In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane.

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C - PKC; phospholipases A2 and Cζ - PLA2, PLCζ; soluble adenylyl cyclase 10 - sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 - Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.