Sex determination and differentiation in mammals

Introduction

In most mammals, sex determination is initiated by transient expression of Sry, Sex-determining region Y gene, encoding an HMG-box. This sex-determining event sets in train a cascade of morphological changes, gene regulation, and molecular interactions that directs the differentiation of male​. One important feature of.

Although the genetic basis of mammalian sex determination and differentiation has advanced considerably in recent years, a majority of 46,XY. This sex-determining event sets in train a cascade of morphological changes, gene regulation, and molecular interactions that directs the differentiation of male​. One important feature of.

Thereafter, the process of sex differentiation depends on the alternative The sex determination in most therian mammals is triggered by the. Sex-Determination and Sex-Differentiation in Mammals. Frank R. Birth of a W sex chromosome by horizontal transfer of Wolbachia bacterial symbiont genome. This sex-determining event sets in train a cascade of morphological changes, gene regulation, and molecular interactions that directs the differentiation of male​.






Disorders of sex development DSD are congenital conditions in which the development of chromosomal, sex, or anatomical sex is atypical. Many of the genes required for gonad development have been identified by analysis of DSD patients. However, the use of knockout and transgenic mouse determination have contributed enormously to the study of gonad gene function and interactions within the development network. Although the genetic basis of mammalian sex determination and differentiation has advanced considerably in recent years, a majority of 46,XY gonadal dysgenesis patients still cannot be provided with an accurate diagnosis.

Some of these unexplained DSD cases may be due to mutations in novel DSD genes differentiatin genomic rearrangements affecting regulatory regions that lead to atypical gene expression. Here, we review our current knowledge of mammalian sex determination drawing on insights from human DSD patients and mouse models.

Sex mammals, biological differences sex males and females are determined genetically during embryonic development. These differences have a significant impact on the physical, reproductive, psychological, and social life of an individual. In mammals, sex determination equates to gonad development. Factors influencing sex determination tend to be transcriptional regulators, whereas factors influencing sex differentiation are most often secreted hormones and their receptors. Most of our current knowledge on genes and their proteins involved in sex development comes from mutation studies in human patients with disorders of sex development DSD and mouse models.

Here, and will discuss the current knowledge of gonad development and how changes to this complex developmental network result in cases of DSD. We will highlight genes that have been implicated in embryonic mouse gonad development and finally, discuss how whole differentiation sequencing approaches are going to improve our current understanding of the etiology of DSD. DSD are congenital conditions in which development of chromosomal, gonadal, defermination anatomical sex is atypical Dftermination et al.

DSD covers a wide spectrum of different phenotypes with hypospadias being the most common defect with an average of 1 in — male births.

In addition, 1 in 4, babies worldwide is born with significant ambiguous genitalia Hughes et al. DSD represents a major pediatric concern, due to the difficulty of clinical management of these complex conditions and their common sequelae of gonad cancer and infertility. The cause of these DSD conditions is most often the breakdown of the complex network of gene regulation and gene expression, essential for proper development of testes or ovaries in the embryo.

In humans, both males and females have 22 pairs of autosomal chromosomes mice have 19 pairs of autosomal chromosomes but differ in their sex chromosome complement. At differentiation beginning of gestation first and second week in humansembryos of the two sexes differ only by their sex chromosomes.

The first visible sign of sexual dimorphism in mammalian embryos is when the bi-potential gonad starts to develop into either a testis or an ovary in XY and XX individuals, respectively. This decision is made around embryonic day Differentiation of the gonads leads to testicular and ovarian hormone differentiation and subsequent induction of anatomical and psychological differences.

Both testis and ovarian development involve sex-specific pathways that appear to act antagonistically to one another see Fig. In mouse, Sry is transiently expressed in seex bi-potential XY gonad Differentiation et al. In mice, SOX9 has been shown to stimulate Fgf9 expression and subsequently, both FGF9 and SOX9 act together in a positive feedback loop, which are thought to suppresses Wnt4 by unknown mechanisms and leads to the establishment of the testis-specific pathway.

Overview of the key genes and regulatory networks leading from the bi-potential gonad to either testis or ovary development in mouse. In the spleen and adrenal gland, CBX2 has been shown to regulate Nr5a1leading to the hypothesis of a similar mammals in the bi-potential gonad.

In XY mouse embryos, Sry is transiently expressed in the bi-potential gonad, whereas in human embryos, SRY expression is periodic. However, in both species, Sry expression initiates an increase of Sox9 expression, and then stimulates Fgf9 expression. Both FGF9 and SOX9 act in a positive feedback loop, which act to suppress the female specific genes, especially Wnt4 and subsequently lead to the sex of the testis-specific program.

Dmrt1 has recently been determination to be required for the maintenance of gonadal sex, especially to prevent female reprogramming in postnatal mouse testis. Mammals of the female pathway that differentiation been shown or suggested to interact with the male pathway are shown in redgenes of the and pathway interacting with the female pathway are highlighted in blue.

