Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies

Abstract

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.

Tssk6 is required for Izumo relocalization and gamete fusion in the mouse

“…In addition to exocytosis, it is well established that many proteins change their immunofluorescence pattern during the acrosome reaction (Saxena et al, 1986;Kawai et al, 1989). Results in the present work indicate that Tssk6-null sperm are capable of undergoing the acrosome reaction spontaneously or after treatment with Ca 2+ ionophore; however, antigen relocation (e.g.…”

“…Deficient actin polymerization in Tssk6-null sperm Changes in the immunofluorescence pattern of sperm surface proteins during capacitation and acrosome reaction have been described previously (Miranda et al, 2009;Saxena et al, 1986;Primakoff and Myles, 2007;Selvaraj et al, 2007). It has been shown that changes in protein localization are at least in part due to actincytoskeleton dynamics (Saxena et al, 1986;Hernandez-Gonzalez et al, 2000).…”

“…It has been shown that changes in protein localization are at least in part due to actincytoskeleton dynamics (Saxena et al, 1986;Hernandez-Gonzalez et al, 2000). Because of the failure of Izumo to relocate following the acrosome reaction and the defects observed in the head-tail connection in Tssk6-null sperm, the distribution of the actin cytoskeleton in WT and Tssk6-null sperm was investigated.…”

Induced hyperactivity in boar spermatozoa and its evaluation by computer-assisted sperm analysis

“…Hyperactivation in boar spermatoza has been mentioned several times (Saxena et al 1986, Blottner et al 1989, Hamano et al 1989, but an analysis of movement patterns in ejaculated boar spermatozoa and in spermatozoa flushed from the oviducts of pigs has only been reported by . A detailed investigation of hyperactivated boar spermatozoa using CASA has not yet been reported.…”

Mammalian Male Gametogenesis: Growth, Differentiation and Maturation of Germ Cells

“…On the other hand, Saxena eta/. (63) reported that an antigen located on the pig sperm head migrates to the tail region during capacitation. In some cases, the migration of the antigen is directly linked with the appearance of fertilizing ability.…”