Showing papers on "Sperm plasma membrane published in 1994"

Reactive oxygen species generation and human spermatozoa: The balance of benefit and risk

Abstract: Although the generation of reactive oxygen species is an activity normally associated with phagocytic leucocytes, mammalian spermatozoa were, in fact, the first cell type in which this activity was described. In recent years it has become apparent that spermatozoa are not the only nonphagocytic cells to exhibit a capacity for reactive oxygen species production, because this activity has been detected in a wide variety of different cells including fibroblasts, mesangial cells, oocytes, Leydig cells, endothelial cells, thyroid cells, adipocytes, tumour cells and platelets. Since the capacity to generate reactive oxygen species is apparently so widespread, the risk-benefit equation for these potentially pernicious molecules becomes a matter of intense interest. In the case of human spermatozoa, the risk of manufacturing reactive oxygen metabolites is considerable because these cells are particularly vulnerable to lipid peroxidation. Indeed, there is now good evidence to indicate that oxygen radicals are involved in the initiation of peroxidative damage to the sperm plasma membrane, seen in many cases of male infertility. This risk is off-set by recent data suggesting that superoxide anions and hydrogen peroxide also participate in the induction of key biological events such as hyperactivated motility and the acrosome reaction. Thus, human spermatozoa appear to use reactive oxygen species for a physiological purpose and have the difficult task of ensuring the balanced generation of these potentially harmful, but biologically important, modulators of cellular function.

A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg.

Abstract: A typical mammalian egg is surrounded by an outer layer of about 3,000 cumulus cells embedded in an extracellular matrix rich in hyaluronic acid. A current, widely proposed model is that the fertilizing sperm, while it is acrosome intact, passes through the cumulus cell layer and binds to the egg zona pellucida. This current model lacks a well-supported explanation for how sperm penetrate the cumulus layer. We report that the sperm protein PH-20 has a hyaluronidase activity and is present on the plasma membrane of mouse and human sperm. Brief treatment with purified, recombinant PH-20 can release all the cumulus cells surrounding mouse eggs. Acrosome intact mouse sperm incubated with anti-PH-20 antibodies can not pass through the cumulus layer and thus can not reach the zona pellucida. These results, indicating that PH-20 enables acrosome intact sperm to penetrate the cumulus barrier, reveal a mechanism for cumulus penetration, and thus provide the missing element in the current model.

A free radical theory of male infertility

Abstract: There is a growing body of evidence to indicate that a significant factor in the aetiology of male infertility involves a loss of sperm function as a consequence of oxidative stress. This stress originates from the excessive generation of reactive oxygen species by the spermatozoa and results in the peroxidation of unsaturated fatty acids in the sperm plasma membrane. It is possible that reactive oxygen species originating from infiltrating leucocytes could also stress the spermatozoa although the protective properties of seminal plasma would render this unlikely in vivo. Whatever the source of the reactive oxygen species, the lipid peroxides thereby generated exhibit powerful negative correlations with the movement characteristics of the spermatozoa and their capacity for sperm-oocyte fusion. These findings should have important implications for the development of rational techniques for the diagnosis and treatment of male infertility.

Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida, monitored with the calcium fluorescent indicator, fluo-3. inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: Tyrphostin A48, pertussis toxin, and 3-quinuclidinyl benzilate

Abstract: The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25 degrees C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca2+]i. Initial [Ca2+]i was 231 +/- 58 nM (+/- SE, n = 43). Addition of heat-solubilized mouse zonae pellucidae to capacitated sperm increased [Ca2+]i by 106 +/- 19 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 +/- 18 nM (+/- SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca2+]i induced by solubilized zonae pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB), an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121-130). This [Ca2+]i increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G1 proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida-induced increase in [Ca2+]i, all three inhibitors also blocked the zona pellucida-induced acrosome reaction. These results indicate that [Ca2+]i increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca2+]i suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding.

Visualization and quantification of glycolipid polarity dynamics in the plasma membrane of the mammalian spermatozoon

Abstract: Seminolipid (sulphogalactosylalkylacylglycerol), the gly colipid that is specific for mammalian germ cells, is located exclusively in the outer leaflet of the sperm plasma membrane. In this study the lateral distribution of semi nolipid on sperm heads has been investigated by indirect immunofluorescence labelling and detection with digital imaging fluorescence microscopy. In freshly ejaculated sperm cells this glycolipid was present primarily at the apical ridge subdomain of the plasma membrane of the sperm head. After binding the sperm cells to zona-coated coverslips seminolipid migrated, in 40 minutes, from the apical ridge to the equatorial subdomain of the plasma membrane. A similar redistribution of seminolipid was observed during capacitation of sperm cells in vitro induced by Ca 2+ or bovine serum albumin. Comparable migration of seminolipid was also found after prolonged storage of ejaculated sperm cells, albeit at a much slower rate. Addition of arylsulphatase A, an enzyme present in seminal plasma that desulphates seminolipid, significantly enhanced the migration of seminolipid during storage of sperm cells. Its breakdown product desulphoseminolipid (galactosylalkylacylglycerol) appeared highly specifically at the equatorial segment. The measured fluorescence intensity over the sperm head surface correlated linearly with the spatial probe distribution as was checked by fl uorescence lifetime imaging microscopy. This paper demonstrates and quantifies for the first time the polarity of seminolipid on the surface of the sperm cell and the dynamic alterations that occur in this polarity during post-ejacula tory events.

