iv Spermatogenesis is a highly coordinated and productive process that maintains continuous sperm production in post-pubertal males. Spermatogonial stem cells (SSCs) are the basis of spermatogenesis and balance self-renewal with differentiation to maintain the stem cell pool while producing differentiating progenitors that give rise, through several transit-amplifying mitotic divisions, two meiotic divisions and morphological differentiation to sperm. The molecular mechanisms driving these fate

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... decisions are poorly understood, though numerous RNA regulatory pathways are thought to be critical. We used microarray analysis of distinct testis models to identify genes whose expression correlates with SSC activity. Fourteen RNA binding proteins were identified, and their testis expression was validated using RT-PCR and immunohistochemistry. The multi-functional RNA regulator YBX1 was expressed only by cells located on the basement membrane of seminiferous tubules, consistent with the location of SSCs. YBX1 was of special interest because of its heretofore unexplored role in the testis, as well as its possible phosphorylation by AKT. The AKT signaling pathway is activated in mouse SSCs by GDNF, a factor essential for SSC survival and self-renewal. We hypothesized that YBX1 was phosphorylated by AKT signaling downstream of GDNF, and that this phosphorylation would affect YBX1 interaction with target messenger RNAs (mRNAs). We show that YBX1 is phosphorylated in spermatogonia, and that GDNF signaling modulates YBX1 phosphorylation. Inhibiting either PI3K or AKT in SSC cultures also causes a loss of YBX1 phosphorylation, suggesting that GDNF works through the PI3K/AKT signaling pathway to phosphorylate YBX1. To identify mRNA targets of YBX1, we immunoprecipitated YBX1 from SSC cultures grown in the presence or absence of GDNF, and isolated co-immunoprecipitated mRNAs. In normal growth conditions (containing GDNF), YBX1 associated with mRNA for genes expressed by all GDNF SIGNALING REGULATES YBX1/mRNA INTERACTIONS IN MOUSE SPERMATOGONIA