Asthenozoospermia accounts for almost 50% of the cases of male infertility. Our study investigating phosphoproteins differentially expressed in asthenozoosperm has identified the phosphoproteins relevant to sperm motility and the signature molecules likely to be altered in asthenozoospermia. The 66 phosphoproteins differentially expressed included four alpha tubulin isoforms which were differential expressed in individuals with poor sperm motility; an increase in TUBA3E and TUBA4A and decrease in TUBA3C and TUBA8. Isoforms TUBA3C, TUBA4A and TUBA8 are reported to be abundantly present in the testis. Of these, TUBA3C and TUBA4A are known to be acetylable isoforms. Their differential expression in asthenozoosperm prompted us to investigate the role of reversible acetylation of alpha tubulin in sperm motility.
Materials and methods
Alpha tubulin, acetylated α tubulin, and isoforms TUBA3C, TUBA4A, and TUBA8 were investigated in Percoll separated human sperm and HDAC6 in rat sperm by Western blot analyses, Flow cytometry, Realtime RT-PCR and IIF localization and data statistically analyzed. The observations were analyzed in silico for obtaining further insights. Interaction between HDAC6 and α Tubulin was elucidated by IIF co-localization and Co-immunoprecipitation studies. Sperm HDAC6 activity, motility and status of Ac α-tubulin was investigated in the presence of HDAC inhibitors Trichostatin A, Tubastatin A and Sodium Butyrate.
The differential expression of these isoforms was validated in the normal- and asthenozoosperm at protein and transcript level. Investigation of acetyl α tubulin expression revealed a reduction in tubulin acetylation in asthenozoosperm. The decrease in TUBA3C and increase in TUBA4A transcripts, both being acetylable isoforms of alpha tubulin, could be elucidated on the basis of transcription factors binding to the promoters of the respective isoforms while the reduction in acetyl alpha tubulin in asthenozoosperm could be explained on the basis of decreased TUBA3C and the association of HDAC6 with TUBA4A. However the presence of HDAC6 on sperm had not been hitherto reported. In this study we have demonstrated for the first time the presence of HDAC6 transcript and protein in testicular- and caudal-sperm of rat and further by co-localization and coimmunoprecipitation studies we showed that HDAC6 interacts with alphatubulin and they colocalize in the mid piece and principal piece of sperm flagella. Using HDAC inhibitors we further demonstrated that HDAC6 in sperm is catalytically active and inhibitors of HDAC6 increase acetylation and restrict sperm motility.
Our data suggests an association between reversible α tubulin acetylation and sperm motility. We show that alpha tubulin acetylation is reduced in sperm of asthenozoospermic individuals. Paradoxically, our experiments in the rat sperm show that inhibition of HDAC6 increase alpha tubulin acetylation but restrict sperm motility. The persistent expression of HDAC6 on the sperm flagella in the presence of HDAC6 inhibitor hints at a possible role for HDAC6 possibly as a microtubule associated protein(MAP). On the basis of our observations with the human and rat sperm and the available literature on microtubule stability in atat1 KO mice, we conclude that HDAC6 may act as a MAP and maintain dynamic instability in sperm flagella, and that dynamic instability may be a pre-requisite for normal sperm motility.