Left-Right Asymmetry in Chicken Embryonic Gonads

≪Review≫ Left-Right Asymmetry in Chicken Embryonic Gonads

Sittipon Intarapat1 andClaudioD.Stern2

1 Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 2 Department of Cell and Developmental Biology and UCL Centre for Stem Cells and Regenerative Medicine, University College London, Gower Street, London WC1E 6BT, UK.

Avian embryos have reproductive organs with unique characteristics. In female, the gonads develop asymmetrically: the left gonad generates a functional ovary, whereas the right gonad and associated embryonic oviduct (Müllerian duct) regress. In males, however, both left and right gonads develop into testes. Recent evidence, however, revealed that left-right asymmetry can be detected in both sexes. Even male embryos have a greater number of germ cells in the left gonad. Moreover, pluripotency-associated markers, as well as SSEA1, the surface antigen that is strongly expressed in chick embryonic stem cells, also show asymmetric expression in both sexes both in germ cells and in stromal cells of the gonad. This review provides an update of the state of the field.

Key words: chick, gonads, left-right asymmetry, PGCs, pluripotent stem cell markers J. Poult. Sci., 51: 352-358, 2014

Introduction Gonadogenesis and Left-right Asymmetry of Embryonic Gonads During embryonic development, left-right asymmetry of some organs is associated with several processes such as During development of the gonads, the intermediate embryonic turning, forming and patterning of visceral organs mesoderm starts to form a rod-shaped structure situated (Levin, 2005; Raya and Izpisua Belmonte, 2006). A number between the paraxial and lateral plate mesoderm. A pair of of genes encoding transcription factors and secreted growth rod-shaped-structure then rapidly proliferate giving rise to factors play a crucial role in these processes; some differ paired bulged structures called “the genital or gonadal among different species whereas a few are strongly ridges”. These ridges locate medioventrally on the meso- conserved. Two of the key factors, Pitx2 and Nodal, are nephros (Romanoff, 1960; McCarrey and Abbott, 1979; conserved in all vertebrates and perhaps even in all events Rodemer et al., 1986). It has been reported that thickening that establish left-right asymmetry in embryogenesis (Levin of the mesenchymal blastema of the genital ridges, with et al., 1995; Ryan et al., 1998; Yoshioka et al., 1998; Zhu et contributions from the coelomic epithelium and mesonephros al., 1999; Levin, 2005). PITX2 is a homeobox-containing resulted in a group of differentiated cells in the gonads transcription factor with a bicoid-type homeodomain, (Bishop-Calame, 1966; Carlon et al., 1983; Merchant- whereas Nodal is a secreted protein of the TGFβ superfamily. Larios et al., 1984; Carlon and Stahl, 1985; Ukeshima et al., Their expression patterns have been found in the left lateral 1987; Martineau et al., 1997). At the beginning of plate mesoderm during the development of heart and head gonadogenesis, the gonads of both sexes are not yet to be (Gage et al., 1999a, b; Zhu et al., 1999) and generally on the distinguished by gonadal morphology so called the “indiffer- left side of every organ system that show left-right asym- ent stage” (McCarrey and Abbott, 1979; Clinton, 1998; metry in vertebrates. Pitx2-knockout mice have abnormal- Clinton and Haines, 2001). Thereafter, the indifferent ities of internal organ asymmetry (Lin et al., 1999; Lu et al., gonads enter to sexual differentiation in which male and 1999). Recently, Pitx2 was reported to play a role in em- female gonadal ridges differentiate into the testes and bryonic gonad asymmetry in both sexes (Guioli and Lovell- ovaries, respectively (Clinton, 1998; Clinton and Haines, Badge, 2007; Ishimaru et al., 2008; Rodriguez-Leon et al., 2001; Smith and Sinclair, 2004; Smith et al., 2007; Smith, 2008). 