurring in Egr4-de®cient male mice was p53-independent (Tourtellotte et al., 1999). Egr4-Egr1 double mutants have permitted the elucidation of the level of redundancy between these Egr family members in regulating luteinizing hormone production in male mice (Tourtellotte et al., 2000). Double mutants suggest that Bmp8b and Bmp4 function as heterodimers and homodimers in primordial germ cell speci®cation in the mouse (Ying et al., 2000). The similar meiotic phenotypes of Msh5±/± mice and of double mutant Msh4±/±/Msh5±/± have Introduction Many mutant mice obtained by genetic engineering exhibit spermatogenesis anomalies (for reviews see Simoni, 1994; Grootegoed et al., 1998; Okabe et al., 1998; Lamb, 1999). In a previous review, the various germ cell phenotypes obtained by genetic engineering were classi®ed with regard to the spermatogenesis step affected. This previous review also considered the impact of germ cell genetic alterations on Sertoli cell behaviour and the in¯uence of genetic background on the extent of spermatogenesis failure (Escalier, 1999a). The present review presents subsequently published data on the subject, and an attempt is made to enlighten on the gains brought by genetic engineering to the knowledge of spermatogenesis and the biology of reproduction. Ó European Society of Human Reproduction and Embryology 191 D.Escalier Table I. Overview of the genes found to be involved in spermatogenesis by genetic engineeringa Gene Spermatogenesis anomaly Reference(s) Recombination, caretaker genes (safeguard genomic integrity) Mlh1 DNA mismatch repair Msh5 DNA mismatch repair Msh4 DNA mismatch repair Pms2 DNA mismatch repair Dmc1 recombinase Scp3 synaptonemal complex protein 3 FAC Fanconi anaemia C gene Fanca Fanconi anaemia A gene Baker, S. et al., 1996; Edelmann et al., 1996 Edelmann et al., 1999 Kneitz et al., 2000 Baker et al., 1995 Pittman et al., 1998; Yoshida et al., 1998 Yuan et al., 2000 Whitney et al., 1996 Cheng et al., 2000 Cell cycle Ccna1 Ccnd2 Cdk4 INK4d P27Kip1 cyclin A1 cyclin D2 cyclin-dependent kinase cyclin D-dependent holoenzyme cyclin-dependent kinase inhibitor Liu et al., 1998 Sicinski et al., 1996 Rane et al., 1999 Zindy et al., 2000 Teixeira et al., 2000 Lawson et al., 1999 Il-2 (ecto) PDGF-A Follistatin (over) ActRIIA bone morphogenetic proteins (intercellular signalling proteins) bone morphogenetic proteins bone morphogenetic proteins inhibin a epidermal growth factor vascular endothelial insulin-like neurotrophic factor stem cell growth factor receptor Kit receptor tyrosine kinase interleukin-2 platelet-derived growth factor activin-binding activin type IIA receptor Zhao et al., 1998; Ying et al., 2000 Zhao et al., 1996 Matzuk et al., 1992; Pierson et al., 2000 Wong et al., 2000 Korpelainen et al., 1998 Baker, J. et al., 1996 Meng et al., 2000 Blume-Jensen et al., 2000 Kissel et al., 2000 Ohta et al., 1990 Gnessi et al., 2000 Guo et al., 1998 Matzuk et al.; 1995 Regulatory transcription Egr4 A-myb c-fos c-myc (exo; over) Jun D Tarbp2 Crem Lhx9 Sprm-1 mOvo1 Nhlh2 HSF1 (active form) zinc ®nger, down-regulation substrate for cdk response to mitogens activator activator protein 1 complex activator activator homeobox Pou-homeodomain zinc ®nger, possible regulator basic helix±loop±helix heat shock transcription factor 1 Tourtellotte et al., 1999 Toscani et al., 1997 Johnson et al., 1992 Suzuki et al., 1996; Kodaira et al., 1996 TheÂpot et al., 2000 Zhong et al., 1999 Blendy et al., 1996; Nantel et al., 1996 Birk et al., 2000 Pearse et al., 1997 Dai et al., 1998 Good et al., 1997 Nakai et al., 2000 DNA, RNA-binding proteins Cenpb TIAR TSL Mvh N-ras DNA-binding (centromeric) RNA-binding RNA-binding RNA helicase GDP/GTP binding Hudson et al., 1998 Beck et al., 1998 Kuroda et al., 2000 Tanaka et al., 2000 Mangues et al., 1990 Signalling intermediate Hsp70-2 Telomerase Nek1 Pp1cg MAT (over) Dhh IRS4 chaperone (protein folding, assembly) holoenzyme NIMA-related kinase phosphatase catalytic subunit matrix metalloproteinase Desert hedgehog insulin receptor substrates Dix et al., 1997; Zhu et al., 1997 Rudolph et al., 1999; Lee, H.-W. et al., 1998 Upadhya et al., 2000 Varmuza et al., 1999; Jurisicova et al., 1999 Rudolph-Owen et al., 1998 Bitgood et al., 1996 Fantin et al., 2000 Growth factors TGFb Bmp4 Bmp8a Bmp8b EGF (exo) VEGF (over) IGF1 GDNF (+/±; over) Kit/SCF-R 192 Genetic engineering and spermatogenesis Gene Spermatogenesis anomaly Hormones FSH-R ER-a ER-a+b Dax1 (Ahch) (over) ABP (over) SRC-1 ACE Rxrb PLR Insl3 Cyp19 Apoptosis Bcl-2 Bcl-w Bax Apaf FSH receptor oestrogen receptor orphan receptor androgen-binding protein steroid receptor coactivator-1 angiotensin-converting enzyme steroid receptor prolactin, pituitary insulin-like hormone superfamily aromatase cytochrome P450 (converts androgens to estrogens) Kinases c-mos (mis) v-mos (over) c-abl c-ros R Tyro-3 Csnk2a2 Camk4 Histones, protamines H3.3A (hypomorphic)histone Tnp1 Tnp2 Protamines (mis) Other membrane-related factors Osp/claudin-11 Gja1 Nectin-2 Basigin Fertilin Cyritestin b1 integrin Slc12a2 Oncostatin Other enzymes HR6B HSL Proacrosin Proacrosin (exo) PC4 GT ODC (over; mis) Dierich et al., 1998; Krishnamurthy et al., 2000; Abel et al., 2000 Hess et al., 1997; Couse and Korach, 1999 Couse et al., 1999 Yu et al., 1998 Joseph et al., 1997; Selva et al., 2000 Xu et al., 1998 Hagaman et al., 1998; Ramaraj et al., 1998 Kastner et al., 1996 Gof®n et al., 1998; Steger et al., 1998 Zimmermann et al., 1999; Nef and Parada, 1999 Fisher et al., 1998; Robertson et al., 1999 pro-survival family pro-survival family apoptosis promoter apoptosis activating factor Furuchi et al., 1996; Knudson et al., 1997 Ross et al., 1998; Print et al., 1998 Knudson et al., 1995; Rucker et al., 2000 Honarpour et al., 2000 transcription activator Matsui et al., 2000; Rotter et al., 1993; Shao et al., 2000 Holmberg et al., 1998; Yamasaki et al.,1996 Tumour suppressor p53 E2F-1 (exo) Reference(s) serine threonine kinase serine threonine kinase tyrosine kinase tyrosine kinase receptor tyrosine kinase receptor casein kinase II a¢ Ca2+/calmodulin-dependent protein kinase IV (serine/threonine kinase) Higgy et al., 1995 Rosenberg et al., 1995 Kharbanda et al., 1998 Yeung et al., 1999, 2000 Lu et al., 1999 Xu et al., 1999 Wu et al., 2000 transition protein 1 transition protein 2 Couldrey et al., 1999 Yu et al., 2000 Yu et al., 2000 Lee et al., 1995; Maleszewski et al., 1998 transmembrane protein connexin 43 Ig gene superfamilly Ig gene superfamily, transmembrane ADAM family (transmembrane protein) ADAM family (membrane-anchored) cell surface receptor Na+-K+-2Cl±cotransporter NKCC1 haematopoietin cytokine Gow et al., 1999 Juneja et al., 1999 Bouchard et al., 2000 Igakura et al., 1998 Cho et al., 2000 Shamsadin et al., 1999 Anderson et al., 1999 Pace et al., 2000 Malik et al., 1995 ubiquitin-conjugating enzyme hormone-sensitive lipase serine proteinase Roest et al., 1996 Osuga et al., 2000 Baba et al., 1994; Adham et al., 1997 O'Brien et al., 1996 Mbikay et al., 1997 Lu and Shur, 1997 Halmekyto et al., 1991 serine protease, proprotein convertase serine protease, galactosyltransferase ornithine decarboxylase Table I continued overleaf 193 D.Escalier Gene Spermatogenesis anomaly Reference(s) Others Calmegin IFN-b BrcA1 THEG (insertion) E-MAP-15 chaperone (folding control) interferon-b nuclear phosphoprotein testicular haploid expressed gene epithelial microtubule-associated protein Ikawa et al., 1997 Iwakura et al., 1988 Cressman et al., 1999 Yanaka et al., 2000 Komada et al., 2000 a Most were knockout mice, except: over = overexpression of the gene; mis = misexpression, either premature or delayed; exo = exogenous expression of a gene from another species; ecto = ectopic.) indicated that Msh5 might be epistatic to Msh4 (Kneitz et al., 2000). Even triple mutants have been produced to study the function of Sertoli cell receptors with tyrosine kinase activity (Tyro3, Axl and Mer) and their simultaneous inactivation had led to azoospermia (Lu et al., 1999). Alternatives are to create conditional genome alterations by controlling their onset, frequency, spatial location and tissue/cell type speci®city. To avoid potential transcriptional interference, point mutation by the Cre-lox P system is largely used in other ®elds. Of particular interest is the control of transgene expression by endogenous regulatory sequence of the gene of interest (socalled knock-in). The latter enables the study of gene complementation, such as a possible rescue of a disease by a member of the same gene family. Gene-trap mutagenesis in embryonic stem cells (ES) is a powerful forward genetic approach in mice as it allows the preselection or prescreening of the gene trap of interest. This method has been used to identify retinoic acidresponsive genes during spermatogenesis, and simultaneously to mutate them. The trapped gene was E-MAP-115 (encoding an epithelial microtubule-associated protein of 115 kDa) and was identi®ed as a retinoic-inducible gene (Komada et al., 2000). Germ cell transplantation has been used in combination with genetically modi®ed mice. Oestrogen receptor a-null mutant males (ERa±/±) had been transplanted to testes of recipient wildtype mice expressing the receptor (ERa) but depleted of germ cells. The recipients served as surrogate fathers for the infertile ERa±/± males. Data showed that male germ cells do not require ERa for development or to function in fertilization, suggesting that male ERa±/± mice were infertile due to disruption of oestrogen action within somatic cells of the male reproductive system (Mahato et al., 2000). Toxic transgenesis for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation. Expression of diphtheria toxin A-chain was directed to the male germ line by fusion to the rat histone H1t gene. This resulted in the total elimination of male germ cells in two lines of mice. This experiment has demonstrated the complete