Characterization of a Monoclonal Antibody to a Human Intra-Acrosomal Antigen that Inhibits Fertilization1

Abstract

Among monoclonal antibodies (mAb) selected after the immunization of mice with human ejaculated spermatozoa, mAb I9G9 (IgG1 kappa) was found by immunoperoxidase staining to label most of the acrosome of human spermatozoa permeabilized with methanol-acetone. The antigen was poorly expressed on the surface of fresh ejaculated sperm, but was detectable on most viable sperm after 5-h incubation in medium containing human serum albumin (HSA) followed by 30-min incubation with the calcium ionophore A23187. This treatment resulted in acrosomal loss. Immunoelectron microscopy labeling with I9G9 mAb localized the antigen within the acrosome. Immunocytochemistry on testis sections showed that antigen was located in the round spermatids within the adluminal compartment of the seminiferous epithelium. Western blotting of sperm extract proteins showed that sperm intra-acrosomal (SIAA) recognized by I9G9 mAb had a polymorphism of immunogenic peptides from 16 to 35 kDa. Most of the antigenic peptides possessed an isoelectric point of approximately 5. When spermatozoa were treated with a series of protease inhibitors, the polymorphism of immunogenic peptides was reduced, suggesting that the multiple form of the antigen was due, at least in part, to proteolytic processing. In the testis, only a single peptide band of 35 kDa was detected with mAb I9G9. Studies of human tissue specificity by Western blotting showed that the epitope recognized by I9G9 mAb was present solely in ejaculated spermatozoa and the testis. I9G9 mAb did not agglutinate or immobilize sperm but inhibited the penetration of zona-free hamster ova by human sperm.

Germ cell-specific localization of immunoreactive riboflavin carrier protein in the male golden hamster: appearance during spermatogenesis and role in sperm function

“…The presence of strong immunoreactivity in the Golgi/acrosomal region, coinciding with the onset of acrosomal biogenesis, is consistent with the earlier report on the association of this protein with the head region of a few mammalian spermatozoa (Bhat et al 1995). Such a pattern of immunolocalization that we observed is shared by several other acrosomal-specific proteins, such as Tep22 (Neesen et al 2002), proacrosin (Phi- Van et al 1983), protein disulfide isomerase (Ohtani et al 1993), SP-10 (Kurth et al 1993), SIAA (Jimenez et al 1994), rat 2B1 (Jones et al 1996), ACIII (Gautier-Courteille et al 1998), Kir3.2d (Inanobe et al 1999), Acrin1 (MN7) (OhOka et al 2001) and CYP51 (Cotman et al 2001). However, the site of biosynthesis of immunoreactive RCP in various mammalian species (Subramanian & Adiga 1996) remains to be investigated.…”