Jocelyn A. van den Bergen

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 p27Kip1, p15INK4b, and p16INK4a; 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.

Copy Number Variation in Patients with Disorders of Sex Development Due to 46,XY Gonadal Dysgenesis

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases.

Dppa2 and Dppa4 Are Closely Linked SAP Motif Genes Restricted to Pluripotent Cells and the Germ Line

Despite the enormous medical potential of ESCs, the molecular mechanisms conferring the ability to differentiate into all cell types of the embryo remain elusive. We used an in silico approach to identify genes expressed exclusively in mouse preimplantation embryos and pluripotent cell lines. Two of these genes were developmental pluripotency‐associated gene 2 (Dppa2) and Dppa4, which we show are closely linked genes encoding putative nuclear SAP domain proteins expressed in human and mouse pluripotent stem cells and germ cell tumor‐derived embryonal carcinoma cells. In the mouse, these genes are transcribed in germinal vesicle‐stage oocytes and throughout the cleavage stages of embryogenesis. They then become restricted to the pluripotent inner cell mass of blastocysts and are subsequently downregulated. After gastrulation, Dppa2 and Dppa4 are expressed only in the developing germ line, showing that these genes mark cells of the pluripotent cycle. In the germ line, both genes are downregulated as the germ cells commit to the oogenic pathway or soon after commitment to the spermatogenic pathway. We have observed similar germ line expression profiles for other pluripotent markers, and these results are consistent with the hypothesis that pluripotent markers must be downregulated during fetal germ line development, a process that may be required to facilitate appropriate germ line differentiation. The study of expression and function of pluripotent markers such as Dppa2 and Dppa4 is likely to unveil new aspects of the regulation of pluripotency and germ line development in mammals.

Analysis of Esg1 expression in pluripotent cells and the germline reveals similarities with Oct4 and Sox2 and differences between human pluripotent cell lines.

Establishment of pluripotent epiblast cells is a critical event during early mammalian development because all somatic lineages and the primordial germ cells (PGCs) are derived from them. The epiblast and PGCs are in turn the precursors of pluripotent embryonic stem cells and embryonic germ cells, respectively. Although PGCs are specialized cells, they express several key pluripotency‐related genes, such as Oct4 and Sox2. We have analyzed Esg1 expression in mouse and human cells and shown that in the mouse the gene is specifically expressed in preimplantation embryos, stem cells, and the germline. Moreover, Esg1 coexpresses with Oct4 and Sox2, confirming its identity as a marker of the pluripotent cycle. Esg1 is also expressed with Oct4 and Sox2 by human embryonic stem cells and in germ cell carcinoma tissue but not by all human embryonal carcinoma cell lines. These data suggest that together with Oct4 and Sox2, Esg1 plays a conserved role in the pluripotent pathway of mouse and human stem and germ cells.

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.—Western, P. S., van den Bergen, J. A., Miles, D. C., Sinclair, A. H. Male fetal germ cell differentiation involves complex repression of the regulatory network controlling pluripotency. FASEB J. 24, 3026–3035 (2010). www.fasebj.org

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 Tgfβ family members has indicated that Tgfβs 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 Activins, TGFβs, 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 TGFβ 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/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development.

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

BackgroundDisorders 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.ResultsWe 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.ConclusionsOur 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.

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 G2/M cell cycle proteins revealed that entry into meiosis and cell cycle exit at G2/M involves repression of G2/M promoting Cyclin B1, coincident upregulation of G2/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 G2/M surveillance mechanism operates during germ cell proliferation and that passage into meiotic G2/M involves the combined repression of G2/M through Cyclin B3 and activation of the key G2/M checkpoint regulatory network modulated through ATM and CHK2. This work shows that the core regulatory machinery that controls G2/M progression in mitotic cells is activated in female mouse germ cells as they enter meiosis.

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 p27KIP1, p15INK4B, 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.

Normalizing gene expression levels in mouse fetal germ cells.

Abstract 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.