A quantitative assay for capacitation: Evaluation of multiple sperm penetration through the zona pellucida of salt‐stored hamster eggs

Abstract

The endpoint for sperm capacitation occurs when spermatozoa become able to penetrate intact zonae pellucidae of unfertilized homologous eggs. Activation of eggs stimulated by sperm fusion or by gamete aging initiates changes in zonae pellucidae that bar further sperm entry. This zona block mechanism reduces the usefulness of eggs as indicators for sperm capacitation. Egg storage in concentrated salt solutions destroys the zona block mechanism while retaining the biological sperm receptor/activator functions of the zona pellucida [Yanagimachi et al., Fertil Steril 31:562-574, 1979]. We have developed techniques for the quantitative assay of capacitation using multiple sperm penetration into zonae and have evaluated the sperm-response characteristics of hamster eggs stored in a modified salt solution (STOR). Sperm, preincubated in capacitating or noncapacitating treatments (CAP or NOCAP, respectively), were coincubated with unfertilized and fertilized STOR-treated zonae for 4 hr. Then zonae were stripped of externally bound sperm and the sperm heads in the perivitelline space (PVS) were fluorescently labeled with Hoechst dye 33342. Entry of CAP sperm into the PVS of STOR-treated unfertilized eggs was highly correlated with sperm concentration (Rw = .859) and was log linear from 1 X 10(3)-2 X 10(5) CAP sperm/ml. At 2 X 10(5) CAP sperm/ml, the mean number of PVS sperm was 63.5 and the maximum observed was 158. NOCAP sperm very rarely penetrated unfertilized zonae (2 sperm into 75 eggs). Few CAP sperm entered the PVS of STOR-treated fertilized eggs (two sperm into 54 eggs at 2 X 10(5) sperm/ml) unless the sperm concentration was raised to high levels. Differences between replicates were due to male but not female (ie, egg) differences. We conclude that 1) STOR-treated unfertilized zonae can be used to accurately quantitate differences between sperm capacitation and/or fertility states and 2) the availability of large numbers of homogeneous "shelf-stable" zonae will make it feasible to perform hamster sperm capacitation bioassays on a large scale.