The RNA binding protein Sam68 transiently localizes to the chromatoid body and interacts with the RNA helicase MVH in meiotic and post-meiotic germ cells

INTRODUCTION The chromatoid body (CB) is a unique structure of male germ cells. It is composed of thin filaments that condense into a compact perinuclear granule after meiosis and it remains a distinctive feature in the cytoplasm of elongating spermatids (1). Although the function of CB remains elusive, an involvement in RNA processing and storing has been recently proposed. The CB of round spermatids was shown to gather in close proximity proteins involved in different steps of RNA metabolism, like the RNA helicase VASA, the decapping enzyme DCP1a, regulators of small non coding RNAs, like AGO2/3, Dicer, MIWI, with specific miRNAs (2). These observations have suggested a role for CB in the control of mRNA quality and in translational regulation. We have recently shown that the nuclear RNA-binding protein Sam68 translocates to the cytoplasm in secondary spermatocytes and round spermatids, where it regulates translation of a subset of mRNAs required for spermiogenesis. Notably, in some cells Sam68 accumulated in granules resembling the CB. The aim of this study was to investigate the role of Sam68 in the CB and its possible involvement in the miRNA pathway. MATERIAL AND METHODS Immunofluorescence analysis: after elutriation, isolated male germ cells where stained with specific antibodies directed against Sam68 or VASA. Immunoprecipitation: VASA was immunoprecipitated from total extracts of secondary spermatocytes with an anti-Sam68 polyclonal antibody. RESULTS AND DISCUSSION Immunofluorescence analysis revealed that Sam68 localizes in the CB (Fig.1). Moreover, we observed that Sam68 colocalizes in the CB with a germ cell-specific chromatoid body component, the RNA helicase MVH (mouse VASA homologue), as indicated by the arrows. This occurs only in the secondary spermatocytes, when Sam68 translocates into the cytoplasm (3), but not in round spermatids, where Sam68 is predominantly nuclear (fig.2). Immunoprecipitation experiments, performed from lysates of secondary spermatocytes, demonstrated that Sam68 directly interacts with VASA (Fig.3). A microarray screen has revealed several miRNAs whose expression is altered in Sam68 knockout meiotic and post-meiotic germ cells. These results suggest the involvenment of Sam68 in the miRNA pathway in male germ cells.