Generation of heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout human iPSC lines.

The transcription factor SOX9 plays a critical role in several embryonic developmental processes such as gonadogenesis, chrondrogenesis, and cardiac development. We generated heterozygous (MCRIi031-A-1) and homozygous (MCRIi031-A-2) SOX9 knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers. These cell lines will allow us to further explore the role of SOX9 in critical developmental processes.

Generation of heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2/COUP-TFII knockout human iPSC lines.

The NR2F2 gene encodes the transcription factor COUP-TFII, which is upregulated in embryonic mesoderm. Heterozygous variants in NR2F2 cause a spectrum of congenital anomalies including cardiac and gonadal phenotypes. We generated heterozygous (MCRIi030-A-1) and homozygous (MCRIi030-A-2) NR2F2-knockout induced pluripotent stem cell (iPSC) lines from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. Both iPSC lines exhibited a normal karyotype, typical pluripotent cell morphology, pluripotency marker expression, and the capacity to differentiate into the three embryonic germ layers. These lines will allow us to explore the role of NR2F2 during development and disease.

Analysis of variants in GATA4 and FOG2/ZFPM2 demonstrates benign contribution to 46,XY disorders of sex development.

BACKGROUND: GATA-binding protein 4 (GATA4) and Friend of GATA 2 protein (FOG2, also known as ZFPM2) form a heterodimer complex that has been shown to influence transcription of genes in a number of developmental systems. Recent evidence has also shown these genes play a role in gonadal sexual differentiation in humans. Previously we identified four variants in GATA4 and an unexpectedly large number of variants in ZFPM2 in a cohort of individuals with 46,XY Differences/Disorders of Sex Development (DSD) (Eggers et al, Genome Biology, 2016; 17: 243). METHOD: Here, we review variant curation and test the functional activity of GATA4 and ZFPM2 variants. We assess variant transcriptional activity on gonadal specific promoters (Sox9 and AMH) and variant protein-protein interactions. RESULTS: Our findings support that the majority of GATA4 and ZFPM2 variants we identified are benign in their contribution to 46,XY DSD. Indeed, only one variant, in the conserved N-terminal zinc finger of GATA4, was considered pathogenic, with functional analysis confirming differences in its ability to regulate Sox9 and AMH and in protein interaction with ZFPM2. CONCLUSIONS: Our study helps define the genetic factors contributing to 46,XY DSD and suggests that the majority of variants we identified in GATA4 and ZFPM2/FOG2 are not causative.

NR5A1 gene variants repress the ovarian-specific WNT signaling pathway in 46,XX disorders of sex development patients.

Several recent reports have described a missense variant in the gene NR5A1 (c.274C>T; p.Arg92Trp) in a significant number of 46,XX ovotesticular or testicular disorders of sex development (DSDs) cases. The affected residue falls within the DNA-binding domain of the NR5A1 protein, however the exact mechanism by which it causes testicular development in 46,XX individuals remains unclear. We have screened a cohort of 26 patients with 46,XX (ovo)testicular DSD and identified three unrelated individuals with this NR5A1 variant (p.Arg92Trp), as well as one patient with a novel NR5A1 variant (c.779C>T; p.Ala260Val). We examined the functional effect of these changes, finding that while protein levels and localization were unaffected, variant NR5A1 proteins repress the WNT signaling pathway and have less ability to upregulate the anti-testis gene NR0B1. These findings highlight how NR5A1 variants impact ovarian differentiation across multiple pathways, resulting in a switch from ovarian to testis development in genetic females.

A novel, homozygous mutation in desert hedgehog (DHH) in a 46, XY patient with dysgenetic testes presenting with primary amenorrhoea: a case report.

