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

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