Chapter1 Introduction 1.1 Germ cell development Reproduction in most animal species involves determination and differentiation of germ cells and sexes From flies to humans, reproduction begins early development with the segregation of primordial germ cells (PGCs) from the somatic lineage and the allocation of these cells to the germ cell lineage It continues with mitotic proliferation, entry and progression through meiosis, and finally culminates with maturation of immature germ cells into fully-differentiated and functional sperm and eggs that fertilize to generate a new life The commitment to PGCs is either induced by tissues in mammals (Tam and Zhou, 1996; Lawson et al., 1999) or pre-determined by maternal germplasm, an oosplasmic region containing polarized determinants for the germline which is asymmetrically localized after fertilization in most of the non-mammals (Saffman and Lasko, 1999) The germ plasm is characterized by the presence of polar granules – electron dense structures associated with mitochondria, fibrils and RNA proteins Because of its cloudy appearance under electron microscope, the germ plasm is also termed nuage Nuage exists not only in PGCs, but also in oogonia, oocytes, spermatogonia, spermatocytes and spermatids and has been documented in at least animal species including the insects, fishes, amphibians and mammals (Eddy, 1975) In Drosophila, C.elegans and Xenopus the germ plasm plays a critical role in germ cell determination and the early specification of the germline Transplantation experiments in Drosophila and Xenopus together with genetic studies in Drosophila and C.elegans demonstrated that germ plasm is required for germ cell specification (Illmensee and Mahowald, 1974; Okada et al., 1974; Nieuwkoop and Sutasurya, 1979; Ephrussi et al., 1991; Seydoux and Strome, 1999) In contrast, morphologically distinct germplasm has so far not been identified in early mammalian embryos In the mammals, germ cell determination occurs later during development, and is not directly dependent on maternal molecules In these species germ cell determination is governed by an induction mode involving cell – cell interactions In the mouse, transplantation experiments showed that cells are not initially committed to the germline, and its cell-cell interactions just before gastrulation induce cells to become PGCs (Tam and Zhou, 1996) ) The formation of PGCs was shown to depend on the bone morphogenetic protein (Bmp4) in the mouse embryos (Ying and Zhao, 2001; Ying et al., 2001) Embryological experiments also suggest that polarity does not contribute to germ cell specification (Zernicka-Goetz, 1998) Most of the non-mammalian species have a pre-deterimined germ cell lineage that is specified by cytoplasmic germ cell determinants laid in the oocyte In the Xenopus, germ plasm is synthesized and transported to the vegetal cortex during oogenesis Germ plasm is inherited by the vegetal-most blastmomeres of newly fertilized embryos, within the presumptive endoderm, and segregated into a few cells at the blastula stage (Houston and King 2000b; Kloc et al., 2001) Those cells that inherit germ plasm will give rise to PGCs, and sister cells that not inherit germ plasm become somatic endoderm Similarly, germ cells are pre-determined by maternally inherited germ plasm in the chicken (Tsunekawa et al., 2000) In fish, the specification of germ cells was controversial before the discovery of germ cell specific markers Nuage-like structures are detected during oogenesis and late embryogenesis, and the origin of germ cells has been traced to different germ layers in different fish (Selman et al 1993) Therefore, it was difficult to assign a specific time point and event to germ cell specification in fish The recent identification of the zebrafish vasa orthologue indicates that germ cell precursors are separated early from somatic cells, and they can be traced throughout embryogenesis (Olsen et al 1997; Yoon et al 1997; Weidinger et al 1999; Braat et al 1999) Recently, Knaut et al (2000) has shown that vasa RNA is a component of a germ plasmlike structure, and maternal signals induce a change in germ plasm segregation, which results in germ plasm activation and consequently in germline specification 1.1.1 Germ cell genes in Drosophila An important mechanism for the specification of the animal germline is the localization of specific molecules to the germ plasm The germplasm components include RNA binding proteins or transcription factors and their RNAs RNA maternal germplasm and its regulations are achieved via translation of vegetally-localized mRNAs (Micklem, 1995; Grunert and St Johnston, 1996; Macdonald and Smibert, 1996) Germ plasm components, or germ cell genes, have been best studied in Drosophila where twelve genes belonging to the “posterior group” maternal effect genes (cappuccino, spire, staufen, oskar, vasa, valois, mago nashi, tudor, nanos, pumilio, par-1 and pipsqueak) have been identified for the formation of the pole cells Mutations in these genes could result in loss of polar granules, disruption of the pole plasm and lack of germ cells In the Drosophila embryo, ectopic germ cells are formed if oskar RNA is mislocalised to the anterior pole (Ephrussi and Lehmann, 1992) Ectopic Oskar protein causes the accumulation of nanos mRNA, which is both a zinc finger protein and translational regulator that is required for germcell migration (Kobayashi et al., 1986; Forbes and Lehmann, 1998) Vasa and Tudor proteins are required for nanos RNA localization in PGC determination (Schupbach and Wieschaus, 1986; Wang et al., 1994; Boswell and Mahowald, 1985) Immunocytological studies have shown that Oskar, Vasa and Tudor proteins colocalised with the polar granules (Bardsley et al., 1993; Breitwieser et al., 1996; Hay et al., 1988a, b) Nanos protein was shown to be involved in establishing and maintaining germline stem cells (GSCs) by preventing their precocious entry into oogenesis in Drosophila ovaries (Wang and Lin, 2004) Several additional maternal genes which encode RNA-binding proteins are well characterized in non-mammalian organisms including teleost species Tong et al (2000) has identified the first maternal gene identified in the mouse; female mice homozygous for a mutation at the mater (maternal antigen that embryos require) locus developed normally, and produced normal eggs Following fertilization, eggs of mater -/- females developed up to two cell stage Mater is a single-copy gene expressed only in oocytes 1.1.2 Identification of germ cells Many approaches have been used to investigate the origin of germline In teleost, PGCs were previously recognized histologically through their morphological characteristics and cells with such traits have been identified after gastrula stage In birds, PGCs at early developmental stages were identified by the presence of high glycogen content PGCs in mouse embryos have been distinguished by their characteristic alkaline phosphatase (AP) activity (Hahnel et al., 1990; Ginsburg et al., 1990) Although AP is described as a classical marker for PGCs, positive reaction was shown in different tissues of mouse embryos (MacGregor, 1995) Other methods to detect the presence of PGCs include Oct- 3/4 expression (Okazawa et al., 1991; Yeom et al., 1996), several cell-surface antigens recognized by monoclonal antibodies such as SSEA-1 (Fox et al., 1981), 4C9 (Yoshinaga et al., 1991) and EMA-1 (Hahnel and Eddy 1986) However, all these methods are not specific enough to distinguish cells destined to a germ cell fate from pluripotent stem cells remaining in the undifferentiated state The subsequent discovery of vasa gene and its homologs in many organisms have facilitated the identification of germ cells In the zebrafish, studies of vasa have allowed tracing of the germ cell lineage early in development (Yoon et al., 1997) Zebrafish PGCs were characterized (Braat et al., 2000) and the asymmetric segregation of vasa RNA which distinguishes germ cell precursors from somatic cells and germ plasm segregation patterns in cleavage stage embryos were described (Knaut et al., 2000) Additional studies using zebrafish as a model have greatly enhanced our understanding of the mode of specification of PGCs, cell-fate maintenance and the migration of these cells towards the gonads, where they differentiate into gamates (Raz, 2003) In the chicken, the isolation of vasa homolog gene (Cvh) has enabled a more precise tracking for the origin of PGCs Immunohistochemical analyses using specific antibody raised against CVH protein indicate that CVH protein is localized in cytoplasm of germ cells ranging from presumptive PGCs in uterine-stages embryos to spermatids and oocytes in adult gonads (Tsunekawa et al., 2000) The identification of PGCs in the development of pre-implantation rabbit embryos using mouse Vasa homologue protein in young rabbit embryos indicate that MH-vasa is more specific than current AP staining (Montiel et al., 2001) The human VASA gene is specifically expressed in the germ cell lineage and VASA protein expression in human germ cells has been characterized at various stages of development, suggesting that VASA is a reliable marker of germ cells (Castrillon et al., 2000) 1.1.3 Vasa family genes The vasa gene was initially identified in Drosophila by genetic screens for maternaleffect mutations that affected anterior-posterior polarity and caused a deficiency in pole cells formation in embryos from mutant mothers (Hay et al., 1988; Lasko and Ashburner, 1988) The Vasa protein (Vas) can be detected in the germline cells of Drosophila throughout their development, and in early embryos it is specifically localized to pole granules