BACKGROUND: Desert hedgehog (DHH) mutations have been described in only a limited number of individuals with 46, XY disorders of sex development (DSD) presenting as either partial or complete gonadal dysgenesis. Gonadal tumours and peripheral neuropathy have been associated with DHH mutations. Herein we report a novel, homozygous mutation of DHH identified through a targeted, massively parallel sequencing (MPS) DSD panel, in a patient presenting with partial gonadal dysgenesis. This novel mutation is two amino acids away from a previously described mutation in a patient who presented with complete gonadal dysgenesis. Adding to the complexity of work-up, our patient also expressed gender identity concern. CASE PRESENTATION: A 14-year-old, phenotypic female presented with primary amenorrhoea and absent secondary sex characteristics. Investigations revealed elevated gonadotrophins with low oestradiol, testosterone of 0.6 nmol/L and a 46, XY karyotype. Müllerian structures were not seen on pelvic ultrasound or laparoscopically and gonadal biopsies demonstrated dysgenetic testes without neoplasia (partial gonadal dysgenesis). The patient expressed gender identity confusion upon initial notification of investigation findings. Formal psychiatric evaluation excluded gender dysphoria. Genetic analysis was performed using a targeted, MPS DSD panel of 64 diagnostic and 927 research candidate genes. This identified a novel, homozygous mutation in exon 2 of DHH (DHH:NM_021044:exon2:c.G491C:p.R164P). With this finding our patient was screened for the possibility of peripheral neuropathy which was not evident clinically nor on investigation. She was commenced on oestrogen for pubertal induction. CONCLUSION: The evaluation of patients with DSD is associated with considerable psychological distress. Targeted MPS enables an affordable and efficient method for diagnosis of 46, XY DSD cases. Identifying a genetic diagnosis may inform clinical management and in this case directed screening for peripheral neuropathy. In addition to the structural location of the mutation other interacting factors may influence phenotypic expression in homozygous DHH mutations.

Functional characterization of novel NR5A1 variants reveals multiple complex roles in disorders of sex development.

Variants in the NR5A1 gene encoding SF1 have been described in a diverse spectrum of disorders of sex development (DSD). Recently, we reported the use of a targeted gene panel for DSD where we identified 15 individuals with a variant in NR5A1, nine of which are novel. Here, we examine the functional effect of these changes in relation to the patient phenotype. All novel variants tested had reduced trans-activational activity, while several had altered protein level, localization, or conformation. In addition, we found evidence of new roles for SF1 protein domains including a region within the ligand binding domain that appears to contribute to SF1 regulation of Müllerian development. There was little correlation between the severity of the phenotype and the nature of the NR5A1 variant. We report two familial cases of NR5A1 deficiency with evidence of variable expressivity; we also report on individuals with oligogenic inheritance. Finally, we found that the nature of the NR5A1 variant does not inform patient outcomes (including pubertal androgenization and malignancy risk). This study adds nine novel pathogenic NR5A1 variants to the pool of diagnostic variants. It highlights a greater need for understanding the complexity of SF1 function and the additional factors that contribute.

Variants in congenital hypogonadotrophic hypogonadism genes identified in an Indonesian cohort of 46,XY under-virilised boys.

BACKGROUND: Congenital hypogonadotrophic hypogonadism (CHH) and Kallmann syndrome (KS) are caused by disruption to the hypothalamic-pituitary-gonadal (H-P-G) axis. In particular, reduced production, secretion or action of gonadotrophin-releasing hormone (GnRH) is often responsible. Various genes, many of which play a role in the development and function of the GnRH neurons, have been implicated in these disorders. Clinically, CHH and KS are heterogeneous; however, in 46,XY patients, they can be characterised by under-virilisation phenotypes such as cryptorchidism and micropenis or delayed puberty. In rare cases, hypospadias may also be present. RESULTS: Here, we describe genetic mutational analysis of CHH genes in Indonesian 46,XY disorder of sex development patients with under-virilisation. We present 11 male patients with varying degrees of under-virilisation who have rare variants in known CHH genes. Interestingly, many of these patients had hypospadias. CONCLUSIONS: We postulate that variants in CHH genes, in particular PROKR2, PROK2, WDR11 and FGFR1 with CHD7, may contribute to under-virilisation phenotypes including hypospadias in Indonesia.

Disorders of sex development: insights from targeted gene sequencing of a large international patient cohort.

BACKGROUND: Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. RESULTS: We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. CONCLUSIONS: Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Signaling through the TGF beta-activin receptors ALK4/5/7 regulates testis formation and male germ cell development.