tissue speci®city of the H1t promoter (Bartell et al., 2000). In another ®eld, a living marker of germ cells has been generated using a transgenic strain of mice in which green ¯uorescent protein (GFP) is driven by part of the promoter of the Oct4 gene (Anderson et al., 1999). This facility has enabled visualization of living primordial germ cells (PGC) and their active migration at the time they exit the primitive streak, their migration into the allantois, and then directly into the endoderm to 194 be embedded in the hind-gut epithelium (Anderson et al., 2000). In addition, this method has allowed germ-cell puri®cation by ¯ow cytometry to be used to prepare cDNA and identify both cell±cell and cell±matrix adhesion molecules expressed by germ cells during and after migration. Using this method, functional studies have shown that E-cadherin is required for compaction of the germ cells into the genital ridges (reviewed in Wylie, 2000). A mouse strain expressing enhanced GFP (EGFP) under chicken beta-actin promoter showed that beta-actin is highly expressed in spermatogonia and elongating spermatids, and that its expression is regulated by FSH (Ventela et al., 2000). Finally, the generation of transgenic animals has been made by gene transfer into male germ line stem cells using a retroviral vector. At least one in 300 stem cells could be infected. The expression of the retrovirally delivered reporter lacZ transgene persisted in the testis for more than 6 months. This study provides a method to address the potential of human somatic cell therapy DNA constructs to enter a patient's germ line, but also to introduce accidentally permanent genetic changes by gene therapy (Nagano et al., 2000). Spermatogenesis revisited by means of knockout mice To date, about 100 genes expressed in the male reproductive system have been studied by genetic engineering (Table I). Many genes found to be involved in spermatogenesis following genetic engineering have already been described and listed in reviews (Okabe et al., 1998; Escalier, 1999a; Lamb, 1999; Layman and McDonough, 2000); those published later are presented in Table II. Spermatogenesis, a very sensitive function to genetic damages Null mutation is a powerful method to reveal genes that can be involved in failure of spermatogenesis since, in most cases, the ®nding of the involvement of a gene in spermatogenesis was fortuitous and unexpected. Three examples can be given. The casein kinase IIa¢ catalytic subunit (CkIIa¢ was known to be expressed in brain. Mice with inactivation of the Csnk2a2 gene (encoding CkIIa¢) were sterile due to severe anomalies of spermiogenesis, while brain functions were apparently unaffected (Xu et al., 1999). The zinc-®nger transcription factor Egr4 was known to be expressed at high levels in the brain and not in other tissues. Null mutants for Egr4 appeared neurologically normal, but males were infertile due to an arrest of the early stages of meiosis for most of spermatocytes and to premature germ cell Genetic engineering and spermatogenesis Table II. Spermatogenesis failure following genetic engineering in the mouse. (For data prior to February 1999, see Escalier, 1999a.) Gene ±/± Gja1 (Cx43) BMP4±/± b1 integrin±/± Kit/SCF-R±/±a INK4d±/± Fanca±/± Apaf-1±/± GDNF+/± (over) Mvh±/± Scp3±/± Msh5±/± Msh4±/± TLS±/± Tyro±/± Axl±/± Mer±/± EGF (exo) HSF1 (active form) Insl3±/± Osp/claudin-11±/± PDGF-A±/± Pp1cg±/± Tnp 1 (TP1)±/± FSHR±/± Tarbp2±/± E-MAP-115±/± EGR4±/± Jun D±/± THEG (insertion) cyp 19±/± (ArKO) Csnk2a2±/± Camk4±/± Slc12a2±/± (NKCC1) nectin-2±/± c-ros±/± Nek1(kat mutations) HSL±/± Cyritestin Fertilin b±/± Spermatogenesis anomaly Reference(s) primordial germ cell de®ciency absence of germ cells absence of gonad colonization by germ cells early spermatogonial stem cell block testis atrophy, germ cell apoptosis testis atrophy, germ cell loss spermatogonia degeneration some spermatogonia depletion spermatogonia accumulation and degeneration zygotene step arrest, apoptosis zygotene arrest late zygotene arrest zygotene arrest, chromosome condensation and pairing failure unpaired, mispaired spermatocytes spermatocytes, spermatid degeneration partial pachytene arrest, no spermatozoa late pachytene step arrest and apoptosis spermatocytes I arrest spermatocyte lysis spermatocyte arrest, absence of adult Leydig cells round spermatid arrested and polyploid round spermatid depletion spermatid chromatin condensation anomaly, very poor sperm motility hypospermatogenesis, cytoplasmic droplet, reduced sperm chromatin compaction abnormal sperm nuclear morphogenesis manchette and nuclear shape abnormalities ¯agellar defects sperm head misshapen; ¯agellum misassembled spermatid arrest, head misshapen, ¯agellum misassembled spermatid arrest, acrosomal abnormalities (4.5 months±1 year) sperm nuclear elongation defect malformed and detached acrosome abnormally heads and tail improper attachment nuclear shape and acrosomal anomalies sperm nuclear and cytoskeletal anomalies sperm tail angulation; unable to enter the oviduct testicular hypoplasia oligospermia, Sertoli injuries zona pellucida binding inability reduced sperm-egg fusion Juneja et al., 1999 Lawson et al., 1999 Anderson et al., 1999 Kissel et al., 2000 Zindy et al., 2000 Cheng et al., 2000 Honarpour et al., 2000 Meng et al., 2000 Tanaka et al., 2000 Yuan et al., 2000 Edelmann et al., 1999 Kneitz et al., 2000 Kuroda et al., 2000 Lu et al., 1999 Wong et al., 2000 Nakai et al., 2000 Nef and Parada, 1999 Gow et al., 1999 Gnessi et al., 2000 Varmuza et al., 1999 Jurisicova et al., 1999 Yu et al., 2000 Krishnamurthy et al., 2000 Zhong et al., 1999 Komada et al., 2000 Tourtellotte et al., 1999 Thepot et al., 2000 Yanaka et al, 2000 Fisher et al., 1998; Robertson et al., 1999 Xu et al., 1999 Wu et al., 2000 Pace et al., 2000 Bouchard et al., 2000 Yeung et al., 2000 Upadhya et al., 2000 Osuga et al., 2000 Shamsadin et al., 1999 Cho et al., 1998, 2000 exo = exogenous expression of a gene from another species. a Point mutation. death (Tourtellotte et al., 1999). Inactivation of the apoptotic protease-activating factor-1 gene (Apaf-1) induced degeneration of spermatogonia and, inversely, an excess of neuronal progenitor cells (Honarpour et al., 2000). In other cases, widespread disturbances were expected due to the ubiquitous expression of the gene, but it was the testis that was primarily affected. This was the case for the cyclin D-dependent holoenzyme, INK4d, suspected to be a tumour suppressor but whose Ink4d gene inactivation led to testicular atrophy and germ cell apoptosis (Zindy et al., 2000). Similarly, the inactivation of Slc12a2 (encoding the Na+-K+-2Cl± cotransporter NKCC1) did not lead to kidney or blood pressure disturbances, but to failure of spermatogenesis and inner-ear defects (Pace et al., 2000). Nectin2 is a component of adherens junctions, the expression of which is ubiquitous. However, nectin-2±/± mice had no overt defects other than infertility in males. Nectin-2 was found expressed in the 195 D.Escalier testes only during the later stages of spermatogenesis and the structural defects observed in spermatozoa of nectin-2±/± mice suggested a role of the protein in sperm cytoskeletal organization (Bouchard et al., 2000). The kit receptor tyrosine kinase was known to have a role in proliferation, survival, adhesion, secretion and differentiation. Point mutation in this gene had shown that it is critical in spermatogenesis at premeiotic stages, and that other factors compensate for haematopoiesis, melanogenesis and primordial germ cell development (Kissel et al., 2000). Such examples are numerous (see in Escalier, 1999a), and show that many genes common to germ cells and somatic cells are critical for spermatogenesis. This could also be the case for oogenesis, since Cd9-de®cient mice exhibited only female sterility, although Cd9 is known to participate in cell migration and adhesion (Kaji et al., 2000). Finally, genetic engineering reveals that some genes thought to be essential for female reproduction are more critical to spermatogenesis. Dax1 was considered as an ovarian-determining gene, but Dax1-deleted mice showed testicular epithelium degeneration and normal ovarian development (Yu et al., 1998). Conversely, genetic engineering has revealed that some gene products thought to be determinants for spermatogenesis or fertilization have, in fact, a less important function. This was the case following proacrosin gene inactivation, as this did not lead to sterility or sperm phenotype anomalies but only to a delay in fertilization (Baba et al., 1994; Adham et al., 1997). Also, inactivation of the genes of the transition proteins (Tp) involved in the replacement of histones during sperm chromatin compaction did not lead to sterility of Tp2±/± mice or impaired only partially the male fertility of Tp1±/± mice (Yu et al., 2000) (see also Gene redundancy). Xist is implicated in female X-inactivation and thought to be involved in X inactivation during meiosis. However, Xist-de®cient mice did not exhibit testis anomalies and were fully fertile, demonstrating that Xist is not implicated in the reversible XY inactivation in spermatocytes (Marahrens et al., 1997). Mesothelin (a glycoprotein that is highly expressed in mesothelial cells) is expressed in many tissues, including the testis. Unexpectedly, null mutant mice for this gene developed normally and produced offspring normally (Bera and Pastan, 2000). A widely proposed model suggested that fertilin promotes spermegg fusion. In mice lacking the fertilin b-subunit, fertilin a is also absent from mature spermatozoa. Nevertheless, sperm lacking both fertilin subunits can fuse with eggs at 50% of the wild-type rate. Data suggested that other gamete surface molecules act to promote membrane fusion, and that fertilin's role is in spermatozoon±egg plasma membrane adhesion (Cho et al., 2000). Very sensitive steps: germ cell proliferation