Human sperm bound to the zona pellucida have normal nuclear chromatin as assessed by acridine orange fluorescence

Abstract

The aim of this study was to determine if only sperm with double stranded DNA could bind to the human zona pellucida (ZP). Sperm samples from 124 infertile men with a range of semen abnormalities were studied. Oocytes that had failed to fertilize in IVF or ICSI were used for the sperm-ZP binding test. A group of four oocytes were incubated for 2 h with 2 x 10(6)/ml motile sperm selected by colloidal silica gradient centrifugation (CSGC). After assessing the number of sperm bound per ZP, all sperm bound to the surface of the ZP of four oocytes were dislodged and placed on a glass slide. The double (green fluorescence) or single stranded (denatured, red fluorescence) DNA of sperm in semen, motile sperm selected by CSGC and ZP-bound sperm, was assessed by acridine orange (AO) fluorescence. The percentage of sperm with green fluorescence was significantly correlated with normal sperm morphology in semen and after CSGC preparation. The proportion of sperm with green fluorescence was significantly higher in motile sperm selected by CSGC than ejaculated sperm. There were very few sperm (average <8%) with red fluorescence bound to the ZP, even in men who had very high (>70%) proportions of sperm with red fluorescence in their semen. Sperm binding to human ZP is highly selective for double stranded DNA. Sperm with single stranded or denatured DNA bind less or do not bind at all to the ZP, probably because of defects of motility and, more especially, morphology.