Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova

Abstract

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39 degrees C) in a modified Tyrode's solution (TALP) containing 1) Chol-free liposomes (-Chol, 50 x 10(6) sperm and 600 nmol phospholipid/ml); 2) liposomes containing 30 mol% Chol (+Chol, 2 x 10(8) sperm and 300 nmol total lipid/ml); or 3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with -Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 x 10(6)) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-microliters droplets of TALP under medical fluid in an atmosphere of 5% CO2 in air (3 h, 39 degrees C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined.(ABSTRACT TRUNCATED AT 250 WORDS)