and survival and meiotic prophase I Primordial germ cell and spermatogonia Knockout mice are also models used to study several factors involved in the migration (Anderson et al., 2000) and maintenance of germ cells. Bmp4 null mutant embryos contain no PGC and lack an allantois, showing that BMP4 is a cell signalling factor required for the formation of the germline during gastrulation. Beta-galactosidase activity in Bmp4 (lacZneo) embryos revealed that before gastrulation, Bmp4 is expressed ®rst in the extraembryonic ectoderm and later in the extraem- 196 bryonic mesoderm. Bmp4 extraembryonic ectoderm expression regulates the formation of allantois and PGC precursors and the size of the founding population of PGC (Lawson et al., 1999). Bmp8b is found to be expressed in the extraembryonic ectoderm in pregastrula and gastrula stage mouse embryos. The absence of PGC in 43% of Bmp8b null mutants showed that Bmp8b is required for PGC generation (Ying et al., 2000). Besides, integrin b1±/± PGC have shown that b1 integrin is required for gonad colonization by germ cells (Anderson et al., 1999). Knockout mice have shown that Tiar (Beck et al., 1998) and p53 (Matsui et al., 2000) are involved in regulation by apoptosis of maintenance of fetal germ cells. Tiar is a RNA-binding protein whose role might be in the expression of a survival factor or survival factor receptor that is essential for PGC development (Beck et al., 1998). Both body growth and the number of PGC can be affected by inactivation of Tiar (Beck et al., 1998) and Zfx genes (Luoh et al., 1997). The growth factor gene GDNF (Meng et al., 2000) and the TR gene encoding the telomerase holoenzyme TR (Lee, H.-W. et al., 1998) play a role in spermatogonia proliferation. GDNF is involved in DNA synthesis in spermatogonia, and is expressed by Sertoli cells, while the GDNF receptor and the Ret receptor tyrosine kinase are expressed in testicular germ cells. GDNF interacts with membrane receptors which mediate stimulation of the Ret receptor tyrosine kinase (Viglietto et al., 2000). Dazla is essential for the survival of spermatogonia (Ruggiu et al., 1997). Germ cell proliferation and initiation of spermatogenesis are under the control of the transforming growth factor gene BMP8b (Zhao et al., 1996), while the mouse homologue of Drosophila Vasa (Mvh) operates on the proliferation and differentiation of male germ cells (Tanaka et al., 2000). Bcl-2 family members (Knudson and Korsmeyer, 1997) are involved in control of the number of spermatogonia (see Escalier, 1999a). The localization of Bcl-x(l), the long transcript of Bcl-x, suggests that it may regulate germ cell density, possibly in cooperation with the apoptotic inducer Bax (reviewed in Beumer et al., 2000). Mice containing two copies of a hypomorphic allele of Bcl-x lacked spermatogonia and were sterile, the primordial germ cells being depleted by day 15.5 embryos. The loss of Bcl-x function in the hypomorph was corrected by deletion of both copies of the bax gene, showing that the balance of Bcl-x and Bax control PGC survival and apoptosis (Rucker et al., 2000). Primary spermatocytes Primary spermatocytes are very sensitive to genetic manipulation, particularly when genes are involved in recombination. In most cases this leads to meiotic arrest that reveals the existence of meiotic checkpoints for DNA damage in mammals, particularly in primary spermatocytes at the pachytene step (reviewed in Escalier, 1999b). Other meiotic prophase I stages can be affected by a null mutation, as found in null mutant mice with an arrest at the leptotene step for Atm (Barlow et al., 1998), at the zygotene step for Dmc1 (Pittman et al., 1998; Yoshida et al., 1998), Scp3 (Yuan et al., 2000), Msh5 (Edelmann et al., 1999), Msh4 (Kneitz et al., 2000) and Mvh (Tanaka et al., 2000), and at the diplotene step for cyclin A1 (Liu et al., 1998). Inactivation of DNA mismatch repair genes, thought to be involved in recombination, can lead to various spermatogenesis impairments. Mlh1-de®cient mice showed meiotic arrest (Edelmann et al., 1996), while Pms2- Genetic engineering and spermatogenesis de®cient mice could produce spermatids, although abnormal (Baker et al., 1995). Spermatocytes arrested in the meiotic prophase were targets for apoptotic events leading to their elimination and seminiferous tubule depletion. At advanced stages, Sertoli cells showed vacuolation or sloughing, demonstrating the importance of somatic and germinal cell interactions for their survival (review in Escalier, 1999a). The in¯uence of Sertoli cells on germ cell survival has been shown by inactivation of three Sertoli receptor genes of the Tyro3 family (Tyro 3, Axl, Mer). These Sertoli cell receptors are normally expressed during postnatal development, and their ligands (related to sex hormone-binding globulin) are expressed by Leydig cells before sexual maturity. Mice lacking any single receptor, or any combination of two receptors, were fertile while male triple mutants were sterile. They exhibited germ cell death which did not result from the death of Sertoli cells. Data showed that Tyro 3 family receptors and their ligands might act as essential regulators of Sertoli-cell function. The combined activation of the receptors in Sertoli cells may be required for the production of germ-cell trophins and thus to the role of Sertoli cells as nurturers of developing germ cells (Lu et al., 1999). The in¯uence of Sertoli cells on spermiogenesis is shown in Rxrb (retinoid 3 receptor) mutant mice. Rxr is normally expressed in Sertoli cells and, in Rxrb null mice, Sertoli cells accumulated unsaturated triglycerides while spermatid release did not occur. The epididymis contained few spermatozoa with abnormal acrosomes and tails. In old mutant males, progressive degeneration of the germinal epithelium occurred, ending with acellular lipid-®lled tubules (Kastner et al., 1996). Another main gain of genetic engineering is knowledge of the importance in germ cells of tumour suppressor genes, such as p53, and of genes involved in apoptosis induction, such as Bax, or involved in protection against apoptosis, such as members of the bcl2 family (Escalier, 1999a; Print and Loveland, 2000). Of interest is the ®nding that p53 promotes apoptosis of fetal testicular cells only after day 16.5 postcoitum, but not before (Matsui et al., 2000) (see also section, Diseases and male infertility). How the Bcl-2 gene and its pro-survival relatives prevent activation of the cytoplasmic proteases (called caspases) that mediate apoptosis is unknown. Bcl-w is an important prosurvival protein that participates in the regulation of apoptosis by binding pro-apoptotic factors Bax and Bak (Yan et al., 2000). The apoptotic protease-activating factor-1 (Apaf-1) is a key trigger of apoptotic events through cytochrome c-mediated apoptosis. The complex composed of Apaf-1, dATP and cytochrome c activates a series of caspases, leading to apoptotic cell death. Degeneration of spermatogonia resulting in the virtual absence of spermatozoa in surviving Apaf-1 null mutants suggested that alternative apoptotic pathways work in conjunction with and parallel to Apaf-1 and can modify its effect on programmed cell death in testis (Honarpour et al., 2000). Pachytene spermatocyte: a target to thermal injury response Another aspect of data obtained by genetic engineering is related to the vulnerable nature of spermatogenesis to thermal insult. Heat shock induces activation of heat shock transcription factor (HSF), which regulates expression of heat shock genes, the major products of which act as molecular chaperones by facilitating protein folding and assembly (Hartl, 1996). HSF1 is a major heat stress-responsive factor expressed ubiquitously to protect cells. Mice expressing the active form of HSF1 (Nakai et al., 2000) were infertile as a result of blockage of spermatogenesis at the spermatocyte I stage. Spermatocyte apoptosis appeared by 3 weeks. At 4 weeks, 25±30% of the seminiferous tubules contained clusters of apoptotic cells compared with 30±35% after mild heat exposure (43°C for 15 min). A much lower cyclin A1 level than that in wild-type mice was found (cyclin A1 normally increases in late pachytene and reaches a peak in the diplotene stage). The transgene was found to be highly expressed in spermatogenic cells, but not in Sertoli cells or interstitial Leydig cells. HSF1 would be a major trigger for the induction of apoptosis of germ cells. Late pachytene spermatocytes were revealed to be target cells for HSF1-induced cell death. It was suggested that HSF1 is a good candidate for acceleration of apoptosis of germ cells because it can sense thermal stress directly as well as indirectly. In isolated pachytene spermatocytes, HSF1 was activated by a lower temperature threshold than somatic cells in the testis. HSF1 active form expression by transgenesis did not affect cell growth, differentiation or deathÐan important ®nding considering the therapeutic use of heat shock response. Another important repercussion of these data should be in the ®eld of cryptorchidism, because experimental cryptorchidism induces apoptosis of almost the same subset of cells (Nakai et al., 2000). It can be noted that the function of molecular chaperone assumed by heat shock proteins may be used for other functions than thermal injury during spermatogenesis. This is well illustrated by Hsp70-2-de®cient mice, showing that HSP70-2 is required for assembly of a functional CDC2/cyclin B1 complex in pachytene spermatocytes, and thus that HSP70-2 plays a role in synaptonemal complex desynapsis which necessitates phosphorylation of the synaptonemal protein-1 (SCP1) by CDC2 (Zhu et al., 1997; for review, see Escalier,1999a). Unexpected sex-speci®c factors The use of knockout mice allows a list to be compiled of genes involved in meiotic recombination and chromosome pairing. As expected, many genes involved in recombination