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfß family members has indicated that Tgfß's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFß's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFß signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFß receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFß and Activin signaling during testis formation and male germ cell development.

The proto-oncogene Ret is required for male foetal germ cell survival.

The spermatogenic and oogenic lineages originate from bipotential primordial germ cells in response to signalling in the foetal testis or ovary, respectively. The signals required for male germ cell commitment and their entry into mitotic arrest remain largely unknown. Recent data show that the ligand GDNF is up regulated in the foetal testis indicating that it may be involved in male germ cell development. In this study genetic analysis of GDNF-RET signalling shows that RET is required for germ cell survival. Affected germ cells in Ret-/- mice lose expression of key germ cell markers, abnormally express cell cycle markers and undergo apoptosis. Surprisingly, a similar phenotype was not detected in Gdnf-/- mice indicating that either redundancy with a Gdnf related gene might compensate for its loss, or that RET operates in a GDNF independent manner in mouse foetal germ cells. Either way, this study identifies the proto-oncogene RET as a novel component of the foetal male germ cell development pathway.

Mitotic arrest in teratoma susceptible fetal male germ cells.

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27(KIP1), p15(INK4B), activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility.

Male fetal germ cell differentiation involves complex repression of the regulatory network controlling pluripotency.

Mammalian germ cells are derived from the pluripotent epiblast and share features with pluripotent stem cells, including the expression of key genes that regulate developmental potency. The core genes Oct4, Sox2, and Nanog that regulate pluripotency in stem cells also perform important roles in regulating germ cell development and potentially in occurrence of germ line tumors in humans. Despite this, our understanding of the regulation of these genes during germ cell development remains limited. In this study we examine the regulation of pluripotency in the mouse fetal germ line. We show that male-specific methylation occurs in key functional elements of the Nanog and Sox2 promoters, and these genes are suppressed during early male germ cell differentiation. Furthermore, Oct4 translation is suppressed post-transcriptionally as germ cells differentiate down the male lineage and enter mitotic arrest. Combined, our data strongly support the conclusion that repression of the core machinery regulating pluripotency is a robust and early event involved in the differentiation of the male germ cell lineage. We hypothesize that active repression of pluripotency is required for fetal male germ cell differentiation and that failure of this mechanism may render germ cells susceptible to tumor formation.

Regulation of the female mouse germ cell cycle during entry into meiosis.

During mouse embryonic development germ cells proliferate extensively until they commit to the male or female pathway and arrest in mitosis or meiosis respectively. Whilst the transition of female germ cells exiting the mitotic cell cycle and entering meiosis is well defined histologically, the essential cell cycle proteins involved in this process have remained unresolved. Using flow cytometry we have examined the entry of female germ cells into meiosis, their termination of DNA synthesis and entry into prophase I. Analysis of key G(2)/M cell cycle proteins revealed that entry into meiosis and cell cycle exit at G(2)/M involves repression of G(2)/M promoting Cyclin B1, coincident upregulation of G(2)/M repressing Cyclin B3 and robust establishment of the ATM/CHK2 pathway. By contrast we show that the ATR/CHK1 pathway is activated in male and female germ cells. This data indicates that an important G(2)/M surveillance mechanism operates during germ cell proliferation and that passage into meiotic G(2)/M involves the combined repression of G(2)/M through Cyclin B3 and activation of the key G(2)/M checkpoint regulatory network modulated through ATM and CHK2. This work shows that the core regulatory machinery that controls G(2)/M progression in mitotic cells is activated in female mouse germ cells as they enter meiosis.

Normalizing gene expression levels in mouse fetal germ cells.

