Analysis of the dynamic process of sperm penetration of the human oocyte using time-lapse cinematography

Abstract

OBJECTIVE: Using time-lapse cinematography (TLC), we analyzed the dynamic process of human sperm penetration to investigate the mechanisms blocking polyspermy after sperm attachment. DESIGN: Retrospective visual analysis. MATERIALS AND METHODS: Between July 2004 and April 2008, TLC was performed on single, randomly selected oocytes after insemination, as previously described (Mio, 2008). The study focused on oocytes where the movements of the leading sperm (the one that penetrated the zona pellucida (ZP) most deeply) and the following sperm were captured in the same field. Sperm movements were then analyzed from the time of entry into the ZP to the incorporation of the leading sperm. RESULTS: We observed and recorded the process of sperm penetration and the sperm entry point in 22 embryos. In 3 of these embryos (target embryos), we were able to capture the movements of the leading and following sperm. Sperm attached to the oocyte membrane approx 96 minutes after insemination and 37 minutes later, the sperm head decondensed and disappeared. There was no difference in the time-course of the fertilization process of the target embryos and the other embryos observed. In the 3 target embryos, both leading and following sperm proceeded forward until the leading sperm attached on the ooplasmic membrane. The following sperm penetrated 12.9 μm (68.9%) of the ZP on average and, despite their active tail movements, stopped their progression within 10 seconds of the leading sperm attaching on the membrane. CONCLUSIONS: To date, the primary block to polyspermy in the mammalian oocyte is described as a zona reaction induced approximately 5 to 8 minutes after oocyte activation by sperm incorporation. In this study, 10 seconds after sperm-oocyte attachment, the following sperm were unable to continue further despite their sustained tail motility. This suggests that an unknown process has prevented the progression of the following sperm. Further investigations using TLC are underway to elucidate this mechanism of polyspermy prevention.