are common to both sexes, such as Dmc1 (Pittman et al., 1998; Yoshida et al., 1998), Msh5 (Edelmann et al., 1999) and Atm (Barlow et al., 1996; Xu et al., 1996). Moreover, some of the genes are also involved in somatic recombination, such as Mlh1 and Atm. It is noteworthy that there are sex-speci®c factors for apparently similar events. The products of the DNA mismatch repair genes Mlh1 and Pms2 are known to form heterodimers. Inactivation of Pms2 has led to meiotic arrest in the male only, although Pms2 might be involved in DNA mismatch repair in a variety of tissues (Baker et al., 1995). In contrast, inactivation of Mlh1 (Baker, et al., 1996; Edelmann et al., 1996) and Msh4 (Kneitz et al., 2000) has led to meiotic arrest in both sexes (Edelmann et al., 1996). Chromosome pairing is also related to sex-speci®c factors. SCP3 is a major component of the synaptonemal complexes, and Scp3 inactivation disrupted only male meiosis (Yuan et al., 2000). The same phenomenon is found for the Xlr family whose members are expressed in lineages undergoing recombination (Escalier et al., 1999). Xmr is the testicular member of this family that is expressed in spermatocytes during the meiotic recombination (Calenda et al., 1994). Unexpectedly, it is the lymphoid Xlr member, and not Xmr, that it is expressed during the female 197 D.Escalier meiotic recombination (unpublished data). This fact supported data suggesting that Xmr could be involved in male Xchromosome inactivation in mammals (Escalier and Garchon, 2000). A similar divergence in germ cell processes between sexes is also observed at earlier stages, as seen following inactivation of the cKit receptor/stem cell factor (kit/SCF-R) gene leading to only male infertility (Blume-Jensen et al., 2000). By contrast, point mutation in the binding site for phosphatidylinositol 3'-kinase in the kit gene reduced female fertility due to development of ovarian cysts and ovarian tubular hyperplasia, while spermatogenesis was arrested at the premeiotic stages, leading to male sterility (Kissel et al., 2000). Also a targeted mutation of the mouse Vasa homologue gene (Mvh) affected only male reproduction, although Mvh expression was ®rst detected in primordial germ cells after colonization of the genital ridges (Tanaka et al., 2000) and in germ cells undergoing gametogenic processes in both the male and female (Toyooka et al., 2000). On the contrary, both sexes were affected in other genes involved in spermatogonia maintenance such as Tiar, Tr (telomerase) and Dazla (see Escalier, 1999a). Functional analysis of genes involved in sperm chromatin compaction Many germ cell differentiation events can also be shown by mouse models. An example is histone replacement by transition proteins (TP) and protamines during spermiogenesis (Baarends et al., 1999; Zhong et al., 1999). It has been shown that the doublestranded RNA binding protein PRBP (encoded by the gene Tarbp2) is required for correct translational activation of the mRNAs encoding the protamines. Disruption of the Tarbp2 gene has led to sterility and oligospermia. A failure to synthesize the protamines resulted in delayed replacement of TP. Spermatids showed abnormal nuclear morphogenesis, developmental arrest and degeneration. PRBP is suspected to be a chaperone in the assembly of speci®c, translationally regulated ribonucleoproteins (Zhong et al., 1999). Pre-existing protamine-1 (Prm1) mRNAs are stored for several days, and their temporal translation or messenger RNA stability are under the control of regulatory elements located in the 3¢ untranslated region (3¢UTR) of mRNAs. Prm1 mRNA is stored in spermatids as a cytoplasmic ribonucleoprotein particles that contain Y box proteins. Y box family members are nucleic acid binding proteins that can bind to the Prm1 3'UTR in vitro. Data suggest that Y box proteins could function as translational repressors or could protect mRNAs from degradation during their extended period of storage (reviewed in Braun, 2000). Premature translation of Prm1 mRNA caused precocious condensation of spermatid nuclear DNA and abnormal head morphogenesis. Round spermatids were arrested and intercellular bridges were relaxed, leading to multinucleated cells. The incomplete processing of protamine-2 protein suggested that its processing is coupled with protamine-2 codeposition with protamine-1 (Lee et al., 1995). The sequential deposition of sperm basic nuclear proteins on chromatin was disrupted in Camk4±/± mice which had sperm counts less than 4% of those of wild-type males and abnormal spermatozoa with failure to develop hook-shaped heads and improper attachment of the ¯agellum. Camk4 is the Ca2+/ 198 calmodulin-dependent protein kinase IV implicated in transcriptional regulation and associated with chromatin and nuclear matrix. Step-15 spermatids in Camk4±/± mice showed loss of protamine-2 and prolonged retention of TP2. However, transcription of Prm2 was unaffected, suggesting that protamine-2 was prematurely degraded. Protamine-2 was phosphorylated by Camk4 in vitro, indicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in male germ cells. The results suggested that successful interaction of mature protamine-2 with chromatin is required for displacement of TP2 (Wu et al., 2000). In vitro, transition protein 1 (TP1) reduces the interaction of DNA with the nucleosome core, while transition protein 2 (TP2) compacts the DNA in nucleosomal cores. Mice lacking the major TP1 have been obtained following targeted deletion of the Tnp1 gene. Only 60% of Tnp1-null males were infertile, and the fertile Tnp1±/± mice showed a decrease in the number of litters produced. Sperm production was normal in Tnp1±/± mice, but only 23% of the spermatozoa showed any movement, and most of those did not show forward progression. Abnormal rod-shaped chromatin condensation units appeared in the nuclei of condensing spermatids at step 12, but all the chromatin condensed from step 14. Sperm heads with a blunted or bent tip were seen in 16% of epididymal spermatozoa, possibly generated by the abnormal chromatin condensation that could reduce the rigidity of the ®ne apex of the spermatozoon. In Tnp1±/± mice, TP2Ðindividually or in concert with the precursor of protamine-2 (pP2)Ðwere able to initiate chromatin condensation, and these proteins completely replaced histone without involvement of TP1. Data suggested that the signals that normally activate TP1 translation can, in the absence of TP1, act on TP2 and P2 mRNA and that TP1 is functionally related to TP2 and the protamines (Yu et al., 2000). Moreover, most Tnp2±/± male mice had normal numbers of spermatozoa and normal fertility, although the litter size was reduced and some spermatozoa presented blunt-tipped heads (Yu et al., 2000). In Tnp2 mutants, protamine-2 precursors were found in epididymal spermatozoa, and no changes were detected in TP1 concentrations (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 Tnp1 gene expression, and human CREM protein is synthesized in steps 1±3 round spermatids. This could explain why a reduction of Crem expression and a lack of both CREM and TP1 were found in human arrested spermatids at step 3 (Steger et al., 1999), but it is unclear whether this anomaly is secondary or not to the arrest. Mice deleted in Crem presented a spermatogenesis arrest at the round spermatid step (reviewed in De Cesare et al., 1999). Recent data have shown that CREM requires a tissue-speci®c coactivator, ACT (Activator of CREM in Testis) to elicit its regulatory function in the testis (Sassone-Corsi, 2000). Disruption of mHR6B normally involved in histone 2A and 2B ubiquitination for their degradation did not lead to blockage of the synthesis of TP, but to an anomaly of their distribution leading to abnormally shaped sperm heads (Roest et al., 1996). The reduction in size of the testis, seminal vesicles and epididymis found in follicle-stimulating hormone receptor knockout (FORKO) mice that lack the FSH receptor gene (FSHR) could be due to a decrease in the level of testosterone. The 23% increase in propidium iodide stainability of elongated spermatids and the Genetic engineering and spermatogenesis increased sperm head size in FORKO mice suggested a disturbance in the normal replacement of histones by protamines during spermiogenesis, leading to poor condensation of spermatid nuclei. The decreased motility was associated with a bent tail, and 80% of the ¯agella had retention of the cytoplasmic droplet (Krishnamurthy et al., 2000). A disturbance of protamine deposition in mice expressing the galline protamine led to heterogeneous chromatin and instability of the head-¯agellar attachment (Maleszewski et al., 1998) (for the examples in this paragraph, see Escalier, 1999a). Taken together, these data show that disturbances of expression timing of either TP or protamines lead to spermatid arrest. It is noteworthy that anomalies of the spermatid chromatin compaction result in ¯agellar anomalies, showing the importance of nuclear integrity on the ¯agellar anchorage. That the ¯agellar anomalies result from defects in the shape of the implantation fossa did not seem to be the explanation in all cases, suggesting that disturbances of the chromosome territories and/or the nucleoskeleton might also be involved. Unexpected ¯agellar morphogenetic factors Knockout mice help to detect factors involved in the organization and reorganization of the cytoskeleton during spermiogenesis. Nectin-2 is a component of cell±cell anchoring junctions (adherens junctions) and is linked to F-actin through the actin ®lament-binding protein l-afadin. Anchoring junctions connect the cytoskeletal elements of neighbouring cells, and are also believed to regulate cell shape and differentiation through signalling pathways. Nectin-null mutant males produced morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and in mitochondrial localization. Sperm nuclei were