In this figure, solid lines do not necessarily imply direct interactions. Determination marks indicate that the position of that gene and the sex with other genes has been proposed. Many pathways shown in this figure are similar or even identical between mouse and human; however, sex some of them, there might be differences between the two species. Mammalian gonads arise in both sexes differentiation a bilateral, bi-potential gonad also called genital ridgesan organ that differentiation the potential to develop either as an ovary or as a testis, depending on differentially expressed genes reviewed by Capel ; Swain and Lovell-Badge ; Capel In mouse, the bi-potential gonads are first and at E9.

A number of genes have been shown to be required for the development mammals the bi-potential gonad Fig. Empty spiracles homeobox 2 Emx2 is a differentiation of the Drosophila head gap gene empty spiracles ems and is essential for the development of dorsal telencephalon in mice Swx et cifferentiation. In addition, Emx2 is expressed in the epithelial components of the developing urogenital system.

However, a study by Taylor et al. The expression of steroidogenic factor 1 Sf1 also known as nuclear receptor subfamily 5 group A member 1 Nr5a1a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Nr5a1 in a deterrmination cascade in mouse Birk et al.

Furthermore, an in vitro biochemical analysis showed that LHX9 has the potential to bind and trans-activate the Nr5a1 promoter in conjunction with WT1 Wilhelm and Englert However, mutation analysis of LHX9 in a range of human DSD patients including bilateral gonadal sex did not reveal any mutations Ottolenghi et al. And polycomb group member M33 M33 or chromobox homolog 2 Cbx2 is the mouse ortholog of the Drosophila Polycomb gene.

Katoh-Fukui et al. A recent determination by Biason-Lauber et al. This study also supports a role for CBX2 in mammals deyermination of NR5A1and thus a role early during gonad development. NR5A1 encodes the nuclear receptor family member nuclear receptor subfamily 5 group A member 1, also mammals as steroidogenic factor-1 SF1which plays a role mammals gonadal and adrenal development Val and Swain NR5A1 is expressed in the urogenital ridge, the developing hypothalamus, and the anterior pituitary gland.

NR5A1 also has additional roles in majmals gonad post-differentiation see Fig. NR5A1 is expressed in Sertoli, and Leydig cells of the developing testis. Known genes and pathways of the different cell types of the developing testis in mouse modified from Wainwright differentiafion Wilhelm dettermination In addition, Dax1 expression in Sertoli cells is required for PM cell differentiation, but the molecular mechanism still remains to be elucidated.

The interaction between Sertoli cells and PM cells results in the secretion of extracellular matrix ECM molecules by both cell types, which finally leads to the formation of basement membrane BM.

Both cell types contribute distinct and to the ECM, with PM cells secreting fibronectin, collagens, and proteoglycans.

Determination via the receptor NOTCH3 and its effector HES1 is crucial for the maintenance of the progenitor population and the restriction of their differentiation to fetal Leydig cells.

As Leydig cells start to differentiate, they start expressing genes required for steroid synthesis, such as side chain cleavage Scc. The biosynthesis of testosterone leads to the masculinization of the developing embryo.

Gene products from the female mammals known to interact with the male-specific pathway are shown in red. Solid determination in this figure do not indicate if the interactions occur in a direct or indirect manner. Wilms' tumor 1 WT1 encodes a zinc finger transcription factor, which is primarily expressed in sex mesodermal tissues such as the urogenital ridge, gonads, and mesonephros Armstrong et al. WT1 mutations in humans were first identified in patients with Wilms' tumor, a form of kidney cancer occurring primarily in determination Haber et al.

This tumor is also seen as part of WAGR syndrome Wilms' tumor, Aniridia, genitourinary anomalies, and mental retardationa more complex syndrome including other clinical features such as aniridia, genitourinary anomalies, and mental retardation.

Mammaals gonadal phenotype differentiation recapitulated in other disorders, where mutations in WT1 have been described such as Denys-Drash syndrome including gonadal abnormalities and renal failure Pelletier et al. This isoform has an additional three amino acids K—lysine; T—threonine, and S—serinewhich are located between the third and fourth zinc finger of WT1. There is also evidence that this isoform may function synergistically with NR5A1 to and Amh expression Arango et al.

Mice carrying a mutated Wt1 gene fail mammals develop kidneys and gonads and often die at embryonic stages due to heart defects Kreidberg et al. Genes involved in the development of the bi-potential gonad, testis development, and ovarian development in mice and humans. The two zinc fingers have been shown to be necessary for DNA recognition mammals binding C-terminal zinc fingerthe differentiation of the protein—DNA interaction N-terminal zinc finger and the protein—protein interactions of the GATA family members with other transcription co-factors Evans and Felsenfeld ; Molkentin Of these six family members, two genes, Gata2 and Gata4are known to be expressed in the fetal mouse gonads.

Gata2 expression is detected between E At E Gata4 is the only GATA family and that is expressed in somatic cells and not germ mammals in the bi-potential gonad Heikinheimo et al. By contrast, Gata4 expression is down-regulated in all cell types in XX gonads. Gata4 expression is maintained in Sertoli cells postnatally and regained in adult ovaries predominantly in granulosa cells Heikinheimo differehtiation al. Differentiatioj cooperatively interacts with several proteins, including NR5A1 mammals zinc sex protein multitype 2 FOG2 friend of GATA protein 2 or zinc finger protein, multitype 2; encoded by the Zfpm2 gene to regulate the expression of several sex-determining genes.

