Genetic and Epigenetic Effects in Sex Determination Sezgin Ozgur Gunes1,2, Asli Metin Mahmutoglu1, and Ashok Agarwal*3
Genetic and Epigenetic Effects in Sex Determination Sezgin Ozgur Gunes1,2, Asli Metin Mahmutoglu1, and Ashok Agarwal*3
Sex determination is a complex and dynamic process with multiple genetic cascade are not completely understood. This review aims at discussing and environmental causes, in which germ and somatic cells receive various current data on the genetic effects via genes and epigenetic mechanisms that sex-specific features. During the fifth week of fetal life, the bipotential affect the regulation of sex determination. embryonic gonad starts to develop in humans. In the bipotential gonadal tissue, certain cell groups start to differentiate to form the ovaries or testes. Birth Defects Research (Part C) 108:321–336, 2016. Despite considerable efforts and advances in identifying the mechanisms VC 2016 Wiley Periodicals, Inc. playing a role in sex determination and differentiation, the underlying mechanisms of the exact functions of many genes, gene–gene interactions, Key words: sex determination; SRY; SOXE; NR5A1; GATA4; WT1; epigenetics and epigenetic modifications that are involved in different stages of this
Introduction formation via inducing a different set of genes (Sekido and Sex determination is a biological process determining the Lovell-Badge, 2008; Rigby and Kulathinal, 2015). development of the primordial gonad into male (testes) or Animal experiments and genetic analyses in patients female (ovary) gonads (Herpin and Schartl, 2011). During with developmental sex disorders (DSDs) have demonstrat- the sex determination cascade, the initial event is the forma- ed that many genes and pathways, such as GATA4, SOX9, tion of the gonadal primordium, also known as gonadal or NR5A1, FOG2, Hedgehog, and the Map Kinase signaling path- genital ridge (Ronfani and Bianchi, 2004). The gonadal pri- way, play critical roles in the sex determination process mordium derives from the mesonephros that is one of the (Cotinot et al., 2002). Recent studies have proposed that epi- tubular nephric structures of the urogenital ridges in mam- genetic mechanisms are also involved in the regulation of mals (Lucas-Herald and Bashamboo, 2014), and is coated by sex determination (Kuroki et al., 2013; Mulvey et al., 2014; coelomic epithelium (Wilhelm et al., 2013). Epithelial cells Skinner et al., 2015). In this review, current and past litera- derived from the proliferation of the epithelium of the ture on genetic effects on the regulation of sex determina- gonadal primordium enter the mesenchyme and result in tion, with special emphasis on male sex determination, is the formation of primitive sex cords. These sex cords lose discussed. In addition, the contribution of epigenetic mecha- nisms to the process of sex determination is reviewed. connections with the epithelial surface, share the same morphology in fetuses with XX or XY sex chromosomes, and SRY are called bipotential gonads (Matzuk and Lamb, 2008; Sex Determining Region Y (SRY) (OMIM *480000) is a single Sadler, 2011). exon gene and encodes a DNA binding protein, a member of A variety of genes including WT1, FOG2, and NR5A1 the high mobility group (HMG)-box family. The SRY gene enco- are known to play a role in the bipotential gonad forma- des a transcription factor and is located on the position p11.3 tion (Barrionuevo et al., 2012). The differentiation of bipo- of the Y-chromosome (Premi et al., 2006). This transcription tential gonads along the testis- or ovary-specific pathway factor is called the testis-determining factor (TDF), induces is induced by SRY gene expression (Wijchers and Festen- male sex determination, and has been proposed to play a piv- stein, 2011). The expression of the SRY gene gives rise to otal role in sex determination in males (Page et al., 1987). The the activation of downstream genes involved in testis for- SRY transcript has many transcription starting sites, with the