irregularly shaped, with translucent vesicles and indentations. The formation of a tightly packed helical sheath of mitochondria was impaired, and mitochondria were frequently present alongside the nucleus. The outer dense ®bres were jumbled and extended into the head. While F-actin was found to be prominent in the middle piece in wild-type spermatozoa, in nectin±/± mice, F-actin was mainly present in the head and poorly present in the middle piece. It was suggested that nectin-2 on the middle piece is required for proper localization of F-actin, through interaction with l-afadin. Nectin-2 might bring the lafadin±F-actin complex to the middle piece and mediate structural changes of actin. These changes may be important in nuclear shaping, mitochondrial relocation and dense ®bre organization during spermiogenesis (Bouchard et al., 2000). JunD contributes to the AP-1 transcription factor complex whose function is complex, including tumour suppression. JunDde®cient mice presented anomalies of both spermatogenesis and mating behaviour, suggesting that functions of JunD dimers in the pituitary and the testis are essential to maintain normal testis function. A speci®c block in spermiogenesis was observed in homozygous JunD±/±, and an absence of ¯agella in the lumen of the seminiferous tubules correlated with a marked decrease in the number of late spermatid heads located near the lumen. There were two classes of sperm phenotypes, either asthenoteratozoospermia or asthenozoospermia alone. In the ®rst case, the sperm head showed an abnormal hammer-like shape, and misassemblage of the microtubules and of the periaxonemal structures of the ¯agellum. When the ¯agellum merged out of the cell cytoplasm, the periaxonemal cytoplasm bulged out, forming irregular cytoplasmic masses (TheÂpot et al., 2000). Therefore, the absence of JunD led to sperm ¯agellar growth impairment and, as in other tissues, JunD is thought to be a growth inhibitor. Ki/ki (kisimo) mice had disruption of THEG (testicular haploid expressed gene) by insertional mutation. THEG is expressed in round and elongated spermatids, and needs Sertoli cells for its continued expression in spermatids (Nayernia et al., 1999). Ki/ki mice had virtually no spermatozoa, and the spermatids exhibited misshapen nuclei. At the ¯agellar level, microtubules and coarse ®bres were arranged in a whirl. THEG interacts in vitro with chaperin containing TCP-1 (t-complex poly peptide Ie) (CCTe), which is known to be required for the correct folding or assembly of cytoskeletal proteins. Data have suggested that abnormal or absent ¯agella in ki/ki mice may be due to impairment of the assembly of cytoskeletal proteins such as the tubulins (Yanaka et al., 2000). Homozygous c-ros knockout mice are sterile, and the epididymal spermatozoa exhibit bent tails. After mating, 46± 86% of knockout spermatozoa in the uterus were bent at the cytoplasmic droplet even when motile, and motile spermatozoa showed a reduction in their straightline and curvilinear velocities. No spermatozoa were recovered from the oviduct or observed within the uterotubal junction. The infertility of c-ros knockout male mice can be explained by the inability of the spermatozoa to enter the oviduct, as a result of their bent tails and their compromised ¯agellar vigour within the uterus (Yeung et al., 2000). Discovery of other genes involved in spermiogenesis Null mutants for the zinc-®nger transcription factor Egr4 presented a maturation arrest with 70% of spermatocytes undergoing apoptosis, and these results suggested that spermatocytes required Egr4 to transgress the early to mid-pachytene stage of meiotic prophase. The maturation arrest was incomplete, and some Egr4-de®cient animals exhibited spermatozoa with heads that were either separated entirely or bent sharply back on the ¯agella. In addition, the ¯agella were often fragmented, sharply kinked, or had tightly coiled distal ends. Widespread alterations in testicular gene expression were found, although no de®nitive target genes were identi®ed to account for these anomalies. Crem and A-myb were markedly up-regulated in Egr4-null testis, as were several genes that participate in steroidogenesis or steroid signalling. These alterations possibly re¯ected compensatory responses to ineffective pachytene spermatocyte maturation and inadequate levels of spermatogenesis (Tourtellotte et al., 1999). Null mice in the Sla12a2 gene (normally expressing the Na++ K -2Cl± co-transporter NKCC1) had delayed formation of the lumen of the seminiferous tubules. By 1.5 months of age, marked abnormalities in spermatogenesis were observed and cellular loss increased with advancing age. Few spermatids were present, but defects were particularly striking when spermatids gradually acquired the polarized shape and structural features of spermatozoa. Spermatids showed anomalies of the cap-phase acrosomal vesicle and of the nuclear shape. Acrosomal defects included abnormal placement of the acrosomal granule and vesicular structures resembling two acrosomes at opposite poles of one nucleus. Normally, the seminiferous tubule ¯uid contains 10-fold higher concentrations of K+, and 10-fold lower concentrations of 199 D.Escalier Table III. Various other disorders of the reproductive functions after gene inactivation Gene Anomalies Reference(s) mOvo1 IGF1 FSHR urogenital defects vestigial ducts, seminal vesicles and prostate smaller epididymis and seminal vesicles; reduced testicular weight; follicular development arrest; vagina imperforate, atrophic uterus efferent ductule ¯uid reabsorption failure smaller epididymis epididymal degeneration cryptorchidism; deregulation oestrus cycle failure initial epididymis segment lack of genital ridges and gonads failure of sperm-egg fusion premature arrest of female fertility block in folliculogenesis (early antral stage) impaired fertility; reduced primary follicles ovulation of pre-meiotic oocytes lack of embryo implantation seminal vesicles and prostate anomalies embryo implantation defects ovarian teratomas ovary sex reversal uterus disruption (epithelium, glands) atretic follicles; external genitalia, uteri, mammary gland underdevelopment defective decidualization absence of an allantois reduced placenta; de®cit in mounting capacity breast underdevelopment; impaired nursing developmental impairment embryonic lethality embryonic lethality defects in mating behaviour defects in mating behaviour Dai et al., 1998 Baker, J. et al., 1996 Krishnamurthy et al., 2000; Abel et al., 2000 ER-a E-MAP-115 Bmp8b Insl3 c-ros Lhx9 Cd9 Fanca Inhibin a (over) Bcl-x (hypomorphic allele) PLR basigin Mos ab ER centromere protein B cyp19 interleukin-11 BMP4 c-fos A-myb cyclin B1 Rad 51 BLM Jun D EGF (exo) Hess et al., 1997 Komada et al., 2000 Zhao et al., 1998 Zimmermann et al., 1999; Nef and Parada, 1999 Yeung et al., 1999 Birk et al., 2000 Kaji et al., 2000 Cheng et al., 2000 Pierson et al., 2000 Rucker et al., 2000 Gof®n et al., 1998 Steger et al., 1998 Igakura et al., 1998 Hirao and Eppig, 1997 Couse et al., 1999 Fowler et al., 2000 Fisher et al., 1998 Robb et al., 1998 Lawson et al., 1999 Johnson et al., 1992 Baum et al., 1994 Toscani et al., 1997 Brandeis et al., 1998 Tsuzuki et al., 1996 Chester et al., 1998 Thepot et al., 2000 Wong et al., 2000 ER = oestrogen receptor; exo = exogenous expression of a gene from another species. Na+ than does blood plasma, and is thought to play a vital role in normal germ cell development and Sertoli cell function. It was suggested that K+ can be pumped into the Sertoli cell from the interstitial ¯uid by an active process, probably involving a Na+, K+ translocating ATPase, and that NKCC1 transports K+ at the basolateral surface of the Sertoli cells (Pace et al., 2000). Protein kinase casein kinase II (Ck2) is a cyclic-AMP and calcium-independent serine-threonine kinase. Ck2 has been implicated in DNA replication, regulation of transcription, translation and control metabolism. Ck2 has been found to be associated with the nuclear matrix, and also with chromatin itself. Mice de®cient for Csnk2a2 encoding the casein kinase IIa¢ catalytic subunit (CkIIa¢) showed anomalies of spermatid nuclear morphogenesis with nuclear and acrosomal shaping anomalies. Elongating spermatids developed abnormalities of the anterior head, and the nuclear and acrosomal morphology became increasingly deformed, with many acrosomes becoming detached from the nucleus or exhibiting an excrescence. In the epididymis, sperm heads were round or ovoid. It was suggested that CkIIa¢ may play a role in regulating the development of the nuclear 200 architecture, and that CsnK2a2 could be a candidate globozoospermia gene. This study was the ®rst case in which a unique role for one of the isoforms of Ck2 was demonstrated (Xu et al., 1999). Aromatase enzyme cyp19 converts C19 steroids (androgens) to C18 steroids (oestrogens), and is active in Sertoli cells and Leydig cells. Aromatase is present in spermatocytes and spermatids. ArKO mice with targeted disruption of cyp19 were initially fertile, but developed Leydig hyperplasia/hypertrophy and progressively disrupted spermatogenesis, despite no decrease in gonadotrophins or androgens. Female ArKO mice also showed a progressive phenotype. Spermatogenesis disruption was evident from 18 weeks of age and affected all 1-year-old ArKO mice. Spermiogenesis was arrested at early stages in ArKO mice. Round spermatids did not complete elongation and spermiation, and degenerating and multinucleated round spermatids were seen in tubules exhibiting spermiogenic arrest. Abnormal acrosomes often were observed in which uneven spreading over the nuclear envelope was apparent, and more than one acrosomal granule was frequently visible. The phenotype was different from ERKO mice Genetic engineering and spermatogenesis Table IV. Genes involved in spermatogenesis and tumour susceptibility Gene de®ciency Tumour susceptibility