Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of male gamete, of which the underlying molecular mechanisms are poorly understood. AKAP3 (A-Kinase Anchoring Protein 3) is one of the major components of fibrous sheath of sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about two weeks. Nascent AKAP3 was found to form prot... More
Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of male gamete, of which the underlying molecular mechanisms are poorly understood. AKAP3 (A-Kinase Anchoring Protein 3) is one of the major components of fibrous sheath of sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about two weeks. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs) including PIWIL1, PABPC1 and NONO, as revealed by co-immunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs of which proteins are synthesized during elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1 and NONO interact with each other and co-localize in spermatids' RNA granule, the chromatoid body (CB). In addition, NONO was found in extra cytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits co-localize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and co-sediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in post-meiotic male germ cells, underscoring molecular mechanisms that regulate protein synthesis during mouse spermiogenesis.
