Role In Gamete Interaction Research Articles

Identification of Cell Surface Proteins of the Sea Star Mature Egg using Complimentary Methods

Multiple events occur between the sperm and egg membranes at fertilization, including sperm-egg binding, species-specific discrimination, membrane fusion, and egg activation. To date, only a handful of egg membrane proteins have been identified, and none of these are known to be involved in the egg activation pathway at fertilization. An excellent model organism to address how these events take place is the Pacific bat star, Patiria miniata, due to the quantity of synchronized oocytes that are large in size, nearly transparent, and amenable to many different techniques. To address this lack of knowledge regarding the egg cell surface, three databases were developed to explore the membrane proteome of the mature sea star egg. These databases utilized the first sea star mature egg transcriptome for the sea star, which has 96,723 unigenes and is available on NCBI (BioProject ID: 389668). The first database is a prediction of all mature egg transcripts that have putative transmembrane alpha-helical sequences using two different software programs: Phobius and TMHMM, which use different methods for prediction. After combining results from both programs, 15,501 transcripts were predicted to encode a transmembrane domain. The majority possessed a single transmembrane domain (9744); however, many were predicted to have multiple transmembrane domains. The second database categorized proteins into five major signaling families, each of which have been hypothesized to play a role in gamete interaction in different species. This database identified unique representatives of 26 Tetraspannins, 28 Receptor tyrosine kinases, 8 A Disintegrin and Metalloproteases (ADAMS), 140 G-protein coupled receptors, and 9 Integrins. The final database represents the membrane proteins currently present in the mature egg as identified by mass spectrometry. In the mature egg membrane preparation, 951 proteins were identified by mass spectrometry. Of these, 167 matched with the transcripts identified using the topology prediction software (Database 1) and 272 were matched with transcripts belonging to the five families of interest (Database 2). Interestingly, overlap between all three databases identified five proteins that are present in the unfertilized egg and belong to one of the signaling families predicted to be involved at the surface. These proteins were an integrin alpha and beta-like isoform, and three different tetraspannins. A major goal of this work is to provide targets for future functional studies of receptors involved in activation of fertilization at the egg surface.

Mannosylated glycoconjugates on the surface of activated sperm in the giant freshwater prawn are crucial for sperm binding with the egg vitelline envelop.

The unusual morphology and poorlydefined acrosome-like structure in the mature sperm of the giant freshwater prawn Macrobrachium rosenbergii has led to difficulties in identifying the state of sperm activation. Mature distal vas deferens sperm (dVSp) can be activated by the calcium ionophore A23187 to show acrosome reaction-like enzymatic activities that increase their binding and penetration capabilities. However, these short-lived enzymatic activities limit their usefulness as a marker of sperm activation for further qualitative and quantitative analyses, leading to our examining the alterations in the exposureof sperm surface glycoconjugates both as markers of sperm activation and for their role in gamete interaction. Our results showed that after A23187 treatment, there was an increased exposure of mannosylated glycoconjugates on the sperm surface revealed by significant Concanavalin A (Con A) staining. Furthermore, sodium metaperiodate pre-treatment, Con A pre-incubation, or co-incubation with α-mannose monosaccharides all significantly reduced A23187-induced dVSp binding to the egg vitelline envelop, demonstrating the importance of sperm surface mannosylated glycoconjugates in the binding process. These same pre-treatments of sperm also resulted in the inhibition of thebinding of soluble vitelline envelop proteins (MrVE) to both the sperm surface and to mannosylated dVSp protein bands. Therefore, the present study demonstrated the importance of the exposure of mannosylated glycoconjugates on the surface of activated dVSp, both as a reliable marker of sperm activation and as a binding factor in the gamete interaction process. Furthermore, these findings allow for a better understanding of the surface glycoconjugate-mediated interaction process between gametes in this species of prawn.

Mapping of the N-Linked Glycoproteome of Human Spermatozoa

N-Linked glycosylation, a type of post-translational modification, plays important roles in cell-cell recognition, adhesion, and interactions. Although N-linked glycosylated proteins in sperm are known to be important for gamete binding, little is known about the composition of these proteins, particularly glycosylation sites, in humans. In the present study, the use of glyco-FASP, coupled with the tandem mass spectrometry (MS/MS) method, led to the identification of 554 N-glycosylation sites and 297 N-glycosylated proteins in human sperm. Bioinformatics analysis revealed enrichment of proteins with functions in cell recognition and fertilization. Overall, about 91% of the human sperm N-glycosylated proteins were classified into "membrane", "extracellular region", and "lysosome" groups, based on subcellular localization annotation. Furthermore, glutathione peroxidase 4 (GPX4), a membrane glycoprotein identified in our glycoproteome, was shown to play a significant role in gamete interactions using the in vitro fertilization assay. Accordingly, we propose that characterization of the human sperm glycoproteome should effectively aid in clarifying the mechanisms of fertilization and provide a valuable resource for the future development of male contraceptives and diagnosis of male infertility.

Gamete interactions in teleost fish: the egg envelope. Basic studies and perspectives as environmental biomonitor.

The current knowledge about teleost fish egg envelope is summarized. The paper analyzes the organization and deposition process of the protein composition and genes involved in the synthesis of teleost fish egg envelopes and their role in gamete interaction during fertilization. Pelagic and demersal species that our research group is working with are especially considered. The vertebrate ZP family of proteins, the evolution and relationship among the different genes and their expression are taken into account. We consider fish envelope as a possible biomonitor for ecological contaminants. The biotechnological applications for aquaculture and genomic and post-genomic approaches are auspicious.