mation by inducing Sertoli-cell differentiation, whereas the size of the transcript being approximately 900 nucleotides dysfunction or absence of SRY expression results in ovary (Clepet et al., 1993). The SRY protein is constituted of N- terminal, central, and C-terminal domains containing the HMG box which is an evolutionary conserved DNA binding 1 Department of Medical Biology, Faculty of Medicine, Ondokuz Mayis and bending domain spanning over 79 amino acids. The HMG University, Samsun, Turkey 2Department of Multidisciplinary Molecular Medicine, Ondokuz Mayis box motif provides binding to a specific DNA sequence via University, Health Science Institute, Samsun, Turkey interaction with the minor groove and causes bending of the 3American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, DNA helix up to a 908 angle. This bending enables the forma- Ohio tion of an active transcriptional complex on the DNA. Protein–
*Correspondence to: Ashok Agarwal, Lerner College of Medicine, Andrology protein interactions and post-translational modifications also Center and the American Center for Reproductive Medicine, Cleveland take place in the HMG domain. Thus, since most of the muta- Clinic, Mail Code X-11, 10681 Carnegie Avenue, Cleveland, OH 44195. tions occur de novo in the DNA sequence encoding this E-mail: [email protected] domain, and these mutations cause to abnormalities in DNA Published online in Wiley Online Library (wileyonlinelibrary.com). Doi: 10.1002/bdrc. binding and sex reversal, the HMG box domain has a very crit- 21146 ical role (Helszer et al., 2013). HMG box domain mutations
VC 2016 Wiley Periodicals, Inc. 322 MOLECULAR MECHANISMS IN SEX DETERMINATION
also change nuclear importation and are involved in the pancreas specification, gliogenesis, and neural crest devel- minority of human sex reversal cases (Sim et al., 2008). opment (Huang et al., 2015). SOXE proteins contain the Alterations in the DNA sequence of the SRY gene can transcription activation domain, as well as the DNA- result in gonadal dysgenesis, which is one of the DSDs binding HMG domain, similar to all other SOX proteins. (Schlessinger et al., 2010). DSDs are congenital abnormities Moreover, the DNA-dependent dimerization domain is a with numerical or structural chromosomal aberrations, unique structure of SOXE proteins (Barrionuevo and gonadal, or anatomic sex development abnormalities (Ohne- Scherer, 2010). In mammals, SOXE genes are involved in sorg et al., 2014). 46,XX, 46,XY and sex chromosome DSDs sex determination (She and Yang, in press) via enhancing are categories of developmental sex disorders, according to the activities of testis-specific enhancer (TES) of Sox9 core the Chicago Consensus (Hughes et al., 2006). The aneuploi- element (TESCO) and anti-Mullerian Hormone (Amh)pro- dies of X- and/or Y-chromosomes cause sex chromosome moter (Otake and Kuroiwa, 2016). DSDs, including Turner syndrome (TS), Klinefelter syn- In 46,XY embryos, gonadal differentiation is initiated by drome (KS), and 45,X/46,XY mosaicism. Children with KS or SRY expression between 41 and 44 days postovulation TS have no genital differences in their early lives; whereas, (d.p.o.)/Carnegie stages 18 (CS18). Once SRY expression children with the mosaic 45,X/46,XY karyotype have ambig- peaks at 44 d.p.o./CS18, a period of time when sex cords are uous genitalia in their newborn period. Although 46,XX first seen, SOX9 gene activation is initiated in the bipotential DSDs and 46,XY DSDs result from different conditions, high gonad and triggers Sertoli cell differentiation. The expres- levels of androgen exposure during the fetal period and sion of the SRY gene continues up to 18-weeks gestation incomplete intrauterine virilization, respectively, they all (Hanley et al., 2000; Eggers et al., 2014). SOX9 gene expres- cause the generation of external genitalia improper to sex sion is promoted by not only SRY expression, but also chromosome constitution (Achermann et al., 2015). enhancers located in different regions upstream of SOX9 To date, more than 50 mutations of the SRY gene have gene, depending on tissue type and time (Lybaek et al., been associated with sex reversal (Andonova et al., 2015). 