Integrity of the germline is important for the transfer of genetic and epigenetic information to offspring and for fertility. The PIWI/piRNA pathway, consisting of piRNAs and PIWI, TDRD and DNMT3 proteins, plays a crucial role in embryonic gonads by repressing retrotransposon activity through the recruitment of de novo methyltransferases. However, how PIWI/piRNA machinery is regulated in mammalian germ cells is unknown. In embryonic mouse gonads, signalling by TGFβ superfamily members is highly dynamic and important for testis development, including through influence on germ cell proliferation, differentiation and maturation. Our preliminary data indicates that changes in Activin A bioactivity alter piRNA machinery synthesis in the mouse.
The human TCam-2 seminoma cell line is a useful model of fetal germ cells as it shares many functional markers and is responsive to TGFβ superfamily ligands1. TCam-2 cells were cultured for 48 hours with varying Activin A doses (1.25 – 25 ng/mL) or for 6, 24 and 48 hours with 5 ng/mL Activin A in serum-free conditions, then collected for transcript analysis by qRT-PCR. Each experiment had duplicate samples with results compared to vehicle controls and were repeated at least 3 times.
KIT, previously shown to be elevated in TCam-2 cells following 24 hours of Activin A exposure1 was increased at 24 and 48 hours, while TDRD1 was upregulated only at 24 hours. NODAL is significantly upregulated at 6, 24 and 48 hours, while its co-receptor TDGF1 is upregulated at 48 hours. Two markers of male germ cell differentiation were significantly decreased in Activin A-treated samples, the de novo methyltransferase co-factor DNMT3L (at 24 and 48 hours) and NANOS2 (all timepoints). These data reinforce the proposal that Activin A bioactivity influences synthesis of piRNA machinery and TGFβ pathway crosstalk in fetal male germ cells.