Project description:Undifferentiated spermatogonia were sorted from Miwi2Tom/FL; ERT-Cre/+ (Miwi2-iKO) and Miwi2Tom/+; ERT-Cre/+ (Miwi2-CTL) mice injected with tamoxifen two weeks after last tamoxifen injection to understand an impact of acute Miwi2 deletion on spermatogenic homeostasis.
Project description:Spermatogonial stem cells are responsible for sustaining gametes production and male fertiliy in men. However, germ cells including SSCs are highly sensitive to chemotherapies and radiations, placing male cancer patients at high risk of treatment-induce infertility. We have previously shown that the undifferentiated spermatogonial population of mouse testis is functionally heterogeneous and contain both stem cells and committed progenitor cells. Using mouse model of chemotherapy-induced germline damage and recovery, we aim to define molecular charactersitics, dynamics and heterogeneity of undifferentiated spermatogonia during germline regeneration compared to homeostatic undifferentiated spermatogonia.
Project description:To identify the transcripts preferentially expressed in type A spermatogonia in rainbow trout immature testis, we compared the transcriptome between type A spermatogonia and testicular somatic cells by microarry analysis. We used fluorescence-activated cell sorting (FACS) to isolate type A spermatogonia or testicular somatic cells from the fishes carrying the transgene, pvasa-GFP, which expresses GFP in spermatogonia. RNA from type A spermatogonia and from testicular somatic cells were hybridized to a microarray after Cy3 labeling.
Project description:ID4 belongs to a family of transcriptional regulators involved in stem cell function. ID family proteins act by binding and inhibiting activity of basic-HLH (bHLH) transcription factors, including E-proteins and class II factors. ID function is also mediated through interaction with non-bHLH proteins. Spermatogonial stem cells (SSCs) are found within a population of undifferentiated spermatogonia in the testis and are essential for the sustained production of sperm over the lifespan. It is reported that ID4 is involved in SSC maintenance but the downstream targets of ID4 in SSCs are unclear. To gain insight into the function of ID4 in SSCs we identified interacting partners in cultures of mouse undifferentiated spermatogonia by immunoprecipitation using a specific ID4 antibody and mass spectrometry analysis.
Project description:DDX5, or PLZF co-immunoprecipitation in lysates from cultured undifferentiated spermatogonia followed by identification of eluted proteins using mass spectrometry. IgG control IPs included.
Project description:Microarray analysis was performed in order to detail the gene expression profiles in murine b-2M-SPa-6+c-kit-undifferentiated and b-2M-SPa-6+c-kit+ differentiating spermatogonia. These data were used to compare human and mouse transcriptomes of undifferentiated spermatogonia.
Project description:The environmental exposures and lifestyle of parents can alter the development of offspring. How this environmental information is coded into heritable messages to be transmitted by gametes remains unknown, but epigenetic mechanisms have been implicated. We recently determined that disruption of histone H3 di-methylation at lysine 4 (H3K4me2) in sperm has transgenerational consequences in the development of offspring. However, little is known about when in spermatogenesis histone methylation is established and whether epimutations induced in developing sperm are permanent. We hypothesize that epigenetic modifications to histones established in spermatogonia persist through spermatogenesis and can be transmitted to offspring via the sperm. Our objective was to determine what genomic regions bearing histone H3K4me2 in spermatogonia are also present in sperm. Methods: Using transgenic mice expressing Oct4-GFP, we isolated an enriched spermatogonia population and performed ChIP-seq for H3K4me2. Results: Our analysis revealed that H3K4me2 is located throughout the genome in spermatogonia and particularly at the transcription start site region (TSS) of more than 16,000 genes. Remarkably 44% of H3K4me2 peaks present in spermatogonia are conserved in sperm. The greatest overlap between spermatogonia and sperm occurred at the TSS with 83% similarity. Finally, we assessed the expression level of genes enriched in H3K4me2 in spermatogonia and sperm. We observed that genes with the highest enrichment in H3K4me2 in sperm are expressed at higher levels in spermatogenesis and during development. Conclusion: These findings suggest that if epimutations are induced in spermatogonia they may persist in sperm and influence the health and development of offspring.
