Boar Sperm Plasma Membranes Research Articles

Characterization of human sperm plasma membrane: glycolipids and polypeptides.

The plasma membranes from ejaculated human spermatozoa were removed by nitrogen cavitation (600 PSI for 10 min) and isolated by centrifugation followed by a discontinuous sucrose density gradient centrifugation. Glycolipid analysis of the plasma membrane revealed a three-fold enrichment in gangliosides: GM3 and GD1a/GD1b and neutral glycolipids: globoside and sulfatide as compared to that of whole human sperm. Two dimensional electrophoresis of human sperm plasma membranes revealed about 75 polypeptides. Several of these polypeptides were similar in migration and in display of shape and color to that found in boar sperm plasma membranes.

Modulation of the lipid composition of boar sperm plasma membranes during an acrosome reaction in vitro

An in vitro acrosome-like reaction was induced in spermatozoa from the boar cauda epididymis by incubation in Tyrode's solution containing 1 mg/ml fatty acid-free bovine serum albumin. Plasma membranes were isolated from the spermatozoa at different times during the incubation and analyzed for their lipid composition. The total lipid, phospholipid, and glycolipid content of the membranes did not change during the acrosome-like reaction, whereas the amount of diacylglycerols and free fatty acids increased. Within the phospholipid class, a decrease of the inositol phospholipid and sphingomyelin content was observed, whereas the other phospholipids of the plasma membranes did not decrease significantly after 2 h of incubation. Changes in the sterol composition of the membranes were also observed. The onset of the lipid changes was correlated with the uptake of extracellular calcium by the spermatozoa. These results for the lipid changes in isolated sperm plasma membranes during an in vitro acrosome reaction provide the first direct evidence that a modulation of the plasma membrane lipid composition is involved in an acrosome-like reaction of mammalian spermatozoa.

Increase in the concentration of major boar sperm surface proteins during maturation in the epididymis.

High-resolution two-dimensional polyacrylamide gel electrophoresis analyses have indicated that several major boar sperm plasma membrane polypeptides (PMPs) increased in concentration during maturation in the epididymis. To investigate this further, monoclonal antibodies (MAbs) to two of these PMPs/glycoproteins referenced as 4.85 and 5.0 and polyclonal antisera (PCA) raised against PMP 5.0 were used to quantify these changes. ELISA assays for both PMPs, using solubilized plasma membrane (PM) and intact PM as antigens, indicated that both PMPs were present in greater concentration in cauda PM than in caput PM. ELISA assay using PCA against 5.0 and whole sperm from the cauda and caput epididymis also showed an increase in the concentration of this protein during transit through the epididymis. Indirect FITC fluorescence microscopy showed that MAbs reacted specifically with the luminal aspect of secretory cells of the corpus epididymis and, with greater intensity, with secretory cells of the cauda segment. When MAb tags were used the fluorescence technique indicated that PMP 5.0 was restricted to a narrow area of PM overlying the principal segment of the head of caput sperm but fluorescence extended further into the principal segment and into the flagellum of cauda PM. Since MAb binding sites appear to be partially masked, PCA were also used to localize antigenic sites. PCA to PMP 5.0 showed antigen localization in both head and flagellar PM of cauda and caput sperm. The fluorescence of cauda sperm PM, however, was significantly more intense than that of caput sperm PM. MAb to PMP 4.85 also bound to antigen more extensively on cauda sperm. The sum of these analyses suggests that major sperm PMPs are secreted by the epididymis and absorbed by sperm. The magnitude of surface protein changes occurring during epididymal transit in boar sperm appears to be greater than heretofore recognized.

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.

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.