Reference(s) N-ras P53 mammary and salivary glands tumours lymphoma, sarcoma, adenoma Leydig cell tumour, gonadoblastoma chromosome loss/duplication, interstitial deletion human Bloom syndrome thymic lymphomas increased incidence of malignancies various tumours ovarian and testicular tumours ovarian teratomas lymphomas; intestinal adenomas; intestinal adenomas; adenocarcinoma lymphomas; sarcomas telomerase teratocarcinomas (germ cells) lymphomas, carcinomas known to be involved in human liposarcomas and leukaemias testicular tumours Mangues et al., 1990 Donehower et al., 1992 BLM Atm FAC E2F-1 Cyclin D2 Mos Mlh1 Pms2 Tls (RNA-binding) GDNF (over) Shao et al., 2000 German, 1993 Barlow et al., 1996; Xu et al., 1996 Whitney et al., 1996 Yamasaki et al., 1996 Sicinski et al., 1996 Hirao and Eppig, 1997 Prolla et al., 1998 Prolla et al., 1998 Rudolph et al., 1999 Kuroda et al., 2000 Meng et al., 2000 over = overexpression. that lack ERa and exhibit disruption of spermatogenesis caused by abnormal ¯uid reabsorption. These ®ndings suggest that oestrogen plays a hitherto unsuspected role in spermatid differentiation and spermatogenesis (Robertson et al., 1999). Use of the gene trap screen has allowed the isolation of ROSA63 mutant mice exhibiting a phenotype similar to those in vitamin A-de®cient animals and retinoic acid receptor a (RARa) knockout mice. ROSA63 mice shared a mutation in the gene encoding an epithelial, microtubule-associated protein of 115 kDa (E-MAP-115), that is highly expressed in testis and kidney and whose expression is induced by retinoic acid. E-MAP-115 is a MAP implicated in stabilizing and reorganizing microtubules (MT), and is found in the perinuclear subdomain of MT in cultured cell lines of epithelial origin. In ROSA63 mutant mice, the testes and epididymis were smaller. As early as the ®rst wave of spermatogenesis, all condensed spermatids demonstrated abnormal shape, underwent progressive degeneration, and were phagocytosed by Sertoli cells. Spermatids were ®rst lost by 3 months of age, followed by loss of spermatocytes and spermatogonia by 12 months. The nuclear shape was slightly abnormal at steps 8 and 9, and became more abnormal in mid-step 9±11. About 80% of spermatid sections showed anomalies of the microtubular manchette (absent, reduced, ectopic). Regions of the spermatid nucleus without manchette were rounded, and regions of the nucleus impacted by ectopic manchette were indentedÐa phenotype that could be compared with azh mutants. By contrast, the ¯agella appeared normal. Fewer MT bundles were seen in Sertoli cells, and these MT bundles were thinner and poorly developed. These data suggested that impaired MT functions in the Sertoli cells in the absence of E-MAP-115 led to depletion of germ cells in the mutant. E-MAP-115 could also be required in the Sertoli cells for secreting proteins necessary for survival and differentiation of germ cells (Komada et al., 2000). Impairment of spermiogenesis beginning at the round spermatid stage and production of polyploid spermatids was seen after disruption of the protein phosphatase catalytic subunit Pp1cg (Varmuza et al., 1999). Moreover, spermatids presented high rates of DNA fragmentation, as are found in spermatids from men with non-obstructive azoospermia (Jurisicova et al., 1999). What about the blood±testicular barrier? In this ®eld, the ®ndings are surprising. The inactivation of the Osp/claudin-11 gene, a component of the Sertoli cell-occluding junctions, led to Sertoli cell release. It was expected that this disruption of the blood±testicular barrier could lead to an autoimmunity process, but neither blood cells nor in¯ammation were found in testes of these mice (Gow et al., 1999). Misexpression of the human interleukin-2 gene in mice has led to lymphocyte in®ltration in cerebellar tissues and skin, but not in testes (although these testes were affected, as seen by the depletion of later stages of spermatogenesis; Ohta et al., 1990). Other data relative to reproduction Some knockout mice presented anomalies of different components of the male reproductive system (Table III; see also Nishimori and Matzuk, 1996). Mice de®cient for the growth factor Bmp8b showed germ cells and epididymal epithelium degenerations (Zhao et al., 1998). Mice with a targeted mutation of the insulin-like growth factor gene Igf1 had reduced spermatogenesis at 18% of the normal level and vestigial ducts, seminal vesicles and prostate (Baker, J. et al., 1996). In mice de®cient for the nuclear ERa, the testes were atrophic and the efferent ductules showed defective ¯uid reabsorption, leading to sperm dilution and infertility (Hess et al., 1997). Insl3, a member of the insulin-like hormone superfamily, was seen to be expressed 201 D.Escalier in Leydig cells and postnatal ovary. Inactivation of Insl3 led to bilateral cryptorchidism (Zimmermann et al., 1999) and deregulation of the oestrus cycle with subsequent female infertility (Nef and Parada, 1999). Homozygous c-ros knockout male mice presented an epididymal anomaly and angulation of the sperm ¯agella due to an impaired volume regulation in the epididymis (Yeung et al., 1999). Other studies not directly related to spermatogenesis, but nevertheless concerning reproduction, are worth mentioning. Transgenic mice overexpressing Dax1 have contributed to knowledge on the factors involved in sex determination (Hanley et al., 2000). Dax1 is responsible for dosage-sensitive sex reversal. XY mice carrying extra copies of mouse DAX1 as a transgene show delayed testis development when the transgene is expressed at high levels. Complete sex reversal occurs when the transgene is tested against weak alleles of the sex-determining Ychromosome gene Sry (Swain et al., 1998). Mice lacking the LIM homeobox gene Lhx9 failed to develop genital ridges and gonads failed to form. Genetically male mice were phenotypically female due to an absence of anti-MuÈllerian hormone and testosterone. It was suggested that some human gonadal agenesis can be related to Lhx9 mutations (Birk et al., 2000). Cd9, a member of the transmembrane-4 superfamily, interacts with integrin proteins and possibly participates in cell migration and adhesion. Cd9 is present on egg microvilli and, in its absence, females were infertile due to a failure of sperm-egg fusion while sperm-egg binding was normal (Kaji et al., 2000). A null mutation in Basigin, an immunoglobulin superfamily gene member, affected embryo implantation and male meiosis (Igakura et al., 1998). Mice with a targeted disruption of the prolactin gene shared male and female reproduction anomalies. Half of the homozygous males were infertile or showed reduced fertility (Gof®n et al., 1998), and the growth of seminal vesicles and ventral prostate was affected (Steger et al., 1998). Homozygous females were infertile as a result of multiple reproductive abnormalities, including ovulation of premeiotic oocytes, reduced pre-implantation oocyte development, lack of embryo implantation, and absence of pseudopregnancy (Gof®n et al., 1998). Mos-de®cient mice developed ovarian teratomas that possibly were derived from parthenogenetically activated oocytes undergoing early embryonic development within the ovaries (Hirao and Eppig, 1997). Null mice for the centromere protein B gene exhibited testis weight reduction and disrupted luminal and glandular epithelium of the uterus (Fowler et al., 2000). Mice with a null mutation in the interleukin-11 receptor-a chain gene had defective decidualization, leading to female infertility (Robb et al., 1998). Null A-myb female mice had underdevelopment of the breast epithelial compartment following pregnancy and were unable to nurse their newborn pups, while males exhibited arrest of spermatogenesis (Toscani et al., 1997). The ®nding that mice de®cient for the Fanca gene (Fanconi anaemia complementation group A) did not develop anaemia, but sterility is of particular interest. Male mice exhibited hypospermatogenesis and rarely produced offspring, while female mice produced smaller litters and ceased to breed between 10 and 21 weeks of age. Therefore, at the gonad level, these mice can be compared with Fanconi patients, who often display hypogonadism, reduced sperm count and premature menopause (Cheng et al., 2000). 202 Diseases and male infertility Knowledge of the genes involved in spermatogenesis and cancer DNA mismatch repair genes appear to be responsible for most cases of hereditary non-polyposis colorectal cancer cells (Arnheim and Shibata, 1997). Use of knockout mice has led to the identi®cation of which genes are involved in the maintenance of the genome integrity of both somatic and meiotic cells (Table IV) and which protect somatic cells only. This was the case for the DNA mismatch repair gene Msh2 expressed in spermatogonia and spermatocytes (Richardson et al., 2000) whose inactivation induced susceptibility to lymphoid tumours and fully conserved fertility (De Wind et al., 1995; Reitmair et al., 1995). Inversely, inactivation of the DNA mismatch repair gene Msh5 led only to sterility in both sexes (Edelmann et al., 1999). Therefore tissuespeci®c genes, genes common to actively proliferating cells, and genes common to cells undergoing recombination can be distinguished. This leads to the identi®cation of genes involved in both cancer predisposition and sterility (Table IV). An outstanding model was the Atm gene due to its involvement in human ataxia telangiectasia (Hawley and Friend, 1996). Mechanisms of tumorigenesis can be analysed by means of knockout and transgenic mice. Inhibin-de®cient mice developed gonadal stromal tumours, showing that inhibin is a negative regulator of gonadal stromal cell proliferation and a tumoursuppressor gene with gonadal speci®city (Matzuk et al., 1992). In cell lines, loss of function of the p53 tumour suppressor protein is associated with increased genetic instability including aneuploidy, gene