Requirement of sperm‐oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida–free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5 ± 0.6 and 4.3 ± 0.6 for ICSI; 3.0 ± 0.3 and 3.8 ± 0.4 for activated and 0.2 ± 0.1 and 0.6 ± 0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore‐puromycin‐activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes. Mol. Reprod. Dev. 52:183–188, 1999. © 1999 Wiley‐Liss, Inc.     

Involvement of calcium signaling and the actin cytoskeleton in the membrane block to polyspermy in mouse eggs

This study examines the effects of actin microfilament-disrupting drugs on events of fertilization, with emphasis on gamete membrane interactions. Mouse eggs, freed of their zonae pellucidae, were treated with drugs that perturb the actin cytoskeleton by different mechanisms (cytochalasin B, cytochalasin D, jasplakinolide, latrunculin B) and then inseminated. Cytochalasin B, jasplakinolide, and latrunculin B treatments resulted in a decrease in the percentage of eggs fertilized and the average number of sperm fused per egg. However, cytochalasin D treatment resulted in an increase in the average number of sperm fused per egg and the percentage of polyspermic eggs. This increase in polyspermy occurred despite the observation that cytochalasin D treatment caused a decrease in sperm-egg binding and did not affect spontaneous acrosome reactions or sperm motility. This suggested that cytochalasin D-treated eggs had an impaired ability to establish a block to polyspermy at the level of the plasma membrane. The effect of cytochalasin D on the block to polyspermy was not due to a general disruption of egg activation because sperm-induced calcium oscillations and cortical granule exocytosis were similar in cytochalasin D-treated and control eggs. However, buffering of intracellular calcium levels with the calcium chelator BAPTA-AM resulted in an increase in polyspermy. Together, these data suggest that a postfertilization decrease in egg membrane receptivity to sperm requires functions of the egg actin cytoskeleton that are disrupted by cytochalasin D. Furthermore, egg activation-associated increased intracellular calcium levels are necessary but not sufficient to affect postfertilization membrane dynamics that contribute to a membrane block to polyspermy.     

Isolation of 125I-concanavalin A-labeled plasma membrane from unfertilized mouse eggs

A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona-free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213–222, 1986) were labeled with ¹²⁵I-concanavalin A (ConA) prior to sonication and fractionation on iso-osmotic self-generated Percoll density gradients. Experiments using the ConA-specific sugar α-methylmannoside (αMM) indicated that ¹²⁵I-ConA bound specifically to the egg PM. Greater than 95% of ¹²⁵I-ConA binding to zona-free eggs was blocked in the presence of 0.1 M αMM, and incubation of eggs in αMM after ¹²⁵I-ConA labeling caused release of 85–90% of bound label. Fractionation of ¹²⁵I-ConA-labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of ¹²⁵I-ConA-labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that ¹²⁵I-ConA did not label other organelles. As a control, human erythrocytes were labeled with ¹²⁵I-ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for ¹²⁵I-ConA-labeled eggs. These results indicate that ¹²⁵I-ConA can be used as a specific marker to support PM isolation from small numbers of zona-free mouse eggs.     

Preventing polyspermy in mammalian eggs—Contributions of the membrane block and other mechanisms

The egg's blocks to polyspermy (fertilization of an egg by more than one sperm) were originally identified in marine and aquatic species with external fertilization, but polyspermy matters in mammalian reproduction too. Embryonic triploidy is a noteworthy event associated with pregnancy complications and loss. Polyspermy is a major cause of triploidy with up to 80% of triploid conceptuses being the result of dispermic fertilization. The mammalian female reproductive tract regulates the number of sperm that reach the site of fertilization, but mammals also utilize egg‐based blocks to polyspermy. The egg‐based blocks occur on the mammalian egg coat (the zona pellucida) and the egg plasma membrane, with apparent variation between different mammalian species regarding the extent to which one or both are used. The zona pellucida block to polyspermy has some similarities to the slow block in water‐dwelling species, but the mammalian membrane block to polyspermy differs substantially from the fast electrical block that has been characterized in marine and aquatic species. This review discusses what is known about the incidence of polyspermy in mammals and about the mammalian membrane block to polyspermy, as well as notes some lesser‐characterized potential mechanisms contributing to polyspermy prevention in mammals.     