2010; Chue and Smith, 2011; Ayers et al., 2013). The somatic cells in the gonadal ridges become steroidogenic or Received: February 22, 2014, Accepted: April 15, 2014 Released Online Advance Publication: May 25, 2014 hormone producing cells and supporting cells in both sexes Correspondence: Dr. S. Intarapat, Department of Anatomy, Faculty of (Merchant-Larios et al., 1984; Merchant-Larios et al., 1993). Science, Mahidol University, Bangkok 10400, Thailand. Primordial germ cells (PGCs) are surrounded by these (E-mail: [email protected]) somatic cells and therefore further develop into primary sex Intarapat and Stern: Gonadal Asymmetry in Chicken Embryos 353 cords (Romanoff, 1960; Stahl and Carlon, 1973; McCarrey ovary (Romanoff, 1960; Smith and Sinclair, 2001; Smith and Abbott, 1979). Like mammalian gonads, chicken and Sinclair, 2004). There is no further development of the embryonic gonads consist of two layers, the outer cortex and right gonad and this leads to the formation of vestigial inner medulla (Maraud et al., 1987; Clinton, 1998; Clinton structures (Carlon and Stahl, 1985) or complete regression and Haines, 2001; Smith and Sinclair, 2004). Development (Romanoff, 1960) at the adult stage. The molecular of the primary sex cords differs in the two sexes: embryonic mechanism underlying asymmetrical development and the ovaries develop from the cortex, while the testes develop degeneration of the right ovary in female birds relies on from the medulla (Romanoff, 1960; McCarrey and Abbott, PITX2 (Guioli and Lovell-Badge, 2007; Ishimaru et al., 1979; Maraud et al., 1987). In developing testes, cell divi- 2008; Rodriguez-Leon et al., 2008). Guioli and Lovell- sion in the medulla is faster than that in the cortex leading Badge (2007) demonstrated that misexpression of Pitx2 in to the thinning of the cortex in male gonads and vice versa the right gonad using transfection with the RCAS retroviral (Romanoff, 1960; McCarrey and Abbott, 1979; Clinton, vector is sufficient not only to induce symmetric develop- 1998). Furthermore, the medullary cords further develop ment of the gonads but also to rescue the degeneration of the into secondary sex cords (testicular cords in male) right ovary. A later experiment using a similar method (Romanoff, 1960; Stahl and Carlon, 1973; McCarrey and demonstrated that RCAS-Pitx2 transfected gonads exhibits Abbott, 1979). Conversely, in developing ovaries, the cor- symmetric distribution of a number of Vasa and Oct4 tical cords further develop; whereas the medullary cords positive cells in both left and right genital ridges compared to regress resulted in secondary sex cord development giving wild-type embryos at stage 20 HH (Rodriguez-Leon et al., rise to the thick cortex in female left ovary (Romanoff, 1960; 2008), along with retention of the right ovary in female Stahl and Carlon, 1973; McCarrey and Abbott, 1979). embryos at stage 34 HH (Rodriguez-Leon et al., 2008). Gonadal differentiation is controlled by the genes located on These findings reveal a pivotal role of PITX2 in germ cell sex chromosomes (Stevens, 1997; Smith and Sinclair, 2001; survival in the right gonad as well as restoration of the right Smith and Sinclair, 2004; Smith et al., 2007; Smith, 2010; gonad from degeneration in female embryos. Chue and Smith, 2011; Ayers et al., 2013). In addition, two Asymmetric Germ Cell Distribution in independent mechanisms including the