2014). TESCO is one of the regulatory elements of the SOX9 These mutations are summarized in Table 1. Most of the gene and engages an important role as an enhancer in sex mutations of SRY gene associated with gonadal dysgenesis determination of the mammalian organisms (Kimura et al., have been detected in the HMG box. Helszer et al. (2013) 2014). DNA lesions leading to changes in the upstream regu- detected a novel missense mutation (c.341A>G) causing the lation of SOX9 have been suggested to be related to SRY neg- substitution of asparagine by aspartic acid at codon 65 ative XX DSDs (Kojima et al., 2008). Benko et al. (2011) have (p.N65D) in the HMG box of SRY gene. The binding ability of reported duplications and deletions in the SOX9 regulatory the N65D variant of the SRY was analyzed by gel retardation region in patients with isolated 46,XX and 46,XY DSDs, analysis. These authors suggested that the c.341A>Gmuta- respectively. Although duplication in the upstream region of tion may also contribute to the pathogenesis of 46,XY gonadal SOX9 is known to cause SRY2 XX DSDs (Hyon et al., 2015), dysgenesis (Helszer et al., 2013). Although M64I is a classical polymorphisms of SOX9 do not result in SRY2 XX DSDs (Xia sex reversal mutation at position 9 in the HMG box, it has no et al., 2015). Recently, a study has proposed that the regula- adverse effect on DNA binding (McElreavey et al., 1992). tory regions of the SOX9 gene span two different regions, XY Another mutation with no effect on DNA binding activi- and XX sex reversal regions. Therefore, deletions or duplica- ty is the F108S mutation, a familial mutation in the SRY tions cause distinct forms of DSDs (Kim et al., 2015). gene (J€ager et al., 1992). More recently, the F108S mutation SOX9 has been found to be associated with elevated polyubiquiti- SOX9 (OMIM *608160) is an autosomal gene located on nation. This process is a modification correlated with an 17q24.3, and is a primary downstream target of SRY (Foster increase in proteosomal degradation. Polyubiquitination of et al., 1994). SOX9 protein has very critical and diverse func- mutant SRY has been suggested to lead to an increase in tions in embryonic development (Katoh-Fukui et al., 2015), proteosomal degradation, resulting in a reduction of intra- such as sex determination by initiating male-specific sex cellular level of SRY. The alteration of intracellular SRY lev- determination in the default of Sry in Sertoli cells (Kent els caused a reduction in transcriptional activation of Sox9, et al., 1996; Vidal et al., 2001; Chaboissier et al., 2004), carti- which can obstruct normal gonadogenesis (Gerhardt, 2016; lage formation (Bi et al., 1999), and neural crest (Spokony Racca et al., 2016). These mutations also demonstrate that et al., 2002; Cheung and Briscoe, 2003), skeletal (Foster SRY is a cornerstone in sex determination. However, apart et al., 1994, Wagner et al., 1994), brain (Pompolo and Harley, from SRY, other genes are also essential for gonadal devel- 2001), and craniofacial development (Lee and Saint-Jeannet, opment in males, such as SOX9, NR5A1,andGATA4 (Fig. 1). 2011). SOX9 heterozygous mutations are involved in Campo- SOXE GENES melic Dysplasia identified with defects in bone morphology SRY-related HMG box (SOX)8, SOX9, and SOX10 proteins (Gordon et al., 2014) and sex reversal (Berta et al., 1990). are SOXE transcription factors which are implicated in var- Ortego et al. (2015) colleagues have shown that overexpres- ious developmental processes including sex determination, sion of Sox9 promotes testis determination in male mice IT EET EERH(ATC 0:2–3 (2016) 108:321–336 C) (PART RESEARCH DEFECTS BIRTH
TABLE 1. Studies Reporting Mutations of SRY Gene
Type of Amino acid Location of mutation change mutation in SRY Effects of mutation Transmission Phenotype Method Reference
