PARTIAL CHARACTERIZATION OF THE CALCIUM-RELEASING ACTIVITY OF PORCINESPERM CYTOSOLIC EXTRACTS

Injection of sperm cytosolic extracts into mammalian eggs has been sho wn to elicit intracellular calcium ([Ca2+](i)) oscillations that are s imilar in amplitude, duration, and frequency to those observed followi ng fertilization. Thus, to characterize the Ca2+-release component(s) in porcine sperm cytosolic extracts, a combination of fractionation te chniques was used. The fraction with Ca2+ releasing activity was preci pitated by 50% saturating solutions of ammonium sulfate and Western bl ot analysis showed that the pellets contained glucosamine-6-phosphate deaminase (gpd)/oscillin, a protein which has been suggested to be the sperm's active component. Single and double isoelectrofocusing (IEF) of porcine sperm extracts generated fractions with different Ca2+-rele asing activities. Fractions with maximal Ca2+-releasing activity did n ot contain material that was immunoreactive with antibodies against gp d/oscillin; adjacent fractions containing gpd/oscillin had no Ca2+-rel easing activity. These findings were confirmed by IEF coupled with siz e exclusion chromatography on Superose 12 and with hydroxyapatite chro matography. These procedures predict an isoelectric point of our activ e component of 6.5-7.0 and a relative molecular weight ranging from 29 to 68 kDa. In summary, the data show that the Ca2+ release-inducing c omponent(s) of porcine sperm extracts can be fractionated and that gpd /oscillin is not the pig sperm Ca2+ oscillogen. (C) 1998 Academic Pres s.