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.
Volume 176, no. 10, p. 3036: Figure 1 should appear as shown below. Sequence from amino acids 601 to 644 was omitted in the original figure.
To better understand, to optimize, and to validate the technique of intratesticular (i.t.) injection, several parameters related to i.t. injection were examined. Volumes exceeding 50 microliters could be injected i.t.; however, testes frequently became excessively turgid and backflow of injected fluids occurred. Thus, a volume of 50 microliters or less was deemed optimal for injection. To determine the rate of distribution of substances throughout the testis, trypan blue was injected i.t. near the caudal pole of the testis, and the movement of dye was monitored. Within 2 min, the dye had spread approximately 1 cm from the site of injection, and in 5 min it had spread twice that distance. In 2 h, the dye had become distributed throughout the testis except at its extreme cranial pole. Seminiferous tubules did not take up dye, indicating that the spread of dye was via peritubular lymphatics. Seminiferous tubule histology appeared virtually unaffected by i.t. injection, even at regions adjacent to the site of injection, when a sterile 26-gauge or smaller bore needle was utilized. To determine disappearance from the testis, radiolabeled inulin was injected i.t. Half time for absorption was achieved at 1.75 h. Potential vehicles were explored in which compounds with a variety of physical properties could be injected. Gum tragacanth, normal saline, ethylene glycol, dimethyl sulfoxide (DMSO) mixed 1:1 with normal saline, sesame oil, and propylene glycol were found to be suitable injection vehicles, whereas ethanol, dissolved in normal saline in concentrations as low as 0.5% was found unsuitable. To assess vehicle efficiency, various vehicles were utilized with a known testicular toxin (taxol) and injected into one testis, and the histology was compared with the contralateral testis injected with vehicle alone. All vehicles, found suitable above, allowed dispersion of taxol to influence areas distant from the site of injection. Intratesticular injection assesses the potential of agents to directly affect the testis, and systemic metabolism is avoided. Their rapid spread throughout the lymphatics of the testes allows seminiferous tubules to be exposed to agents in innocuous vehicles more rapidly and in higher concentration than is often possible when using systemic injections.
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.
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