An MN9 antigenic molecule, equatorin, is required for successful sperm-oocyte fusion in mice.

The acrosome plays an important role in fertilization. This study was designed to examine the role and behavior of a molecule, equatorin (the antigenic molecule of the monoclonal antibody mMN9), localized at the equatorial segment of the acrosome. In vitro fertilization (IVF) investigation was conducted to examine the role of this molecule, by assessing the effect of mMN9 in TYH medium (a modified Krebs Ringer bicarbonate solution) containing mMN9 at 0 (control), 25, 50, and 100 microg/ml. Under these conditions, the IVF investigation was divided into two experiments: 1) the zona pellucida (zona)-intact experiment, in which capacitated sperm inseminated cumulus- and zona-intact oocytes; and 2) the zona-free experiment, in which acrosome-reacted sperm inseminated zona-free oocytes. It was found that mMN9 did not affect sperm motility, zona binding, or zona penetration, but it significantly inhibited fertilization, reducing the rates of pronucleus and two-cell embryo formation in both the zona-intact and zona-free oocyte experiments. In addition, when judged at 5 h after insemination in the zona-intact experiment, nearly half of the unfertilized oocytes had accumulated sperm in the perivitelline space (perivitelline sperm), and concurrently we confirmed by electron microscopy the presence of many unreleased cortical granules preserved beneath the oolemma, indicating no occurrence of sperm-oocyte fusion. Confocal laser scanning light microscopy with indirect immunofluorescence demonstrated that equatorin was localized at the equatorial segment in both capacitated and perivitelline sperm (acrosome-reacted sperm). These results suggest that equatorin that is preserved at the equatorial segment is involved in the process of sperm-oocyte fusion in mice.