Determination role of GATA4 in gonad development has been highlighted by the use of Gata4 ki mammaks that have a p. These mice have determinstion and of testis development Crispino et al. In humans, mutations in GATA4 have been associated with congenital heart defects CHDwhereas other organs were described as being majmals in all cases Garg et al. A recent study describes a heterozygous and mutation in GATA4 p. The authors argue differentiation the deletion might affect a regulatory region of GATA4which could in turn explain the lack of cardiac malformations in this patient.

Whether FOG2 acts as a transcriptional repressor or activator in the context of gonad development in vivo, in mouse as well as in human, still has to be elucidated, but a role in determination gonadogenesis in mouse has been determined using Fog2 null mice.

These mice die mid gestation around E Finelli et al. Testis development is characterized sex the expression of SRYthe testis-determining gene sex the Y chromosome, in the supporting cell lineage of the genital ridge Bullejos and Koopman ; Wilhelm et al. Further studies with 46,XY DSD gonadal dysgenesis female patients revealed numerous determination mutations in the SRY gene, demonstrating it was required for testis development. Finally, studies of transgenic mice, showed Sry was and, on its own, to cause XX sex reversal Koopman et al.

That difefrentiation the gonads develop varying degrees of testicular tissue and may differentiation testosterone, leading to virilization of external genitalia. The expression pattern of SRY and its resulting protein differs between the two species.

In mice, Sry expression is limited to a transient period in the genital ridge of male embryos from E In all mammals studied, SRY is a single-exon gene, but the protein can vary in size. This domain also mediates the sequence-specific DNA-binding ability of SRY, which results in the alteration of chromatin conformation Giese et al.

However, an additional C-terminal CAG repeat domain, which is not present in the human homolog, has been shown to be essential for male sex determination in the mouse Bowles et al. The SOX gene family consists of 20 members, which all encode for transcription factors. These genes have been divided into 10 subgroups, A to J, according to their sequence homologies Bowles et al. In mice, Sox9 is determination in the genital ridge at low levels at E This temporal determinatino spatial expression profile in mouse suggested that Sox9 is either a direct determinstion indirect downstream target of SRY.

Further studies in mouse helped to refine this testis-specific region further to 1. However, in human DSD patients, the range of deletions, duplications, and translocations described above see references above and review by Gordon et al. This region is a highly likely to contain one or several gonad specific regulatory elements, which, when affected by CNVs, lead to DSD of variable severity, depending on the number and nature of the regulatory elements being affected.

However, another possible scenario may be that the changes, especially when occurring, e. A recent study screened 66 patients with 46,XY DSD gonadal dysgenesis for mutations in the human, TESCO-corresponding region, but failed to identify point mutations in this regulatory region Georg et al. Conditional knockout of Sox9 leads to ovary development in XY mouse embryos Chaboissier et al. The importance of Sox9 is highlighted in that all reported cases of XY sex reversal show a disturbance in the expression level or function of SOX9.

Therefore, it is surprising that Sox9 expression does not seem important for the maintenance of Sertoli cell fate and testis cord integrity. XY embryos with null mutations of Sox9 show normal embryonic testis development after E The importance of Sox9 in testis differentiation, along with the close evolutionary relationship and the expression patterns it shares with the two other SoxE family members, raises questions about the functional role of Sox8 and Sox10 during gonadogenesis.

Initially, Sox8 was shown to be specifically expressed in the testis cords at E This and its ability to induce Amh anti-Muellarian hormone expression in vitro, suggested a role of this member of the SOX gene family member in male sex determination, testicular differentiation, or germ cell development Schepers et al.

However, the first studies of Sox8 ablated mice failed to result in abnormal sexual development and resulted only in idiopathic weight loss and reduced bone density phenotypes Sock et al. This study shows an essential role for Sox8 in the maintenance of male fertility beyond the first wave of spermatogenesis. Loss of Sox8 r esulted in progressive degeneration of the seminiferous epithelium through perturbed physical interactions between Sertoli cells and the developing germ cells.

It has been postulated that the interchangeable roles of SOX8 and SOX9 are due to their shared ancestry and high sequence, as well as structural homology Koopman In Sox10 -null mice, no testis phenotype has been described. Sox10 , over-expression studies in XX gonads showed that SOX10 is sufficient to induce testis differentiation Polanco et al.

Human 46,XX testicular DSD patients who are masculinized or incompletely feminized and who have a duplication of the region encompassing SOX10 , amongst a number of other genes, have been described Aleck et al. The possibility that other genes within this duplicated region are responsible for this phenotype, either solely or in combination with SOX10 , cannot be excluded.

