Heat Shock Proteins as Markers for Cellular Stress in Mammalian Sperm

The cryosurvival of sperm requires cell signaling mechanisms to adapt to anisotonic conditions during the freezing and thawing process. Sperm were evaluated for their cellular expression and levels of phosphorylation of both heat shock proteins (HSPs) 70 and 90 under anisotonic conditions as a potential model for cell signaling during osmotic stress in macaque sperm (Chapters 1 and 2) and cryopreservation in equine sperm (Chapter 3). In Chapters 1 and 2, osmotically stressed sperm were either prepared for phosphoprotein enrichment, immunoprecipitation by HSP 70, or processed for flow cytometry. The enrichment of phosphoproteins and western immunoblotting revealed an increase in the phosphorylation of HSP 70 but not HSP 90 under osmotic stress conditions. There was a significant increase in the level of tyrosine phosphorylation of HSP 70 after hypertonic treatments. In Chapter 2 the pre-treatment of sperm with an inhibitor of HSP 90 prior to osmotic stress resulted in an overall increase in the tyrosine phosphorylation of proteins under anisotonic conditions. This data confirms the importance of HSP 70 and HSP 90 during osmotic stress conditions in rhesus macaque sperm. In Chapter 3, we measured the level of phosphorylation, acrosome reaction rates, and mitochondrial membrane potential (MMP) in cryopreserved equine sperm. To evaluate the effect of HSP 90 inhibition on the cell stress response, sperm were pre-incubated and cryopreserved with increasing levels of geldanamycin. Following cryopreservation, post-thaw membrane integrity was monitored with Syber-14 and PI, and fluorescence was measured by flow cytometry. Tyrosine phosphorylation rates were measured by flow cytometry and confirmed by western blot. Lastly the acrosome reaction was induced with either calcium ionophore or progesterone and rates were measured by flow cytometry and indirect fluorescence microscopy. There was a significant effect on 30 and 60 minute progressive motility, MMP and tyrosine phosphorylation rates at 4.5 and 8.4 & mu;M concentrations of geldanamycin. In addition, the progesterone induced acrosomal loss rates were significantly increased with increasing doses of geldanamycin suggesting HSP 90 inhibition may play a physiological role in the post-thaw period of equine cryopreserved sperm. Taken together these experiments suggest HSP 70 and 90 play an important role in the cell stress response of mammalian sperm.