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Showing papers on "Sperm plasma membrane published in 1995"


Journal ArticleDOI
TL;DR: An analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.
Abstract: Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

325 citations


Journal ArticleDOI
TL;DR: The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.
Abstract: In order to extend the static information of immunola belling sulphogalactolipids in fixed boar spermatozoa, a fluorescent sulphogalactolipid analogue, galactose(3sulphate)-β1-1′[( N-lissamine rhodaminyl)-12-aminododecanoyl]-sphingosine, was incorporated into plasma membranes of living spermatozoa and its lateral distribu tion over the sperm head was studied. The fluorescent lipid was enriched in the apical ridge subdomain of freshly ejaculated sperm cells. After sperm binding to the zona pellucida the lipid redistributed to the equatorial segment of the sperm surface. A similar shift occurred during capacitation in vitro with 2 mM CaCl2 or with 4% (w/v) bovine serum albumin. The desulphated derivative galactose-β1-1′[(N-lissamine rhodaminyl)-12-aminododecanoyl]-sphingosine was also incorporated into the plasma membrane of freshly ejaculated sperm cells and clearly stained the apical ridge subdomain and the (pre)-equatorial subdomains of the sperm heads. The desulphogalactolipid analogue showed a slightly faster migration to the equatori al segment of the sperm plasma membrane than did its sulphated counterpart. The measured fluorescence intensity distributions correlated linearly with the spatial probe dis tribution, which was checked by fluorescence lifetime imaging microscopy. The observed migration of the incorporated glycolipids precedes the acrosome reaction and is one of the underlying molecular events likely to be important in the process of sperm capacitation. The results of this study suggest that lipid phase segregation is an important driving force for the organization of the sperm head plasma membrane into subdomains.

120 citations


Journal ArticleDOI
TL;DR: Sperm immobilization prior to ICSI damages the sperm plasma membrane, this damage is sufficient for thiol-reducing agents to gain access to the sperm nucleus, and PVP possibly interferes with sperm nucleus decondensation.
Abstract: In the present study we investigated the relevance of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) in the fertilization process. Using supravital staining of the spermatozoa with eosin and studying sperm decondensation with 2 mM dithiothreitol (DTT) in conditions imitating sperm handling during ICSI, we demonstrated that immobilization of the spermatozoon by squeezing its tail between the glass pipette and the bottom of the dish damages the sperm plasma membrane. Polyvinylpyrrolidone (PVP), which is usually present in the drop with the spermatozoon to facilitate its handling, was found to impede the access of both eosin and DTT to the sperm nucleus. We conclude that (i) sperm immobilization prior to ICSI damages the sperm plasma membrane, that (ii) this damage is sufficient for thiol-reducing agents to gain access to the sperm nucleus, and finally that (iii) PVP possibly interferes with sperm nucleus decondensation.

117 citations


Journal ArticleDOI
Norio Suzuki1
TL;DR: Seventy-four peptides producing similar biological effects, named sperm-activating peptide (SAP), have since been purified from the solubilized jelly layer of seventeen species of sea urchins distributed over five taxonomic orders.
Abstract: A decapeptide (GFDLNGGGVG) isolated from the solubilized jelly layer of the sea urchin Hemicentrotus pulcherrimus stimulates the respiration and motility of H. pulcherrimus spermatozoa and, in addition, produces a number of biological effects on H. pulcherrimus spermatozoa including increases in cAMP and cGMP levels, activation of a Na+/H+ exchange system, and increases in intracellular pH (pHi) and [Ca2+] ([Ca2+]i). The peptide activates the metabolism of endogenous phosphatidylcholine and promotes the acrosome reaction as a specific co-factor of a major acrosome reaction-inducing substance, fucose sulfate glycoconjugate. The peptide also induces an electrophoretic mobility change in the guanylate cyclase of the sperm plasma membrane with concomitant dephosphorylation and inactivation of the enzyme. Seventy-four peptides producing similar biological effects, named sperm-activating peptide (SAP), have since been purified from the solubilized jelly layer of seventeen species of sea urchins distributed over five taxonomic orders. These peptides show essentially the same biological effects on sea urchin spermatozoa although their activity and structures are specific at the ordinal level. Equilibrium binding experiments using a radioiodinated SAP-I analogue [GGGY(125I)GFDLNGGGVG] to H. pulcherrimus spermatozoa suggests the presence of two classes of receptors (high affinity and low affinity) specific for SAP-I binding. Based on the Kd values and EC50's for SAP-I's biological activity, we presume that the high affinity receptor is associated with respiration-stimulating activity and elevations in pHi, while the low affinity receptor is coupled to elevations in cGMP and [Ca2+]i. The radioiodinated SAP-I analogue crosslinks to a 71 kDa protein which contains a single membrane-spanning domain at almost near C-terminus. A SAP-I precursor which is synthesized in the accessory cells contains five SAP-I and seven SAP-I-like decapeptides, each separated by a single lysine residue.

