Effect of modified membrane vesicles from seminal plasma on the fertilizing capacity of rabbit spermatozoa.

Partial extraction of cholesterol and phospholipid from membrane vesicles in rabbit seminal plasma decreased their inhibitory effect on fertilizing capacity in rabbit spermatozoa. Pronase digestion, to remove surface proteins, had no pronounced effect on vesicle decapacitation activity. Evidence of fusion between these vesicles and spermatozoa was obtained using [³H] galactose labelled vesicles. The results are consistent with addition of vesicle lipid (cholesterol) to the sperm plasma membrane causing an inhibition of fertilizing capacity.     

Occurrence of prostasome-like membrane vesicles in equine seminal plasma

Equine seminal plasma was shown to contain membrane vesicles that are similar to the well characterized prostasomes in human seminal plasma. Determination of nucleoside and nucleotide concentrations of these particles have shown that ATP, ADP and adenosine are the main components of the nucleotidic pool. 5' nucleotidase, endopeptidase and dipeptidyl peptidase i.v. activities have been found on the surface of the particles. The interaction between these prostasome-like vesicles and spermatozoa was demonstrated by electron micrograph scans which revealed the steps of a fusion-like process leading to mixing of the membranes. In addition, endopeptidase activity, a marker enzyme of these seminal vesicles that is normally absent from equine spermatozoa, was shown to be acquired by these cells after interaction with the vesicles. The addition of these vesicles to equine spermatozoa resulted in the modification of adenylate catabolism. Therefore, a role in stabilizing the energy charge of the spermatozoa thus allowing longer viability is proposed for these organelles.     

Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.     

Effect of seminal plasma on the characteristics and fertility of rabbit spermatozoa

The effects of different dilutions of seminal plasma (SP) on the qualitative characteristics of rabbit spermatozoa and on their fertilising ability were analysed. Ejaculated semen was centrifuged twice and the sperm resuspended in media with decreasing ratios of SP/Tris: (1/2; 1/5; 1/10; 1/20; 1/30; 1/100) until the complete substitution was with SP. The control constituted sperm in undiluted SP. Samples were maintained at 37 degrees C and kinetic analysis done at fixed intervals (0-6h). Also the thiobarbituric reactive substances (TBA-RS) values were determined. Rabbit sperm suspended in Tris, or with extremely low content of SP, lost motility and viability within 1-3h, while sperm suspended in SP either undiluted or diluted up to 10-fold, showed similar motility during the 6h period (from 39 to 49%). Further dilutions of SP (1/20-1/30) had no effect during the initial 2h of storage but thereafter the decline of motility was more marked (after 6h: from 0 to 17%). Kinetic parameters followed the same trend and differences were particularly marked after storage: the highest values were in samples diluted up to 1/10; a sharp decline in motility characteristics was observed at higher dilutions. The addition of SP (1/2 v/v) to immotile sperm reactivated 35.5% of cells. However, SP did not significantly affect fertility rate or litter size possible involving an interaction with the female reproductive tract. SP reduced lipid oxidation (TBA-RS) of semen only after storage. A positive correlation between final TBA-RS and cell viability indicated that peroxidation was one of the cause of rabbit sperm deterioration during conservation.     

Seminal plasma proteins and the reaction of spermatozoa from intact boars and from boars without seminal vesicles to cooling.

Spermatozoa from intact boars and from boars without seminal vesicles were resuspected in diluent and cooled at different rates to 0 degrees C. Glutamic oxaloacetic transaminase and lactate dehydrogenase activities were greater in the diluents which had contained spermatozoa from intact boars than in those which contained spermatozoa from animals without seminal vesicles. The incubation of seminal plasma from an intact boar with spermatozoa from a vesiculectomized animal before cooling also increased the enzyme activity in the diluent. The factors responsible for this effect were associated with the basic protein fractions of boar seminal plasma, in particular the proteins with haemagglutinating activity which may have been adsorbed onto the spermatozoa. Spermatozoa were exposed to colloidal Fe(OH)2+ to determine by electron microscopy the charge on the surface of the plasma membrane of washed epididymal spermatozoa and ejaculated spermatozoa from intact and vesiculectomized boars. Epididymal spermatozoa bound the positively charged particles more readily than the ejaculated spermatozoa from the intact boars, due to the absence of membrane-bound protein.     

Aprotinin and a seminal plasma factor inhibit the motility of demembranated reactivated rabbit spermatozoa

The demembranation and reactivation of ejaculated rabbit spermatozoa have been studied. ATP, Mg, glutamate, dithiothreitol (DTT), and Tris-HCl were found to be essential for a good reactivation. With this experimental model, we investigated the effects of protease inhibitors on the reinitiation of movement by ATP and on the movement of already motile spermatozoa. Soybean trypsin inhibitor (STI) prolonged the length of reactivation by 7- to 8-fold, whereas pepstatin, antithrombin III, phenylmethylsulfonyl fluoride (PMSF), and alpha-1-antitrypsin had no significant effect. Aprotinin (1.5 micrograms/ml) and leupeptin (50 micrograms/ml) completely prevented the reinitiation of movement by ATP; aprotinin at the same concentration even blocked the movement of motile spermatozoa. A tissue-specific seminal plasma factor could also prevent both the reinitiation of movement and the movement of motile spermatozoa. However, it took 2-3 times the amount of seminal plasma to stop the movement than to prevent the reinitiation of movement. The inhibitor in the seminal plasma is most probably not a protease or an aprotinin-like protease inhibitor since a partially purified preparation of the seminal plasma inhibitor does not hydrolyze a trypsin substrate, is not inhibited by protease inhibitors and has no significant capacity to inhibit trypsin. The data suggest that aprotinin and the seminal plasma inhibitor block movement through different mechanisms. Aprotinin and the seminal plasma inhibitor represent two new tools to study the regulation of sperm movement.     

Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa

Summary The seminal vesicles synthesize in an androgen-dependent manner a neutral protein of 13.5 kDa molecular weight that makes up about 40% of their secretion (major protein). An antiserum against this protein raised in rabbits was used to localize the antigen within the seminal vesicles. In addition to intraluminal secretion of the seminal vesicles and the ampulla of the vas deferens, ejaculated and ampullary spermatozoa revealed an intense immunoreaction, which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Comparison of spermatozoa taken from the tail of the epididymis with ampullary spermatozoa showed that about 90% of the latter, but only 10–20% of the former presented this distributional pattern of immunoreactive sites. Epididymal epithelium as well as calf seminal vesicle epithelium showed no immunoreactivity with major protein antiserum. Using a pre-embedding staining technique with gold-labeled primary or secondary antibodies, respectively, no immunostaining could be achieved at the ultrastructural level. Incubation experiments of epididymal spermatozoa in EGTA-containing solutions in the absence of calcium resulted in a gradual labilization and eventual loss of the plasma membrane of the sperm middle piece. After removal of (at least part of) the plasma membrane, bound major protein could be visualized immunohistochemically close to the mitochondria of the middle piece using a gold-labeled primary or secondary antibody. The acceptor site for major protein therefore seems to reside inside the plasma membrane of the sperm middle piece. Incubation of epididymal spermatozoa in phospholipase-containing solutions removed the acceptor site from the spermatozoa. Separation by polyacrylamide treatment of proteins from epididymal sperm cells extracted by sodium hydroxide or phospholipase treatment, subsequently transblotted on nitrocellulose sheets and directly labeled with gold-tagged major protein, demonstrated a protein duplet with a molecular weight of 65 and 67 kDa, respectively, which appears to represent the specific binder of major protein underneath the sperm surface. Binding of major protein to this 66 kDa acceptor site is regarded as a physiological event that may be related to the onset of hyperactivated sperm motility.Dedicated to Professor Dr. Th.H. Schiebler on the occasion of his 65^th birthdayThis study was supported by the Deutsche Forschungsgemeinschaft (grant Au 48/7-8)     

Metabolism of phospholipids by spermatozoa and seminal plasma

1. The hydrolysis of added (32)P-labelled phospholipids by whole ram and bull semen and the separated spermatozoal and plasma components was examined. 2. The ethanolamine phosphoglycerides were rapidly attacked by washed spermatozoa, forming predominantly glycerylphosphorylethanolamine, but with whole semen and seminal plasma a lysophosphatidylethanolamine was also detected. 3. The hydrolysis of lecithin by spermatozoa and plasma was very slow, and glycerylphosphorylcholine was the sole product detected. 4. Ram testicular spermatozoa were comparatively inactive in metabolizing both phospholipids, but ampulla contents showed the same activity as ejaculated semen. 5. Phosphatidylinositol was metabolized by spermatozoa obtained from any portion of the ram reproductive tract and also by seminal plasma. With testicular components, ampulla contents and washed ejaculated spermatozoa, inositol monophosphate, an unidentified phosphate ester and inorganic phosphate were the main products. In contrast, with whole semen and seminal plasma, glycerylphosphorylinositol was the predominant water-soluble phosphate ester. 6. Accessory-gland secretion obtained from vasectomized rams showed a pronounced phospholipase A activity towards ethanolamine phosphoglyceride. 7. On aerobic incubation of whole ram semen there was a decrease in the concentration of all phospholipid classes, although cardiolipin showed the greatest percentage decrease. In the choline phosphoglyceride fraction, this loss was confined to the plasmalogen component. This breakdown of phospholipids was decreased considerably when the spermatozoa were washed, and was not observed when whole bull semen was incubated under similar conditions.     