The electrical block to polyspermy induced by an intracellular Ca2+ increase at fertilization of the clawed frogs,Xenopus laevis and Xenopus tropicalis

Polyspermy blocking, to ensure monospermic fertilization, is necessary for normal diploid development in most animals. We have demonstrated here that monospermy in the clawed frog, Xenopus tropicalis, as well as in X. laevis, is ensured by a fast, electrical block to polyspermy on the egg plasma membrane after the entry of the first sperm, which is mediated by the positive‐going fertilization potential. An intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the sperm entry site was propagated as a Ca²⁺ wave over the whole egg cytoplasm. In the X. tropicalis eggs fertilized in 10% Steinberg's solution, the positive‐going fertilization potential of +27 mV was generated by opening of Ca²⁺‐activated Cl^?‐channels (CaCCs). The fertilization was completely inhibited when the egg's membrane potential was clamped at +10 mV and 0 mV in X. tropicalis and X. laevis, respectively. In X. tropicalis, a small number of eggs were fertilized at 0 mV. In the eggs whose membrane potential was clamped below ?10 mV, a large increase in inward current, the fertilization current, was recorded and allowed polyspermy to occur. A small initial step‐like current (IS current) was observed at the beginning of the increase in the fertilization current. As the IS current was elicited soon after a small increase in [Ca²⁺]_i, this is probably mediated by the opening of CaCCs. This study not only characterized the fast and electrical polyspermy in X. tropicalis, but also explained that the initial phase of [Ca²⁺]_i increase causes IS current during the early phase of egg activation of Xenopus fertilization.     

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane,the other involving the follicle cells

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137-143, 1997. © 1997 Wiley-Liss, Inc.     

CaMKII can participate in but is not sufficient for the establishment of the membrane block to polyspermy in mouse eggs

Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).     

Possible involvement of a sialylated component of the sperm plasma membrane in sperm-zona interaction in the mouse

We have studied the molecular mechanisms of gamete interaction in vitro in the laboratory mouse, Mus musculus. In particular, we were interested in whether this interaction is similar to a lectin-hapten-mediated process. Inhibition of sperm-zona binding was examined using various concentrations (.25 mM to 50 mM) of different sugars (sialic acid α-methylmannose, glucose, fucose, galactose, and N-acetyl-glucosamine). Sperm-zona binding was significantly decreased when eggs were pretreated with 10 mM of sialic acid or α-methylmannose but not by other sugars tested. Furthermore, treatment of capacitated sperm with neuraminidase destroyed their ability to bind and fertilize eggs. We have also used a specific lectin for sialic acid from the horseshoe crab (Limulus polyphemus) to agglutinate mouse sperm. The lectin (.120 ng/ml) mediated agglutination of mouse sperm (10⁵ sperm/ml) was routinely observed to increase from a 10% agglutination immediately following their isolation from the epididymis to 100% agglutination 90 minutes later. Collectively, these results suggest the appearance of specific sugar moieties on the surface of the sperm plasma membrane which, in this particular species of mouse, are sialylated glycoproteins acting as ligands for specific receptors on the surface of the egg. These are the first data to indicate that sperm-egg recognition and attachment is a lectin-hapten-mediated process in the mouse.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

Rapid,nonradioactive, and quantitative method to analyze zona pellucida modifications in single mouse eggs

A rapid, nonradioactive method to monitor the ZP2 to ZP2_f conversion in the zona pellucida of single mouse eggs has been developed. This assay is based on the chemiluminescent detection of biotinylated ZP2 and ZP2_f following electrophoresis under reducing conditions and electrophoretic transfer to Immobilon P. This method is about 10 times faster and detects similar extents of ZP2 to ZP2_f conversion following A23187-induced egg activation, when compared to the commonly used radioiodination procedures. © 1994 Wiley-Liss, Inc.     

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.     

Steroid hormone specifically binds to rat kidney plasma membrane

A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using anin vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 µM corticosterone. Under standardized conditions of pH 7.4 at 2°C and 30 min incubation time, the binding was saturable and showedK _d=13±3 nM andB _max=616±34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane.     

The lymphocyte plasma membrane: Locus of control in the immune response

Summary The lymphocyte plasma membrane is the locus of events which control the immune response. T and B lymphocytes, which mediate cellular and humoral immunity respectively, show distinctive plasma membrane morphologies and cell surface receptors. The dynamic state of these plasma membrane components is emphasized by their lateral mobility in the fluid plane of the membrane, as well as variation in their structure or expression as the lymphocyte proliferates and differentiates in response to stimulation by antigen or mitogens. The best understood membrane glycoproteins are surface membrane immunoglobulins that serve as antigen receptors on B cells, and the histocompatability-β ₂ microglobulin complex that has an immunoglobulin-like structure. Other less well defined surface structures showing modulation during the cell cycle may affect growth regulation of proliferating lymphocytes. Some of these are shared by fetal and neoplastic cells. Major theories of lymphocyte signaling are discussed, and the early events in lymphocyte activation are reviewed. While a complete model encompassing all these early events is not yet possible, the central issues can be usefully discussed in terms of receptor-transducer-effector concepts derived by strong parallels from a knowledge of hormone-membrane interactions. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975. Supported by: Medical Research Council of Canada and National Cancer Institute of Canada. Scholar of the Medical Research Council of Canada.     