The vasa gene of Drosophila has been shown to be a member of the DEAD (Asp-Glu-Ala-Asp) protein family of ATP-dependent RNA helicases (Liang et al., 1994) The DEAD box proteins share eight characteristic sequence motifs, and are involved in nuclear and mitochondria splicing processes, RNA editing, rRNA processing, translational initiation, nuclear mRNA transport and mRNA degradation (Luking et al., 1998) Among the DEAD box proteins, the Vas- and PL10-related proteins are very similar to each other Vas-related genes have been reported in 18 animal species including Hydra (Kazufumi et al., 2001), C.elegans (Roussel and Bennett, 1993), planarian (Shibita et al., 1999), oyster (Fabioux et al., 2004), Xenopus laevis (Komiya et al., 1994), chicken (Tsunekawa et al., 2000), mouse (Fujiwara et al., 1994), human (Castrillon et al., 2000), zebrafish (Yoon et al., 1997; Olsen et al., 1997), medaka (Shinomiya et al., 2000), tilapia (Kobayashi et al., 2000), trout (Yoshizaki et al., 2000), and seabream (Cardinali et al., 2004) The sequence similarity of the Vas- and PL-10 related proteins and the restricted existence of the vas-related genes to metazoans appear to suggest an ancestor of the vas-related genes was derived from an ancient PL-10 related gene and thereafter acquired the specificity in germline cells (Mochizuki et al., 2001) The distribution of vasa RNA and Vasa protein has been determined at different developmental stages in many animals (Raz, 2000) Vasa RNA is expressed exclusively in the germ cells In Drosophila, vasa RNA is uniformly distributed in early embryos, but the protein is localized to its posterior pole, where it is associated with the polar granules The Vasa protein can bind target mRNAs such as oskar and nanos, and control their translation (Hay et al., 1990; Lasko and Ashburner, 1990) The Vasa homologs of Canenorhabditis elegans, glh-1, glh-2 and glh-4, have been shown to play a role in germcell proliferation and gametogenesis (Gruidl et al., 2000) In the mouse, where germ cells are induced through cellular interactions rather than inheritance of maternal cytoplasm determinants, the expression of vasa is in the PGCs as they arrive at the gonad, and expression is induced by interaction between the germ cells and somatic cells of the developing gonad (Toyooka et al., 2000) Further studies in animals lacking vasa gene provided evidence for the role in the development of germ cells and gametogenesis in these organisms In Drosophila, vasa null mutants fail to develop mature oocytes, indicating that in mature gonads the vasa gene is involved in oocyte maturation (Styhlet et al., 1998) and there could be interplay between oogenesis and the activity of the Vasa protein (Ghabrial and Schupbach, 1999) In Canenorhabditis elegans, injection of antisense RNA disrupted oogenesis and spermatogenesis, leading to arrest at the pachytene stage (Kuznicki et al., 2000) In Xenopus, microinjection of antibodies against the Vasa homolog protein (XVLG) into blastomeres of 32-cell stage caused a reduction in the number of PGCs at the tadpole stage (Ikenishi and Tanaka, 1997) In the mouse, loss of vasa function affects differentiation of male germ cells, resulting in male sterility (Tanaka et al., 2000) 1.1.4 Daz family genes The founder member of the family is DAZ that was identified as the essential locus deleted in azoospermia in human Azoospermia affects approximately 2% of men worldwide (Shinka and Nakahori, 1996) The DAZ locus on human Y chromosome, the azoospermic factor (AZF), is vital for proper differentiation of male germ cells DAZ is present as a multicopy gene cluster (Saxena et al., 1996), and deletion of the DAZ clusters has been correlated with azoospermia and oligospermia, which makes DAZ a strong candidate for the AZF DAZL (DAZ-like), a human autosomal DAZ gene was found to map to human chromosome (Shan et al., 1996) and to mouse chromosome 17 (Cooke et al., 1996; Reijo et al., 1996) Subsequently, BOULE was mapped to human chromosome and was proposed to be a meiotic regulator which is highly conserved throughout metazoans (Xu et al., 2001) It has been proposed that BOULE is the ancestral gene that is conserved from flies to humans, whereas DAZL arose in the early vertebrate lineage and DAZ arrived on Y-chromosome during primate evolution, possibly from the transposition and repeat amplification of the ancestral autosomal gene DAZL (Yen et al., 1996) DAZ and DAZL1 share a similar genomic structure and sequence (Chai et al., 1997a) DAZ and DAZL differ in copy number and overall protein structure; DAZ protein has several DAZ isoforms with a polymorphic DAZ repeat region of 8-24 amino acid residues, compared to DAZL protein which contains only one DAZ repeat (Reijio et al., 1995; Yen et al., 1997) DAZ family proteins carry two conserved domains, namely the ribonucleoprotein (RNP)-type RNA recognition motif (RRM) and the DAZ motif, and exist in both vertebrates and invertebrates (Cookie et al., 1996; Eberhart et al., 1996; Reijo et al., 1995) These genes are thought to be involved in cell cycle switch from mitotic to meiotic cell division (Gromoll et al., 1999); this cell cycle switch is controlled by RNA-binding proteins in the yeast (Watanabe et al., 1997) DAZ, DAZL and BOULE share the RRM that is not shared with other RNA binding protein and they are highly conserved from fruitfly to humans (Burd and Dreyfuss, 1994) The expression of Daz genes appears to vary in different organisms (Johnson et al., 2003) Human DAZ and Drosophila boule are transcribed specifically in the male germ line (Eberhart et al., 1996; Reijo et al., 1995; Saxena et al., 1996), while human DAZLA/DAZH and its homologs in mouse, zebrafish, and Xenopus are expressed in the germline of both sexes (Houston et al., 1998; Maegawa et al., 1999; Ruggiu et al., 1997; Seligman and Page, 1998) Interestingly, expression product differs in Xenopus and zebrafish In Xenopus, xdazl RNA is localized to the germ plasm in eggs and embryos Zebrafish germ cells are found to originate at the periphery of the developing blastoderm (Olsen et al., 1997; Yoon et al., 1997; Knaut et al., 2000) Despite variation in the localization patterns, conservation of sequences among DAZ homologs may translate into conservation in functional homology; Drosophila mutants for boule, in which germ cells arrest during meiotic progression (Eberhart et al., 1996), can be rescued by the product of Xenopus Dazl (Houston et al., 1998) The ability of a human DAZ transgene to rescue partially the spermatogenic defects of Dazl1 knock-out mice (Slee et al., 1999) supports functional conservation between DAZ and DAZL Daz and Dazl are expressed exclusively in germ cells, suggesting a pivotal role in the formation of PGCs and also differentiation of germ cells in the mouse (Cooke et al., 1996; Ruggiu et al., 1997; Gromoll et al., 1999) Further studies in the phenotypic analyses of animals lacking dazl gene provided direct evidences for the role of dazl in the development of germ cells and gametogenesis in these organisms In Xenopus, Xdazl controls the formation of the PGCs during embryogenesis and inhibition of Xdazl leads to loss of the PGCs (Houston et al., 1998; Houston and King, 2000b) In Dazla-defective mouse, female germ cells are arrested at the prophase of meiosis I, whereas development of male germ cells is terminated after the spermatogonial stage (Ruggui et al., 1997; Reijio et al., 1996) Highly variable testicular defects are associated with the absence of Daz gene in human, ranging from complete absence of germ cells to spermatogenic arrest with occasional production of condensed spermatids in infertile male (Reijo et al., 1995) In the developing male/female genital ridges, PGCs change morphologically and become gonocytes Gonocytes proliferate and give rise to spermatogonia/oogonia, which are male/female germ stem cells (GSCs) Upon sexual maturation, GSCs differentiate and undergo meiosis to form gametes Gametogenesis represents a second fundamental process in animal reproduction 1.2 Sexual development – genetic sex determination Organisms displaying sexual dimorphism have evolved different sex determining mechanisms; in many species with 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5:2013-2017 Yen PH, Chai NN, Salido EC 1997 The human DAZ genes, a putative male infertility factor on the Y chromosome, are highly polymorphic in the DAZ repeat region Mamm Genome 8:756-759 Zernicka-Goetz.M 1998 Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles Development 125:4803-4808 Zirkin BR 1998 Spermatogenesis:its regulation by testosterone and FSH Semin Cell Dev Biol 9:412-422 Zhu L, Wilken J, Phillips NB, Narendra U, Chan G, Stratton SM, Kent SB, Weiss MA 2000 Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers Genes Dev 14:1750–1764 95 Appendices A Plasmid maps EcoRI (53) lcvasa pvasa 4939 bp EcoRI (1995) The 1937 bp lcvasa cDNA is inserted between EcoRI sites The arrow indicates the ORF T7 EcoR I (53) VASA-ISH EcoR I (487) SP6 pV-ISH 3431 bp Plasmid map of vasa probe for ISH The arrow indicates the sense strand used for ISH 96 EcoR I (53) lcd azl EcoR I (717) pdazl 366 bp The 654 bp lcdazl cDNA is inserted between EcoRI sites The arrow indicates the ORF T7 EcoRI (53) D AZL -ISH EcoRI (522) SP6 pD-ISH 3466 bp Plasmid map of dazl probe for ISH The arrow indicates the sense strand used for ISH 97 EcoR I (53) lcso x3 psox3 EcoR I (956) 3900 bp The 900 bp lcsox3 cDNA is inserted between EcoRI sites The arrow indicates the ORF T7 EcoR I (53) SOX3-ISH EcoR I (469) SP6 pS-IS H 3413 bp Plasmid map of sox3 probe for ISH The arrow indicates the sense used for ISH 98 Developmental stages of seabass embryos at different intervals Oil globule Blastoderm 2h 4h 6h 8h Head tail Embryonic streak 10h 12h optical lobe yolk sac somites 14h 17h 99 [...]