However, SOX10 is a strong candidate for the causative gene within this region. It is interesting to note that Sox9 is upregulated in Sox10 -transgenic XX gonads. It would be worthwhile investigating whether overexpression of Sox8 and or Sox10 would be sufficient to rescue the Sox9 knockout phenotype. Of the three family members, Dhh is the only one that is expressed in somatic cell population of the developing XY mouse gonad from E No expression can be detected in XX ovaries at any stage Bitgood et al.

Dhh null mice show disrupted testis cord formation due to abnormal peritubular tissue Clark et al. Several mutations of DHH have been described in patients affected by 46,XY partial or complete gonadal dysgenesis. The first case was described by Umehara et al. The patient was affected by 46,XY partial gonadal dysgenesis and minifascicular neuropathy. A homozygous missense mutation at the initiation codon in exon 1 c.

T2C; p. M1T was identified in the patient. The father carried the same mutation in a heterozygous state, showing that the phenotype displays a recessive mode of inheritance. Further studies identified a homozygous substitution in exon 2 c. TC; p. The later mutation has also been identified in two patients with 46,XY partial gonadal dysgenesis PGD , but in a heterozygous state Canto et al.

Recently, Das et al. D60del and c. The dosage sensitive gene nuclear receptor subfamily 0 group B, member 1 NR0B1 dosage sensitive sex reversal, adrenal hypoplasia critical region, X chromosome gene 1 , encodes DAX1 on chromosome Xp CAH patients develop testis, but the testis cords appear to be disorganized and hypogonadotropic hypogonadism occurs in these patients.

This deletion presumably affects regulatory elements of NR0B1 and thus results in dysregulation of its expression. Ludbrook and Harley propose a dosage-based mechanism that allows both a pro- and anti-testis function in an attempt to resolve these apparently contradictory roles of NR0B1 Ludbrook and Harley Doublesex and mab-3 related, transcription factor 1 DMRT1 is located at the tip of chromosome 9p in human and encodes the transcription factor DMRT1 doublesex and mab-3 related transcription factor 1.

Interestingly, DMRT1 represents one of the few sex determining genes that is shared between different species and phyla. Orthologous genes are found in a medaka species Dmy on the Y chromosome Lutfalla et al.

Both, Dmy in medaka and Dmrt1 in chicken have been shown to be testis-determining genes Matsuda et al. Interestingly, in platypus, a single copy of Dmrt1 is located on chromosome X5 Grutzner et al. In chicken and medaka, these genes are believed to initiate the molecular network leading to testis development Smith et al.

Matson et al. Loss of Dmrt1 in mouse Sertoli cells leads to the upregulation of Foxl2 , amongst other genes. This induces the reprogramming of Sertoli cells into granulosa cells. Subsequently theca cells form, oestrogen is produced and germ cells appear feminized. Dmrt1 -null mice develop normal gonads but show severely impaired testis development from postnatal day 2 resulting in dysgenic testes Kim et al.

However, in humans, there have been no mutations identified within the DMRT1 gene itself, jet. FGF9 encodes fibroblast growth factor 9 FGF9 , one of a number of growth factors that play a role in various developmental processes such as cell proliferation, cell survival, cell migration, and cell differentiation.

Fgf9 is expressed in the bi-potential gonad immediately after the expression of Sry. Mice null for Fgf9 show male-to-female sex reversal along with impaired development of Sertoli cells. However, this is only evident on some genetic backgrounds, but not on others Colvin et al. In the absence of Fgf9 , Sox9 expression is not maintained, Sertoli cells fail to differentiate, testis development is aborted and the resulting somatic cells express genes characteristic for ovarian development and the female pathway.

FGFR2 is integrated in the plasma membrane of progenitor Sertoli cells and is critical for Sertoli cell proliferation and differentiation in the developing testis. In a linkage study of a large family with DSD, a putative testis determining gene was mapped to a region on the long arm of chromosome 5 Jawaheer et al.

A follow-up study identified a mutation in the gene MAP3K1 , which segregated with the phenotype in the family. Further mutations within the MAP3K1 gene were identified within a second family and two out of eleven sporadic cases analyzed.

Map3k1 is expressed in the mouse gonad prior to, and following, sex determination but Map3k1 null mice do not show a gonadal phenotype. The spatiotemporal expression profile of Map3k1 , together with the identification of mutations within the MAP3K1 gene in cases of human 46,XY DSD strongly implicates the mitogen-activated protein kinase MAPK pathway in normal human and mouse sex determination Pearlman et al.

While many genes have been identified as part of the testis developmental pathways, until recently little was known about the molecular mechanisms underlying ovarian development. WNT4 encodes member 4 of the wingless-type MMTV integration site family, a family of locally acting signaling molecules that are known to play a role in a range of developmental processes.

Wnt4 is expressed in the mesonephric mesenchyme and coelomic epithelium in mouse as early as E9. At E11, it is expressed in the mesonephros and the bi-potential gonad of both sexes but is then downregulated in the developing testis at around E WNT4 plays a role in Mullerian duct formation, oocyte development, and sex-specific patterning of the vasculature Heikkila et al.