Changes in the lipid content of boar sperm plasma membranes during epididymal maturation

Plasma membranes of boar sperm from caput, corpus and cauda of the epididymis were purified by differential- and sucrose-density equilibrium centrifugation and were found to yield a single band at a density of 1.13 g/cm 3. This fraction was enriched in acid and alkaline phosphatase, 5′-nucleotidase and (Na + + K +)-ATPase activities, whereas it contained minimal amounts of hyaluronidase and N-acetylglucosaminidase and no succinic acid dehydrogenase activities. The plasma membrane of caput, corpus and cauda sperm had the same phospholipid/protein and cholesterol/phospholipid ratios but yielded different amounts of protein and individual lipid classes. Several changes in the plasma membrane were observed during transit of sperm through the epididymis. Within the phospholipid class a decrease in the percentage of phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol was detected accompanied by an increase in amount of phosphatidylcholine, sphingomyelin and polyphosphoinositides. In the other lipid classes there was a decrease in the amount of free fatty acid and the major glycolipid. The amount of cholesterol decreased, while the amount of desmosterol and cholesterol sulfate increased. There was an increase in the amount of diacylglycerol. In addition, the changes in the fatty acid composition of the total membrane lipid and each phospholipid were determined. The above changes in the lipid composition of the plasma membrane during epididymal maturation may help to explain the decreased resistance to cold shock and changes in membrane fluidity of sperm during transit in the epididymis.

Isolation of major proteins from boar sperm plasma membranes by preparative gel electrophoresis and localization of a major polypeptide using specific monoclonal antibodies.

A preparative procedure using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is described for isolating major boar sperm plasma membrane polypeptides (PMPs) in soluble form. Proteins were first separated on 6 mm diameter gels using a pH gradient expanded in the acidic region. The second dimension used 6 mm thick, 10% acrylamide gels. Major proteins identified by Coomassie staining were excised and electroeluted. The procedure was applied to the isolation of a group of proteins in the molecular weight range 40K-50K which comprise a major fraction of the total integral membrane protein in these cells (groups 4 and 5). Yields of electrophoretically pure soluble polypeptides from these groups were between 0.3 mg - 0.5 mg from the processing of 16 gels per week. Electroeluted proteins were also used to elicit monoclonal antibodies to major proteins. Monoclonal antibodies to the major plasma membrane protein referenced as 4.85 were isolated and shown to be specific to this protein by transblotting procedures. This protein was primarily localized over the anterior portion of the principal segment of ejaculated sperm by indirect FITC fluorescence microscopy. The ability to isolate 60-100 mg of plasma membranes per week from the cauda epididymides of boars also permitted developing a procedure for the rapid fractionation of large amounts of detergent solubilized plasma membranes by isoelectric focusing in flatbeds of Biogel P200. For the first time, individual proteins of sperm surface proteins can be isolated in large enough amounts to begin detailed biochemical characterization, localization, and functional testing.

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

AbstractBoar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm‐zona attachments.

Properties of boar sperm plasma membranes (PM): Proteins released by washing and differential solubility in salts, detergents, and sensitivity to surface radiolabelling