Missense Gly ! Arg HMG Box DNA binding De novo Gonadal Dysgenesis SSCP assay and DNA Hawkins et al. (1992a) activity may change Sequencing
Nonsense NA HMG Box DNA binding De novo Gonadal Dysgenesis SSCP assay and DNA Hawkins et al. (1992a)
activity may change Sequencing
Missense Phe ! Ser HMG Box No differences in Familial Gonadal Dysgenesis DNA Sequencing Jager€ et al. (1992) DNA binding
activity
Nonsense Gln ! Stop ORF DNA binding De novo Pure Gonadal Dysgenesis Asymetric PCR and DGGE McElreavey et al. (1992) activity may change
Deletion – 5’ to the HMG Box NA De novo Pure Gonadal Dysgenesis Southern Blot Analysis McElreavey et al. (1992)
Missense Ile ! Thr HMG Box NA De novo Pure Gonadal Dysgenesis DGGE and DNA Sequencing McElreavey et al. (1992)
Nonsense Tyr ! Stop HMG Box NA De novo Pure Gonadal Dysgenesis DGGE and DNA Sequencing McElreavey et al. (1992)
Nonsense Gln ! Stop HMG Box NA De novo Pure Gonadal Dysgenesis DGGE and DNA Sequencing McElreavey et al. (1992) Missense Met ! Ile HMG Box NA De novo Pure Gonadal Dysgenesis DGGE and DNA Sequencing McElreavey et al. (1992)
Missense Val ! Leu ORF NA Familial Gonadal Dysgenesis DNA Sequencing Vilain et al. () 46, XY Male
Nonsense Lys ! Stop ORF NA De novo Gonadal Dysgenesis DNA Sequencing Muller€ et al. (1992)
Missense Ile ! Met ORF NA Familial Complete Gonadal SSCP assay and Hawkins et al. (1992b) Dysgenesis DNA Sequencing
Missense Leu ! Ile ORF NA NA Complete Gonadal SSCP assay and Hawkins et al. (1992b) Dysgenesis DNA Sequencing
Deletion – ORF NA De novo Complete Gonadal Dysgenesis SSCP assay and Hawkins et al. (1992b)
DNA Sequencing
Missense Arg ! Gly HMG Box NA De novo Pure Gonadal Dysgenesis DNA Sequencing Affara et al. (1993)
Missense Met ! Trp HMG Box NA De novo Pure Gonadal Dysgenesis DNA Sequencing Affara et al. (1993)
Missense Arg ! Trp HMG Box NA NA Pure Gonadal Dysgenesis DNA Sequencing Affara et al. (1993)
Nonsense Gln ! Stop HMG Box NA NA Pure Gonadal Dysgenesis DNA Sequencing Affara et al. (1993)
Silent Leu ! His SRY NA Post Zigotic True Hermaphroditism DNA Sequencing Braun et al. (1993) Deletion – Outside of TDF NA Induced (Mice) Gonadal Dysgenesis Southern Blot Analysis Capel et al. (1993) 323 324
TABLE 1. Continued
Type of Amino acid Location of mutation change mutation in SRY Effects of mutation Transmission Phenotype Method Reference
Missense Ala ! Thr HMG Box No or reduced De novo Gonadal Dysgenesis DNA Sequencing Zeng et al. (1993) DNA binding
activity
Nonsense Trp ! Stop HMG Box NA De novo Gonadal Dysgenesis SSCP assay and DNA Iida et al. (1994) Sequencing
Nonsense Leu ! Stop 30 Non-HMG Box NA Familial Gonadal Dysgenesis SSCP assay and DNA Tajima et al. (1994) Sequencing
Missense Ser ! Gly HMG Box Reduced DNA De novo Gonadal Dysgenesis SSCP Assay Schmitt-Ney binding activity et al. (1995)
Missense Pro ! Leu HMG Box Reduced DNA Familial Gonadal Dysgenesis SSCP Assay Schmitt-Ney binding activity et al. (1995)
Interstitial –30 to SRY NA De novo Partial Gonadal Southern Blot Analysis McElreavey
Deletion Dysgenesis et al. (1996)
Nonsense Gln ! Stop ORF Incomplete DNA Familial Gonadal Dysgenesis DNA Sequencing Bilbao et al. (1996) binding
Nonsense Trp ! Stop ORF NA De novo Complete Gonadal Dysgenesis FAMA Veitia et al. (1997)
Missense Arg ! Trp ORF NA De novo Complete Gonadal Dysgenesis DNA Sequencing Veitia et al. (1997)
Missense Arn ! Asn HMG Box NA De novo Gonadal Dysgenesis SSCP Assay and DNA Battiloro et al. (1997) Sequencing OEUA EHNSSI E DETERMINATION SEX IN MECHANISMS MOLECULAR Point – 50 to SRY NA NA Pure Gonadal Dysgenesis SSCP Assay and DNA Poulat et al. (1998)
Sequencing
Nonsense Lys ! Stop 50 to HMG Box NA De novo Gonadal Dysgenesis SSCP Assay and DNA Scherer et al. (1998) Sequencing
Missense Met ! Arg HMG Reduced DNA De novo Gonadal Dysgenesis SSCP Assay and DNA Scherer et al. (1998) binding activity Sequencing
Missense Phe ! Val HMG Reduced DNA De novo Gonadal Dysgenesis SSCP Assay and DNA Scherer et al. (1998) binding activity Sequencing