Role of the vitellus in the block to polyspermy in golden hamster eggs

The block to polyspermy in golden hamster eggs is believed to operate only at the zona pellucida. However, changes in the egg vitellus also prevent further entry of capacitated sperm. When zona-free hamster eggs spontaneously activated in vitro, and in vivo fertilized eggs at pronuclear stage were inseminated with capacitated human sperm, penetration did not occur. In the case of a homologous system using hamster sperm and in vivo fertilized hamster eggs, slight attachment of sperm was observed but no penetration. The cortical granules were found to be released in spontaneously activated and in fertilized eggs as observed by phase contrast microscopy. These observations suggest that the egg vitellus plays a role in the block to poiyspermy in addition to that of the zona block.     

Inhibition of plasma membrane and mitochondrial transmembrane potentials by ethanol

The actions of ethanol and its primary oxidative metabolite, acetaldehyde, on plasma membrane and mitochondrial transmembrane potentials were examined in rat brain using fluorescence techniques. Subchronic treatment of adult rats with ethanol resulted in a significant depolarization of both the plasma and mitochondrial membranes when the mean blood ethanol level of the rats was 59±11 mM (mean±SEM, n=6). Acute dosing of animals (4.5 g/kg, i.p.) failed to show any significant alterations. Various concentrations of ethanol, added in vitro to a crude synaptosomal preparation isolated from the rat cerebrocortex (P₂) from untreated animals, depolarized both the plasma and mitochondrial transmembrane potentials in a dose-related manner. Addition of acetaldehyde in vitro did not reveal any significant effects on plasma or mitochondrial transmembrane potential.     

Changes in exposed membrane proteins during in vitro capacitation of boar sperm

Exposed plasma membrane proteins were labeled with 125I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.     

A sodium-dependent, fast block to polyspermy occurs in eggs of fucoid algae

More than 70% of Pelvetia fastigiata eggs and about 15% of Fucus distichus eggs become polyspermic when fertilized at natural sperm concentrations in a low-sodium (2.5 mM Na+, 450 mM N-methyl glucamine) artificial seawater. Natural levels of polyspermy are 1-3% for both species. Polyspermic eggs germinate and respond to photopolarization, but do not develop beyond an abnormal, "stumpy," four-cell stage. They die within 1-1.5 weeks. The sodium-dependent block is a fast block, and it is replaced by a second block (probably cell wall formation) no later than 9 min (Pelvetia) after eggs are shed. The sodium-dependent block in Pelvetia is very efficient; when external sodium is raised to only 47.5 mM, the level of polyspermy drops to about 25%. These results are compared with data on marine invertebrates in the context of factors such as the sperm/egg concentration at fertilization and natural, osmotic (salinity) stress.     

Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization

Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca²⁺ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCζ in generating changes in egg intracellular Ca²⁺ despite the fact that PLCζ constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca²⁺ oscillations has not been examined. Reducing egg PLCβ1 by a transgenic RNAi approach resulted in a significant decrease in Ca²⁺ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCβ1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca²⁺ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCζ in egg activation.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Distribution of filipin-sterol complexes in the plasma membrane of stallion spermatozoa during the epididymal maturation process

The presence and distribution of cholesterol in mature and immature epididymal spermatozoa was analyzed using filipin as a cytochemical tool in freeze-fracture replicas and thin section preparations. The polyenic-antibiotic filipin formed complexes with 3, beta -OH sterols, producing characteristic protrusions, or pits, that were heterogeneously distributed in the plasma membrane of stallion spermatozoa, revealing a specific organization in a functionally specialized area of the gamete. The acrosomal region of the sperm head presented a significantly higher density of filipin sterol complexes than the postacrosomal region, which was usually free of these complexes. The plasma membrane of the flagellum also showed filipin sterol complexes randomly distributed in freeze-fracture replicas. The strong filipin labeling observed in the membrane of spermatozoa obtained from the caput region of the epididymis decreased significantly during epididymal passage. The significance of these changes is not completely understood, but they might contribute to establishing the molecular organization necessary for sperm transit and storage in the epididymis as well as to development of motile spermatozoa that are able to fertilize the oocyte and induce normal embryonic development.