107 citations


Journal ArticleDOI
01 Jan 1995-Lipids
TL;DR: It is concluded that trout spermatozoa do not display any homeoviscous adaptations in these conditions and the dietary fatty acid intake greatly modified the fatty acid profile of plasma membrane phospholipids.
Abstract: The effect of a long-term adaptation of rainbow trout to 8 and 18°C combined with a corn oil-or a fish oil-supplemented diet on the characteristics of the spermatozoan plasma membrane was investigated. The experiment lasted up to 22 mon during which spermatozoa were collected from the mature males. Spermatozoan plasma membranes were isolated by nitrogen cavitation, and the cholesterol content, phospholipid composition and fatty acid pattern were investigated. Membrane viscosity was assessed on whole cells by electron spin resonance using spin-labeled phospholipids. Neither diet nor rearing temperature influenced the cholesterol content of the plasma membrane nor the phospholipid class distribution. The rearing temperature of the broodstock only slightly affected the phospholipid fatty acids. A minor decrease in 18∶0 and increase in monounsaturated fatty acids was observed for the cold-adapted fish. These modifications were not sufficient to affect membrane fluidity, and we conclude that trout spermatozoa do not display any homeoviscous adaptations in these conditions. On the contrary, the dietary fatty acid intake greatly modified the fatty acid profile of plasma membrane phospholipids. The fish oil-fed trout displayed a much higher n−3/n−6 fatty acid ratio than did the corn oil-fed ones, but the 22∶6n−3 levels remained unchanged. Modifications in plasma membrane composition by the diet were obtained although neither of the two diets was deficient in essential fatty acids. The enrichment in n−3 fatty acids, however, did not affect plasma membrane fluidity which was unchanged by the diets.

79 citations


Journal ArticleDOI
TL;DR: It is demonstrated that the sperm mannosidase is an integral plasma membrane component of the rat sperm and is localized on the periacrosomal region of the sperm head, and proteolytic processing of the membrane-bound alpha-D-mannosidase during maturation of spermatozoa is demonstrated.

70 citations


Journal ArticleDOI
TL;DR: Examination of mouse sperm in hyposmotic media by microscopy revealed little swelling of the cells, indicating that the low-amplitude swelling model may be the one more applicable to these cells, consistent with the observed marked increase in Osmcrit and decrease in t at 0 degrees C compared to the other temperatures.

