Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Proteomic and functional analysis of human sperm detergent resistant membranes

Mammalian spermatozoa attain the ability to fertilize an oocyte as they negotiate the female reproductive tract. This acquisition of functional competence is preceded by an intricate cascade of biochemical and functional changes collectively known as "capacitation." Among the universal correlates of the capacitation process is a remarkable remodeling of the lipid and protein architecture of the sperm plasma membrane. While the mechanisms that underpin this dynamic reorganization remain enigmatic, emerging evidence has raised the prospect that it may be coordinated, in part, by specialized membrane microdomains, or rafts. In the present study we have demonstrated that human spermatozoa express recognized markers of membrane rafts. Further, upon depletion of membrane cholesterol through either physiological (capacitation) or pharmacological (methyl-β-cyclodextrin) intervention, these membrane rafts appear to undergo a polarized redistribution to the peri-acrosomal region of the sperm head. This finding encourages speculation that membrane rafts represent platforms for the organization of proteins involved in sperm-oocyte interactions. Support for this notion rests with the demonstration that membrane rafts isolated on the basis of their biochemical composition in the form of detergent resistant membranes (DRMs), possess the ability to adhere to homologous zona pellucidae. Furthermore a comprehensive proteomic analysis of the DRMs identified a number of proteins known for their affinity for the zona pellucida in addition to other candidates putatively involved in the mediation of downstream binding and/or fusion with the oolemma. Collectively these data afford novel insights into the subcellular localization and potential functions of membrane rafts in human spermatozoa.     

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.     

Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes

Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

Isolation and proteomic analysis of mouse sperm detergent-resistant membrane fractions: evidence for dissociation of lipid rafts during capacitation

Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DiIC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation.     

Composition and significance of detergent resistant membranes in mouse spermatozoa

Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as "capacitation." The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization. Emerging evidence indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, the mechanisms responsible for gathering key recognition molecules within this putative complex have yet to be defined. In this study, we provide the first evidence that chaperones partition into detergent resistant membrane fractions (DRMs) within capacitated mouse spermatozoa and co-localize in membrane microdomains enriched with the lipid raft marker, G(M1) ganglioside. During capacitation, these microdomains coalesce within the apical region of the sperm head, a location compatible with a role in sperm-zona pellucida interaction. Significantly, DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. A comprehensive proteomic analysis of the DRM fractions identified a total of 100 proteins, a number of which have previously been implicated in sperm-oocyte interaction. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface and thus present an important mechanistic insight into the fundamental cell biological process of sperm-oocyte interaction.     

Annexin A6 is recruited into lipid rafts of Niemann–Pick type C disease fibroblasts in a Ca-dependent manner

Niemann–Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

Annexin A6 is recruited into lipid rafts of Niemann-Pick type C disease fibroblasts in a Ca2+-dependent manner

Niemann-Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

Membrane rafts of the human red blood cell

The cell type of election for the study of cell membranes, the mammalian non-nucleated erythrocyte, has been scarcely considered in the research of membrane rafts of the plasma membrane. However, detergent-resistant-membranes (DRM) were actually first described in human erythrocytes, as a fraction resisting solubilization by the nonionic detergent Triton X-100. These DRMs were insoluble entities of high density, easily pelleted by centrifugation, as opposed to the now accepted concept of lipid raft-like membrane fractions as material floating in low-density regions of sucrose gradients. The present article reviews the available literature on membrane rafts/DRMs in human erythrocytes from an historical point of view, describing the experiments that provided the solution to the above described discrepancy and suggesting possible avenue of research in the field of membrane rafts that, moving from the most studied model of living cell membrane, the erythrocyte’s, could be relevant also for other cell types.     