AbstractIn this study a variety of properties of boar sperm plasma membrane proteins were examined. A qualitative and quantitative analysis of proteins washed from boar sperm revealed that large numbers and a variety of polypeptides (Ps) are easily removed from sperm upon washing. Initially (by the second wash), Ps are released from the plasma membrane (PM) of epididymal sperm and primarily correspond to those in epididymal fluid, but eventually (fifth wash) Ps are released that are not seen in epididymal fluid nor as components of the PM. These Ps appear to originate from the sperm cytosol and signal the damaging effects of extensive washing on sperm. Upon washing, ejaculated sperm release Ps characteristic of both epididymal fluid and accessory sex glands. Epididymal Ps are almost completely released by the fourth wash; accessory gland proteins appear to be more tenaciously bound and continue to be released with further washing. Most basic accessory gland Ps bind strongly enough to resist the series of washes necessary for the preparation of PM vesicles. About one‐half of ejaculated sperm lose motility after five washes, but evidence of massive release of internal Ps, such as seen in epididymal sperm, is not noted. In the epididymis and after ejaculation, sperm are coated with numerous Ps which are released upon washing; many are released nonspecifically and rapidly, others are more firmly bound. These analyses extend the surface map of boar spermatozoa to include a description of loosely bound proteins and their origin. These results also indicate that the qualitative and quantitative changes in surface membrane protein composition, occurring after simple washing, are significant and may confound the interpretation of surface composition changes in studies which rely solely on immunological or radiolabelling procedures.In order to determine the nature of the binding of major polypeptides (Ps) to the lipid bilayer of boar sperm plasma membranes (PMs), the solubility of Ps in solutions of different ionic strength and in detergents was examined. Several major polypeptides (identified in previously published surface maps) were extracted by hypotonic and hypertonic salt solutions, suggesting that electrostatic interactions play a major role in their binding to the bilayer. Other major proteins were extracted only by detergents, suggesting that these proteins are embedded deeply into the bilayer. These extraction procedures also provided a new strategy for isolating specific Ps in large quantities. Radiolabelling procedures identified about 80 surface‐exposed Ps, some of which are major constituents of the PM and others which are quantitatively minor components. Labelling of PM vesicles reveals about sixfold more Ps than does labelling of whole sperm. Increased labelling appears to be the result of surface accessibility of PM constituents after removal of loosely bound Ps from epididymal fluid and seminal plasma during the washings which accompany the preparation of PM vesicles from whole sperm. These results prescribe caution when interpreting changes in surface organization and membrane structure which are dependent solely on the use of radiolabels.

Characterization of (Ca2+ + Mg2+) adenosine triphosphatase activity and calcium transport in boar sperm plasma membrane vesicles and their relation to phosphorylation of plasma membrane proteins.

Properties of (Ca2+ + Mg2+) adenosine triphosphatase (ATPase) in plasma membranes from boar epididymal spermatozoa are described. Enzyme activity is optimum at high pH and has a high affinity for Ca2+. It is not inhibited by the calmodulin antagonist trifluoperazine (TFP), but it is inhibited by low concentrations of Ca2+. Plasma membrane vesicles obtained by hypotonic lysis of intact sperm [mixed inside-out (IOV) and right side-out (ROV) vesicles] transport 45Ca2+ in the presence of oxalate. Similar to the Ca2+-stimulated Mg ATPase activity, transport is unaffected by TFP, but unlike the ATPase, transport is at an optimum rate near neutral pH and is completely inhibited by p-chloromercurphenylsulfonate (pCMS). When plasma membranes are labeled in the presence and absence of Ca2+ and Mg2+ with [gamma-32P]ATP, differences in the intensity of labeling and lability of bound 32P to alkali and hydroxylamine suggest that two polypeptides between 100-120K may be related to a transport ATPase. The addition of TFP at concentrations which stimulate net Ca2+ uptake in intact cells causes intense labeling of a single neutrally charged protein near 68K. These labeling patterns and the properties of (Ca2+ + Mg2+) ATPase identify particular plasma membrane proteins (PMPs) from the complex surface of these cells that may be involved in Ca2+-dependent functions and support the view that calmodulin is not directly involved in the regulation of ATP-driven Ca2+ efflux from boar spermatozoa.

Boar sperm cytoplasmic droplets: Their ultrastructure, their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes

Cytoplasmic droplets of the boar are progressively lost from the flagellum of boar spermatozoa during epididymal transit, at ejaculation and during the nitrogen cavitation technique for isolation of plasma membranes. Apparently very fragile, these structures are broken up in the fluids of the reproductive tract and in the buffer used during the nitrogen cavitation procedure. The maximal potential contamination of cytoplasmic droplet internal vesicular membranes in plasma membrane fractions was determined to be 2.2% of the entire membrane surface area collected. The highly sensitive silver-stained, two-dimensional (2-D) polyacrylamide (PAGE) gels of boar sperm plasma membranes did not reveal cytoplasmic droplet, internal membrane, marker polypeptides, further demonstrating the high purity of plasma membrane preparations. In addition, freeze-fracture demonstrates that the internal membranes of the cytoplasmic droplet show few intramembranous particles and these may contribute little protein to plasma membrane preparations. The presence of two forms of vesicular elements in boar sperm Cytoplasmic droplets (typical vesicles and collapsed vesicles) is described.

