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S. Villarroya and R. Scholler

Summary. The regional antigenic heterogeneity of human spermatozoa is confirmed with 6 monoclonal antibodies raised against ejaculated human spermatozoa. The topographical localization of the antigenic determinants suggests the existence of at least 6 domains on the human spermatozoon. Different fixatives had severe detrimental effects on the antigen—antibody binding. On live human spermatozoa, each antibody bound to a distinct region: acrosome, equatorial segment, entire tail, neck, midpiece and terminal piece. The antigens detected on the acrosome, equatorial segment and entire tail were surface components, whereas the other three were intracellular structures. The determinant present along the entire tail was a sperm-coating antigen. The molecular weights of the recognized antigens were estimated with the Western blot technique.

Immunostaining of individual ejaculates established that the percentages of positive cells were 12–56% for the acrosome, 8–35% for the equatorial segment, 90–100% for the entire tail, 20–52% for the neck, 9–35% for the midpiece and 36–90% for the terminal piece. In addition, labelling of motile and immotile spermatozoa showed differences in the percentages of positive cells, with 5 out of 6 monoclonal antibodies, or in the fluorescence intensity, with the last one labelling the entire tail.

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S. Villarroya and R. Scholler

Summary. An integral component of human spermatozoa, a glycoprotein of Mr 143 000 (two subunits of Mr 76 000 and 67 000) was recognized by the a-HS 1A.1 monoclonal antibody. The antigen was localized on the plasma membrane over the sperm head, as demonstrated by transmission electron microscopy. The antigen– antibody binding on gametes during changes in their functional state was followed by an indirect immunofluorescence assay of live human spermatozoa. In freshly ejaculated spermatozoa the antibody binding pattern revealed a patchwork quilt-like topography of the plasma membrane over the acrosome; the percentage of positive cells varied from 20 to 78% with a mean of 50% (n = 82). Incubation in a capacitation medium could increase this percentage up to 98%, revealing new epitopes in an energy-dependent and temperature-independent manner; concomitantly, a part of the antigen migrated in energy-independent and temperature-dependent manner and accumulated in a ring over the postacrosome. When an acrosome reaction was induced in vitro in the presence of Ca2+ with either A23187, ionomycin or human follicular fluid, the HS 1A.1 antigen migrated until immobilization in a well defined pattern around the equatorial segment (single band) or around the equatorial and postacrosomal segments (2 or, seldom, 3 bands). The new antigen localization resulted from a lateral diffusion of pre-existing molecules, occurred in only a few minutes, did not require energy and was temperature-dependent. At the same time, the well outlined large patch burst into a multitude of small spots before vanishing. This veil-like labelling was often observed in spermatozoa kept in the seminal plasma or treated with a metabolic poison.

The HS 1A.1 antigen localization reflects surface changes induced by the incubation in a capacitation medium and the acrosome reaction. Apart from the regional heterogeneity of the plasma membrane of a single cell, as noted above, there were differences in the plasma membrane changes in individual spermatozoa from the same ejaculate as well as in semen samples from different donors. The new antibody binding pattern was often alike in successive ejaculates of the same donor. In patients consulting for infertility the percentage of positive cells was often low and migration of the antigen was slight or absent.