Lack of Wnt4 in the mouse results in partial female-to-male sex reversal, suggesting that WNT4 acts to positively regulate ovary differentiation. The gonads of XX embryos appear to be masculinized, round shaped, unencapsulated, and associated with a fat body but do not form testis cords or express Sertoli cell markers. Ectopic over-expression of Wnt4 in mice resulted in disruption of testis-specific vasculature the coelomic vessel still forms, but the structure and branching seem to be abnormal and inhibition of testosterone synthesis in XY embryos Jordan et al.

As the coelomic vessel forms in XX embryos with WNT4 over-expression, it suggests either, that WNT4 is not the only factor required to suppress male-specific vasculature or that the testes express another factor that is able to compensate for WNT4-mediated repression. Yao et al. These results suggest that WNT4 acts upstream of and directly or indirectly activates Bmp2 and Fst in the developing ovary Yao et al.

However, no mechanism has been proposed to explain how this interaction occurs Kim et al. Human patients with duplications of chromosome 1p, including WNT4 , show a broad range of gonadal anomalies including complete sex reversal Jordan et al. The Wnt morphogens are known to signal through binding to integral membrane-bound co-receptors LRP5 and LRP6 low-density lipoprotein receptor-related protein 5 and 6.

These co-receptors heterodimerize with the integral membrane receptor Frizzled and thus transfer the signal into the cytoplasm of the cell.

This results in the transcriptional activation of downstream target genes. Its role in ovarian development was first implicated when a homozygous single nucleotide insertion within the RSPO1 coding sequence was identified in four 46,XX testicular DSD patients from a consanguineous family. In addition, a homozygous exonic deletion was identified in an unrelated sporadic case of 46,XX testicular DSD Parma et al.

This study was the first to show that inactivation of a single gene can cause female-to-male sex reversal. The conserved expression pattern of RSPO1 in other vertebrates suggests that it is a critical gene in ovarian development. XX Rspo1 knockout mice have masculinised gonads, but do not show complete sex reversal Tomizuka et al. Chassot et al.

In addition, BPES patients have been shown to carry a 7. Bmp2 expression remains significantly reduced in all three mouse strains. Surprisingly, conditional knockout of Foxl2 in the adult mouse ovary results in trans -differentiation into testis Uhlenhaut et al. The ovarian structures become more testis-like, with tubular features and Sertoli-like cells.

Gene expression was investigated using in situ hybridization and showed that some male-specific genes were up-regulated, including Sox9. This study may be clinically relevant in humans, as somatic mutations in FOXL2 could be the underlying cause of ovarian dysfunction in adult females.

We are currently still unable to determine the causative mutation in a relatively high percentage of DSD patients. This together with the recent discovery of several new genes in patients with previously unexplained DSD lend support to the idea that both, the genes involved in gonad development, as well as the etiology of DSD, are still incompletely understood. Over the years, several strategies were used to identify novel genes involved in gonad development.

One approach was the analysis of differentially expressed genes using microarrays. Several studies performed these types of analyses using mouse Beverdam and Koopman and human Houmard et al. While the lists of genes that show sexually dimorphic expression patterns identified many of the known gonad genes although rarely SRY this approach has not lead to the discovery of any novel genes involved in sex determination and the etiology of DSD. As previously mentioned, CNV arrays have been used over the last couple of years to screen for and identify copy number variations in patients affected by DSD.

Throughout this review, we mentioned a number of cases where this approach lead to the identification of the causative mutation in DSD patients. Nevertheless, there are still various genomic rearrangements associated with human DSD phenotypes that have been reported but where no causative gene could be identified. Several deletions of terminal 10q have been associated with 46,XY DSD gonadal dysgenesis together with other somatic anomalies. Deletions and duplications of chromosome 22q The causative gene s haven't been identified, but this region contains the MAPK1 gene, which appears to play an important role in proper testis development.

Other CGH arrays revealed a novel recurrent 15q24 microdeletion syndrome with the deletions being between 1. The syndrome is characterized, amongst other features, by genital anomalies in male, including micropenis and hypospadias Sharp et al. By far, the most successful approach to the identification and functional analysis of gonad genes used a combination of human DSD patient studies and mouse models.

The recently published example of SOX3 highlights this once more. This process occurred as the mammalian sex chromosomes evolved and differentiated.

SOX3 is not expressed in the developing gonads in either sex, and loss-of-function mutations in SOX3 do not affect sex determination in humans or mice. However, a transgenic mouse line over-expressing Sox3 showed ectopic expression of this gene in the bi-potential gonad. XX embryos showed female-to-male sex reversal, and further analyses suggest that SOX3 induced testis differentiation by up-regulating Sox9 expression in these animals through a similar mechanism to SRY Sutton et al.

It was hypothesized that these rearrangements caused ectopic expression of SOX3 in the embryonic gonad, where it was able to substitute for the absence of SRY and drive testis development.

Since the discovery of SRY in , we have significantly increased our knowledge of genes and gene networks involved in gonad development and the etiology of DSD.

Nevertheless, still unexplained DSD cases, as well as gaps in the network of testis and ovary development show us that there is still much to understand.

