Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the a23187-induced acrosome reaction of guinea pig sperm
Authors: Allen C. A. , Green D. P. L. 
Published: April 2021

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 immunogoldlabelling. 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 Mrs of 34, 46, 48 and 51×103. 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 Mr of 30×103. Production of this band was inhibited in the combined presence of 100 μM phenylmethylsulphonyl fluoride and 100 μM 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 Mr 46, 48 and 51×103 and replaced them with a strong band of Mr 44×103 and two minor bands of Mr 43 and 45×103. 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. One pool allows sperm to present epitopes de novo on the equatorial segment at the time the acrosome reaction occurs. The possible location of at least part of the precursor pool for these epitopes was established using a third monoclonal antibody, G3. This antibody binds to the equatorial segment of acrosome-reacted sperm and cross-reacts with the 34 kDa antigen recognized by antibody G11. In acrosome-intact sperm, antibody G3 binds to the extracellular face of the anterior plasma membrane of the head. It follows that the 34 kDa antigen cannot be recognized by antibodies G11 and M13 in this location, even though it is recognized by these antibodies in detergent extracts. Trypsinization of acrosomeintact sperm was without effect in generating the epitopes recognized by antibodies G11 and M13. Expression of the epitopes was not inhibited when the acrosome reaction was induced in the presence of 1 mM p-aminobenzamidine. The evidence suggests that the equatorial segment antigen recognized by antibodies G11 and M13 is either 34 kDa protein which has undergone a conformational rearrangement during the acrosome reaction, or a smaller protein derived from the 34 kDa protein by enzymic processing. All three antibodies (G3, G11, M13) were able to block sperm/oocyte fusion in heterologous fusion assays between guinea pig sperm and hamster oocytes. The evidence suggests that the external 34 kDa antigen (and possibly some product derived from it) may play some role in the fusion of sperm and oocyte.