Catalase activity in human spermatozoa and seminal plasma

Catalase activity was determined in human semen by measuring the oxygen burst with a Clark electrode, after H₂O₂ addition. Significant catalase activities (mean ± SD) were found in migrated, motile spermatozoa (44 ± 17 nmoles O₂/min/10⁸ cells) and in seminal plasma of normozoospermic men (129 ± 59 nmoles O₂/min/ml). It has been demonstrated that seminal catalase originated from prostate; however, its activity was not correlated with the usual prostatic markers (such as citric acid and zinc). Our data suggest a multiglandular function secreted by this organ. The catalase activities measured in seminal samples from asthenozo-ospermic, infertile men were found lower than those from normozoospermic subjects. The understanding of the relative contribution of the different enzyme systems against O₂ toxicity (superoxide dismutase, catalase, glutathione peroxidase) seem to be a priority area of research to understand disturbances of sperm function.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Osmotically reactive plasma membrane vesicles prepared from rabbit kidney tubules by mild hypotonic lysis

Plasma membrane vesicles were obtained by hypotonic lysis in an ice-cold medium containing EDTA and MgCl₂. The vesicles were isolated by differential centrifugation. Compared to a total kidney homogenate, a 10–12-fold enrichment of trehalase and alkaline phosphatase (marker enzymes for renal brush border), and a 6-fold enrichment of (Na⁺---:K⁺)-stimulated ATPase, (a marker enzyme for the basal and lateral plasma membrane of the tubule cell), was achieved. Contamination by other cell organelles was very low. The plasma membrane vesicles enclosed small amounts of the cytoplasmic enzymes lactate dehydrogenase and malate dehydrogenase, which exhibited full activity only after their release into the medium by sonication.Electron micrographs of this preparation showed microvilli with drumstick-like expansions, but also spherical vesicles. By measuring the distribution of radio-actively labelled compounds of different molecular weight in a packed sediment of the plasma membranes under isotonic conditions, an intravesicular volume of 82% or 9 μl/mg of membrane protein was estimated. The intravesicular volume decreased when the osmolality of the medium was augmented by raffinose. The scattering of light by the vesicular suspension could be used to monitor rapid volume changes. By this method, the following sequence of flux rates was established: glycerol>erythritol> adonitol>mannitol. The fluxes of LiCl, NaCl, and KCl were almost identical, but faster than those of adonitol and mannitol. The data indicate, that a large fraction of the plasma membrane isolated in this preparation have formed vesicles, and also that they have retained, as far as investigated, the permeability characteristics of the plasma membrane.     

Lipids of plasma membrane and outer acrosomal membrane from bovine spermatozoa

Plasma membrane (PM), primarily from the anterior sperm head, and outer acrosomal membrane (OAM), were isolated from ejaculated bovine spermatozoa, and the major lipid classes were characterized. Whole sperm (WS) lipids were analyzed for comparison. PM was removed by nitrogen cavitation and purified by sucrose density-gradient centrifugation. The OAM was removed by centrifugation through hyperosmotic sucrose and recovered by sucrose density-gradient centrifugation. The PM contained primarily spherical vesicles from the region overlying the OAM and was enriched 9- and 13-fold in 5'-nucleotidase and alkaline phosphatase activity, respectively, compared to the original cavitate. The OAM was recovered as caplike structures with associated ground substance. Protein, phospholipid, and cholesterol (PR, PL, and CH as micrograms/5 x 10(9) sperm) were 300, 467, and 93 for PM and 276, 111, and 25 for OAM, respectively. Corresponding values for WS (mg/5 x 10(9) sperm) were 31.4, 6.63, and 0.72. The PR/PL (w/w) and CH/PL (mol/mol) ratios were 0.66 and 0.38 for PM; 2.48 and 0.26 for OAM; and 4.39 and 0.22 for WS. Cholesterol was the only free sterol detected by gas/liquid chromatography in WS, PM, and OAM, with traces of CH sulfate present in all three preparations. Glycolipid tentatively identified as sulfogalactolipid was detected by thin-layer chromatography (TLC) in PM but not OAM. Phospholipid composition of WS and membranes was determined by TLC. Cardiolipin (3% of total PL) was present in WS only. Choline, ethanolamine, and inositol phosphoglycerides (CP, EP, PI, PIP, PIPP); sphingomyelin (SP); phosphatidylserine (PS); and lysophosphatidylcholine (LPC) were present in WS, PM, and OAM. Approximately 50% of total PL was CP in all preparations; SP was 13% of PL in PM and 17% in OAM (p less than 0.05); EP was 7% of PL in PM and 10% in OAM (p less than 0.05). The differences in composition between PM and OAM is discussed with respect to capacitation and ability of sperm to undergo the acrosome reaction.     

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.     

Adsorption of seminal plasma proteins by boar spermatozoa

Seminal plasma protein adsorption by boar spermatozoa was examined using ejaculated sperm from vesiculectomized boars and seminal plasma from vasectomized boars. Sperm adsorbed 14 pg protein/sperm in 10 min. When seminal plasma proteins were radiolabeled, most of the adsorbed radiolabel was present in low M(r) proteins, particularly a 12700 M(r) protein. A 349300 M(r) seminal plasma protein was also readily adsorbed. Three radiolabeled seminal plasma proteins (307600, 165400 and 7400 M(r)) were not detected on the sperm; either they are not adsorbed by the sperm or the sperm were previously exposed to these proteins in other accessory sex gland fluids and had already adsorbed them. A 29100 M(r) sperm protein was also radiolabeled (4.9% of the adsorbed radiolabel), although there was no corresponding seminal plasma protein. Large quantities of seminal plasma protein (particularly low M(r) proteins) are adsorbed by sperm not previously exposed to seminal vesicle secretion. The functions of these proteins are yet to be determined.