Rabbit Sperm Reversible Decapacitation by Membrane Stabilization with a Highly Purified Glycoprotein from Seminal Plasma

Abstract

A substance has been purified from rabbit seminal plasma which reversibly inhibits fertilization in vivo and blocks the acrosome reaction in a concentration dependent manner. It is likely that the inhibition of fertilization is a direct effect of what may be its primary property of blocking the acrosome reaction. For this reason we have named the substance Acrosome Stabilizing Factor (ASF). Major purification steps include column chromatography on Sephadex G-200 and then on Ultragel AcA 22, followed by disc electrophoresis using a 20-50% sucrose gradient as a support medium. An ASF concentration of 28.5 µg/ml was sufficient to inhibit the in vitro acrosome reaction by 50%, in comparison to the seminal plasma concentration of 2 mg/ml which was required to achieve the same level of biological activity. Native molecular weight estimates of ASF by Ultrogel AcA column chromatography and polyacrylamide disc gel electrophoresis are 355,000 daltons and 370,000 daltons, respectively. The subunit molecular weight is 121,000 daltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). ASF has a carbohydrate: protein ration of 1:1.6 as determined by the anthrone and Lowry assays. No lipid was detectable in a chloroform-methanol (2:1, v/v) extract of the purified material. It is anticipated that further characterization of ASF and elucidation of its specific mechanism of action will provide new insight into the capacitation process.