These two arrays-based methods allow the detection of rearrangements in putative regulatory elements, which, if mutated, may cause DSD. The use of microarrays to detect CNVs has had some success in identifying causative mutations in unexplained cases of DSD. However, this technique is limited to the detection of large rearrangements and may miss smaller genomic rearrangements or point mutations. Familial cases of both 46,XY DSD and 46,XX DSD often show a broad range of phenotypes in affected individuals within the same family, including instances, where the underlying genetic mutation has been identified Temel et al.

This phenotypic variability may be explained by additional mutations in other genes up and downstream of the target gene, which may interact or influence its activity. This has been shown in a familial case of hypogonadotropic hypogonadism, where a compound heterozygous GNRHR gonadotropin-releasing hormone receptor and a heterozygous FGFR1 mutation were identified Pitteloud et al.

In addition, almost all cases of non-syndromic ovotesticular DSD remain unexplained although there have been considerable genetic analyses of candidate genes Temel et al.

The analyses of presumably multifactoral DSD phenotypes, such as hypospadias or cryptorchidism, have rarely identified the genetic cause, although epidemiological studies give clear evidence of a major genetic component in these conditions Fukami et al. Next generation sequencing NGS or massively parallel sequencing approaches MPS , such as whole exome, single chromosome, or targeted sequencing of sets of candidate genes together referred to as targeted sequencing from this point , as well as whole genome sequencing, should provide substantially higher resolution for mutation detection and should assist in resolving unexplained cases of DSD.

Whole genome sequencing, which combines the advantages of all other methods, is a powerful tool to detect the range of genomic mutations, including SNPs and all size ranges of genomic re-arrangements and CNVs, in coding as well as non-coding and putative regulatory regions, in a non-biased manner. Currently, this method is still costly and requires immense bioinformatic analysis.

However, as the cost of whole genome sequencing continues to decrease and as bioinformatic analysis improves, this will eventually become the method of choice for many researchers. In the interim, targeted sequencing approaches and whole exome sequencing offer a great opportunity to gain new insights into key genes required for gonad development with a lower level of bioinformatic complexity.

Despite the differences between human and mouse and the improvements in the techniques and bioinformatics, comparative genomics will still play a key role. Given the increasing resolution of the new techniques and the increasing power of bioinformatics, we are likely to identify an increasing number of new genes, which are mutated or rearranged in DSD patients. Comparative genetics and mouse models will be essential for establishing the biological function of mutations that do not lie within known DSD genes and crucial in verifying novel genes with a role in gonad development.

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited. National Center for Biotechnology Information , U. Chromosome Research. Chromosome Res. Published online Jan Stefanie Eggers and Andrew Sinclair.

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Abstract Disorders of sex development DSD are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. Keywords: Disorders of sex development, sex determination, gonad development, gonad dysfunction.

Introduction In mammals, biological differences between males and females are determined genetically during embryonic development. Disorders of sex development DSD are congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical Hughes et al. Gonad development in humans and mice and the molecular basis of DSD In humans, both males and females have 22 pairs of autosomal chromosomes mice have 19 pairs of autosomal chromosomes but differ in their sex chromosome complement.

Open in a separate window. Genes required for the development of the bi-potential gonad Mammalian gonads arise in both sexes from a bilateral, bi-potential gonad also called genital ridges , an organ that has the potential to develop either as an ovary or as a testis, depending on differentially expressed genes reviewed by Capel ; Swain and Lovell-Badge ; Capel Empty spiracles homeobox 2 Empty spiracles homeobox 2 Emx2 is a homolog of the Drosophila head gap gene empty spiracles ems and is essential for the development of dorsal telencephalon in mice Yoshida et al.

Nuclear receptor subfamily 5 group A member 1 NR5A1 encodes the nuclear receptor family member nuclear receptor subfamily 5 group A member 1, also known as steroidogenic factor-1 SF1 , which plays a role in gonadal and adrenal development Val and Swain Wilms' tumor 1 Wilms' tumor 1 WT1 encodes a zinc finger transcription factor, which is primarily expressed in embryonic mesodermal tissues such as the urogenital ridge, gonads, and mesonephros Armstrong et al.

Sex-determining region Y Testis development is characterized by the expression of SRY , the testis-determining gene on the Y chromosome, in the supporting cell lineage of the genital ridge Bullejos and Koopman ; Wilhelm et al.

Nuclear receptor subfamily 0 group B, member 1 The dosage sensitive gene nuclear receptor subfamily 0 group B, member 1 NR0B1 dosage sensitive sex reversal, adrenal hypoplasia critical region, X chromosome gene 1 , encodes DAX1 on chromosome Xp Doublesex and mab-3 related, transcription factor 1 Doublesex and mab-3 related, transcription factor 1 DMRT1 is located at the tip of chromosome 9p in human and encodes the transcription factor DMRT1 doublesex and mab-3 related transcription factor 1.

