Mammalian male or female development depends on testis or ovary formation, involving sex-specific transcription and signalling in embryonic gonads. Disrupted testis and germline development is strongly associated with testis cancer in humans. In mice, Sry and Sox9 promote testis cord formation, committing the fetus to male development. SRY and SOX9 induce Sertoli cell Fgf9 expression, essential for testis development and is thought to promote male germline differentiation. As FGFs signal through Mitogen-Activated Protein Kinase (MAPK) in other tissues, we explored whether FGF9 regulates male germline development through MAPK by inhibiting FGF or MEK1/2 signalling.
Embryonic day (E)11.5-12.5 Oct4GFP transgenic mouse testes were cultured with FGF receptor (FGFRi) or MEK1/2 (MEKi) inhibitors for 24-96h. Impacts on testis cords and Sertoli, somatic and germ cell development were determined using immunofluorescence, flow cytometry and RNA sequencing.
E12.5+72h FGFRi or MEKi culture reduced Sertoli cell proliferation and disrupted testis cord formation; characterised by mis-location of Sertoli cells throughout testis cords and germ cells mis-localised outside testis cords. Assessment of DPPA4 and DNMT3L expression and mitotic arrest indicated that male germline differentiation was prevented by MEKi. Moreover, some germ cells expressed female germline markers but did not enter meiosis. Meanwhile, E12.5+72h FGFRi culture did not affect male germline differentiation, though E11.5+96h FGFRi culture had a slight, but insignificant, impact on mitotic arrest. To identify developmental pathways disrupted by FGFRi or MEKi, global transcriptional regulation is currently being assessed in germ and somatic cells.
Together, our data indicate essential roles for MEK1/2 signalling in male germline development, but a surprisingly limited role for FGF signalling. While FGF9 may partially promote male germline development, our data strongly indicate that additional ligands acting through MEK1/2 play a significant role. Our work highlights a need for further understanding of mechanisms underlying gonad development and testis cancer in humans.