genetic cascade Female and Male Embryonic Gonads regulating cellular differentiation and the sex-determining mechanism controlling the differentiation of the gonadal Asymmetric germ cell distribution in chicken embryos ridges have been proposed (Clinton, 1998; Clinton and was first reported in 1935 by Witschi (Witschi, 1935); since Haines, 2001; Smith and Sinclair, 2001; Smith and Sinclair, then, it has been described that the number of PGCs in the 2004; Smith et al., 2007; Smith, 2010; Chue and Smith, left gonad is greater than that in the right gonad. Other 2011; Ayers et al., 2013). In summary, gonadogenesis in previous studies also supported that the left gonadal ridges male and female embryos is so called medullary and cortical contain more germ cells than the right side in female em- development, respectively (Romanoff, 1960; McCarrey and bryos (Van Limborgh, 1954; Van Limborgh, 1968; Van Abbott, 1979). Limborgh, 1970; Vallisneri et al., 1990; Zaccanti et al., 1990). Unlike mammals, which have apparently symmetric It is not yet clear what actually controls asymmetric germ cell gonads, most female bird species develop asymmetrically, distribution. It is possible that chemotactic and/or mitogenic generating a functional ovary only on the left side, whereas factors play a role in germ cells migration, and it has been males develop bilateral testes (Romanoff, 1960). Gonadal proposed that the left gonadal ridge secretes such factors asymmetry has been reported in several avian species more than the right, leading to increased PGCs migration including duck (Van Limborgh and Van Faassen, 1960; Van and/or division in the left gonad (Witschi, 1935; Swartz and Limborgh, 1970), turkey (Burke, 1973) and chicken (Van Domm, 1972). One study supported this by showing that Limborgh, 1954; Van Limborgh, 1960; Hocking, 1992; engrafted quail PGCs differentially colonized the left and Calhim and Birkhead, 2009). This asymmetry can be related right presumptive gonads at limb bud stages (HH 18-24) to the results of gonad growth (Mittwoch et al., 1971; (Didier and Fargeix, 1976). This phenomenon was also seen Mittwoch and Mahadevaiah, 1980). Before sexual differen- at later stages, when presumptive gonads are differentiated tiation (the “indifferent stage”), there is no detectable into the ovary since the number of oogonia in the left ovary morphological asymmetry between left and right embryonic was higher than that in the right and germ cell death was also gonads in either sex. Morphological differences in embry- much lower (Vallisneri et al., 1990; Ukeshima and Fujimoto, onic gonads appear after sexual differentiation; male 1991). However, the nature of such factors, or even whether embryos (which are the homogametic sex, ZZ) develop or not they exist, remains to be elucidated. bilateral testes, while female embryos (heterogametic, ZW) Early differences between female and male embryos were develop a functional left ovary and the right ovary regresses thought to include a greater number and size of female germ (Romanoff, 1960; Smith and Sinclair, 2001; Smith and cells at an earlier stage than in males (Dubois and Croisille, Sinclair, 2004). Gonadal differentiation from bipotential 1970; Vallisneri et al., 1990; Zaccanti et al., 1990), based on gonads into differentiated gonads is molecularly orchestrated the localization of PAS positive cells (Fuyuta et al., 1974; by several sex-determining genes (summarized in Table 1). Fujimoto et al., 1976). Another possible factor contributing In female birds, only the left gonad becomes a functional to germ cells reduction in chicken embryonic ovaries is 354 Journal of Poultry Science, 51 (4)