Electrophoretic map of boar sperm plasma membrane polypeptides and localization and fractionation of specific polypeptide subclasses.

A high resolution, two-dimensional (2-D) electrophoretic map of the plasma membrane (PM) polypeptides from the ejaculated boar spermatozoon is described. 2-D silver-stained polyacrylamide gel electrophoresis (PAGE) gels revealed over 250 polypeptides; Coomassie blue staining revealed more than 100. Fifty Coomassie-staining polypeptides were catalogued and biochemically characterized, with twenty of these designated major sperm PM polypeptides. Cavitation pressures ranging from 50 PSI to 1000 PSI were used to enrich 2-D maps either in head PM (50 PSI) or in flagellar PM (1000 PSI) and provided tentative localization of certain PM polypeptides. Immunoabsorption chromatography showed that most major polypeptides seen in the 2-D map were antigenic. Major polypeptide bands from single dimensional (1-D) gels were excised, antibodies against them were produced in rabbits, and the polypeptides were localized via indirect fluorescein isothiocyanate (FITC) technique. Cross-reacting antigenic determinants in the PAGE PM polypeptides were determined by transblotting and staining the transblots by an indirect peroxidase technique. Cross-reactivity was extensive with several polypeptide groups, but specific enough with others to allow tentative localization. Lectin affinity chromatography using Con A, WGA, RCA-1, PNA, and DBA indicated the lectin specificity of PM polypeptides. These data together with periodic acid-Schiff (PAS) and carbohydrate-specific silver staining permitted identification of glycoproteins in the 2-D maps. FITC coupled to specific lectins showed the regional distribution of these lectins on the sperm surface. The 2-D polypeptide map and the catalogue of properties of major Coomassie-stained PM polypeptides provides a reference for future studies in the boar and other species.

Characterization of boar sperm plasma membranes by two-dimensional PAGE and isolation of specific groups of polypeptides by anion exchange chromatography and lectin affinity chromatography.

High resolution, two-dimensional PAGE was used to characterize purified plasma membranes (PM) from boar spermatozoa. In addition to the abundance of polypeptides resolved, unusual features of PM discerned in gel patterns include a large number of basic polypeptides and the presence of at least three polypeptides with molecular weights in excess of 200 K daltons. In order to reduce these large numbers of polypeptides into smaller fractions, an important step in developing a surface map of these membranes, detergent-solubilized membranes were fractionated by the techniques of ion exchange chromatography and lectin affinity chromatography. PM polypeptides, solubilized in the nonionic detergents, were fractionated on DEAE Sephadex using phosphate ion gradients containing EDTA. Most of the major acidic polypeptides were eluted as a group at high phosphate ion concentrations. Membrane polypeptides can be separated into smaller populations by affinity chromatography on wheat germ agglutinin-agarose (WGA) or by Concanavalin A-agarose. Concanavalin A-agarose binds most PM glycoproteins, but partial purification of an important group of very basic proteins was obtained by repeated chromatography of polypeptides that failed to bind to this lectin. Since biologic activity (including immunologic potency) is often retained in the detergents used, the ability to fractionate the major polypeptide of the boar sperm PM into different and much smaller populations in large quantity and to characterize these proteins by electrophoresis provides a starting point for determining the functional significance and location of the major surface proteins of the boar sperm PM.

The effects of antisperm plasma membrane antibodies on sperm-egg binding, penetration, and fertilization in the pig.

