ACROSOME-INTACT BOAR SPERMATOZOA INITIATE BINDING TO THE HOMOLOGOUS ZONA-PELLUCIDA IN-VITRO

The acrosome reaction is a prerequisite for zona pellucida penetration by mammalian spermatozoa, In some species, the sperm undergo the acro some reaction before binding to the zona pellucida, and in other speci es only acrosome-intact sperm can initiate binding to the zona, In the present investigation, we addressed the question whether acrosome-int act or acrosome-reacted boar sperm initiate binding to the pig zona pe llucida by studying the acrosomal status of sperm bound to zonae pellu cidae. Our approach was to vary the percentage of acrosome-intact sper m in suspension by long preincubation before incubation with hemizonae for 1 min. We hypothesized that if only acrosome-intact sperm are abl e to initiate binding to the zona pellucida, the majority of the sperm on the zona surface would be acrosome-intact regardless of the percen tage of acrosome-reacted sperm in suspension, Fluorescein isothiocyana te-conjugated peanut agglutinin (Arachis hypogaea; FITC-PNA) in combin ation with optical sectioning by confocal laser-scanning microscopy wa s used to study the acrosomal status of sperm bound to the hemizona, E lectron microscope studies showed that the FITC-PNA binding site is ma inly limited to the outer acrosomal membrane of boar sperm, thus valid ating the use of FITC-PNA as an accurate probe for studying boar sperm acrosome reaction. Over 90% of the sperm bound to a hemizona were acr osome-intact irrespective of whether the majority of sperm in the susp ension were acrosome-intact, acrosome-reacting, or acrosome-reacted. T here was a significant difference (Kruskal-Wallis test, p < 0.05) in t he mean +/- SEM number of sperm bound to the outer side, inner side, a nd edge of a hemizona (48 +/- 8, 14 +/- 3, and 7 +/- 2; n = 58; respec tively). The acrosomal status of sperm bound to the various surfaces o f hemizonae was similar, Taking the respective zona pellucida surface area into consideration, it was calculated that an average of 1.9 +/- 0.3, 1.0 +/- 0.2, and 1.5 +/- 0.3 spermatozoa were bound per 1000 mu m (2) of outer side, inner side, and edge of a hemizona, respectively (m ean +/- SEM, n = 38). These observations indicate that acrosome-intact boar spermatozoa initiate binding to the pig zona pellucida. A gradie nt of sperm binding sites also exists, decreasing from the outside to the inside of the zona pellucida.