Changes on the heat shock protein 70 (HSP70) in water buffalo spermatozoa revealed the capacitation like event in cryopreservation

Abstract

Heat Shock Protein 70 (HSP70) induction helps alleviate the adverse effects of several types of stress in the cells. It can also play a significant role in sperm protection and survival during the extreme conditions of the semen cryopreservation process. The present study was conducted to investigate the role of this protein in spermatozoa of water buffalo by analyzing the changes in its gene expression, protein induction, and distribution after cryopreservation. Differences in HSP70 mRNA and protein levels between fresh and frozen semen samples were determined through real-time PCR and western blot analysis, respectively. Cellular localization patterns of HSP70 in the different sample groups were then detected in an indirect immunofluorescence assay. Lastly, the average sperm motilities of the semen sample groups were evaluated and compared. Results showed that the HSP70 mRNA expression decreased by fifty percent (P<0.05) while the protein level significantly rose by sixty percent (P<0.05) in the frozen sample group. The localization of HSP70 protein in the sperm head also shifted from the equatorial segment in the fresh sample group to the post-acrosomal region in frozen semen samples. Moreover, the average sperm motility appeared to fluctuate with the mRNA level and comparably decreased by fifty percent (P<0.05) in the frozen sample group. These changes on the HSP70 in spermatozoa exhibited the adverse effects of the underlying capacitation-like event in the cryopreservation process. Overall, these findings presented a valuable insight into the current buffalo semen cryopreservation technique and a potential approach to its improvement.