Localization and regulation of plasma membrane Ca(2+)-ATPase in bovine spermatozoa

Calcium (Ca(2+)) signals, produced by the opening of plasma membrane entry channels, regulate a number of functions in spermatozoa such as capacitation and motility. The mechanisms of Ca(2+) removal from the sperm, required to restore resting [Ca(2+)](i), include plasma membrane Ca(2+)-dependent ATPase (PMCA) isoenzymes as well as a plasma membrane Na(+)-Ca(2+) exchanger. We have recently shown that bovine sperm PMCA is stimulated by PDC-109, a secretory protein of bovine seminal vesicles. To demonstrate the subcellular localization and regulation of bovine sperm PMCA, we have performed cell fractionation, enzyme activity determination and Western blotting studies of PMCA in spermatozoa removed from the cauda epididymidis of bull. Fractionation of sperm heads and tails resulted in a distinct association of ATPase activity with the tail membrane fraction. In vitro stimulation studies with PDC-109 using intact and fractionated sperm showed an increase in enzyme activity up to 105% in sperm tail membranes. Furthermore, thapsigargin inhibition did not alter the stimulatory effect of PDC-109 on ATPase activity, indicating that no sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), but only PMCA isoenzymes are involved in this effect. Western blotting studies using a polyvalent PMCA antibody showed the exclusive presence of a 135 kDa band in the tail plasma membrane fraction. To elucidate whether or not the stimulatory effect was a direct one or indirectly mediated through PKA and PKC activation, PKA and PKC inhibitors, respectively, were used in the Ca(2+)-ATPase activity assays, which was followed by PDC-109 stimulation. The stimulatory effect of PDC-109 on PMCA was still observed under these conditions, while no phosphotyrosine proteins could be detected by Western blotting in sperm extracts following PDC-109 treatment. Co-immunoprecipitation studies, PDC-109 affinity chromatography as well as overlay blots failed to show a strong association of both PMCA and PDC-109, pointing to an indirect, perhaps phospholipid-mediated effect.