Immunofluorescence antigen localization on boar sperm plasma membranes: Monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during sperm maturation and at ejaculation

Abstract

Purified boar sperm plasma membranes (PM) and PM proteins were used as antigens to produce 58 monoclonal antibodies against surface antigens. Fluorescence labelling (biotin-avidin-FITC) was used to determine the distribution of antigens in caput and cauda epididymal and in ejaculated spermatozoa with hybridoma supernatants and/or 1:100 diluted ascites fluid after subcloning. Sixteen areas (subdomains) of apparent restricted antigen mobility were identified and significant differences in the localization of most antigens in caput, cauda, and ejaculated PM were recognized. While localization patterns were highly reproducible with a given protocol for sample preparation and immunolabelling, localization patterns were markedly affected by changes in protocols. Fluorescence patterns were affected by the manner in which sperm were labelled (live sperm or sperm labelled at various steps), by washing, and by temperature or by addition of seminal plasma. These results indicate that the dynamic properties of the sperm PM or the surrounding fluids can easily mask or unmask or reconfigure binding sites for highly site-specific monoclonal antibodies and that antigen distribution is probably under-estimated when these labelling techniques are used. Such changes in the accessibility of antigenic sites to monoclonal antibodies limited determining the extent of distribution of a given antigen on epididymal sperm. However, the reproducibility of patterns when a given protocol is used and the large number of antibodies (39/42) displaying marked differences in localization on caput, cauda, and ejaculated PM suggest that changes in the organization of the PM constituents, whether by addition or subtraction of antigen or through configurational changes in proteins, are a major consequence of sperm maturation in the epididymis.

Rabbit Epididymal Secretory Proteins. II. Immunolocalization and Sperm Association of REP381

“…The failure to induce this change in localization by permeabilization of the sperm plasmalemma or by treatment in high ionic strength medium argues that the redistribution is the result of protein migration rather than exposure of a second population of REP38 previously cryptic on intact sperm. The migration of REP38 under capacitating conditions is consistent with the finding that sperm surface antigens migrate during capacitation and the acrosome reaction in a number of other species [30][31][32][33][34] and also with findings of an earlier report that the plasmalemma of rabbit sperm is remodeled after ejaculation [35].…”

Maturation of mammalian spermatozoa: modifications of the acrosome and plasma membrane leading to fertilization

“…The boar spermatozoa P86/5-antigen (78 K) is distributed over the entire head, except for the rostral region, but is localized in the rostral region during capacitation and the acrosome reaction (Töpfer-Petersen et al 1990). Distribution changes have also been shown in boar spermatozoa (Saxena et al 1986b).…”

Two Rat Middle-Piece Coating Antigens Are Lost upon in vitro Capacitation

“…This apparent discrepancy may depend on the fact that in vivo sperm-coating substances from the seminal plasma might possess higher af finity than CFS for binding sites of the distal tail. Similar differences for the binding of other proteins to the sperm cell in the pres ence or absence of soluble seminal factors have been reported [46][47][48]. Plasma mem brane modifications occurring during sperm maturation or at ejaculation [2.…”