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

Abstract

This study is a detailed investigation ofchanges in epididymal protein anti-agglutinin on ejaculated boar spermatozoaduring an incubation designed to promote capacitationin vitro. Ejaculated spermatozoa were collected from sixmature boars, washed, and incubated to promote capacitation. Sperm sampleswere subjected to Western blotting-densitometric analyses, flow cytometryafter immunostaining and immunocytochemical observation by indirectimmunofluorescence. An antiserum to anti-agglutinin was raised in a rabbit bysubcutaneous injection of a purified antigen, as described previously(Harayama et al. 1999). Western blotting-densitometricanalyses revealed an approximate halving of the amount of sperm-boundanti-agglutinin during the first 45-min incubation, followed by a gradualdecrease thereafter. Comparison between immunostained sperm samples by flowcytometry before and after incubation confirmed this decrease in sperm-boundanti-agglutinin during the incubation. Microscopic characterizationestablished 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 conclusionthat a large portion of the anti-agglutinin bound to sperm acrosomes isreleased at an early stage of the capacitation processin vitro.