58 citations


Journal ArticleDOI
TL;DR: In the guinea-pig, a sperm plasma membrane protein is a strong candidate for the mediator of the fusion process by which the egg engulfs the sperm, and in the mouse, a 94-kDa protein appears essential for this binding.
Abstract: Sexual reproduction requires that the gamete carrying the male-derived haploid chromatin join with the gamete carrying the female-derived haploid chromatin during fertilization to produce the diploid zygote. To accomplish this feat, the sperm must not only meet the egg, it must recognize the egg and be recognized in turn by the egg, and in the end must enter and be engulfed by the egg. In this selective overview of gamete interactions that lead to fertilization, encounters of three kinds, followed by the finale of gamete fusion, are considered from the sperm's viewpoint, with particular emphasis on the mammalian species with the mouse as the principal model. The first encounter is with the zona pellucida of the egg, to whose surface the sperm must bind. Mouse sperm appear to have four binding sites for zona ligands. Three interact with sugar moieties of the oligosaccharide chains of the mouse zona glycoprotein ZP3; the fourth binds a peptide backbone arginine. Capacitation is not required for this encounter, but is obligate for the second encounter--induction of the acrosome reaction in the bound sperm. The acrosome reaction is an exocytotic process that makes available the enzymatic machinery needed for sperm penetration the zona which is the end point of a sequence of reactions directed by intracellular signalling systems. In mouse sperm, these systems are presumed to be activated by ligands on ZP3 binding to ligand-specific sperm receptors with consequent aggregation of receptors. No receptor has been identified with certainty, nor have candidates for putative ZP3 ligands been identified. Completion of the acrosome reaction allows the sperm to penetrate the zona and, bind to the egg plasma membrane, thereby completing the third encounter. In the mouse, a 94-kDa protein appears essential for this binding. In the guinea-pig, a sperm plasma membrane protein (formerly PH-30, now fertilin), is a strong candidate for the mediator of the fusion process by which the egg engulfs the sperm. Decondensation of the sperm chromatin reverses the remarkable packing of DNA organized by sperm protamines. Mitochondrial DNA is also engulfed by the egg; the question of whether this DNA makes a small finite, or null, contribution to cytosolic inheritance is still in debate. The puzzles attending these encounters are presented as reminders of the intricacy and fascination, as well as of the vital necessity, of gamete interaction.

53 citations


Journal ArticleDOI
TL;DR: The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase.
Abstract: Previous studies from this laboratory have identified rat epididymal luminal fluid acid beta-D-galactosidase activity which also optimally hydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separated the luminal fluid beta-D-galactosidase into two molecular forms by ion-exchange chromatography on a column of DE-52. The separated enzyme activities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immobilized p-nitrophenyl beta-D-thiogalactopyranoside. The purified forms, when resolved by SDS/PAGE under reducing conditions, showed apparent molecular masses of 84 and 97 kDa. Kinetic studies, including a pH-dependent substrate preference and pH-dependent association/dissociation, disclosed no differences between these two forms. The two forms had identical N-terminal amino acid sequences. However, the 97 kDa form contained much more total carbohydrate and sialic acid than the 84 kDa form. The carbohydrate moieties in the two forms were assessed by comparing their size on SDS/PAGE before and after treatment with endo-enzymes. The removal of N-linked glycans by treatment with N-glycanase or endoglycosidase F generated de-N-glycosylated polypeptides of an apparent molecular mass of 70 kDa, and indicated that the two forms contained varying amounts of asparagine (N)-linked high mannose/hybrid-type and biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid sequences indicated that the two forms probably have identical or very similar polypeptides. The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving caput sperm PM proteins (before and after treatment in vitro of the membranes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially binds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput (but not cauda) sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase. This result suggests a possible role for the epididymal luminal fluid beta-D-galactosidases.

49 citations


Journal ArticleDOI
TL;DR: DMA is a suitable probe to identify human sperm mannose-binding sites crucially involved in sperm-zona interaction that appear to require free calcium concentrations to operate, and their expression changes with capacitation and acrosome reaction.
Abstract: A D-mannosylated albumin (DMA) neoglycoprotein was assessed to validate experimentally a probe capable of detecting mannose-binding sperm receptors involved in human sperm-egg interaction. DMA specifically blocked zona binding of swim-up human spermatozoa in a concentration-dependent manner. While no considerable effect was observed on sperm-zona initial contact, almost 50% of spermatozoa bound to the zona during a 2-hour period detached from it when DMA was introduced in the incubation medium. DMA inhibition was evident when 10% fetal bovine serum, but not 3.5% human serum albumin (HSA), was used as Ham's F10 medium supplementation. This may be due to the amount of free calcium in the medium since addition of 40 mM CaCl2 to F10-HSA restored DMA inhibition. Furthermore, the higher the calcium concentration in the incubation buffer, the greater the DMA blockage of sperm-zona binding. Unfixed sperm presented fluorescent DMA label over the entire acrosomal area (cap pattern), or concentrated at the equatorial segment (bar pattern). These patterns increased during capacitation, appearing on an average of 20% of the sperm after overnight incubation. They also increased, especially the bar pattern, following calcium ionophore treatment. Nearly all of methanol-fixed spermatozoa displayed the fluorescent label at the head level. Concomitant assessment of sperm membrane integrity and DMA fluorescent patterns revealed that DMA fluorescence coincided mostly with permeabilized or altered sperm plasma membrane. In conclusion, DMA is a suitable probe to identify human sperm mannose-binding sites crucially involved in sperm-zona interaction. These sites appear to require free calcium concentrations to operate, and their expression changes with capacitation and acrosome reaction. Precise location on human spermatozoa, however, warrants further investigation.