Missense Ser ! Asn 50 Non-HMG Box DNA binding Familial Partial Gonadal Dysgenesis DNA Sequencing Domenice activity may change et al. (1998)
Nonsense Tyr ! Stop De novo 45,X/47,XYY Mosaicism DNA Sequencing Takagi et al. (1999) IT EET EERH(ATC 0:2–3 (2016) 108:321–336 C) (PART RESEARCH DEFECTS BIRTH
TABLE 1. Continued
Type of Amino acid Location of mutation change mutation in SRY Effects of mutation Transmission Phenotype Method Reference
Upstream of Lack of DNA
HMG Box binding motif
Missense Ser!Asn 50 Non-HMG Box NA De novo Ullrich-Turner Syndrome DNA Sequencing Canto et al. (2000)
Missense Gly ! Gln HMG Box NA De novo Complete Gonadal DNA Sequencing Schaffler et al. (2000) Dysgenesis with Yolk-Sac Tumor
Missense Arg ! Ile Non-HMG Box NA Familial Partial Gonadal DNA Sequencing Assumpcao et al. (2002) Dysgenesis
46, XY Male
Missense Asn ! His HMG Box Altered DNA De novo Pure Gonadal DNA Sequencing Assumpcao et al. (2002) binding activity Dysgenesis
Missense Arg ! Asn HMG Box No DNA binding De novo Complete Gonadal – Mitchell and Harley (2002) activity Dysgenesis
Missense Leu ! Pro HMG Box No DNA binding NA Complete Gonadal – Mitchell and Harley (2002) activity Dysgenesis
Missense Phe ! Val HMG Box Disrupted DNA De novo Complete Gonadal – Mitchell and Harley (2002) binding activity Dysgenesis
Missense Met ! Arg HMG Box Reduced DNA De novo Complete Gonadal – Mitchell and Harley (2002) binding activity (6 Dysgenesis
Fold)
Missense Arg ! Pro HMG Box Reduced DNA NA Complete Gonadal – Mitchell and Harley (2002) binding activity (4 Dysgenesis
Fold)
Missense Ala ! Thr HMG Box Reduced DNA De novo Complete Gonadal – Mitchell and Harley (2002) binding activity (3 Dysgenesis
Fold)
Missense Met ! Thr HMG Box Reduced DNA NA Complete Gonadal – Mitchell and Harley (2002) binding activity (1.7 Dysgenesis
Fold) 325 326
TABLE 1. Continued
Type of Amino acid Location of mutation change mutation in SRY Effects of mutation Transmission Phenotype Method Reference
Missense Ser ! Asn Outside the Minimal effect on Familial Partial Gonadal – Mitchell and Harley (2002) HMG Box DNA binding Dysgenesis
activity
Missense Tyr ! Phe HMG Box NA Familial Gonadal Dysgenesis DNA Sequencing Jordan et al. (2002) 46, XY Male
Missense Ile ! Met HMG Box DNA binding Familial True Hermophroditism DNA Sequencing Maier et al. (2003) activity may change
Missense Arg ! Trp NLS Reduction in MPB De novo Gonadal Dysgenesis – Harley et al. (2003) binding
Missense Arg ! Gly NLS Reduction in MPB De novo Gonadal Dysgenesis – Harley et al. (2003) binding
Missense Arg ! Asn NLS Normal MPB De novo Gonadal Dysgenesis – Harley et al. (2003) binding
Missense Arg ! Pro NLS NA NA Gonadal Dysgenesis – Harley et al. (2003)
Missense Glu ! Arg ORF NA De novo Gonadal Dysgenesis SSCP assay and DNA Shahid et al. (2004) Sequencing
Missense Glu ! Arg Upstream of NA De novo Gonadal Dysgenesis SSCP assay and DNA Shahid et al. (2004) HMG Box Sequencing
Missense Ser ! Cys Downstrem of NA De novo Gonadal Dysgenesis SSCP assay and DNA Shahid et al. (2004) OEUA EHNSSI E DETERMINATION SEX IN MECHANISMS MOLECULAR HMG Box Sequencing
Nonsense Asn ! Stop HMG Box DNA binding De novo 45,X/46,XY Mosaicism SSCP assay and DNA Shahid et al. (2005) activity may change Sequencing
Nonsense Leu ! Stop Downstrem of DNA binding De novo 45,X/46,XY Mosaicism SSCP assay and DNA Shahid et al. (2005) HMG Box activity may change Sequencing
Missense Gln ! His HMG Box DNA binding De novo 45,X/46,XY Mosaicism SSCP assay and DNA Shahid et al. (2005) activity may change Sequencing
Missense Trp ! Leu NLS Reduction in De novo Gonadal Dysgenesis DNA Sequencing Hersmus et al. (2009) nuclear import with Bilateral
Gonadoblastoma BIRTH DEFECTS RESEARCH (PART C) 108:321–336 (2016) 327
with deleted Sry gene, but also leads to aberrant testicular development, abnormalities in seminiferous epithelium with normal sperm count, motility, morphology, and fertilization capacity (Ortega et al., 2015). In the case of the absence of Sox9 in Sertoli cells, Sox8,orSox10 could be functional (Eggers et al., 2014).