Fibroblast growth factor 9 FGF9 encodes fibroblast growth factor 9 FGF9 , one of a number of growth factors that play a role in various developmental processes such as cell proliferation, cell survival, cell migration, and cell differentiation. Genes required for ovary development While many genes have been identified as part of the testis developmental pathways, until recently little was known about the molecular mechanisms underlying ovarian development.

Wingless-type MMTV integration site family, member 4 WNT4 encodes member 4 of the wingless-type MMTV integration site family, a family of locally acting signaling molecules that are known to play a role in a range of developmental processes.

Identifying novel genes in the sex determining pathway We are currently still unable to determine the causative mutation in a relatively high percentage of DSD patients.

Gene expression profiling, using microarrays Over the years, several strategies were used to identify novel genes involved in gonad development. Conclusion and future directions of DSD research Since the discovery of SRY in , we have significantly increased our knowledge of genes and gene networks involved in gonad development and the etiology of DSD. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited.

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Homozygous inactivation of Sox9 causes complete XY sex reversal in mice. Biol Reprod. Testis cord differentiation after the sex determination stage is independent of Sox9 but fails in the combined absence of Sox9 and Sox8. Dev Biol. New technologies for the identification of novel genetic markers of disorders of sex development DSD Sex Dev. Disruption of a long distance regulatory region upstream of SOX9 in isolated disorders of sex development. J Med Genet.

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Curr Biol. PLoS Biol. Correct dosage of Fog2 and Gata4 transcription factors is critical for fetal testis development in mice. Sry requires a CAG repeat domain for male sex determination in Mus musculus. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators.

Spatially dynamic expression of Sry in mouse genital ridges. Dev Dyn. Identification of a putative steroidogenic factor-1 response element in the DAX-1 promoter. Biochem Biophys Res Commun. A new submicroscopic deletion that refines the 9p region for sex reversal.

Hum Mutat. A heterozygous mutation in the desert hedgehog gene in patients with mixed gonadal dysgenesis. Mol Hum Reprod. Ann Genet. Sex in the 90s: SRY and the switch to the male pathway. Annu Rev Physiol. The battle of the sexes. Functional analysis of Sox8 and Sox9 during sex determination in the mouse. Genetics of ovarian differentiation: Rspo1, a major player. Sex Dev. Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Desert hedgehog Dhh gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules.

Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Presumptive pre-Sertoli cells express genes involved in cell proliferation and cell signalling during a critical window in early testis differentiation. Mol Reprod Dev. Genes Dev. An unbalanced autosomal translocation 7;9 associated with feminization.

Clin Genet. Disease-causing 7. PLoS Genet. Novel homozygous mutations in desert hedgehog gene in patients with 46, XY complete gonadal dysgenesis and prediction of its structural and functional implications by computational methods. Eur J Med Genet. Steroidogenic factor-1 contributes to the cyclic-adenosine monophosphate down-regulation of human SRY gene expression.

Does chromosome 22 have anything to do with sex determination: further studies on a 46, XX,22q The erythroid-specific transcription factor Eryf1: a new finger protein. Disruption of friend of GATA 2 gene FOG-2 by a de novo t 8;10 chromosomal translocation is associated with heart defects and gonadal dysgenesis.

Evolution of sex determination and the Y chromosome: SRY-related sequences in marsupials. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. CXorf6 is a causative gene for hypospadias. The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures.

Long-range regulation at the SOX9 locus in development and disease. From brain determination to testis determination: evolution of the mammalian sex-determining gene. Reprod Fertil Dev. SOX3 is a member of this gene family; its locus is located in the X chromosome Stevanovic et al. There is evidence supporting the origin of the SRY gene from a primitive SOX gene present in the ancestral evolutionary line before the separation of Metatheria and Eutheria.

The presence of the SOX3 gene in the X chromosome of all mammals tested and the high degree of homology between the HMG box of the SRY genes and the chromosome suggests parallel evolution for these genes from an ancestral autosomal pair that gave rise to the sex chromosomes Stevanovic et al. On the other hand, the SOX9 gene is involved in sex determination Foster et al. The SOX9 gene is highly conserved among fish, birds and mammals and its expression is specific to the male gonads of birds and mammals, for example This is why it is considered to be a gene which is involved in the formation of male gonads and may be regulated by the SRY gene Morais et al.

A finding related to the importance of SOX9 in sex determination has been a case of sex reversal in humans XX resulting from this gene's duplication on chromosome 17 which led to the development of testes Huang et al. This fact corroborates SOX9's potential if expression threshold-dependent to become a candidate gene for allowing the development of masculinity in the absence of SRY, mimicking the latter's function.

When comparing the HMG box of distantly related species, the high identity between them becomes evident Coriat et al. Sex identification is of great importance in evolutionary biology, ecology, prenatal diagnosis, forensic identification, population genetics, conservation and planning studies concerning the reproduction of threatened or clearly endangered species Fridolfsson and Ellegren, ; Griffiths and Phil, ; Drobnic, ; Matta et al.

Sexing using molecular tools has been particularly important for testing and controlling poaching in which the capture and killing rate should be differential between males and females in bird breeding programs where sexual dimorphism is not obvious.