48 citations


Journal ArticleDOI
TL;DR: This work adapted the steps of decondensation and visualization of the single sperm cells, and applied a novel detection method, based on enzyme immunocytochemical reactions, with coloured precipitation products, which is possible to discriminate between disomic, diploid and abnormal spermatozoa, somatic cells and spermatozoon that overlap.
Abstract: The detection of some types of aneuploidy in human spermatozoa can be based on the use of the fluorescence in-situ hybridization technique (FISH). One of the crucial steps for FISH is to achieve a proper decondensation and denaturation of the DNA in the specimen, so as to obtain efficient hybridization results. However, after DNA decondensation the morphology of sperm heads is partly distorted and the majority of the tails is lost. This situation leads to problems in the distinction between disomic and diploid spermatozoa, as well as between abnormal spermatozoa and somatic cells. Double- and triple-target FISH can partly solve this discrimination problem. To improve these procedures we adapted the steps of decondensation and visualization of the single sperm cells. Firstly, DNA decondensation with 25 mM dithiothreitol in 1 M Tris at pH 9.5 resulted in sperm cells with intact morphology of both the head and the tail, and allowed efficient single-, double- and triple-target ISH to be performed. Secondly, we applied a novel detection method, based on enzyme immunocytochemical reactions, with coloured precipitation products. Thirdly, this ISH procedure was combined with Diff-Quik staining and bright-field microscopy. This absorption method has the advantage of a permanent signal, and the adapted cytoplasmic staining of the sperm plasma membrane allows the visualization of the outline of the single spermatozoon. Using this approach, therefore, it is possible to discriminate between disomic, diploid and abnormal spermatozoa, somatic cells and spermatozoa that overlap, because the morphology of the cells is not distorted and the tails of the spermatozoa are intact and properly visualized.

Journal ArticleDOI
TL;DR: It is shown that protein-kinase C inhibition inhibits calcium influx activated by progesterone, while leaving the depolarizing effect of this steroid unchanged.

Journal ArticleDOI
TL;DR: These results are the first to demonstrate the importance of HCO3- to mammalian AR initiation by the putative physiological initiator progesterone, and the human sperm AR.
Abstract: Progesterone, a putative in vivo initiator of the human sperm acrosome reaction (AR), has previously been shown to act at the sperm plasma membrane to initiate the AR in vitro. Here, we have investigated whether bicarbonate (HCO 3 ) was required for the progesterone-initiated human AR and whether HCO 3 -dependent cAMP activation might be involved. Capacitated human sperm were suspended in the presence of high (25 mM) or low (1 mM) HCO 3 media. The AR was assayed using fluorescein isothiocyanate (FITC)-conconavalin A with sperm fixed 5 minutes after progesterone or solvent control addition. Progesterone initiated the AR in both high and low HCO 3 - media, but the percentage of AR was significantly lower in the latter medium. In the presence of high HCO 3 - , 20-minute preincubation with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a blocker of HCO 3 - transport, inhibited the progesterone-initiated AR in a dose-dependent manner. The maximum inhibition (85%) was obtained with 18 μM DIDS. Inhibition by DIDS was reversed by washing sperm after treatment. Preincubation of sperm with dibutyryl cAMP (0.1 μM-1 mM) plus DIDS almost completely eliminated the inhibition of the progesterone-initiated AR by DIDS. Dibutyryl cAMP alone did not have a stimulatory effect on the progesterone-initiated AR, when high HCO 3 - was present, but it was able to partially overcome the reduction of AR by low HCO 3 - . These results are the first to demonstrate the importance of HCO 3 - to : 1) mammalian AR initiation by the putative physiological initiator progesterone, and 2) the human sperm AR. Furthermore, they suggest that HCO 3 - transport and HCO 3 - stimulation of adenylate cyclase are involved in the mechanism of the progesterone-initiated human AR.