SOX8 et al. (2012) SOX8 (OMIM *605923), 5.6 kb in size, is located on the petit arm of chromosome 16 and codes the SOXE tran- scription factor that is expressed in branchial arches, pan- creas, brain, heart, limbs, and testis. SOX8 transcription encoded by SOX8 gene is quite similar to other SOXE pro- teins, in terms of the sequence similarity within and out- side of the HMG domain. SOX8 deletion is involved in a- thalassemia/mental retardation syndrome (ATR-16) (Pfei- DNA SequencingDNA Sequencing Paliwal et al. (2011) Paliwal et al. (2011) DNA Sequencing Filges et al. (2011) DNA Sequencing Helszer et al. (2013) DNA Sequencingfer Andonova et al. et (2015) al., 2000; Schepers et al., 2003). Sox8 is one of the definitive markers of Sertoli cells and consolidates Sox9 function in gonad determination. Following the Sox9 acti- vation, Sox8 expression may induce differentiation in a Sertoli cell-specific manner (Chaboissier et al., 2004). In addition, a study conducted with Sox8 knockout mice has shown that Sox8 is vital for male fertility, since the absence of this protein causes an abnormality in the regu- lation of spermatogenesis in an age-dependent manner and progressive degeneration of the seminiferous epitheli- Dysgenesis Dysgenesis Dysgenesis Complete Gonadal Dysgenesis Complete Gonadal Dysgenesis um (O’Bryan et al., 2008). Hence, expression of Sox8 and Sox9 provides for proper testis cord formation (Barrio- nuevo et al., 2009) through maintaining the integrity of the basal lamina of the testis during testis development (Georg et al., 2012). A more recent study has proposed FamilialFamilial Pure Gonadal Pure Gonadal Familial Gonadal Dysgenesis RFLP Stoppa-Vaucher Familial Complete Gonadal De novo De novo that Sox8 and Sox9 provide constant expression of Dmrt1 in adult testis, which is essential to maintain functionality and integrity of seminiferous tubules, suggesting a new function for these genes. Sox8 and Sox9 are antiapoptotic factors in adult Sertoli cells (Barrionuevo et al., 2016).
SOX10
Effects of mutation Transmission Phenotype MethodSOX10 (OMIM Reference *602229), spanning five exons, is one of the activity may change activity may change binding activity binding activity binding activity SOXE genes (She and Yang, in press). Sox10 and SOX10 genes have been cloned from mouse and human, respectively, and characterized in 1998 (Pusch et al., 1998). Furthermore, SRY SOX10 protein shares 59% of its amino acid sequence identi- ty with SOX9 protein (Pusch et al., 1998). SOX10 has a role in
Location of oligodendrocyte development and differentiation of periph- mutation in eral glial cells, as well as sex determination and differentia- tion (Britsch et al., 2001; Cory et al., 2007; Chaoui et al., 2015). Leu HMG Box NA Asp HMG Box Reduced DNA Ser HMG Box Reduced DNA Leu HMG Box DNA binding Ala HMG Box Reduced DNA Stop HMG Box DNA binding SOX10 mutations result in Waardenburg-Hirschsprung ! ! ! ! ! !
