PRIMATE SPERM CONTAIN PROTEIN-PHOSPHATASE-1, A BIOCHEMICAL MEDIATOR OF MOTILITY

Sperm motility initiation, capacitation, and hyperactivation are modul ated by an interplay of intracellular Ca2+, cAMP, and pH. Mechanisms b y which these mediators alter sperm function have not been elucidated but may involve reversible alterations in regulatory protein phosphory lation. Studies were designed 1) to investigate the influence of the p rotein phosphatase (PP) inhibitor calyculin A (CA) on human sperm moti lity and 2) to characterize the CA-sensitive PP and its endogenous reg ulators in human and rhesus monkey sperm. CA (50 nM) treatment of huma n sperm resulted in an increase in percentage motility and an accelera tion in mean path velocity. Inhibition of either protein phosphatase-1 (PP1) or protein phosphatase-2A (PP2A) could be responsible for this motility stimulation, since both of these phosphatases are sensitive t o nanomolar quantities of CA. PP activity in human (n = 4) and rhesus monkey (n = 4) sperm sonicates was measured using [P-32]-phosphorylase -a, the preferred substrate for PP1 and PP2A, in the absence of divale nt cations. Human (6.2 +/- 4.5 x 10(-2) mU/10(8) sperm) and monkey (4. 3 +/- 0.8 x 10(-2) mU/10(6) sperm) sonicates contained activity tentat ively identified as PP1 on the basis of inhibition profiles in okadaic acid (OA) and CA. Western blot analysis with antibodies against vario us isoforms of PP1 subsequently documented the presence of PP1(gamma 2 ) in human and monkey sperm. PP1 activity in most tissues is regulated by the heat-stable inhibitors I1 or I2. Sperm sonicates contained inh ibitor activity similar to I2 as well as glycogen synthase kinase-3 (G SK-3) activity, which is involved in the activation of the PP1-I2 comp lex. These results indicate, for the first time, that human and rhesus monkey sperm contain PP1 and regulators of PP1 and that inhibition of PP1 activity by CA can enhance motility.