Regulated expression of the RNA-binding protein Sam68 during mouse spermatogenesis

Translational control plays a crucial role during both spermatogenesis and oogenesis in organisms as different as worms and mammals. In C. elegans, the RNA-binding protein GLD-1 is essential for meiotic progression and its deletion causes exit from meiosis and gonadal tumors. Sam68 is a mammalian homolog of GLD-1 and it was originally identified as a 68 kDa protein strongly phosphorylated by Src in mitosis (Src associated in mitosis). The exact function of Sam68 is still unknown, even though this protein has been implicated in several aspects of RNA metabolism, such as splicing, nuclear export and translational regulation. Herein, we have investigated the expression and function of Sam68 in mouse spermatogenesis. A time course analysis by Western blot revealed that Sam68 is expressed throughout development of mouse testis, from birth to adulthood. However, immunohistochemistry showed that Sam68 expression and localization within the cells is stage specific. We found that Sam68 is expressed in the nucleus of spermatogonia, it disappears at the onset of meiosis (leptotene/zygotene stages), and it accumulates again in the nucleus of pachytene spermatocytes and round spermatids. Interestingly, Sam68 decored the meiotic spindle in dividing spermatocytes, suggesting a cell-cycle related function. In support of this hypothesis, we found that Sam68 was phosphorylated in a MAPK-dependent manner in pachytene spermatocytes induced to enter metaphase by short-term treatment with the phosphatase inhibitor okadaic acid. Cell fractionation experiments on a sucrose gradient that allows separation of translationally active polysomes from translationally silent RNPs indicated that phosphorylation correlates with a shift of Sam68 from RNPs to polysomes. Moreover, we found that Sam68 was localized in the cytoplasm of round spermatid in specific stages of the seminiferous tubules, suggesting that it can export target RNAs from the nucleus. During spermatogenesis, post-transcriptional control of gene expression plays a crucial role and may allow a timely regulation of protein expression during spermiogenesis, when the genome is silenced. To gain insight into the role of Sam68 during spermatogenesis we set out to identify the RNA molecules specifically bound to it. Immunoprecipitation experiments coupled to RT-PCR have allowed to isolate mRNA molecules specifically interacting with Sam68 in pachytene spermatocytes and round spermatids. These mRNAs have been subcloned and are currently being sequenced. Thus, the stage-specific expression of Sam68 and its regulation by post-translational modification and subcellular localization indicate that this protein plays an important role in post-transcriptional control of spermatogenesis.