Characterization of the antigen recognized by a monoclonal antibody MN9: unique transport pathway to the equatorial segment of sperm head during spermiogenesis

Abstract

MN9, a monoclonal antibody raised against mouse spermatozoa, specifically recognizes the equatorial segment of sperm head in several mammalian species, including humans. Colloidal gold-immuno-electron microscopy of mouse spermatozoa has shown that the antigen is localized in the space between the outer and inner acrosome membranes and on the acrosome membranes at the equatorial segment. Immunoblotting after electrophoresis of spermatozoa from the cauda epididymidis has identified two immunoreactive bands: 38 kDa and 48 kDa in mouse, and 48 kDa in rat. During spermiogenesis in rat, this antigen is transported to the equatorial segment via a unique pathway, first appearing in some cisternae of the endoplasmic reticulum and in the Golgi apparatus of spermatids at around step 3. The antigen can further be found on the vesicles at the trans-side of the Golgi apparatus, in the matrix of the head cap, and on the head cap membrane in step-4 to step-7 spermatids. The antigen appears to be concentrated at the equatorial segment during late spermiogenesis. Neither the (pro-)acrosomic granule nor the surrounding membrane are required in this pathway. This pathway can be termed the 'Golgi-head cap tract'.

A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin

“…Intact sperm were found to be negative to MN9 antibody ( Fig. 2A and B), as previously reported (Toshimori et al 1992, Yamatoya et al 2009). In contrast, MeOH-treated sperm became positive to MN9 antibody; sporadic staining was found at the anterior acrosome, while weak staining was observed at the center of the equatorial segment ( Fig.…”

“…Therefore, the assessment of the acrosome reaction progression by PNA mainly reflects the status of the OAM. This information is important when we consider that the MN9 antibody recognizes the region including carbohydrate moieties on the threonine 138 aa as at least a part of the MN9 epitope (Yamatoya et al 2009), where the functional domain leading to egg activation is supposed to be localized (Toshimori et al 1992).…”

“…To address this issue, we have chosen the acrosomal membrane-anchored molecule equatorin (formerly called MN9 antigen; Toshimori et al 1992, Yamatoya et al 2009); MN9/equatorin is a widely distributed acrosomal protein in mammalian sperm, including human sperm. Equatorin is thought to be involved in the process of fertilization (possibly sperm-egg fusion or an early stage of egg activation) because MN9 antibody inhibited fertilization both in vitro (Toshimori et al 1998) and in vivo (Yoshinaga et al 2001) without inhibiting zona pellucida penetration or sperm-egg binding.…”

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

“…Equatorin was fi rst detected as an MN9 antigen in the mouse, rat, hamster, and human mature spermatozoa using the mouse monoclonal antibody MN9 (Toshimori et al 1992 ). Equatorin is composed of a complex of 48-kDa and 38-kDa proteins located in the acrosome.…”

“…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.…”