SPERM MOTILITY DEVELOPMENT IN THE EPIDIDYMIS IS ASSOCIATED WITH DECREASED GLYCOGEN-SYNTHASE KINASE-3 AND PROTEIN-PHOSPHATASE-1 ACTIVITY

Immotile bovine caput epididymal sperm contain levels of protein phosp hatase activity twofold higher than do mature motile caudal sperm. Com parison of the inhibition profiles of endogenous phosphatase activitie s detected by okadaic acid (OA) and calyculin A (CA) revealed a patter n consistent with the predominance of a type 1 protein phosphatase (PP 1). Immunoblot analysis identified PP1(gamma 2) (the testis-specific i soform of PP1) as the only PP1 isoform in sperm and showed little prot ein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i .e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like ac tivity was detected in sperm. Inhibition of PP1 by the heat-stable I2- like activity purified from sperm could be reversed with purified glyc ogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activ ated by purified GSK-3. The presence of GSK-3 in sperm was demonstrate d by activation of purified PP1I, and quantitation revealed that immot ile caput sperm contained sixfold higher GSK-3 activity than motile ca udal sperm. Immunoblot analysis confirmed the expression of GSK-3 in s perm and revealed the occurrence of both the a and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile spe rm, presumably due to higher GSK-3 activity, is responsible for holdin g motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immo tile bovine caput epididymal sperm and to stimulate the kinetic activi ty of mature caudal sperm. The intrasperm levels of cAMP, pH, and calc ium were unaltered by treatment with these inhibitors. The results sug gest a biochemical basis for the development and regulation of sperm m otility and a possible physiological role for the PP1/I2/GSK-3 system.