Journal ArticleDOI
TL;DR: Results from Western blot analysis on human sperm plasma membrane proteins indicated that human spermatozoa do not express CHIP28 protein on their cell surface, and mercuric chloride (HgCl2), a known water channel blocker, failed to reduce the osmotic water permeability of human spermutozoa.
Abstract: A novel integral membrane protein with an apparent molecular mass of 25 kDa (CHIP28) was first isolated from human erythrocytes and is now recognized as a water channel protein. The expression of this protein has been found in several other cell types that all require high water permeability for their functions. Recent studies have shown that the water permeability (L p ) of human spermatozoa is among the highest reported for mammalian cells. Together with the low activation energy of human spermatozoa for L p , this suggests that CHIP28 water channel may be present in the plasma membrane of human spermatozoa. However, our current studies do not support this hypothesis. Results from Western blot analysis on human sperm plasma membrane proteins, performed through use of an antibody against human erythrocyte CHIP28 protein, indicated that human spermatozoa do not express CHIP28 protein on their cell surface (n=10). Consistent with the Western blot finding, mercuric chloride (HgCl 2 ), a known water channel blocker, failed to reduce the osmotic water permeability of human spermatozoa. The calculated L p values were 1.30±0.29 μm/min/atm (n=16; mean ±SEM) for the control group and 1.31±0.29 (n=9; mean ±SEM), 1.04±0.27 (n=11; mean ±SEM), and 1.34±0.19 (n=6; mean ±SEM), respectively, for the 10 μM, 30 μM, and 50 μM HgCl 2 -treated groups. These L p values are not different (p>0.05). In contrast, the same concentration of HgCl 2 significantly blocked the osmotic water transport across the membrane of human erythrocytes. These data strongly suggest that the high water permeability of human sperm plasma membranes is not due to CHIP but may be mediated by other water channel proteins that are mercury-resistant

Journal ArticleDOI
01 Aug 1995-Zygote
TL;DR: Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types that could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties.
Abstract: Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types. They could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties. The presence of a previously reported large, Ca2+ -selective channel was confirmed. Here, it is reported that the Ca2+ -selective channel from mouse sperm plasma membrane displayed a PNa+/PK+ = 1.6 +/- 0.2 (n = 4) and was blocked by micromolar concentrations of ruthenium red. Fusion yielded also a cation-selective channel (PNa+/PK+ = 2.5 +/- 0.3, n = 3) with a main open conductance substate of 103 pS and a smaller open substate of 51 pS (600 mM K+ cis/100 mM Na+ trans). The channel inserted into bilayers in two stable fashions: a high-activity mode (open probability = 0.57 +/- 0.02, n = 3), and a low activity mode (open probability < 1%, n = 4). In high mode, the channel displayed bursting kinetics and burst length was voltage independent. In addition, a perfectly anion-selective channel, with a slope conductance of 83 pS (600 KCl cis/100 KCl trans), was identified. It displayed a high, nearly constant open probability (approximately 0.90) in the 0 to -80 mV range.

Journal ArticleDOI
TL;DR: Sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G‐proteins, including Ras, which are enriched in flagellar membranes while the other small G'Proteins do not display a preferential distribution along the sea urch in sperm plasma membrane.
Abstract: Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [32P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [32P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [32P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-32P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras. Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.