change disease and Kallmann syndrome (MIM no. 147950) (Kuhl- Amino acid brodt et al., 1998; Pingault et al., 1998; Chaoui et al., 2015). Sox10 is expressed in pre-Sertoli cells in the male genital Continued ridge in mice. However, no abnormality of testis develop- ment has been reported in knockout mice with lost Sox10 Type of mutation Nonsense Tyr Missense Glu Missense Leu NA, not applicable. Missense Asn Missense Glu Missense Phe
TABLE 1. function (Cory et al., 2007). Polanco et al. (2010) have 328 MOLECULAR MECHANISMS IN SEX DETERMINATION
FIGURE 1. Gonadal development in the male. Multiple genes and pathways required for differentiation of bipotential gonad into testis, including NR5A1, GATA4, and WT1 (1KTS isoform), upregulate the master switch gene, SRY. Activation of SRY gene in the bipotential gonad initiates SOX9 expression, and then SOX9 promotes AMH expression in conjunction with NR5A1. FOG2 and GATA4 are also essential for AMH expression and testis development.
FIGURE 2. Epigenetic mechanisms in sex determination. The figure identifies main epigenetic mechanisms involved in sex determination process. Activation of genes involved in this process is regulated by histone modifications and DNA methylation. Gonadal functions are also organized by noncoding RNAs. shown that one of the reasons for sex reversal syndrome is possess DNA sequence similarity in the HMG box domain Sox10 overexpression. Expression levels of the Sox10 gene (Stevanovlc´ et al., 1993). Therefore, the SRY gene has been have been shown to be associated with various degrees of suggested to originate from SOX3 (Foster and Graves, sex reversal in transgenic mice (Polanco et al., 2010). 1994). Sox3 is expressed in the brain and developing gonads, and plays critical roles in neuronal development SOX3 and gonadal function. Weiss et al. (2003) have shown that SOX3 (OMIM *313430) gene has a single exon (Rizzoti Sox3 deletion has no adverse effect on sex determination et al., 2004), belongs to the SOX gene family, and has been in the male or female. However, Sox3 is essential for game- cloned and mapped on the long arm of the X chromosome togenesis in both sexes (Collignon et al., 1996; Bylund in the human. Human and mouse SOX3 genes show high et al., 2003; Weiss et al., 2003). Sox3 runs with steroido- DNA sequence similarity. Moreover, SOX3 and SRY also genic factor 1(Sf1) to transactivate TESCO, similar to Sry. BIRTH DEFECTS RESEARCH (PART C) 108:321–336 (2016) 329
Inactivation or loss-of-function SOX3 mutations have not hormone (AMH) in Sertoli cells, also known as Mullerian been associated with sex determination either in mice or inhibiting substance, causing the regression of the parame- humans, however, are related with growth hormone defi- sonephric (Mullerian) ducts; and therefore, the uterus and ciency or mental retardation. Conversely, chromosomal uterine tube, the upper part of vagina cannot be differenti- rearrangements or gain-of-function mutations may lead to ated from these ducts in the sixth week of gestation. After 46,XX male sex reversal (Sutton et al., 2011; Gunes et al., this time, while expression of SF1 increases in Leydig cells, 2013). Two recent publications, demonstrated that dupli- expression of SF1 decreases in Sertoli cells. Apart from cation of the chromosomal region incorporating the SOX3 SF1 and SOX9, GATA4, and WT1 also participate in the reg- gene led to the sex reversal syndrome in one case (Moa- ulation of AMH secretion (Shen et al., 1994; De Santa Bar- lem et al., 2012), and an 774-kb insertional translocation bara et al., 2000). Furthermore, in Leydig cells, SF1 from chromosome 1 to the distal SOX3 gene led to altera- induces the synthesis of testicular androgens leading to tions in SOX3 expression in a patient with 46,XX male sex differentiation of the mesonephric (Wolffian) ducts into reversal, suggesting that SOX3 can be a switch regulatory male urogenital structures (Parker et al., 1999). gene for promoting sex determination in humans (Haines NR5A1 gene loss-of-function mutations have been con- et al., 2015). nected with various genotypes and phenotypes of DSDs, such as 46,XX ovo-testicular DSD (Baetens et al., in press), NR5A1 46,XY DSD with adrenal failure (Achermann et al., 1999) SF1 is one of the transcription factors identified and and hypospadias (Tantawy et al., 2014), and also 46,XX involved in the modulation of sex determination and ste- DSD with primary amenorrhea (Philibert et al., 2010). In roidogenesis pathways, and development of the reproduc- tive axis and spleen (Oba et al., 1996; Achermann et al., children and adults with a 46,XX karyotype from unrelated 2002; Zangen et al., 2014). SF1 is encoded by the nuclear families, testis development first has been reported to be receptor subfamily 5 group A1 (NR5A1) (OMIM *184757), connected with a missense mutation in the DNA binding also initially known as FTZ-F1 and AD4BP, and has been region of NR5A1 (p.Arg92Trp). The mutation causes non- mapped to chromosome 9q33 by using fluorescence in syndromic 46,XX testicular or ovo-testicular DSD in situ hybridization in the early 1990s (Taketo et al., 1995). humans (Bashamboo et al., 2006). Recently, a novel mater- Its genomic sequence was identified by cloning of human nally inherited NR5A1 mutation (c.195G > A) has also been cDNA (Wong et al., 1996). The NR5A1 gene spans seven found in three siblings with 46,XY DSDs (Fabbri et al., exons with a noncoding exon, and is about 30 kb in length 2014). (Oba et al., 1996). Exon 2 and 3 are the first 2 coding GATA4 AND WT1 exons of NR5A1 and encode two zinc finger domains GATA4 and WT1 are the two transcription factors products which are conserved in rat, mouse, cow, and human, and of having a role in the sex determination process similar necessary for DNA binding. Mutation in these exons to multiple other genes (Table 2). In the sex determination caused NR5A1 haploinsufficiency in a presenting female and differentiation pathways, transcriptions of certain patient with 46,XY gonadal dysgenesis who had external genes have been controlled by the cooperation of GATA4 genitalia with clitoromegaly, absence of the uterus, and a and WT1 (Miyamoto et al., 2008). Wilms’ tumor (WT1) mild form of dysgenetic testes (Barbaro et al., 2011). SF1 expression starts with the sex cord formation and Sertoli binds to the promoters of more than 20 genes, has func- cell differentiation during the early stages of the human tions in male sexual differentiation, steroidogenesis, and embryo (from 6.5 to 8 weeks) and continues with a high reproduction with different roles as a monomer unlike level of expression up to the nineth week of gestation. other transcriptional factors and regulates their transcrip- After sex cord formation, GATA4 and WT1 expression is tion (Achermann et al., 2002). SF1 also binds to the spe- observed in Sertoli cells and consequently these transcrip- cific consensus site of the SRY gene, namely nuclear tion factors are thought not to be involved in the AMH hormone receptor 1 (NHR1) to activate the SRY gene dur- expression onset (De Santa Barbara et al., 2000). To spec- ing the gonadal determination pathway (de Santa Barbara ify the targets of WT1 in the gonad, the gene expression et al., 2001). Interaction between SRY and SF1 provides profile was identified in the urogenital ridges of wild type upregulation of Sox9. In addition, the synthesis of cyto- and Wt1 knockout mice using a microarray. Results have chrome p450 enzymes that are necessary in the produc- shown that alteration in the expression level of Wt1 had tion of steroid hormones is known to be regulated by SF1 no effect on Gata4 expression; therefore, Gata4 has been (Wong et al., 1996). reported to be indirectly regulated by Wt1 (Klattig et al., Tissue-specific expression of NR5A1 starts with the for- 2007). mation of the bipotential gonad primordia. Afterward, this process proceeds in the somatic cells of the sex cords and WT1 Leydig cells (Hanley et al., 1999; Achermann et al., 2002). WT1 (OMIM * 607102) is a gene with ten exons encoding SF1 and SOX9 induce the secretion of anti-Mullerian more than 24 different transcription factor isoforms, due 330 MOLECULAR MECHANISMS IN SEX DETERMINATION
TABLE 2. Genes Involved in Sex Determination Process
Association Gene Location MIM number Function(s) in sex determination process with DSDs References
SRY Yp11.3 480000 Promotes male sex determination 1 Sinclair et al. (1990)