Table 1. Gene expression in the left and right gonads of female and male embryos

Embryonic Embryonic Embryonic Embryonic Genes left ovary right ovary left testis right testis References (ZW) (ZW) (ZZ) (ZZ) AMH --+++ +++ (Oreal et al., 1998) (Andrews et al., 1997; AROM +++ +++ - - Nakabayashi et al., 1998) ASW +++ +++ - - (O’Neill et al., 2000) Bmp7 +++ +++ - - (Hoshino et al., 2005) ERNI +++ + +++ + (Intarapat and Stern, 2013a) cNanog +++ + +++ + (Intarapat and Stern, 2013a) cPouV +++ + +++ + (Intarapat and Stern, 2013a) cSox2 +++ + +++ + (Intarapat and Stern, 2013a) CTNNB1 ++ ++ +++ +++ (Bae et al., 2013) Cvh +++ + +++ + (Intarapat and Stern, 2013a) DAX1 +++ +++ ++ ++ (Smith et al., 2000) (Raymond et al., 1999; DMRT1 + + +++ +++ Guioli and Lovell-Badge, 2007) (Andrews et al., 1997; ER +++ + +++ - Nakabayashi et al., 1998) EY505808 - - + + (Lin et al., 2010) FET1 +++ + - - (Reed and Sinclair, 2002) FOXL2 +++ +++ - - (Hudson et al., 2005b) GET +++ + +++ +++ (Hudson et al., 2005a) HINTW +++ +++ - - (Smith et al., 2009a) 17βHSD ++ ++ - - (Nakabayashi et al., 1998) (Oreal et al., 2002; Lhx9 ++ ++ +++ +++ Guioli and Lovell-Badge, 2007) Ovex1 +++ ++ + - (Carré-Eusèbe et al., 2009) Pitx2 ++ - + - (Guioli and Lovell-Badge, 2007) RFK - - ++ ++ (Lin et al., 2010) RSPO1 +++ - - - (Smith et al., 2008) SF1 +++ +++ ++ ++ (Smith et al., 1999) Sox9 - - +++ +++ (Kent et al., 1996) WDR36 - - +++ +++ (Lin et al., 2010) Wnt4 +++ ++ + + (Smith et al., 2008) Wpkci +++ +++ - - (Hori et al., 2000) Abbreviations: AMH＝Anti-mullerian hormone; AROM＝Aromatase; ASW＝Avian sex-specific W-linked; Bmp7＝ Bone morphogenetic protein 7; ERNI＝Early response to neural induction; cNanog＝Chicken Nanog homeobox; cPouV＝Chicken POU domain class 5 transcription factor 1; cSox2＝Chicken SRY-box containing gene 2; CTNNB1 ＝Catenin (cadherin-associated protein) beta 1; Cvh＝Chicken vasa homolog; DAX1＝dosage-sensitive sex reversal adrenal hypoplasia critical region on chromosome X gene 1; DMRT1＝doublesex-and mab-3-related transcription factor 1; ER＝Estrogen receptor; EY505808＝Gallus gallus cDNA clone ChEST658c21; FET1＝Female expressed transcript 1; FOXL2＝Forkhead box L2; GET＝Gonad expressed transcript; HINTW＝W-linked histidine triad nucleotide binding protein; 17βHSD＝17 beta-hydroxysteroid dehydrogenase; Lhx9＝LIM homeobox 9; Ovex1＝ Ovary expressed 1; Pitx2＝Pairedlike homeodomain transcription factor 2; RFK＝Riboflavin kinase; RSPO1＝R- Spondin1; SF1＝Steroidogenic factor 1; Sox9＝SRY-box containing gene 9; WDR36＝WD repeat domain 36; Wnt4 ＝Wingless-related 4; Wpkci＝W-linked protein kinase C inhibitor. (+++＝strong, ++＝moderate, +＝weak, -＝no expression).

apoptosis of germ cells (Ukeshima and Fujimoto, 1991; factors might play a role. It has been reported that steroids Ukeshima, 1996; Van Nassauw et al., 1996). Previous produced by the presumptive gonads act as germ cell studies reported that there are numerous spaces in the attractants (Baillie et al., 1966; Baillie et al., 1996). One medulla of embryonic ovaries called “lacunae” in which dead experiment supporting this hypothesis involved injection of oogonia are eliminated from both left and right medulla steroid hormones, testosterone cypionate into chicken through the lacunae (Romanoff, 1960; Kannankeril and embryos at 33 hours’ incubation; this resulted in reduction of Domm, 1968; Ukeshima, 1994). PGC migration and number in the gonadal ridges (Swartz, The molecular mechanisms guiding PGC migration in 1975). Another factor reported is a glycoprotein secreted by birds are still unclear. As mentioned above chemotactic the gonadal ridges (Dubois, 1969; Forbes and Lehmann, Intarapat and Stern: Gonadal Asymmetry in Chicken Embryos 355