Structure, composition, and peptide binding properties of detergent soluble bilayers and detergent resistant rafts

Lipid bilayers composed of unsaturated phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol are thought to contain microdomains that have similar detergent insolubility characteristics as rafts isolated from cell plasma membranes. We chemically characterized the fractions corresponding to detergent soluble membranes (DSMs) and detergent resistant membranes (DRMs) from 1:1:1 PC:SM:cholesterol, compared the binding properties of selected peptides to bilayers with the compositions of DSMs and DRMs, used differential scanning calorimetry to identify phase transitions, and determined the structure of DRMs with x-ray diffraction. Compared with the equimolar starting material, DRMs were enriched in both SM and cholesterol. Both transmembrane and interfacial peptides bound to a greater extent to DSM bilayers than to DRM bilayers, likely because of differences in the mechanical properties of the two bilayers. Thermograms from 1:1:1 PC:SM:cholesterol from 3 to 70 degrees C showed no evidence for a liquid-ordered to liquid-disordered phase transition. Over a wide range of osmotic stresses, each x-ray pattern from equimolar PC:SM:cholesterol or DRMs contained a broad wide-angle band at 4.5 A, indicating that the bilayers were in a liquid-crystalline phase, and several sharp low-angle reflections that indexed as orders of a single lamellar repeat period. Electron density profiles showed that the total bilayer thickness was 57 A for DRMs, which was approximately 5 A greater than that of 1:1:1 PC:SM:cholesterol and 10 A greater than the thickness of bilayers with the composition of DSMs. These x-ray data provide accurate values for the widths of raft and nonraft bilayers that should be important in understanding mechanisms of protein sorting by rafts.     

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.     

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.     

Mammalian fertilization: the strange case of sperm protein 56

During mammalian fertilization sperm bind to the egg's zona pellucida (ZP) after undergoing capacitation. Capacitated mouse sperm bind to mZP3 (one of three ZP glycoproteins), undergo the acrosome reaction, penetrate the ZP, and fuse with egg plasma membrane. Sperm protein 56 (sp56), a member of the C3/C4 superfamily of binding proteins, was identified nearly 20 years ago as a binding partner for mZP3 by photoaffinity cross‐linking of acrosome‐intact sperm. However, subsequent research revealed that sp56 is a component of the sperm's acrosomal matrix and, for sperm with an intact acrosome, should be unavailable for binding to mZP3. Recently, this dilemma was resolved when it was recognized that some acrosomal matrix (AM) proteins, including sp56, are released to the sperm surface during capacitation. This may explain why uncapacitated mammalian sperm are unable to bind to the unfertilized egg ZP.     

Dynamics of the mammalian sperm plasma membrane in the process of fertilization

Sexual reproduction requires the fusion of sperm cell and oocyte during fertilization to produce the diploid zygote. In mammals complex changes in the plasma membrane of the sperm cell are involved in this process. Sperm cells have unusual membranes compared to those of somatic cells. After leaving the testes, sperm cells cease plasma membrane lipid and protein synthesis, and vesicle mediated transport. Biophysical studies reveal that lipids and proteins are organized into lateral regions of the sperm head surface. A delicate reorientation and modification of plasma membrane molecules take place in the female tract when sperm cells are activated by so-called capacitation factors. These surface changes enable the sperm cell to bind to the extra cellular matrix of the egg (zona pellucida, ZP). The ZP primes the sperm cell to initiate the acrosome reaction, which is an exocytotic process that makes available the enzymatic machinery required for sperm penetration through the ZP. After complete penetration the sperm cell meets the plasma membrane of the egg cell (oolemma). A specific set of molecules is involved in a disintegrin-integrin type of anchoring of the two gametes which is completed by fusion of the two gamete plasma membranes. The fertilized egg is activated and zygote formation preludes the development of a new living organism. In this review we focus on the involvement of processes that occur at the sperm plasma membrane in the sequence of events that lead to successful fertilization. For this purpose, dynamics in adhesive and fusion properties, molecular composition and architecture of the sperm plasma membrane, as well as membrane derived signalling are reviewed.     

Biochemical characterization of detergent-resistant membranes: a systematic approach

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.     

