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Sexual Differentiation, Gonadal Development, and Development of the External Genitalia: A Review of The Regulation of Sexual Differentiation

Rebecca M. Perrett

Introduction

The development of one's sex comprises 'sex determination' - the development of the undifferentiated gonad into testis or ovaries during embryogenesis, followed by 'sex differentiation' - the determination of phenotypic sex induced by factors produced by the differentiated gonad. This chapter will highlight the molecular mechanisms underpinning these two processes.

During the first 2 weeks of human embryonic development, the only difference between XX and XY embryos is their karyotype. At the two-cell stage of the XX zygote, X chromosome inactivation occurs, enabling males and females to have equal transcript levels from the X chromosome (Huynh and Lee 2001). In developing germ cells, the X is reactivated in the female, so both X chromosomes contribute to oogenesis (Sugimoto and Abe 2007).

The Bipotential Gonad

During the fourth week of human development, the urogenital ridges develop as a thickening of the mesodermic mesonephros covered by coelomic epithelium (CE); it is from this structure that the urogenital system and adrenal cortex originate. In the fifth week, or mouse embryonic day (E) 9.5-10.5, the urogenital ridge divides into a urinary and adreno-gonadal ridge the latter of which forms the gonads and adrenal gland (Swain and Lovell-Badge 1999). Until the sixth week of human development, or mouse E11.5, the undifferentiated gonads of XX and XY individuals are identical and have the potential to form either ovary or testes (bipotential).

Molecular Determinants of Gonadal Development

A number of factors have been shown to be required for the development of the undifferentiated gonad, as illustrated in Figure 1.1. However, due to the limited studies in human development, mouse studies have revealed several more important factors involved in gonadal development, and these are outlined below.

Figure 1.1 Simplistic illustration of the molecular determinants for gonadal differentiation. In the presence of SRY, SOX9 is upregulated and is responsible for the regulation for testicular development. In the absence of SRY, pro-ovarian factors regulate ovarian development (see text for more detail).

Empty spiracles homeobox 2 (Emx2)

Emx2 encodes a homeodomain transcription factor expressed in urogenital epithelial cells. Knockout mice completely lack kidneys, gonads, ureters and genital tracts, but the adrenal glands and bladder are normal (Miyamoto et al. 1997), indicating Emx2 acts after division of the urogenital ridge. It may regulate tight junction assembly, allowing migration of the gonadal epithelia to the mesenchyme (Kusaka et al. 2010).

Paired box gene 2 (Pax2)

Pax2 is a transcriptional regulator expressed within the urogenital system during development, in both ductal and mesenchymal components (Torres et al. 1995). Null mice lack kidneys, ureters, and genital tracts, and the Wolffian and Müllerian tracts degenerate.

Transcription factor 2 (Tcf2)

The POU domain containing Tcf2 gene functions in epithelial differentiation (Coffinier et al. 1999; Kolatsi-Joannou et al. 2001). It is essential for urogenital development, as patients harbouring mutations exhibit genital malformations (Lindner et al. 1999; Bingham et al. 2002).

Steroidogenic factor 1 (Sf1)/Nr5a1

The transcription factor Sf1 is expressed in the hypothalamus, pituitary, gonads, and adrenal glands (Luo et al. 1994; Val et al. 2003). Null mice lack gonads and adrenal glands (Luo et al. 1994; Shinoda et al. 1995). Sf1 also functions later in testis development.

Wilms' tumour 1 (Wt1)

Wt1 encodes multiple isoforms of a zinc finger protein, which act as transcriptional repressors (Menke et al. 1998) or activators (Lee et al. 1999). The -KTS variant promotes cell survival and proliferation in the indifferent gonad, whereas the +KTS isoform functions in testes differentiation (Hammes et al. 2001). The -KTS isoform activates the sex-determining region Y (Sry) and Sf1 promoters (Hossain and Saunders 2001; Wilhelm and Englert 2002). Wt1 is expressed in urogenital ridges (Pritchard-Jones et al. 1990) where it maintains the identity of adreno-gonadal primordium (AGP) the precursor to the gonads and adrenal primordia (Bandiera et al. 2013). Accordingly, null mice lack kidneys and gonads (Kreidberg et al. 1993).