ampli®cation, and point mutation. In null mutants for p53, a signi®cant proportion of ®broblasts and T lymphocytes showed chromosome loss/duplication and interstitial deletion. Increased interstitial deletion was seen also in p53 heterozygous mice. It is suggested that this increased genetic variation starts at the initiation stage of tumorigenesis when functional p53 is absent (Shao et al., 2000). Transgenesis also allows the discovery of genes that might have a role in the formation of testicular tumours (see review in Matzuk et al., 1996) where GDNF, a neurotrophic factor whose receptor is expressed in spermatogonia, is overexpressed. The transgenic spermatogonia began to spread into the interstitium and formed non-metastatic tumours after one year of age. Among 12 old mice, 10 had bilateral and two had unilateral tumours (Meng et al., 2000). GDNF-family ligands may act as paracrine factors in spermatogenesis, and this circuit may be active in germ cell tumours (Viglietto et al., 2000). Various diseases associated with male sterility Male infertility can be associated with other diseases in knockout mice (Table V). This association is not surprising given the numerous laboratory mice with spontaneous mutations leading to male sterility and associated with a panel of diseases. Association of male sterility with reduced body growth is frequently encountered when transcriptional factors are involved (not listed). Deletion of the transcription factor gene Nhlh2 resulted in disruption of the hypothalamic±pituitary axis, leading to obesity and male sterility due to germ cell loss (Good et al., 1997). Inactivation of cyclin-dependent kinase 4 has led to a Genetic engineering and spermatogenesis Table V. Somatic tissues disorders found in null mutant mice with altered spermatogenesis Gene Anomalies Reference(s) c-fos Bax Atm c-Abl FAC E2F-1 Nhlh2 mOvo1 Ahch (Dax1) histone 3.3A connexin 43 Osp/claudin-11 osteopetrosis, lymphopoenia lymphoid hyperplasia immunode®ciency lymphopoenia Fanconi anaemia exocrine gland dysplasia obesity kidney, hair and urogenital defects adrenal hypoplasia congenita neuromuscular de®cit heart abnormality tight junction defects (CNS); conduction velocity defects insulin-de®cient diabetes adipocyte hypertrophy inner ear defects polycystic kidney disease facial dysmorphism, anaemia, cystic choroid plexus exencephaly, cranioschesis lung emphysema; skin and hair defects; defective oligodendrogenesis bone maturation Johnson et al., 1992 Knudson et al., 1995 Xu et al., 1996; Barlow et al., 1996 Kharbanda et al., 1998 Whitney et al., 1996 Yamasaki et al., 1996 Good et al., 1997 Dai et al., 1998 Yu et al., 1998 Couldrey et al., 1999 Juneja et al., 1999 Gow et al., 1999 cyclin-dependent kinase 4 hormone-sensitive lipase Slc12a2a NIMA-related kinase Apaf-1 PDGF-A cyp 19 a Na+-K+-2Cl± cotransporter. Rane et al., 1999 Osuga et al., 2000 Pace et al., 2000 Upadhya et al., 2000 Honarpour et al., 2000 Gnessi et al., 2000 Robertson et al., 1999 CNS = central nervous system. model of insulin-de®cient diabetes and, consequently, to associated male sterility (Rane et al., 1999). Disruption of the hormone-sensitive lipase gene led to oligospermia and male sterility and the adipocytes were 2- to 5-fold enlarged (Osuga et al., 2000). Mutation of the histone 3.3A gene resulted in postnatal death or neuromuscular de®cit, reduced copulatory activity, and poor reproduction rate (Couldrey et al., 1999). Mice lacking the transcription factor E2F-1 were fertile, though with testicular atrophy; in addition, they presented exocrine gland dysplasia, and a broad and unusual spectrum of tumours (Yamasaki et al., 1996). Loss of the Ahch (Dax1) gene led to male sterility, and mutation of Ahch in human caused adrenal hypoplasia congenita (Yu et al., 1998). Male and female germ cell de®ciency and heart abnormalities were found in mice lacking connexin 43, a component of intercellular membrane channels of gap junctions (Juneja et al., 1999). Animals de®cient for the platelet-derived growth factor PDGF-A develop lung emphysema, defective oligodendrogenesis, and skin and hair defects. Most die within a few days, but some individuals survive as long as 6 weeks. Mutant prepubertal testis contained mature fetal Leydig cells, while adult Leydig cells were absent in older ±/± testis, leading to spermatocyte arrest (Gnessi et al., 2000). In some cases, knockout mice with somatic disorders may be compared with human diseases. Homozygous mutants for the Nek1 (a NIMA-related kinase) gene showed polycystic kidney disease (PKD) similar to the human autosomal-dominant PKD. In addition, they showed pleiotropic anomalies such as male sterility, facial dysmorphism, dwar®ng, anaemia and cystic choroid plexus (Upadhya et al., 2000). Mice de®cient for the mOvo1 gene showed kidney, hair and urogenital defects, hypogonadism and reduced sperm production. The phenotype of mOvo±/± mice can be compared with the human Bardet±Bield syndrome (Dai et al., 1998). An animal model with some restraints The challenges of translating knockout phenotypes into gene function has been already discussed for somatic cells: `often a deletion of a gene that has accepted functions from biochemical and cell biology experiments results in an inconsistent phenotype. Frequently this takes the form of no or mild phenotypes and evokes the response that a redundantly functioning gene exists. Equally frequently there is a dramatic phenotype that challenges the investigators' skills to translate it into gene function and draws them into unknown realms' (Ihle, 2000). Transgenic insertions and embryonic stem cell knockouts imply some inherent limitations, and their use as animal models for the understanding of spermatogenesis is still under discussion. Considerations include variations in the phenotype on different genetic backgrounds, confounding effects due to the promoter used, position±effect variegation, and insertion of tandem multi-copy arrays (reviewed in Escalier, 1999a). In addition to these technical limitations, some other parameters constitute restraints for animal models of male reproduction. Gene widespread expression When a gene is involved both in vital processes and spermatogenesis, null mutants may not bring information on its role in 203 D.Escalier Table VI. Expression of the DAZ-like genes in various species Gene Species Male DAZ human DAZL1 human spermatogonia spermatocyte I PGC yes germ cells (embryo) SPGY cynDAZLA DAZL Dazla human monkey marmoset mouse Dazl rat Xdazl Xenopus Boule Drosophila zDazl Dazl zebra®sh C. elegans yes yes spermatogonia B spermatogonia spermatocyte I PGC spermatogonia prospermatogonia spermatocyte I ? spermatogonia? spermatocytes I spermatids PGC (migration) PGC + germ cell spermatogonia spermatocyte I (meiotic divisions) yes no Female PGC, oocyte oocyte oocyte I oogonia, oocytes, granulosa (embryo) no no yes Reference(s) Menke et al., 1997 Lee, J.H. et al., 1998 Seligman and Page, 1998 Dorfman et al., 1999 Tsai et al., 2000 Brekhman et al., 2000 Shan et al., 1996 Carani et al., 1997 Ruggiu et al., 2000 Niederberger et al., 1997 Seligman and Page, 1998 Reijo et al., 1996 oocyte I ? Ruggiu et al., 1997 Rocchietti-March et al., 2000 PGC (migration) PGC+ germ cells no Houston and King, 2000 Mita and Yamashita, 2000 Cheng et al., 1998 yes oocyte I Eberhart et al., 1996 Maegawa et al., 1999 Karashima et al., 2000 PGC = primordial germ cell. spermatogenesis. It is the case when the use of gene knockouts leads to lethality of embryos, of fetus or of newborns such as for cyclin B1 (Brandeis et al., 1998), the recombinase Rad51 (Tsuzuki et al., 1996), the phospholipase cPLA2 (Bonventre et al., 1997) and the Brca2 genes (Sharan et al., 1997). It is also the case for the helicase gene Blm (mutated in Bloom's syndrome), the disruption of which has led to embryonic death (Chester et al., 1998). Nevertheless, it is known that men with Bloom's syndrome are sterile with abnormally small testes and no mature spermatozoa (German, 1993). Gene redundancy Besides indicating candidate genes for infertilities, null mutations demonstrate that some genes expressed in germ cells can be altered without consequence on spermatogenesis and fertilization capacity, although the genes were known to be implicated in spermatogenesis. In most cases this fact is due to the diversity of members of a protein family which can provide a functional overlap and complementation. This is the case for cyclin B1 that compensates cyclin B2 in testis (Brandeis et al., 1998). The same phenomenon is observed for histones (Rabini et al., 2000; see discussion in Couldrey et al., 1999), even for the testis-speci®c linker histone H1t (Lin et al., 2000). H1t is expressed exclusively in pachytene spermatocytes of the testis. H1t-de®cient mice were fertile and reproduced as wild-type mice. The process of spermatogenesis and the testicular morphology remained unchanged. H1.1, H1.2 and H1.4 histone gene expression were enhanced in these mice (Drabent et al., 2000), possibly due 204 to compensatory processes. Compensatory factors are also suspected for the transforming growth factor-alpha (Levine et al., 2000). Anomalies of neuromuscular activity due to a hypomorphic mutation of histone H3.3A may contribute to reduced fertility in mutant mice by compromising their copulatory performance. However, the spermatozoa appeared normal, suggesting a functional redundancy with the H3.3B gene (Couldrey et al., 1999). The presence of redundant mechanisms in the stimulatory control of the gonads by the pituitary is suggested by growth hormone receptor knockout mice. The conservation of sexual maturation and fertility in these mice indicate that their ability of producing prolactin and thyroid-stimulating hormone partially compensates the absence of growth hormone signalling (reviewed in Bartke et al., 1999). Inversely, a gene found to have a redundant function for tissues other