Independent segregation of human immunodeficiency virus type 1 Gag protein complexes and lipid rafts

Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55Gag mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55Gag interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55Gag that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55Gag forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains.     

In situ imaging of detergent-resistant membranes by atomic force microscopy

Purified detergent-resistant membranes (DRMs) are powerful tools for the biochemical study of plasma membrane domains. To what extent these isolated DRMs correspond to native membrane domains remains, however, a matter of debate. The most immediate question to be answered concerns the in situ size range of DRMs, a determination that escapes classical microscopy techniques. In this study we show that in situ three-dimensional images of a material as fragile as Triton X-100-treated cells can be obtained, in buffer, by tapping mode atomic force microscopy. These images establish that, prior to the isolation procedure, the detergent plasma membrane fragments form domains whose size frequently exceeds 15-20 microm(2). This DRMs size range is about 1 order of magnitude higher than that estimated for the larger microdomains of living cells, which strongly suggests that membrane microdomains rearrange into larger DRMs during Triton X-100 treatment. Concomitantly, the images also reveal the presence of the cytoskeleton, which is resistant to detergent extraction, and suggest that, in situ, DRMs are associated with the membrane cytoskeleton.     

Capacitation and the acrosome reaction in equine sperm.

During sexual reproduction, the sperm and oocyte must fuse before the production of a diploid zygote can proceed. In mammals such as equids, fusion depends critically on complex changes in the plasma membrane of the sperm and, not surprisingly, this membrane differs markedly from that of somatic cells. After leaving the testes, sperm cease to synthesize plasma membrane lipids or proteins, and vesicle-mediated transport stops. When the sperm reaches the female reproductive tract, it is activated by so-called capacitation factors that initiate a delicate reorientation and modification of molecules within the plasma membrane. These surface changes enable the sperm to bind to the extracellular matrix of the egg (zona pellucida ZP) and the zona then primes the sperm to initiate the acrosome reaction, an exocytotic event required for the sperm to penetrate the zona. This paper will review the processes that occur at the sperm plasma membrane before and during successful penetration of the equine ZP. It is noted that while several methods have been described for detecting changes that occur during capacitation and the acrosome reaction in bovine and porcine sperm, relatively little has been documented for equine sperm. Special attention will therefore be dedicated to recent attempts to develop and implement new assays for the detection of the capacitation status of live, acrosome-intact and motile equine sperm.     

Distinct membrane fractions from mouse sperm bind different zona pellucida glycoproteins.

Interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized at the molecular level. To identify sperm proteins that recognize ligand ZP3, we used sonicated sperm membrane fractions as competitors in a quantitative binding assay. Sonicated membranes were density fractionated into 4 fractions. Bands 1-3 contained membrane vesicles, and band 4 contained axonemal and midpiece fragments. In competitive binding assays, bands 1, 2, and 3 but not band 4 were able to compete with live, capacitated, intact sperm for soluble 125I-ZP binding. Affinity-purified ZP fractions consisting of a ZP3-enriched fraction (125I-ZP3) and a fraction enriched for ligands ZP1 and ZP2 and depleted of ZP3 (125I-ZP1/2) were obtained by antibody affinity purification of ZP3. In competitive binding assays, bands 2 and 3 competed for 125I-ZP3 binding, but band 1 did not interact with enriched 125I-ZP3. None of the membrane fractions competed for 125I-ZP1/2 binding. These results demonstrate that band 2 and band 3 contain sperm components that interact with ZP3 alone and that components in band 1 interact with ZP3 in conjunction with either ZP1 or ZP2. These data indicate that there must be at least 2 unique sperm plasma membrane components that mediate intact sperm interactions with ZP glycoproteins in mouse. Bands 2 and 3 are likely to contain a primary ZP-binding protein because they interacted directly with ZP3, whereas band 1 may contain sperm proteins involved in later interactions with the ZP, perhaps transitional interactions to maintain sperm contact with the ZP during acrosomal exocytosis.