LIM homeobox 9 (Lhx9)

Knockout of Lhx9, a homeobox protein, causes failure of gonadal development (Birk et al. 2000) and synergizes with Wt1 to regulate Sf1 expression (Birk et al. 2000; Wilhelm and Englert 2002).

Chromobox homologue 2 (Cbx2)

Cbx2 is the mouse homologue of the Drosophila polycomb gene and regulates transcription by altering chromatin structure. Knockout XX mice have small or absent ovaries and XY mice show male-female sex reversal (Katoh-Fukui et al. 1998). Cbx2 may regulate Sf1 expression in the gonad, as it does in the adrenal gland (Katoh-Fukui et al. 2005), or it may alter Sry expression directly (Katoh-Fukui et al. 2012).

CBP/p300 interacting transactivator, with glu/asp-rich c-terminal domain, 2 (Cited2)

Cited2 is a transcriptional regulator expressed in the AGP, and later in the CE and underlying mesenchyme of the genital ridge (Bhattacharya et al. 1999; Braganca et al. 2003). It cooperates with Wt1 to stimulate Sf1 expression in the AGP (Val et al. 2007; Buaas et al. 2009), and also ensures Sry levels are sufficient to trigger testis determination.

Gata binding protein 4 (Gata4)

Gata4 is a transcription factor first detected at E11.5 in somatic cells of XX and XY gonads; at E13.5 it is upregulated in XY Sertoli cells and downregulated in interstitial cells and XX gonads (Viger et al. 1998). It is required for gonadal ridge formation (Hu et al. 2013), along with later functions in testicular and ovarian development.

Primordial Germ Cells

Specification

Primordial germ cells (PGCs), the founder cells of the germ cell lineage, are typically established early during embryonic development. Germ cell specification can either occur through the inheritance of germ cell determinants already present in the egg (preformation), as in Drosophila melanogaster and Caenorhabditis elegans, or in response to inductive signals, as for mice and probably all mammals (epigenesis) (Extavour and Akam 2003; Saitou and Yamaji 2012).

Mouse PGCs (mPGCs) originate in the pluripotent proximal epiblast at about E6.0 when they respond to signals from extraembryonic tissues and express Fragilis/Interferon-induced transmembrane protein 3 (Ifitm3) (Saitou et al. 2002). Bone morphogenetic protein 4 (Bmp4) and 8b from the extraembryonic ectoderm and Bmp2 and wingless-type MMTV integration site family, member 3 (Wnt3) from the visceral endoderm are critical for specification (Lawson et al. 1999; Ying et al. 2000; Ying and Zhao, 2001; Ohinata et al. 2009). At E6.25, about six of these cells express B-lymphocyte-induced maturation protein 1 (Blimp1, also known as PR domain-containing 1, Prdm1): these cells are PGC precursors (Ohinata et al. 2005), although further cells are recruited to become PGCs before E7.25 (Saitou et al. 2002; McLaren and Lawson 2005; Ohinata et al. 2005). Wnt3 acts via ß-catenin to activate the mesodermal factor T (brachyury), which in turn induces Blimp1 and Prdm14 expression (Aramaki et al. 2013); these are transcriptional repressors which suppress the somatic program while allowing establishment of germ cell character (Saitou et al. 2002; Saitou et al. 2005; Ohinata et al. 2005; Vincent et al. 2005; Yabuta et al. 2006; Seki et al. 2007; Kurimoto et al. 2008; Yamaji et al. 2008). The expression of genes which establish/maintain pluripotency are retained via the epiblast, including Sox2, Nanog, Oct4, and Embryonal stem cell gene 1 (Esg1) (Scholer et al. 1990; Ohinata et al. 2005; Western et al. 2005; Yamaguchi et al. 2005; Yabuta et al. 2006; Chambers et al. 2007).

Following establishment of the germ cell lineage, extensive reprogramming of the genome occurs, i.e. erasure of epigenetic marks such as DNA methylation and establishment of new marks (Surani 2001; Hajkova et al. 2002). Imprinting must be reprogrammed in the germ line, as a maternal allele in one generation may be a paternal allele in the next. PGCs do initially acquire genome wide de novo methylation; however, following entry into the gonadal ridge, there is...