A procedure using published surgical techniques is described for determining the effects of antiplasma membrane antibodies on sperm-egg binding, penetration, and fertilization in vivo in the domestic pig. Time of ovulation was controlled and sperm inseminated at precise times relative to ovulation. Sperm were exposed to antibodies (Fab) and then washed free of excess antibody and placed in one uterine horn near the uterotubal junction. Sperm exposed to control material were placed in the opposite horn as a control. One horn was tied with two ligatures near the body of the uterus. Eggs were collected by flushing the oviducts at prescribed times after insemination. in nine animals used for this study, antibodies to boar sperm plasma membranes completely blocked sperm-zona binding, penetration of the zona, and fertilization. In contrast, sperm were bound to the zona pellucida and penetrated eggs in every case on the control side. These results indicate that insemination in vivo is useful in screening antibodies produced against specific plasma membrane antigens to determine their role in fertilization.

Activity and location of cation-dependent ATPases on the plasma membrane of boar spermatozoa

Purified boar sperm plasma membrane vesicles originating primarily from the sperm head, and plasma membrane containing greater amounts of flagellar plasma membrane were assayed for cation-dependent ATPase activity. The ATPase activities were compared with partially purified outer acrosomal membranes and with fused plasma membrane-outer acrosomal membranes prepared from artificially acrosome reacted spermatozoa. Evidence was obtained for the presence of a Ca+2-ATPase in plasma membranes from sperm heads and from flagella. The ATPase may be associated with ATP-dependent calcium extrusion since Mg ATP inhibited calcium binding to plasma membranes. (Na+ + K+) ATPase activity appears to be restricted to the flagellar plasma membrane. No evidence for the presence of a proton translocase such as has been detected in sperm head membranes of the hamster, was detected in either plasma membrane or outer acrosomal membranes.

Electrophoretic and Chromatographic Properties of Boar Sperm Plasma Membranes: Antigens and Polypeptides with Affinity for Isolated Zonae Pellucidae

Two methods were used to assess the ability of plasma membrane (PM) polypeptides to bind to isolated zonae pellucidae (ZP). Crossed immunoelectrophoresis patterns of detergent solubilized PM were compared in the presence and absence of ZP. Two antigens which represented less than 20% of the total antigen response were significantly changed in electrophoretic mobility and precipitin peak height. In the second experiment, detergent solubilized PMS, labeled with iodine‐125 (1125), were absorbed to a column containing purified ZP. Washed columns eluted with SDS‐urea were found to be enriched in several polypeptides which migrate in PAGE‐SDS gels below 22 Kd. Comparison of PAGE patterns of PM originating primarily from the sperm head with those enriched in PM from other regions of the sperm surface suggest that these low molecular weight polypeptides with affinity for isolated ZP are concentrated in the sperm head. These experiments provide a base for determining which PM components are involved in spermegg interactions in vivo.

Evaluation of the purity of boar sperm plasma membranes prepared by nitrogen cavitation.

Quantitative electron microscopy is used to provide new information regarding the purity and the amounts of plasma membrane originating from various regions of the surface of the boar spermatozoon when such membranes are prepared by nitrogen cavitation and sucrose density centrifugation. These data show that the plasma membranes originate primarily from the head of the spermatozoon; they also show that contamination of these plasma membranes by acrosomal and mitochondrial membranes is negligible. Immunolocalization, using plasma membrane vesicles and divalent IgG directed against plasma membrane antigens, shows that plasma membrane vesicles bind extensively to intact sperm plasma membranes but rarely bind to the outer acrosomal membrane. These new data, taken together with that which has been previously reported, indicate that means for obtaining undamaged, highly purified

Characterization and localization of adenylyl cyclase in membrane vesicles and intact boar and human spermatozoa.

The enzymic properties of adenylyl cyclase in purified membrane vesicles from human and boar spermatozoa are described. Plasma membrane vesicles, which appear to be right-side-out, show a marked increase in activity in the presence of the detergent Triton X-100, manganous ion and alkaline pH. Electron-microscope cytochemical assays indicated the presence of adenylyl cyclase in boar and human sperm plasma membranes and also within the axoneme of intact human spermatozoa. The significance and precautions in the evaluation of the cytochemical data are discussed.