Project description:Sustained spermatogenesis in adult males and recovery of fertility following germ cell depletion are dependent on undifferentiated spermatogonia with self-renewal potential. We have previously demonstrated a critical role for the transcription factor Spalt-like 4 (SALL4) in spermatogonial differentiation. However, it remains unclear whether SALL4 has broader roles within the spermatogonial pool despite its ability to co-regulate genes with PLZF, a transcription factor required for undifferentiated cell maintenance. To identify genes regulated by SALL4 in the male germline, we established cultures of undifferentiated spermatogonia from a Sall4 inducible knockout mouse model. Cells were treated with vehicle (as control) or tamoxifen to induce gene deletion, then cells harvested and analysed by microarray to identify genes mis-expressed upon loss of SALL4.
Project description:Maintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. In this manuscript we have identified a key role for the nucleosome remodelling protein Chromodomain Helicase DNA binding protein 4 (CHD4) in regulating SSC function. Gene expression analyses revealed that CHD4 expression is largely restricted to spermatogonia in the mouse testis, and is particularly enriched in SSCs. Using spermatogonial transplantation techniques and RNAi mediated knockdown it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, resulting in a ~50% reduction in colonisation of recipient testes. A single cell RNA-seq comparison depicted reduced expression of ‘self-renewal’ genes such as Gfra1 and Pten following Chd4 knockdown, along with increased expression of signature progenitor genes, Neurog3 and Dazl. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only though its traditional association with the remodelling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated fundamental role for CHD4 in controlling fate decisions in the spermatogonial pool.
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.
