Sex-specific gene expression in embryonic mouse germ cells during commitment to spermatogenesis or oogenesis
Lewis, Luke John Emel
The default developmental choice for both female and male Murine Germ Cells (GCs) is to commit to oogenesis and begin meiosis, which occurs in vivo in the developing ovary, and in vitro, at E12.5-E13.5. Prior to this commitment, female and male GCs are receptive to masculinising signals from the developing male gonad that induce GCs to commit to spermatogenesis at E11.5-E12.5, followed by temporary mitotic arrest. A previous differential expression study identified 2 candidate genes upregulated sex-specifically. Inhibitor of DNA Binding 1 (Id1) mRNA was upregulated in female E13.5 GCs and has not been studied in relation to this developmental paradigm; while Lipocalin-Type Prostaglandin D2-Synthase (Ptgds) mRNA was upregulated in XY E13.5 GC preparations and precious studies show that its high expression by male supporting cells augments masculinisation of cells in the developing male gonad. This thesis aims to firstly confirm sex-specific GC expression of these genes at E13.5, a stage when GCs have committed to spermatogenesis or oogenesis, and secondly, to use this sex-specific expression to shed light upon the molecular events driving GC sexual commitment. Id1 belongs to the Basic Helix-loop-helix (bHLH) family of transcriptional effectors that operate as homo- or heterodimers. Id1 suppresses cellular commitment and exit from the cell cycle partly by binding to and inhibiting the action of pro-differentiation bHLHs. A 40-fold female-specific enrichment of Id1 mRNA and protein in E13.5 XX GCs was confirmed by Quantitative Real-time PCR and by Quantitative Immunofluorescence, respectively. Male GC nuclei had very low levels of Id1 immunofluorescence, whereas female GC nuclei had heterogeneous but significantly higher average Id1 immunofluorescence levels. GCs in both XX and XY E11.5 gonads had heterogeneous Id1 immunofluorescence levels, similar to E13.5 female GCs, but no obvious sex-specific difference was seen at E11.5. However, in E12.5 gonads a sex-specific difference was seen, with XY GCs expressing very low Id1 levels. It appears, therefore, that XY GCs’ Id1 protein expression drops sharply at E11.5-E12.5, coinciding with the time that XY GCs commit to spermatogenesis and arrest mitotically. Female GCs on the other hand continually express heterogeneous Id1 protein levels at E11.5-E13.5. In order to discover candidate Id1-interacting bHLHs in female E13.5 GCs, a Quantitative Real-time PCR expression screen was performed to identify bHLHs enriched sex-specifically in E13.5 GC cDNA. 5 bHLH genes were significantly enriched or detected only in male E13.5 GC cDNA and 16 bHLH genes (including Id2 and Id3, close family members to Id1) were significantly enriched or detected only in female E13.5 GC cDNA. Mnt, a negative regulator of the oncogene Myc, was one of the male-enriched genes and male-specific enrichment of Mnt protein was confirmed by Quantitative Immunofluorescence on E13.5 gonad sections, and qualitatively by immunofluorescence on E12.5 gonad sections. Both female and male GCs expressed high Mnt protein levels at E11.5, so sex-specific Mnt protein expression probably first appears at E11.5-E12.5 due to a downregulation by female GCs. To conclude, 21 genes were enriched or expressed sex-specifically in E13.5 GC cDNA, 16 in female cDNA and are interesting candidates for Id1 interaction in female GCs during commitment to oogenesis and meiotic entry. Mnt protein was enriched male-specifically in E12.5 and E13.5 GC nuclei, when female GCs appear to downregulate Mnt protein levels. Semi-quantitative PCR detected weak Ptgds in purified E13.5 male, but not female, GC cDNA. Also, weak Ptgds immunofluorescence was seen in male, but not female, GC cytoplasm. In conclusion, weak Ptgds mRNA and protein expression was detected sex-specifically in male GCs. A study was then undertaken to test whether molecular signals that are known to affect Ptgds expression in other tissues were able to affect Ptgds expression in developing GCs. E11.5 and E12.5 XX or XY genital ridges were cultured with the phorbol ester Phorbol 12-myristate 13-acetate, Ionomycin, Dexamethoasone or Thyroid Hormone for 3 days and Ptgds expression examined by whole mount in situ hybridisation. All 4 compounds failed to induce or suppress Ptgds expression levels detectably. This suggests that Ptgds expression in GCs may be stimulated by distinct molecular pathways.