Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the A23187-induced acrosome reaction of guinea pig sperm

Abstract

Acrosome-intact mammalian sperm can adhere to zona pellucida-free oocytes but are only capable of fusing if they have previously undergone the acrosome reaction. This suggests that the acrosome reaction results in presentation of at least one novel epitope which plays a role in sperm-oocyte fusion. Monoclonal antibodies were raised against unfixed acrosome-reacted guinea pig sperm and screened by indirect immunofluorescence for binding to the equatorial segment. They were back-screened against unfixed acrosome-intact sperm for absence of binding. Using this approach, two antibodies, G11 and M13, were identified which detect equatorial segment epitopes presented de novo by sperm following an A23187-induced acrosome reaction. The localization of these epitopes to the equatorial segment was confirmed at the ultrastructural level by indirect immunogold-labelling. Fluorescein isothiocyanate-labelled Fab fragments of these two antibodies also localized to the equatorial segment. Affinity chromatography and western blotting established that the two mAbs recognize the same proteins, which have M(r)s of 34, 46, 48 and 51 × 10(3). When sperm were induced to undergo the acrosome reaction with A23187 and incubated with their discharged acrosomal contents, a further band was produced with an M(r) of 30 × 10(3). Production of this band was inhibited in the combined presence of 100 microM phenylmethylsulphonyl fluoride and 100 microM p-aminobenzamidine even though these compounds do not inhibit acrosomal exocytosis. Neuraminidase and O-glycosidase were without effect on the proteins detected by antibodies G11 and M13. Endoglycosidase F, however, eliminated the bands of M(r) 46, 48 and 51 × 10(3) and replaced them with a strong band of M(r) 44 × 10(3) and two minor bands of M(r) 43 and 45 × 10(3). Formaldehyde fixation of acrosome-intact sperm caused partial rupture of the acrosome with loss of the characteristic rouleaux (stacks) of guinea pig sperm. Indirect labelling of these formaldehyde-fixed sperm with fluorescein isothiocyanate- or gold-labelled second antibody, with or without permeabilization with 0.05% Triton X-100, showed dense labelling on the cytoplasmic face of the plasma membrane overlying the convex surface of the acrosome but little labelling elsewhere. Cryosections of acrosome-intact sperm labelled indirectly with immuno-gold showed labelling consistent with the same location, as well as sporadic labelling at other intracellular sites overlying the acrosome. Since there is no evidence that sperm can translocate intact membrane protein from the cytoplasmic face to the extracellular face of the plasma membrane during the acrosome reaction, the evidence suggests that there are two isolated antigen pools.(ABSTRACT TRUNCATED AT 400 WORDS)