Human zona pellucida recognition associated with removal of sialic acid from human sperm surface

Abstract: The ability of human spermatozoa recovered from highly motile sperm fractions to bind wheat germ agglutinin (WGA) after discontinuous Percoll gradient centrifugation was studied. WGA could bind to almost all motile spermatozoa, whereas fewer than 25% of spermatozoa could bind peanut (PNA) and concanavalin A (Con A) agglutinin, two lectins that specifically bind acrosomal membranes. After removal of the plasma membrane with 0.04% Triton X100, WGA, PNA and Con A bound more than 80% of spermatozoa, but binding sites for WGA on the anterior acrosomal region were markedly reduced. The expression of sialic acid on human sperm plasma membrane was demonstrated, since WGA, which specifically recognizes both sialic acid (NeuNAc) and N-acetylglucosamine (GlcNAc), bound almost all intact motile spermatozoa, whereas succinylated WGA, which recognizes only GlcNAc, bound less than 10% of intact motile spermatozoa. Moreover, binding of WGA was compared with that of three other lectins (Sambucus nigra, SNA; Maackia amurensis, MAL and Limulus polyphemus, LPA) with specificity for different NeuNAc linkages. Only SNA, which requires the presence of the disaccharide structure NeuNAc alpha(2,6) Gal/GalNAc, showed a positive correlation with sperm motility as observed with WGA. Moreover, there was a strong inhibition of WGA binding on spermatozoa preincubated with bovine submaxillary mucin containing (2,6)-linked NeuNAc. These results demonstrate the presence of NeuNAc alpha(2,6) Gal/GalNAc glycoconjugate sequences on the plasma membrane of the motile human spermatozoon.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody to human fertilization antigen-1 (FA-1) inhibits bovine fertilization in vitro: application in immunocontraception.

Abstract: A monoclonal antibody (mAb) to the human sperm plasma membrane protein, fertilization antigen-1 (FA-1), was tested for its reactivity with bovine spermatozoa and its effects on bovine fertilization in vitro. Western blot analysis revealed that the FA-1 mAb reacted with proteins of similar molecular mass (53 +/- 2 kDa) in human and bovine sodium deoxycholate (DOC)-solubilized sperm extracts. Indirect immunofluorescence, using epifluorescence microscopy and laser scanning confocal microscopy, revealed that the FA-1 antigen is present in the post-acrosomal region of bovine spermatozoa, which is similar to human FA-1 localization. In bovine in vitro fertilization (IVF) trials, using oocytes obtained from slaughterhouse ovaries, addition of 20, 40, or 80 micrograms/ml of FA-1 mAb to the IVF medium resulted in a linear decrease in the fertilization rate from 86.3% in the controls to 54.6%, 21.6%, and 1.8% in the respective experimental groups (p 0.10) of the FA-1 mAb on percent sperm motility or other motility characteristics tested, suggesting that human FA-1 mAb inhibits bovine sperm cell function at some point after capacitation. In conclusion, the evolutionarily conserved antigen FA-1 has a molecular identity in bovine sperm similar to that in human sperm, and mAb to human sperm FA-1 inhibits fertilization of bovine oocytes. These results indicate that FA-1 is a promising candidate for the development of a contraceptive vaccine. The research also suggests that bovine species could be used as a model for investigating the use of FA-1 as an immunovaccine in ruminants.

Interaction of divalent cations with germ cell specific sulfogalactosylglycerolipid and the effects on lipid chain dynamics.

Abstract: Sulfogalactosylglycerolipid (SGG) is a sulfoglycolipid found ubiquitously in the plasma membrane of mammalian male germ cells. Although its exact cellular function(s) is unknown, it is speculated that SGG may play a role in cation transport, which may be important in sperm-egg interaction. Given the significant role of Ca2+ in many fertilization-related events, the purpose of this study was to determine whether Ca2+ interaction with the negatively charged sulfate group of SGG results in changes to the SGG lipid chain molecular dynamics and to compare these lipid dynamics with those resulting from Na+, Mg2+, or Sr2+ interaction with SGG. Pressure-tuning Fourier transform infrared spectroscopy was used in this study. The results obtained showed that all three divalent cations interacted electrostatically with the sulfate moiety of hydrated SGG, although with varying degrees of strength. It was found that the hydrocarbon chains of hydrated SGG-Na+ multilamellar bilayers were interdigitated, thus increasing disorderedness of the terminal CH3 group of the hydrocarbon chains. The presence of each of the three divalent cations abolished this interdigitation state. Presumably, this is through the cross-linking interaction of each divalent cation with the sulfate groups of neighboring lipid molecules. Moreover, divalent cation interaction was found to increase the lipid chain dynamics of SGG, with Mg2+ inducing the greatest chain disorder followed by Ca2+ and then Sr2+. An increase in chain disorder would increase the bilayer fluidity. Such a phenomenon may prove relevant to the changes observed in the sperm plasma membrane during fertilization-related events.

Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa.

Abstract: We demonstrated previously that pentoxifylline in millimolar concentrations can inhibit superoxide anion production by human spermatozoa. In the present study we have examined the effects of the same concentrations of pentoxifylline on experimentally induced lipid peroxidation, as measured by malondialdehyde formation in the thiobarbituric (TBA) assay. Under the experimental conditions used, preincubation of spermatozoa with pentoxifylline led to a significant dose-dependent stimulation (p < 0.005) of malondialdehyde production amounting to 10.77 +/- 2.35%, 13.45 +/- 2.99% and 17.4 +/- 1.99% (mean +/- SEM) for 1.9, 3.7 and 11.2 mmol/l pentoxifylline, respectively. In the presence of 11.2 mmol/l pentoxifylline, an increase in iron-catalysed lipid peroxidation potential was detected in samples of spermatozoa from 29 infertile men, regardless of their initial levels of malondialdehyde. The results of this study indicate that pentoxifylline might further augment the ferrous ion-stimulated decomposition of pre-accumulation lipid hydroperoxides in the sperm plasma membrane and thus promote malondialdehyde generation in the TBA assay. It is concluded that the stimulatory effect of pentoxifylline on iron-induced lipid peroxidation may have an adverse effect on the quality of sperm suspensions prepared for in vitro fertilization, a possibility which should be investigated further.

Acrosome reaction and fertilization

Abstract: The acrosome reaction that occurs after sperm capacitation, is an exocytotic event induced by a Ca++ influx. It plays an essential role during fertilization, by making spermatozoa able of penetrating the zona and capable of fusing with the egg plasma membrane. Zona pellucida is the natural inducer of the acrosome reaction. Binding of the sperm receptor to ZP3, a zona glycoprotein acting as ligand, triggers the molecular events leading to acrosomal exocytosis. G-proteins may be involved in the signal-transduction pathway during the acrosome reaction. Other known inducers of the human acrosome reaction include follicular fluid, progesterone and the calcium ionophore A23187. Progesterone acts through a receptor on the sperm plasma membrane, while the ionophore promotes non-physiological sperm Ca++ uptake. Several cytochemical procedures have been proposed for evaluating the acrosome reaction: the acrosomal status can be observed after staining or after labeling with lectins or antibodies. These methods both attempt to evaluate sperm viability and to distinguish degenerative acrosomal loss.

Solubilization and partial purification from mouse sperm membranes of the specific binding activity for 3-quinuclidinyl benzilate, a potent inhibitor of the zona pellucida-induced acrosome reaction.

Abstract: 3-Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic acetylcholine receptors, has been demonstrated to inhibit specifically the zona pellucida (ZP)-inducud acrosome reaction (AR) in mouse sperm (Florman and Storey, 1982; Dev Biol 91:121–130). In this study we describe the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative receptor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mouse sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with KD = 7.2 nM and Bmax = 8700 sites/cell. These binding characteristics are similar to those seen with QNB binding to whole cells (Florman and Storey, 1982, J Androl 3:157–164). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent-soluble fraction possessed QNB binding activity similar to that of intact membranes. The detergent-soluble fraction maintained intact ZP receptor(s)–G protein coupling in that treatment of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTPγS binding, respectively. The solubilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fraction. Analysis of this fraction by SDS–PAGE revealed a complex of approximately 5 proteins unique to this fraction. The most prominent protein had a Mr of 72 kDa, which is within the Mr range for muscarinic receptors. A protein with Mr = 41 kDa was also present within this fraction. Subsequent pertussis toxin (PTX)-catalyzed ADP-ribosylation of this fraction revealed this protein to be the α subunit of the Gi class of G proteins. Although the QNB binding activity could not be positively identified, we propose that it is contained in one or more of the proteins unique to this fraction and that these proteins, including Gi, may act as part of a sperm receptor complex for the ZP. © 1994 Wiley-Liss, Inc.

Purification and characterization of a protein kinase from goat sperm plasma membrane

Abstract: A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP(i), is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 microgram/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40-50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (approximately 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies to Human Seminal Plasma Inhibin Adversely Affect Sperm Function Parameters

Abstract: Polyclonal antibodies to intact inhibin (94 amino acids, R-94, 10.5 kDa) and its sequence specific synthetic fragments (R-9, R-17) were evaluated for their effect on various physical and biochemical parameters of sperm function. Intact inhibit had maximum deleterious effect on quantitative motility and mean forward progression of spermatozoa. Antibodies had no effect on sperm fructolysis and sperm nuclear chromatin decondensation reaction. Sperm plasma membrane was damaged in antibodies treated spermatozoa as evidenced by hypoosmotic swelling test and sperm lipid peroxidation reaction.