... differentiation, indicating its association with fish gonadal differentiation (Marchand et al., 2000) In contrast, the mutually exclusive expression of two types of DM domain genes in the testis and ovary of tilapia suggests that both genes play roles in gonadal development and function (Guan et al., 2000) In the protandrous black porgy, differential expression of Dmrt1 transcripts in the gonads suggests... development and cell type specification (Wegner, 1999) The Sry and Sox proteins share a single high mobility group (HMG) domain consisting of 80 amino acids (Sinclair et al., 1990) The HMG domain is a DNA binding motif that is shared by abundant non-histone components of chromatin and specific regulators of transcription and cell differentiation HMG domain is responsible for mediating DNA binding and has... 2000) There are about 30 members of the Sox family, found in insects, amphibians, birds 13 and mammals Sox genes are divided into six groups (A-F) based on the similarity within and outside the HMG box (Pevny and Lovell-Badge, 1997) Despite these variations, chimeric Sry proteins between Sox 3 and 9 HMG domains can bind to and regulate SRY target genes without hampering the development of testis cord and. .. interaction is important in testis development (Matsuzawa-Watanabe Y et al., 2003) 1.2.2 Sry and Sox genes The identification of Sry, the testis determining gene on the mammalian Y chromosome, has enhanced our understanding in the mechanism of sex determination In mice, Sry is expressed in the gonad from 10.5 days post coitum (dpc) to approximately 12.0 dpc, coincident with formation of the onset of. .. al., 1994); and over expression of Sox9 induced female to male sex reversal in mice (Vidal et al., 2001) The presence of Sox17 in ovary, ovotestis and testis in the protogynous rice eel indicates a possible role in gonadal differentiation and a conserved role in spermatogenesis (Wang et al., 2003) Apart from their roles in sexual reproduction, sox genes are also involved in the regulation of diverse... (Foster and Graves, 1994) Sox3 is expressed in the brain and gonads Human patients with X-chromosome deletions spanning SOX3 are mentally retarded, which is consistent with the predominant expression observed in the CNS (Rousseau et al., 1991) Sox3 is detected at the earliest stages of embryonic development (6.5 to 8 dpc) and continues to be expressed throughout the formation of the brain and central... pipetting, and were spun briefly to collect the contents at the bottom The tubes were incubated at 42°C for 1.5 h in an air incubator 100 µl of Tricine-EDTA Buffer was added to both tubes and incubated in a water bath at 72°C for 7 min The newly synthesized first strand cDNAs were then stored in -20 °C freezer for subsequent 3’ and 5’ RACE PCR reactions Simliarly, first strand cDNA (from the other tissues/embryos/... reaction and the mixture were flicked gently and placed on ice for 30 min The cells were then heat shocked for 1 min in a water bath at 42°C, and the tubes were immediately returned to the ice for 2 min 800 µl of Luria-Bertani (LB) medium was added to the tubes containing the cells transformed with ligation reactions and incubated for 1.5 h at 37°C with shaking at approximately 200rpm During the incubation... nervous system Interestingly, Sox3 is expressed in somatic cells of urogenital ridge (Wood and Episkopou, 1999) and the developing gonadal ridge in mouse at the same time as Sry and Sox9 expression (Collignon et al., 1996), suggesting a function for Sox3 in mammalian gonadal development It has been suggested that Sox3 is also involved in testis determination, perhaps by its interaction with the related... complete melting ULTRABIND was resuspended by vortexing at highest speed for 1 min to ensure thorough mixing since ULTRABIND is a mixture of 50:50 pure silica and buffer 4 µl of ULTRABIND was added Following incubation for 5 min with constant inversion to allow DNA binding to silica, the mixture was centrifuged for 5 sec The pellet was resuspended with 1 ml of ULTRA WASH by vortexing for 5-10 sec The mixture ... cytoplasm determinants, the expression of vasa is in the PGCs as they arrive at the gonad, and expression is induced by interaction between the germ cells and somatic cells of the developing gonad... 17h 30h Vasa B BA Fig.2 Vasa expression in the seabass by RT-PCR Expression of lcvasa is specific in the gonads of adult seabass (A) and no expression in other somatic organs Differential expression. .. oocytes, indicating that in mature gonads the vasa gene is involved in oocyte maturation (Styhlet et al., 1998) and there could be interplay between oogenesis and the activity of the Vasa protein (Ghabrial