The growth factor activin functions in testis development at multiple stages that influence the pace of testis development and germ cell differentiation. Activin levels become highly elevated in the fetal testis where its activity regulates gonocyte (male germ cell) and Sertoli cell proliferation. This elevation occurs in a critical period for renewal of DNA methylation in gonocytes, and we considered activin as a candidate for regulating the piRNA machinery that protects the unmethylated gonocyte genome. Piwi-interacting RNAs (piRNA) are crucial for spermatogenesis because they repress activation of transposable elements via recruitment of DNMT3, which catalyzes protective de novo methylation on genomic DNA. In mice, deletion of piRNA synthesis machinery in knockouts results in male-specific sterility; these germ cells exhibit aberrant LINE-1 transposon activation, morphologically abnormal chromatoid bodies in spermatocytes (which also synthesize piRNA) and eventual absence of spermatozoa in adults. However, it remains unclear how the comprehensive activation of genes mediating piRNA activities is achieved. We evaluated activin regulation of piRNA machinery using a human seminoma cell line, TCam-2, derived from a germ cell tumour that is most closely related to gonocytes.
TCam-2 cells were cultured with activin A (5 ng/ml) plus/minus 10% fetal calf serum for 24 hours. Real time RT-PCR (three experiments; n=3 per experiment) was conducted to quantify transcripts relating to piRNA machinery. Amongst the PIWI, TDRD and DNMT3 protein families, activin A significantly down-regulated DNMT3L and TDRD1 which are both male-specific in fetal germ cells, as well as another marker, NANOS2. In contrast, TDRD5 and TDRD7, which are required for chromatoid body assembly in spermatocytes, were up-regulated by activin A, but only under conditions of serum starvation. We propose that activin A influences establishment of piRNA machinery in fetal male germ cells and contributes to chromatoid body assembly via controlling TDRD5 and TDRD7 production during spermatogenesis.