Abstract
Methods were developed to quantitatively reduce the cholesterol (Chol)/phospholipid (PL) ratio of bovine sperm and to determine the effectiveness of this treatment in capacitating sperm. Washed sperm (2 x 10(8)) were incubated in 1.0 ml of modified Tyrode's solution (TS) containing unilamellar liposomes of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and [14C]-Chol (35:35:30 molar ratio, 300 nmol total PL). [3H]-triolein was included as a nonexchangeable marker. After 90 min at 39 degrees C, a 13% net exchange of [14C]-Chol from liposomes to sperm was observed (n = 4), and sperm motility was 80%. Sperm were then washed and 50 x 10(6) sperm were incubated as before with PC/PE liposomes containing no Chol. After 90 min, sperm were separated from liposomes by centrifugation. Measurement of [14C]-Chol in the liposomes (supernatant) and parallel gas chromatographic analysis of extracted, saponified liposomes (n = 4) indicated that 30% of sperm Chol was removed by this procedure. Chol efflux decreased percent motile sperm by less than 10% but reduced sperm velocity by more than 50%. Sperm incubated with no liposomes (control), with liposomes containing Chol (+Chol), and with Chol-free liposomes (-Chol) were washed and resuspended in TS with 0.2% BSA and 30 micrograms lysophosphatidylcholine (LPC)/mg bovine serum albumin (BSA). Percent sperm undergoing the acrosome reaction (AR) upon incubation with LPC-BSA was used as a measure of sperm capacitation. After 60 min of exposure to LPC-BSA at 39 degrees C, the mean (+/- SE) percent motile sperm for control, +Chol, and -Chol treatments was 57.0 +/- 4.9, 60.0 +/- 4.7, and 57.0 +/- 6.8, respectively. Corresponding values for percent AR were 14.0 +/- 3.4, 20.3 +/- 4.4, and 39.7 +/- 1.2. These results suggest that loss of Chol from bovine sperm may be an early step in sperm capacitation in this species.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call