Journal ArticleDOI
TL;DR: It is demonstrated that beta-glucuronidase from rat preputial glands binds with high affinity to spermatozoa from the cauda epididymis, and the existence of phosphomannosyl receptors on the sperm plasma membrane suggests strongly that maturing spermutozoa could be a target for glycosidases secreted into the lumen of the cauds, which then become bound to these cells via different ligand-receptor systems.
Abstract: Summary This study demonstrates that β-glucuronidase from rat preputial glands binds with high affinity to spermatozoa from the cauda epididymis. The binding was calcium-independent and was inhibited by mannose-6-phosphate, but not by other phosphorylated or non-phosphorylated sugars. Binding was also inhibited by α-mannosidase from Dictyostelium discoideum, an enzyme known to have mannose-6-phosphate as the ligand. From solubilized sperm membranes, a protein of >200 kDa and one of 45 kDa, were adsorbed to a column of D. discoideum enzyme and to a phosphomannan column respectively, and eluted with mannose-6-phosphate. According to histochemical observations at the light and the electron microscopic level, gold particles coated with the enzyme became bound to the external surface of the plasmalemma in the acrosomal region of caudal spermatozoa. Similar labelling was observed using gold particles coated with antibodies against the rat 300 kDa phosphomannosyl receptor. The existence of phosphomannosyl receptors on the sperm plasma membrane, and our previous demonstration of the presence of affinity sites for epididymal β-galactosidase on these gametes which is inhibited by phosphofructosyl derivatives, suggest strongly that maturing spermatozoa could be a target for glycosidases secreted into the lumen of the cauda epididymis, which then become bound to these cells via different ligand-receptor systems.

Journal ArticleDOI
TL;DR: This study evaluated the clinical usefulness of one additional parameter for assessment of human sperm cell function in vitro--the hypoosmotic swelling test and found positive correlation exists between HOS results and the outcome of IUI.
Abstract: This study evaluates the clinical usefulness of one additional parameter for assessment of human sperm cell function in vitro--the hypoosmotic swelling test. The hypoosmotic swelling test evaluated the functional integrity of the sperm plasma membrane. The investigation included a comparison of the hypoosmotic swelling test in samples containing motile and immotile spermatozoa and their correlation with the intrauterine insemination outcome. Motile spermatozoa expressed better membrane characteristics, without any importance of the hypoosmotic conditions. Positive correlation exists between HOS results and the outcome of IUI. This test can be a useful addition to the standard battery of semen analyzing tests.

Journal ArticleDOI
TL;DR: The results show that this automated freezing-quick-thawing method results in a small reduction in sperm-zona binding capacity, and that the time-dependent decline in motility parameters observed for both fresh and cryopreserved-thawed samples cannot be related to ATP deficiency under the conditions of the authors' experiments.
Abstract: The objective of the present studies was to assess the functional integrity of the sperm plasma membrane and metabolic and motility characteristics of the recovered motile fraction of human spermatozoa subjected to an automated freezing/quick-thawing method. Sperm membrane features examined included progesterone-induced changes in intracellular levels of calcium ([Ca2+]i), as measured by the fluorescent fura-2 indicator, and the tight binding of spermatozoa to homologous zonae pellucidae as assessed by the hemizona assay (HZA). Basal [Ca2+]i intracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels determined using chemiluminescence with luciferin-luciferase, and motility parameters determined using a computer-aided semen analyser (CASA) were studied concomitantly as an expression of metabolic/functional status. Ejaculates from fertile men (donors) were evaluated after swim-up separation of the motile fraction in both fresh and cryopreserved-thawed samples, and fractions of each ejaculate (fresh and frozen-thawed) were subjected to parallel measurements of the same parameters at the same time frame. Basal and progesterone-induced increase in [Ca2+]i, and ATP levels (up to 24 h) were similar in fresh and frozen-thawed samples. HZA results showed a modest (26%) although significant (p = 0.008) decrease in binding in frozen-thawed samples. The ratios of ATP/ADP in fresh and frozen-thawed samples were also found to be similar. Although post-thaw sperm motility was significantly lower than that of the fresh samples, comparison of the results indicated that the method was capable of preserving > 65% of motile spermatozoa in almost all of the samples cryopreserved. Additionally, the swim-up rescued a motile fraction in the frozen-thawed samples that was not significantly impaired with regard to motility, mean linear velocity or linearity as compared to the fresh fractions in the first 4 h. Our results show that this automated freezing-quick-thawing method results in a small reduction in sperm-zona binding capacity, and that the time-dependent decline in motility parameters observed for both fresh and cryopreserved-thawed samples cannot be related to ATP deficiency under the conditions of our experiments. These in-vitro results are coincident with the maintenance of fertilizing capacity for donor spermatozoa in the in-vitro fertilization (IVF) setting.