Real-time PCR has become a popular method to analyze transcription of genes that are developmentally regulated during organogenesis of the testes and ovaries. However, the heterogenous cell populations and commitment to strikingly different developmental pathways of the germ and somatic cells in these organs complicate analysis of this process. The selection of suitable reference genes for quantifying gene expression in this system is essential, but to date it has not been sufficiently addressed. To rectify this problem, we have used fluorescence-activated cell sorting to purify germ cells from mouse fetal testes and ovaries and examined 16 common housekeeping genes for their suitability as reference genes. In pure populations of germ cells isolated from Embryonic Day 12.5 (E12.5) to E15.5 male and female gonads, Mapk1 and Sdha were identified as the most stable reference genes. Analysis of the heterogenous fraction of gonadal somatic cells revealed that Canx and Top1 were stable in both sexes, whereas a comparative analysis of germ and somatic cell populations identified Canx and Mapk1 as suitable reference genes through these developmental stages. Application of these reference genes to quantification of gene expression in developing gonads revealed that past assays, which employed nonverified reference genes, have in some cases provided misleading gene expression profiles. This study has identified suitable reference genes to directly compare expression profiles of genes expressed in germ and somatic cells of male and female fetal gonads. Application of these reference genes to expression analysis in fetal germ and somatic cells provides a more accurate system in which to profile gene expression in these tissues.

The rhox homeobox gene family shows sexually dimorphic and dynamic expression during mouse embryonic gonad development.

Reproductive capacity is fundamental to the survival of all species. Consequently, much research has been undertaken to better understand gametogenesis and the interplay between germ cells and the somatic cell lineages of the gonads. In this study, we have analyzed the embryonic expression pattern of the X-linked gene family Reproductive homeobox genes on the X chromosome (Rhox) in mice. Our data show that eight members of the Rhox gene family are developmentally regulated in sexually dimorphic and temporally dynamic patterns in the developing germ cells during early gonadogenesis. These changes coincide with critical stages of differentiation where the germ cells enter either mitotic arrest in the testis or meiotic arrest in the ovary. Finally, we show that Rhox8 (Tox) is the only member of the Rhox gene family that is expressed in the somatic compartment of the embryonic gonads. Our results indicate that the regulation of Rhox gene expression and its potential function during embryogenesis are quite distinct from those previously reported for Rhox gene regulation in postnatal gonads.

Dynamic regulation of mitotic arrest in fetal male germ cells.

During fetal mouse development, germ cells enter the developing gonad at embryonic day (E) 10-11. In response to signaling from the male or female gonad, the germ cells commit either to spermatogenesis at E12.5 and enter mitotic arrest or to oogenesis and enter meiotic arrest at E13.5. It is unclear whether male commitment of the germ line and mitotic arrest are directly associated or whether they are developmentally separate. In addition, the published data describing the timing of mitotic arrest are inconsistent, and the molecular processes underlying the control of the cell cycle during mitotic arrest also remain unknown. Using flow cytometric techniques, 5-bromo-2'-deoxyuridine labeling, and immunofluorescent analysis of cell proliferation, we have determined that germ cells in the embryonic mouse testis arrest in G0 during E12.5 and E14.5. This process is gradual and occurs in an unsynchronized manner. We have also purified germ cells and analyzed molecular changes in male germ cells as they exit the cell cycle. This has allowed us to identify a series of molecular events, including activation of p27(Kip1), p15(INK4b), and p16(INK4a); the dephosphorylation and degradation of retinoblastoma protein; and the suppression of CyclinE, which lead to mitotic arrest. For the first time, the data presented here accurately define the mitotic arrest of male germ cells by directly combining the analysis of cell cycle changes with the examination of functionally defined cell cycle regulators.

Annexin XI co-localises with calcyclin in proliferating cells of the embryonic mouse testis.

Mammalian sex determination relies on the expression of SRY, which triggers a tightly regulated cascade of gene expression leading to male differentiation. Many elements of this pathway remain to be identified. Here, we characterise Annexin XI (Anxa11), a gene whose major site of embryonic expression was within the undifferentiated and differentiating testis. Lower level expression was also observed in both sexes in the Müllerian and Wolffian ducts, the somitic dermamyotome, and the dorsal intermediate zone of the neural tube. Anxa11 transcripts were detected in the indifferent gonad from 10.5 days post coitum (dpc), becoming male specific as development proceeded. Expression was within the testis cords, initially in germ cells, and then in both Sertoli and germ cells. Annexin XI protein was seen in the testis cords from 12.5 dpc, localising to the cytoplasm of the Sertoli cells. Expression of calcyclin (S100a6), shown previously to interact with annexin XI in vitro, was also observed in proliferating cells of the embryonic testis, supporting a possible in vivo interaction.