Intracellular pH regulates bovine sperm motility and protein phosphorylation

Bovine sperm in neat caudal epididymal fluid become motile in response to either pH elevation or dilution of the fluid. Buffers containing permeant weak acids at physiologic concentrations are able to mimic these effects of caudal fluid. These observations lead to the hypothesis that a pH-dependent epididymal fluid quiescence factor regulates bovine sperm motility by modulating sperm intracellular pH (pH(i)). Here, we report that sperm pH(i), measured with the fluorescent pH probe carboxyfluorescein, increases by ~ 0.4 units in response to either of these motility-initiating manipulations. At least 26 discrete phosphoprotein bands are distinguishable by sodium dodecylsulfate-polyacrylamide gel electrophoresis after incubation of intact caudal sperm with ³²PO₄. A prominent phosphoprotein, with M(r) ~ 255,000 (pp255) and a relatively high specific radioactivity, is reversibly dephosphorylated in response to elevations in pH(i) that initiate sperm motility. Unlike most of the sperm phosphoproteins, the extraction of pp255 requires reducing agents. This phosphoprotein cosediments with the sperm heads but not the tail, midpiece, soluble, or plasma membrane fractions. No other pH(i)-dependent phosphorylation changes are apparent in gels of whole sperm extracts. However, subcellular fractionation allows the detection of increased phosphorylation of two plasma membrane phosphoproteins (M(r) ~ 105,000 and 97,000) and decreased phosphorylation of another plasma membrane phosphoprotein (M(r) ~ 120,000) in response to increasing pH(i). This is the first report describing changes in endogenous phosphoproteins from intact motile and nonmotile bovine sperm that are regulated by pH(i).