Journal ArticleDOI
TL;DR: In this paper, the authors used a pressure-tuning Fourier transform infrared spectroscopy (FT-IR) technique to detect structural and dynamic effects of gossypol at 5 μ M on a model bilayer membrane of 1,2-dipalmitoyl-sn -glycerol-3-phosphocholine (DPPC).

Journal ArticleDOI
01 May 1995-Zygote
TL;DR: The large natural variation among different porcine sperm populations with regard to their ability to interact with the egg was compared with the relative binding of egg PM material to individual proteins to provide quantitative evidence for possible biological function.
Abstract: Sperm plasma membrane (PM) proteins that demonstrate affinity for egg PM preparations have the potential to be biologically important during sperm-egg binding and/or fusion. In this study four such proteins have been identified. To provide quantitative evidence for possible biological function, the large natural variation among different porcine sperm populations with regard to their ability to interact with the egg was compared with the relative binding of egg PM material to individual proteins. An aliquot from each of 24 porcine ejaculates was evaluated by the zona-free hamster ova bioassay and the remainder processed to yield sperm PM vesicles. Aliquots of sperm PM were solubilised, separated by SDS-PAGE, western blotted and probed with partially purified, biotinylated egg PM protein. Bound egg PM proteins were visualised on western blots by an avidin/biotin/horseradish peroxidase system and analysed by scanning laser densitometry. Four sperm PM proteins (62, 39, 27 and 7 kDa estimated molecular mass) were the predominant binders of egg PM. The amount of egg PM bound to the 62 kDa protein was significantly correlated with the ability of sperm from the 24 ejaculates to penetrate zona-free hamster ova (percentage of ova penetrated, p = 0.01, R = 0.65; number of penetrated sperm per ovum, p = 0.02, R = 0.63).

Journal Article
TL;DR: In this article, an anti-SPM antibody-binding affinity column was used to identify the blood group substance detected on the sperm plasma membrane (SPM), a plasma membrane preparation was obtained from the sperm soluble protein and then injected into a rabbit to produce an anti SPM antibody.
Abstract: In order to identify the blood group substance detected on the sperm plasma membrane (SPM), a plasma membrane preparation was obtained from the sperm soluble protein and then injected into a rabbit to produce an anti-SPM antibody. An anti-SPM antibody-binding affinity column specifically bound 6 polypeptides with molecular masses of 135 kDa, 84 kDa, 78 kDa, 67 kDa, 54 kDa and 20 kDa from seminal plasma. Among these polypeptides, the 84-kDa protein (p 84) showed ABH antigenic activity upon immunoblotting. When viable, motile sperm were incubated at 37 degrees C in the culture medium, they became capacitated and p 84 was released into the medium from the sperm surface after 3 h of incubation, indicating that p 84 is a sperm-coating antigen. Immunoblotting of sexual glands revealed that this protein is originated from the seminal vesicle. Its immunological properties were similar to those of lactoferrin. When seminal plasma was immunoprecipitated with anti-human lactoferrin antiserum, the immunoprecipitates contained both p 84 and ABH antigenic activity. The amino acid sequence of the N-terminus of p 84 was determined to be: G-R-?-R-?-S-V-Q-W-?-A-V-S-Q-P-E-A-D-K-?-F-Q-W-Q-R-N-M-R-K-V-R-G-P-?-V or P-S?-?-I. Although this sequence is highly homologous to lactoferrin, the 18th residue is different (p 84, D; lactoferrin, T). These data suggest that p 84 is the protein which has not been identified and bears the ABH antigen. A sandwich ELISA using anti-SPM antibody was able to bind p 84 and allowed determination of the ABO blood type of semen and saliva, but detected no ABH antigenic activity in breast milk, vaginal fluid, erythrocytes, serum or urine. These results suggest that p 84 is the best candidate for ABO blood typing of semen when contaminating vaginal fluid is present.

