A NOVEL PROTEIN-KINASE-C INHIBITOR IN BOVINE SPERMATOZOA AND ITS EFFECTS ON THE DEVELOPMENT OF FERTILIZED OVA

We found a protein kinase C (PKC) inhibitor in bovine spermatozoa. The inhibitor was purified by Sephadex G-200 gel filtration chromatography, isoelectric focusing and Mono Q chromatography. Inhibition of the PKC activity observed with 2 mu g of the purified inhibitor was comparable with that with 5 mu mol of chlorpromazine, a known potent inhibitor of PKC. Molecular weight of this inhibitor was estimated to be about 63,000, and isoelectric point was pH 4.5. The inhibitor was inactivated when boiled, or digested with trypsin, whereas DNase and RNase were without effect, indicating that the inhibitor is a protein. The inhibitor is neither a protease nor a phosphatase. In fresh bovine spermatozoa, the PKC activity was almost undetectable probably because of the presence of the PKC inhibitor. When spermatozoa were incubated at pH 7.5 at 37 degrees C for 12-16 h, more than half the activity of PKC inhibitor was lost, suggesting that the inhibitor was inactivated by proteolysis. When the purified inhibitor was microinjected into mouse fertilized ova, both the development of one-cell fertilized ova into two-cell stage ova, and two-cell late stage ova into four-cell stage ova were inhibited. In contrast, PKC microinjected into two-cell early stage ova promoted their development into the four-cell and eight-cell stages; two-cell early stage ova injected with saline remained in the same stage. The results suggest a regulatory role of PKC and the PKC inhibitor in the development of fertilized ova.