Changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during capacitation in vitro

Abstract

This study is a detailed investigation of changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during an incubation designed to promote capacitation in vitro. Ejaculated spermatozoa were collected from six mature boars, washed, and incubated to promote capacitation. Sperm samples were subjected to Western blotting-densitometric analyses, flow cytometry after immunostaining and immunocytochemical observation by indirect immunofluorescence. An antiserum to anti-agglutinin was raised in a rabbit by subcutaneous injection of a purified antigen, as described previously (Harayama et al. 1999). Western blotting-densitometric analyses revealed an approximate halving of the amount of sperm-bound anti-agglutinin during the first 45-min incubation, followed by a gradual decrease thereafter. Comparison between immunostained sperm samples by flow cytometry before and after incubation confirmed this decrease in sperm-bound anti-agglutinin during the incubation. Microscopic characterization established that this decrease occurred mainly on the acrosome. Supplementation with seminal plasma (5% or 10%, v/v) attenuated the decrease. These findings are consistent with the conclusion that a large portion of the anti-agglutinin bound to sperm acrosomes is released at an early stage of the capacitation process in vitro.