Journal ArticleDOI
TL;DR: In vitro results confirmed the proteolytic effect of ampullary gland and other ASG on the 43- and 71-kD subunits, despite a reduction in membrane protein concentration, which is the first demonstration that the ampullARY gland is capable of modifying proteins on the sperm surface.
Abstract: Plasma membrane proteins were extracted either from epididymal sperm after incubation with ampullary gland secretion or from uterine sperm derived from surgically treated males belonging to the following groups: TX, excision of all accessory sex glands (ASG); AGX, bilateral excision of ampullary glands; AG, excision of all ASG except ampullary glands; and SH, sham-operated. Total membrane protein, glycoprotein, and SDS-PAGE of individual polypeptide subunits were quantified. After incubation with ampullary gland secretion, both protein and glycoprotein concentrations of epididymal sperm membrane were increased. The protein profile was also significantly altered, with the removal of the 43- and 71-kD subunits and the addition of the 36- and 50-kD subunits. The in vitro results confirmed this proteolytic effect of ampullary gland and other ASG on the 43- and 71-kD subunits, despite a reduction in membrane protein concentration. Modification of the 17-, 20-, 25-, 28-, 56-, and 66-kD proteins were also observ...

Journal ArticleDOI
TL;DR: The in vivo study indicated that interactions of ASG secretions and spermatozoa were complicated by the presence of uterine secretions, and SDS-PAGE profiles showed that both uterine and ASGsecretions could modify proteins on the sperm surface.
Abstract: Plasma membrane proteins were extracted either from epididymal spermatozoa after incubation with ventral prostrate gland secretion or from spermatozoa recovered from uteri of females mated with surgically treated males belonging to the following groups: TX (excision of all accessory sex glands, ASG), VPX (bilateral excision of ventral prostate), VP (bilateral excision of all ASG except the ventral prostate), and SH (sham-operated). Incubation of spermatozoa with ventral prostatic secretion resulted in an 11-fold increase in glycoprotein content of the plasma membrane, but total protein concentration remained unchanged. The in vivo study indicated that interactions of ASG secretions and spermatozoa were complicated by the presence of uterine secretions. Glycoprotein content was reduced in the presence of ventral prostatic secretions. SDS-PAGE profiles showed that both uterine and ASG secretions could modify proteins on the sperm surface. Enrichment of a 25-kD subunit was apparently effected by uterine secr...

Book ChapterDOI
TL;DR: This chapter describes methods for purifying axonemes and the focus is on preparing intact flagella to analyze their membrane proteins and reactivate them to studyAxonemal motility mechanisms in fish spermatozoa.
Abstract: Publisher Summary This chapter describes methods for purifying axonemes and the focus is on preparing intact flagella to analyze their membrane proteins and reactivate them to study axonemal motility mechanisms. Fish spermatozoa offer a good source from which to isolate and purify large amounts of vertebrate flagella and axonemes. Sperm from two teleost species, trout and carp, have been used extensively to study the mechanisms involved in the initiation of sperm motility. During the spawning season, an adult male trout produces a large number of sperm. In carp, sperm production can be induced throughout the year by repetitive injection of pituitary extracts. Trout and carp spermatozoa are quite simple and their flagella contain a classical 9+2 axoneme without periaxonemal structures. The absence of an acrosomal vesicle decreases the risk of contamination of the preparation with proteases. Trout sperm motility can be inhibited by media containing large amounts of potassium or having a low Ph. Carp sperm activation is controlled by a decrease in the external osmotic pressure and is not dependent on the external ions. It is advisable to include different protease inhibitors in extraction buffer (IMEN), as proteasomes are present on trout sperm plasma membrane. For trout, the water should be aerated with bubbling and frequently renewed. Chlorine must be avoided. For injection or sperm collection, fish must be anesthetized. For carp, spermiation can be induced throughout the year by intraabdominal injection of carp pituitary extract. Only a fraction of the sperm obtained from a single trout can be used directly for axoneme isolation. Therefore, for biochemical studies, some of the sperm may be frozen in liquid nitrogen for later use. The chapter discusses isolation of axonemes and flagella, and thereafter the demembranation and ATP reactivation of isolated carp sperm flagella.