An MN9 Antigenic Molecule, Equatorin, Is Required for Successful Sperm-Oocyte Fusion in Mice1

Abstract

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.

Equatorin-Related Subcellular and Molecular Events During Sperm Priming for Fertilization in Mice

“…Equatorin is composed of a complex of 48-kDa and 38-kDa proteins located in the acrosome. The antigen was renamed "equatorin" because the MN9 antibody showed a strong affi nity for the equatorial segment and the MN9 antigen was found to be enriched in the equatorial segment (Toshimori et al 1998 ). Purifi ed MN9 antibody suppresses the events that occur from acrosome reaction to egg activation under in vitro (Toshimori et al 1998 ) and in vivo conditions (Yoshinaga et al 2001 ).…”

“…The antigen was renamed "equatorin" because the MN9 antibody showed a strong affi nity for the equatorial segment and the MN9 antigen was found to be enriched in the equatorial segment (Toshimori et al 1998 ). Purifi ed MN9 antibody suppresses the events that occur from acrosome reaction to egg activation under in vitro (Toshimori et al 1998 ) and in vivo conditions (Yoshinaga et al 2001 ). The MN9 antigen is relocated from the acrosome to the plasma membrane over the equatorial segment during the acrosome reaction (Manandhar and Toshimori 2003 ).…”

“…The outer acrosomal membraneanchored proteins could be involved in the priming process, including the signaling pathway to activate internal molecules, and could also function as components of the platform to maintain the acrosomal structure and other crucial fertilization events. These proteins include ADAM3/cyritestin (Forsbach and Heinlein 1998 ), ZPBP1/SP38/IAM38 (Yu et al 2006 ;Ferrer et al 2012 ), SPACA1/SAMP32 (Hao et al 2002 ;Ferrer et al 2012 ), SPACA4/SAMP14 , OBF13/ Izumo1 (Okabe et al 1987 ;Inoue et al 2005 ), SPESP1/ESP , and MN9/equatorin (Toshimori et al 1992(Toshimori et al , 1998. Because the proteins proposed to be essential for sperm-egg fusion, sperm Izumo1 (Inoue et al 2005 ) and egg CD9 (Kaji et al 2000 ;Le Naour et al 2000 ;Miyado et al 2000 ), are discussed in other chapters; here, we focus on the nature of the acrosomal protein equatorin (Eqtn ) and its related subcellular and molecular events during the sperm priming process in mice.…”

The Mechanism of Sperm–Egg Fusion in Mouse and the Involvement of IZUMO1

“…To verify the acrosome reaction, we stained the spermatozoa with an MN9 monoclonal antibody, which stains only to the equatorial segment of acrosome-reacted spermatozoa (Toshimori et al 1998 ) (Fig. 32.1 ).…”

Fertilization and the oocyte-to-embryo transition in C. elegans

“…During the acrosome exocytosis, there are multiple fusion events between the plasma membrane and outer acrosomal membranes at the anterior region of the sperm head (56). As a result, the contents of the acrosome are released and the inner acrosomal membrane is exposed (7,56,57). This results in a reorganization of the plasma membrane of the sperm head and a change in localization of multiple proteins (56).…”