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    • mSSCs can be maintained and genetically manipulated in

    2018-10-24

    mSSCs can be maintained and genetically manipulated in vitro over extensive periods, providing a valuable resource for in vitro studies of meiosis and post-meiotic development (Kanatsu-Shinohara et al., 2003; Kubota et al., 2004). In the past decade, this culture system has been used to identify key growth factors for the proliferation of mSSCs as well as the signaling pathways and the downstream target genes (Takashima et al., 2015). However, a long-standing and vital question is whether these stem BV6 can be stimulated in culture to complete spermatogenesis. In the present study, we show that RA by itself is sufficient to induce mSSCs to become primary spermatocytes, and that RA together with Sertoli cells from pup mice form a more efficient in vitro model of meiosis. This model can be used to identify genes involved in meiosis. Our work builds the foundation for accomplishing gametogenesis in vitro, which in turn contributes to a better understanding of the mechanisms of gametogenesis and the development of treatment of human infertility.
    Results
    Discussion We report in the present study that RA alone is sufficient for inducing the initiation of meiosis of cultured mSSCs and the derivation of spermatocytes under feeder-free and serum-free conditions. A previous study showed that purified prenatal male PGCs isolated before but not after 14.5 days post coitum (dpc) can be induced by RA to initiate meiosis and to generate spermatocytes in the absence of any somatic cells (Ohta et al., 2010). However, the question of whether the lost co-culture-independent RA responsiveness of the male germ cells after 14.5 dpc can be resumed later in germ cell development remains open. Moreover, the number of isolated PGCs is low and they are refractory to mitotic proliferation even in the presence of 20% serum, thus having limited use in further applications. By taking advantage of cultured mSSCs that can be amplified infinitely while keeping their spermatogenic function, we have been able to address this important question and conduct more mechanistic studies. Our finding is somewhat surprising given that in vivo spermatogenesis, which occurs in the testis containing multiple hormone and/or cytokine-producing cell types, seems to be regulated by diverse factors. That RA plays a key role in meiosis initiation in mammals and other vertebrates has been supported by multiple lines of evidence. However, whether RA is the only essential factor remains unknown, probably due to the lack of an in vitro culture system for germ cells. If RA is indeed the only meiosis-inducing signal in vivo, as suggested by our in vitro data presented here, we can argue that the major roles of other components in the gonads in terms of meiosis initiation are to regulate the production and degradation of this key molecule. This regulation can be complex for reasons that are poorly understood currently. For example, Raverdeau et al. (2012) reported that development of undifferentiated spermatogonia to differentiating A1 spermatogonia in the first spermatogenic cycle was dependent on the Sertoli cell-derived RA but the subsequent spermatogenic cycles were initiated by RA produced by spermatocytes. Few genes regulated by RA in germ cells have been identified despite the important role of RA in meiosis. Stra8 is the best known direct target gene of RA (Oulad-Abdelghani et al., 1996). A recent study indicated that RA regulated two parallel pathways represented by Stra8 and Rec8 (Koubova et al., 2014). Our RNA-seq data of the RA-treated mSSCs not only confirmed these observations but also provided clues for dissecting the mechanisms of meiosis. First, a much larger number of genes are regulated by RA than previously known. Although we are still unable to discern how many of them are direct targets of RA, we think the number should be high based on the 1,041 upregulated and 1,768 downregulated genes in the intersecting sets of the RA-regulated and RA + CHX-regulated genes. More evidently, the binding of RARG to the predicted RAREs in a small panel of RA-regulated genes was mostly confirmed by ChIP-PCR results. Second, RA not only upregulate genes that favor the differentiation of spermatogonia (c-Kit) and the initiation (Stra8) and progression (Rec8, Dmc1, Prdm9, Sycp3) of meiosis, but also downregulate genes beneficial to the self-renewal of mSSCs (Oct4, Plzf, Lin28a, to name a few; see Figure 2 for more), the proliferation of progenitor spermatogonia (Ngn3, Sox3), and the genes safeguarding the premature meiosis (Dmrt1). Third, RA regulates the expression of a diverse family of genes encoding, for example, signaling proteins, metabolizing enzymes, transcription factors, and many others. The results expand far more our recognition of the molecular mechanisms underlying the action of RA as the key regulator of meiosis.