A Complete Localization Pattern and Possible Function for STRA8 in the Postnatal Mouse Testis.

Abstract

Vitamin A, in its active form of retinoic acid (RA), is essential for proper spermatogenesis. When male mice are made vitamin A deficient (VAD), the transition from undifferentiated A spermatogonia to differentiated A1 spermatogonia is blocked and transcript levels of the RA-responsive gene, Stra8, are absent. STRA8 is known to be essential for meiosis in both males and females, however, neither a complete localization study nor a function for STRA8 have yet to be described. We produced full-length mouse STRA8 protein using a bacterial expression system and raised an antibody against this protein in order to complete a full STRA8 localization profile in the developing and adult mouse testis as well as in RA-treated vitamin A sufficient (VAS) and VAD adult male mice. Western blotting experiments determined that the antibody could specifically recognize a protein in testis lysates matching the reported size of STRA8 as well as the recombinant protein and immunohistochemistry demonstrated that STRA8 could be detected in both the nucleus and cytoplasm of differentiated spermatogonia and preleptotene and leptotene spermatocytes and in a stage specific manner in the adult mouse testis. In VAD mice treated for 24 hours after a single injection of either RA or vehicle control, STRA8 positive cells were detected in every tubule within testis cross sections of the RA-treated animal, with very few to no positive cells present in the vehicle control samples. Co-immunofluorescence with STRA8 and GCNA antibodies demonstrated that every germ cell present in the RA-treated samples was STRA8 positive and whole mount immunofluorescence revealed that chains of up to eight STRA8 positive spermatogonia were present in the RA-treated VAD tubules. In addition, treatment of VAS adult male mice with one injection of RA induced the expression of STRA8 in spermatogonia in Stages II-VI, 24 hours after treatment. Given the nuclear localization observed for STRA8, we analyzed the chromatin structure of spermatogonia in Stra8-deficient mice using an antibody against the chromatin methylation mark histone H3K9 trimethylation and found altered patterns of chromatin methylation in the STRA8-deficient spermatogonia compared to wildtype spermatogonia. Taken together, this study describes a complete localization pattern in the postnatal mouse testis for STRA8, demonstrates its expression in response to RA in both VAD and VAS mice, and implies that STRA8 may function to drive changes in chromatin structure in germ cells. This work was supported by the NIH Grants HD 10808 and U54 42454 to MDG. (poster)