Ni Hong, Mingyou Li1, Beiping He and Yunhan Hong,
The adult testis is the male reproductive organ that produces sex hormones for sexual behaviours and sperm in a well-controlled process called spermatogenesis. Spermatogenesis involves self-renewal and differentiation of the male germ stem cells spermatogonia, culminating in the production of sperm for germline transmission. Retinoic acid (RA) is essential for fate decision and meiosis entry of mammalian germ cells. The microphthalmia-associated transcription factor (Mitf) is a newly identified transcriptional activator of germ genes in the fish medaka (Oryzias latipes). Here we report an essential role of RA on Mitf-activated germ gene expression and spermatogenesis. Medaka germ gene promoters DAZ, DND and VAS were found to contain RA-responsive elements and to exhibit little Mitf-activated transcriptional activity in response to RA treatment in both medaka ES cells and male germ stem cells. A procedure for testicular organ culture was established, which allows for test-tube spermatogenesis over a 21-day period. By the ordered testicular architecture, cellular morphology and VAS-driven GFP expression, different spermatogenic stages were identifiable on testicular cross-sections. In organ culture, RA treatment generated testes completely free of spermatogonia, and reduced VAS-driven GFP expression in spermatogonia but enhanced its expression in meiotic and post-meiotic germ cells. Therefore, RA represses Mitf-activated germ gene expression in stem cell cultures and differentially regulates germ gene expression depending upon the stages of spermatogenesis, and represses spermatogonial stem cell maintenance in cultured testes of medaka as a lower vertebrate model. These results demonstrate that RA plays a highly conserved role in germ cell development from fish to mammals.