Functional characterization of human recessive DIS3 variants in premature ovarian insufficiency†.

Premature ovarian insufficiency (POI) is characterized by the loss or complete absence of ovarian activity in women under the age of 40. Clinical presentation of POI varies with phenotypic severity ranging from premature loss of menses to complete gonadal dysgenesis. POI is genetically heterogeneous with >100 causative gene variants identified thus far.

Generation of a homozygous (MCRIi031-A-3) WT1 knockout human iPSC line.

The transcription factor WT1 plays a critical role in several embryonic developmental processes such as gonadogenesis, nephrogenesis, and cardiac development. We generated a homozygous (MCRIi031-A-3) WT1 knockout induced pluripotent stem cell (iPSC) line from human fibroblasts using a one-step protocol for CRISPR/Cas9 gene-editing and episomal-based reprogramming. The cells exhibit a normal karyotype and morphology, express pluripotency markers, and have the capacity to differentiate into the three embryonic germ layers.

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.

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.

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).

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.

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

Background: () 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 mutations. Herein we report a novel, homozygous mutation of identified through a targeted, massively parallel sequencing (MPS) DSD panel, in a patient presenting with partial gonadal dysgenesis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.