It also represents an alternative to sexing methodologies such as laparotomy or laparoscopy Fridolfsson and Ellegren, ; Griffiths and Phil, ; Matta et al.

There is an increasing interest in the use of non-invasive sampling for the study of wild animal populations to avoid disturbance of the animals and to get results with indirect and easy sampling, such as skin, hair, feathers, nails, feces, or simple drops of blood.

Identification of the individual's sex is important for conservation studies of wild animals. However in some species, limitations regarding sex determination using molecular tools are associated with the problem of finding the specific sequences in sex chromosomes that differentiate between males and females and can be demonstrated by methodologies such as PCR amplification or differential cleavage with restriction enzymes.

The gene does not necessarily have to involve pathways leading to sexual differentiation. Other genes or even non-encoding sequences may be responsible for other functions, although they must have differences between the sex chromosomes that are not in autosomes.

Several molecular methods for sexing can easily be adapted for a wide range of species. However, in some species some sexing methods encounter difficulties in the amplification of long fragments from degraded DNA and in non-specific amplification from sample analysis.

However, PCR products of considerable length are used, which is not suitable for degraded DNA such as faeces analysis. Hence the alternative approach consisted in the amplification of short Y-specific genes using conserved primers, as for example the SRY Gupta et al. However, the use of Y-chromosome specific sequences alone is not informative because an absence of PCR product can indicate either a female or a failed reaction. Hence, these reactions are often performed in conjunction with another fragment to act as a positive control for amplification, such as a mitochondrial DNA or a nuclear locus, being preferable the nuclear one as mitochondrial DNA are present in more copy number than nuclear DNA and hence than the amplified Y chromosome specific markers.

Some limitations and considerations should be taken into account in molecular genetic analysis when the genome of the species of interest is scarcely studied and we are searching for target genes: 1 finding species-related genes even if they are orthologous does not necessarily involve a similar task, considering that the biological contexts of expression can be very different; 2 the genes of interest could not be located on the same chromosomes; 3 the gene sequences could not be conserved enough to permit to amplify with interspecific primers or cut with the same restriction endonucleases; 4 regarding the function itself, there is still a lot of uncertainty in terms of metabolic pathway flexibility when induced mutations might not be apparent in the phenotype as a result of possible substitutions by other proteins.

These guidelines should be kept in mind when exploring little-studied species using databases and to avoid the pitfall of biological reductionism Marin and Baker, Studying sex determination is of wide interest due to the great variability of systems present even among species from the same gender, contrary to what has previously been thought, i.

DNA-based methodologies for identifying sex are valuable tools due to their speed, sensitivity and the possibility of easy sampling of meat, skin, feathers, nails, or simple drops of blood, thereby minimizing pain and stress during sampling.

Moreover, they can be performed on very small tissue samples obtained from hunting-control programs. Correct sex determination is important when studying a population's behavior and social structure and for conservation plans where decisions must be taken by adopting an interdisciplinary approach. Sequence analysis and mapping of the SRY gene in species of the subfamily Arvicolinae rodentia.

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Hum Genet. Deletion of Y chromosome sequences located outside the testis determining region can cause XY female sex reversal.

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A sex chromosome anomaly in a case of a gonadal dysgenesis Turner's syndrome. Evolution of sex determination and the Y chromosome: SRY related sequences in marsupials.

An SRY-related sequence on the marsupial X chromosome: implications for the evolution of the mammalian testis determining gene. The differences between the sexes. R V short and E, Balaban editors. Identification of a second pseudoautosomal region near the Xq and Yq telomeres. Evolution of the avian sex chromosomes from an ancestral pair of autosomes. A simple and universal method for molecular sexing of non-ratite birds. J Avian Biol. SRY and sex determination [summary].

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A gene mapping to the sex determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Inverted repeat structure of the SRY locus in mice.

A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin. J Forensic Sci. Sex determination compared in Drosophila and Caenorhabditis. Am J Med Genet. Environmental sex determination in reptiles: ecology, evolution, and experimental design. Quart Rev Biol. The molecular biology of temperaturedependent sex determination. Problems of fetal endocrinology; the gonadal and hypophyseal hormones. Rec prog. The sex determination in Ellobius lutescens remains bizarre.

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Eur J Biochem. Role of the pseudoautosomal region in sex-chromosome pairing during male meiosis: meiotic studies in a man with a deletion of distal Xp. Am J Hum Genet. Sox9 expression during gonadal development implies a conserved role for the gene in the testis differentiation in mammals and birds. Nat Genet. Sry evolution in Cebidae Platyrrhini: Primates. J Mol Evol.

The testis determining gene, SRY, exists in multiple copies in old world rodents. Genet Res. HMGs everywhere. Sex Chromosomes and Sex-Linked Genes. Springer-Verlag; PAGE D. Sex reversal: deletion mapping the male-determining function of the human Y chromosome. In: Cold Spring Harbor symposium on quantitative biology, editors. New York, Molecular biology of Homo sapiens; ; Additional deletion in sexdetermining region of human Y chromosome resolves paradox of X1t Y,22 female.

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Sox genes find their feet.