1999). Transforming growth factor-beta, TGF-β is secreted by the presumptive gonads and acts as chemoattractant (Godin and Wylie, 1991). To date, the chemokine stromal cell-derived factor 1 alpha (SDF-1α), was also suggested to be a chemoattractant to enhance germ cell migration (Stebler et al., 2004). Chemoattractants and their receptors such as SDF-1/CXCR4 and Steel factor/c-Kit play a role in germ cell guidance (Doitsidou et al., 2002) with regard to Dnd (Deadend) gene (Weidinger et al., 2003). These various observations could suggest that several different chemotactic factors could be involved in germ cell migration. In chicken, only female birds show gonadal asymmetry during gonadal development. Recently, Guioli and Lovell- Badge (2007) found asymmetric expression of several Fig. 1. Left-right asymmetric expression of SSEA1 and markers including Pitx2, Fibronectin, N-cadherin, DMRT1 Cvh in male embryonic gonads. (A) Left testis (LT) ex- and LHX9 preferentially in the left gonad in both sexes, and hibits a greater number of SSEA1 positive cells and (B) Cvh Intarapat and Stern (2013a) found that the left gonad has a positive cells than the right testis (RT). greater number of germ cells than the right in both sexes along with differential expression of pluripotency-associated markers (see below). of both sexes (Intarapat and Stern, 2013a). Four genes Asymmetric Gene Expression in Female (cPouV, cNanog, cSox2 and ERNI) are expressed in a greater and Male Embryonic Gonads number of cells on the left than the right gonads (Intarapat Several genes related to gonadal differentiation are and Stern, 2013a). Not only germ cells (cells expressing aymmetrically expressed at different times in both sexes. At Cvh) show asymmetric expression but also somatic cells an early stage, DMRT1 (Raymond et al., 1999; Oreal et al., (Intarapat and Stern, 2013a). This indicates asymmetric 2002; Smith et al., 2003; Yamamoto et al., 2003; Koba et gene expression in stromal cells of the embryonic gonads. al., 2008; Smith et al., 2009b; Yang et al., 2013) and Sox9 Likewise, stage specific embryonic antigen type-1 or SSEA1, (Kent et al., 1996; Morais da Silva et al., 1996; Yamamoto which is strongly expressed in chicken ES and germ cells et al., 2003; Takada et al., 2006) are expressed in male (ZZ) (Pain et al., 1996; Van de Lavoir et al., 2006; Choi et al., embryos, whereas HINTW (Smith, 2007; Smith et al., 2010; Macdonald et al., 2010; Intarapat and Stern, 2013b), is 2009a), FET1 (Reed and Sinclair, 2002), FOXL2 (Hudson et also expressed more abundantly on the left than the right al., 2005b) and aromatase (Andrews et al., 1997; Smith et testis of male (stage 35HH) embryos (Fig. 1). The list of al., 1997; Nakabayashi et al., 1998; Yamamoto et al., 2003) genes showing asymmetric expression in both sexes of are expressed in female (ZW) embryos. chicken embryos is summarized in Table 1. The significance Several factors and molecules are found to be expressed of these expression patterns, including the asymmetric asymmetrically in chicken embryonic gonads. Among them, distribution of germ cells in male gonads, is still unknown. PITX2, expressed in the left embryonic gonads (Guioli and References Lovell-Badge, 2007), plays a role in stimulation of gonadal growth and morphogenesis (Guioli and Lovell-Badge, 2007; Andrews JE, Smith CA and Sinclair AH. Sites of estrogen receptor Ishimaru et al., 2008; Rodriguez-Leon et al., 2008). A and aromatase expression in the chicken embryo. General and signaling molecule, Bmp7 is also expressed asymmetrically: Comparative Endocrinology, 108: 182-190. 1997. it starts bilaterally at the indifferent stage, but at the sexually Ayers KL, Smith CA and Lambeth LS. The molecular genetics of differentiated stage its expression is found in mesenchymal avian sex determination and its manipulation. Genesis, 51: 325-336. 2013. cells of the left ovary (Hoshino et al., 2005; Carre et al., Bae SM, Lim W, Jeong W, Lee JY, Kim J, Bazer FW and Song G. 2011). The protein receptor like oestrogen receptor α (ERα) Sex-specific expression of CTNNB1 in the gonadal morpho- is also shown asymmetric expression in which its expression genesis of the chicken. Reproductive Biology and Endo- is found only in the left cortex of both sexes (Andrews et al., crinology, 11: 89. 2013. 1997; Nakabayashi et al., 1998; Guioli and Lovell-Badge, Baillie AH, Ferguson MM and Hart DM. Histochemical evidence of 2007), as are the matrix and adhesion molecules fibronectin steroid metabolism in the human genital ridge. Journal of and N-cadherin, the transcription factors DMRT1 and LHX9, Clinical Endocrinology and Metabolism, 26: 738-741. 1966. all of which are preferentially expressed in the left gonad of Baillie AH, Ferguson MM and Hart DM. Development in Steroid both sexes (Guioli and Lovell-Badge, 2007). The signifi- Histochemistry. Academic Press. New York. 1996. cance of these asymmetries (especially in the male) is Bishop-Calame S. Experimental study of the organogenesis of the unknown. urogenital system of the chicken embryo. Archives d’Anatomie Microscopique et de Morphologie Experimentale, 55: 215- Genes associated with pluripotency were also recently 309. 1966. found to be expressed asymmetrically in embryonic gonads 356 Journal of Poultry Science, 51 (4)

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