than testis can be essential for spermatogenesis. This is the case for Bcl-w, a pro-survival member of the Bcl-2 family (Print et al., 1998) and for the zinc ®nger transcription factor Egr4 (Tourtellotte et al., 1999). The growth factors Bmp8a and Bmp8b seem to be compensated by other Bmps for the maintenance of female reproduction, while they are essential for male reproduction (Zhao et al., 1998). Null mutants by genetic engineering versus spontaneous mutants Spontaneous mutations can be point mutations, leading to various extents of the disease or disturbances restricted to a tissue. The example of the LMNA gene that encodes lamin A/C, a major Genetic engineering and spermatogenesis constituent of the nuclear matrix, is very demonstrative. In the human, different diseases are found following the localization of a mutation in the gene, including muscular dystrophy, cardiopathy or lipodystrophy (Shackleton et al., 2000). A mutation in the FSH receptor can lead to female infertility, while another inactivating mutation of the same gene has been found in a fertile woman (Tapanainen et al., 1998). Knockout mice with a point mutation demonstrate that a particular phenotype can be obtained by this method. Kit/SCFReceptor is encoded by the mouse dominant white-spotting (W) locus. The loss-of-function W mutation affects all signalling pathways from the receptor, leading to failure of haematopoiesis, melanogenesis and infertility. Knockout mice with a different point mutation in the receptor exhibited only male sterility (Blume-Jensen et al., 2000). Drosophila mutants show how mutations for a gene involved in spermatogenesis and with various penetration lead to different germ cell phenotypes (see Escalier, 1999b). Therefore, it is noteworthy that diseases observed in knockout mice can differ from human diseases following alterations of the same gene. Gene homologies, but functional differences between species Genetic engineering in mammals is the most effective means of controlling the function of a gene. Nevertheless, great differences in the differentiation of male germ cells exist among mammals, including those affecting the recombination steps. For example, the nuclear changes at the pachytene stage differ between the human and the mouse, and even between the mouse and the rat. This can be seen at the level of the granular component of the nucleolus (re¯ecting the level of rRNA storage), which is maximal in the mouse and the rat but not in the human, and this nucleolar compartment is mixed with the XY chromosomes in the mouse but not in the rat (reviewed in Solari, 1974). Many genes involved in mammalian spermatogenesis have been identi®ed ®rst by homology with those known in yeast, particularly genes related to recombination (recombinases, DNA mismatch repair genes) and in Drosophila. Nevertheless, a sequence homology between species does not necessary imply a similar function. For example, the ovo gene is required for survival and differentiation of Drosophila female germ cells, but is required for sperm development in the mouse (Dai et al., 1998) (other examples are given in Escalier, 1999b). The mouse insulinlike hormone 3 (also designated Leydig insulin-like or relaxin-like factor) is expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary, and is essential for establishment of the sexual dimorphic position of the gonads (Adham et al., 2000). Indeed, inactivation of the Insl3 gene leads to cryptorchidism in the mouse (Nef and Parada, 1999; Zimmermann et al., 1999). In the human, a common polymorphism is found in the Insl3 C-peptide region which is not related with idiopathic cryptorchidism (Koskimies et al., 2000; Krausz et al., 2000). The reasons for these functional divergences might be: (i) restriction of homology to only one domain (as found for members of a super-family) while the other domains have a distinct function; (ii) conditioning of the gene function by interaction with other factors; and (iii) differences between species in expression timing and/or gene product cell localization that can be associated to distinct functions. However, other mechanisms can be involved. The gene product can have a function on different factors, regulating their translation or expression, or they can regulate the stability of mRNAs This can be the case for the RNA-binding DAZL (Cheng et al., 1998; Houston and King, 2000), RBM (Venables et al., 2000) and DAX-1 (Lalli et al., 2000). RBM interacts with members of the SR family of splicing factors, and may be a splicing regulator which operates as a germ cell-speci®c cofactor for more ubiquitously expressed pre-mRNA splicing activators (Elliott et al., 2000). The mouse Dazl1 is associated with actively translating ribosomes (Tsui et al., 2000b), and can interact through DAZ repeats with DAZAP1, a RNA-binding protein (Tsui et al., 2000a). Table VI shows that Dazla-like genes are expressed at various stages of germ cell differentiation, depending on the species. Moreover, their expression concerns either only the male or the female, or both. It is noteworthy that the functionality of the Daz gene on the human Y chromosome remains to be demonstrated (Shan et al., 1996), while two Y-chromosomal genes of the AZFa region, USP9Y (Sun et al., 1999) and DBY (Foresta et al., 2000), seem to be implicated in human azoospermia. In contrast to other AZFa genes, DBY produces a short transcript which is only expressed in the testis, suggesting that DBY might be the major AZFa candidate to testiculopathies (Foresta et al., 2000). The homology can be the result of co-option of a gene during evolution for a new function This could be the case of the mouse germ cell-less gene (mgcl-1) which is homologous to the Drosophila germ cell-less (Gcl). Functional homology was demonstrated, since ectopic expression of mgcl-1 in Drosophila partially rescues the gcl-null phenotype. Nevertheless, unlike the case in Drosophila, mgcl-1 is not expressed at the time the primordial germ cells appear, but in adult male germ cells (Leatherman et al., 2000). Other mouse genes homologous to Drosophila germ plasm components involved in the formation of pole cells are highly expressed in mouse spermatocytes. There are the vasa-like family members PL10, P68 and Mvh. Thus, it appears that some mechanisms used in pole cell formation in Drosophila have been co-opted as a group for a later developmental process in mouse spermatogenesis (Leatherman et al., 2000). Genetic status of spermatozoa Intracytoplasmic sperm injection (ICSI) could promote the transgenerational transmission of genetic defects, causing gametogenic failure. Among infertile couples treated with ICSI, 6.4% were shown to have a fertility problem with a de®nite genetic basis, and males displayed a distinct pattern of familial aggregation. The infertile couples had fewer siblings than did fertile controls, suggesting suboptimal fertility in the infertile couples' parents. Therefore, male factor infertility should be considered a potentially heritable condition, and the recurrence risk for infertility in the offspring of couples treated with ICSI might be substantial (Meschede et al., 2000a). A noteworthy ®nding in knockout mice was that only homozygous mice were sterile, supporting the notion that mRNAs and proteins pass through bridges between male germ 205 D.Escalier cells (see Escalier, 1999a). Therefore, sperm cells from heterozygous mice presented a normal phenotype, although half of these spermatozoa shared a genetic defect (Escalier, 1999b). This might be the case for human males heterozygous for the helicase BLM involved in recessive autosomal Bloom's syndrome whose mutation leads to immunode®ciency, cancer predisposition and sterility (German, 1993). Also, spontaneous mutants present male sterility in homozygotes whereas heterozygotes are fertile, as in the case of rat hypodactyly (Krenova et al., 1999). By contrast, the expression of a transgene can cause dominant male sterility at the hemizygous status because of syncitial spermatids. This is found in mice carrying a protamine-1 transgene lacking its normal 3¢ untranslated region, and leads to premature translation of protamine-1 and round spermatid arrest (Lee et al., 1995). Finally, a hemizygous status can lead to Sertoli cell-only syndrome in older GDNF +/± mice, re¯ecting haploinsuf®ciency for this gene product. GDNF is expressed in Sertoli cells, and is essential for undifferentiated spermatogonia (stem cells) (Meng et al., 2000). Conclusions Although there are some limitations inherent to the experimental procedures used, genetic engineering has promoted several major advances in our understanding of mammalian spermatogenesis. The techniques are, however, in their early days, and the emergence of these methodsÐin conjunction with ICSIÐhas generated new interest in the study of spermatogenesis. Moreover, bene®ts have also been derived from advances in cell biology. The ®ndings of mouse genetic engineering studies should be the basis to seek the genes responsible for human male infertility. Male germ cell phenotypes for both infertile men and mouse models should be identi®ed using a rigorous de®nition, the main factor for success being the wide cooperation of laboratories. There is a clear need to create a constitutional DNA `bank' after collecting blood samples from subjects belonging to families with hereditary forms of impaired spermatogenesis, and to create similar banks of pathological germ cells. In humans, although infertile patients show a slight higher prevalence of potentially heritable, non-reproductive diseases (Meschede et al., 2000b), this situation is not studied to any great extent. However, the frequent association of male sterility with diseases and/or tumour predisposition in infertile mice must be mentioned, as this can occur as a result of both activation or overexpression of genes. 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