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 acrosome reaction before binding to the zona pellucida, and in other species only acrosome-intact sperm can initiate binding to the zona. In the present investigation, we addressed the question whether acrosome-intact or acrosome-reacted boar sperm initiate binding to the pig zona pellucida by studying the acrosomal status of sperm bound to zonae pellucidae. Our approach was to vary the percentage of acrosome-intact sperm in suspension by long preincubation before incubation with hemizonae for 1 min. We hypothesized that if only acrosome-intact sperm are able to initiate binding to the zona pellucida, the majority of the sperm on the zona surface would be acrosome-intact regardless of the percentage of acrosome-reacted sperm in suspension. Fluorescein isothiocyanate-conjugated peanut agglutinin (Arachis hypogaea; FITC-PNA) in combination with optical sectioning by confocal laser-scanning microscopy was used to study the acrosomal status of sperm bound to the hemizona. Electron microscope studies showed that the FITC-PNA binding site is mainly limited to the outer acrosomal membrane of boar sperm, thus validating 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 acrosome-intact irrespective of whether the majority of sperm in the suspension were acrosome-intact, acrosome-reacting, or acrosome-reacted. There was a significant difference (Kruskal-Wallis test, p < 0.05) in the mean +/- SEM number of sperm bound to the outer side, inner side, and edge of a hemizona (48 +/- 8, 14 +/- 3, and 7 +/- 2; n = 58; respectively). The acrosomal status of sperm bound to the various surfaces of 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 square micrometers of outer side, inner side, and edge of a hemizona, respectively (mean +/- SEM, n = 38). These observations indicate that acrosome-intact boar spermatozoa initiate binding to the pig zona pellucida. A gradient of sperm binding sites also exists, decreasing from the outside to the inside of the zona pellucida.