Gamete interactions in teleost fish: the egg envelope. Basic studies and perspectives as environmental biomonitor

The current knowledge about teleost fish egg envelope is summarized. The paper analyzes the organization and deposition process of the protein composition and genes involved in the synthesis of teleost fish egg envelopes and their role in gamete interaction during fertilization. Pelagic and demersal species that our research group is working with are especially considered. The vertebrate ZP family of proteins, the evolution and relationship among the different genes and their expression are taken into account. We consider fish envelope as a possible biomonitor for ecological contaminants. The biotechnological applications for aquaculture and genomic and post-genomic approaches are auspicious.

180. CHARACTERIZATION AND FUNCTION OF THE BULL SPERM PROTEIN Spam1: TWO DISTINCT ISOFORMS WITH DISTINCT ROLES

We identified an 80 kDa bull sperm protein (p80) that possesses homology with the Sperm adhesion molecule 1 (Spam1), a GPI-anchored glycoprotein conserved amongst mammals that is required for fertilization. Since bovine Spam1 had not been identified, the aim of this project was to determine if p80 is the bovine Spam1 and to test the hypothesis that it plays a role in gamete interaction during bovine fertilization. Amino acid sequence deduced from 3`/5`Race confirmed that homology between p80 and Spam1 in various species ranged from 47 to 61%.It also revealed specific differences including the absence of a GPI-anchor, the presence of a transmembrane domain, and N- and O- glycosylation sites. By generating and characterising antibodies against p80 N- and C-terminal domains, the protein orientation in the sperm membrane was evaluated. We identified two populations of p80 on the sperm head: one internalised in the anterior region and the second localised on the post-acrosomal region with its hyaluronidase domain exposed to the extracellular environment. Proteomic and immunologic analyses revealed that the p80 post-acrosomal population is a shorter isoform originating from the epididymis while the full length p80 located on the anterior region originates from the testis. Finally, the potential function of p80 during the sperm/zp interaction was evaluated by sperm/zona pellucida (zp) binding assay. The C-terminal extremity of p80 was implicated in sperm binding to the zp by antibody and native protein competition. Furthermore, glycosylation was not required during this interaction since deglycosylated p80 in the incubation medium had the same inhibitory effect on zona binding as the native p80. Collectively, the results demonstrated that p80 is the bovine Spam1, and that two isoforms are present on bull sperm. The hyaluronidase activity of the post-acrosomal isoform is required for cumulus penetration, while the other one participates in sperm/zp binding during fertilization.

Preliminary characterization of multiple hyaluronidase forms in boar reproductive tract

Hyaluronidases play an important role in gamete interaction and fertility in mammals. The objectives of the present study were to investigate multiple forms of the enzyme in boar reproductive tract using electrophoretic methods. Two forms of hyaluronidase (EC 3.2.1.35) were detected in boar seminal plasma (relative molecular masses of 55,000 and 65,000) using hyaluronic acid-substrate polyacrylamide gel electrophoresis in the presence of SDS. These two forms can be separated by means of affinity chromatography on Heparin-Sepharose. They differ, besides their affinity to heparin, also in the pH optimum of their enzymatic activity. The form with relative molecular mass of 55,000 was active both at the acidic (pH 3.7) and the neutral pH (pH 7.4) and was bound to immobilized heparin. The second form (relative molecular mass 65,000) was active only at acidic pH and did not interact with heparin. The same acidic-active form (65,000) was found in seminal vesicle fluids. The hyaluronidase form which is active both at the acidic and the neutral pH (51,000) was detected in epididymal fluid. In the detergent extracts of boar sperm, three active forms of the enzyme were found (relative molecular masses 55,000, 70,000 and 80,000). The form of relative molecular mass 55,000 was active in a wide range of pH (pH 3–8). The forms of relative molecular masses 70,000 and 80,000 were active only at neutral pH.

TheCaenorhabditis elegans spe-38gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization

A mutation in the Caenorhabditis elegans spe-38 gene results in a sperm-specific fertility defect. spe-38 sperm are indistinguishable from wild-type sperm with regards to their morphology, motility and migratory behavior. spe-38 sperm make close contact with oocytes but fail to fertilize them. spe-38 sperm can also stimulate ovulation and engage in sperm competition. The spe-38 gene is predicted to encode a novel four-pass (tetraspan) integral membrane protein. Structurally similar tetraspan molecules have been implicated in processes such as gamete adhesion/fusion in mammals, membrane adhesion/fusion during yeast mating, and the formation/function of tight-junctions in metazoa. In antibody localization experiments, SPE-38 was found to concentrate on the pseudopod of mature sperm, consistent with it playing a direct role in gamete interactions.

Influence of seminal plasma PSP-I/PSP-II spermadhesin on pig gamete interaction

The seminal plasma PSP-I/PSP-II spermadhesin is able to preserve, in vitro, the viability of highly extended boar spermatozoa, suggesting it might be used as a suitable ameliorator for the damaging effects of sperm handling, including in vitro fertilization. However, little is known about the ligand capability of PSP-I/PSP-II as regards the zona pellucida (ZP) or its possible role in gamete interaction. The present study evaluated the effect of the presence of PSP-I/PSP-II (1.5 mg/ml) during in vitro oocyte maturation and also during co-incubation of frozen-thawed boar spermatozoa with either immature (IM) or in vitro matured (IVM) oocytes, either enclosed by cumulus cells or denuded. Exposure of the gametes to the heterodimer during in vitro gamete co-incubation showed a significant blocking effect of sperm penetration rates and a decreased number of spermatozoa per oocyte in both IM and IVM denuded oocytes. Such an effect was not present in cumulus-enclosed oocytes, suggesting the effect could be mediated by exposed ZP receptors. In addition, when PSP-I/PSP-II was added to the IVM medium, oocyte maturation rates were significantly reduced. In conclusion, the results suggest that PSP-I/PSP-II, when present in vitro, blocks sperm-ZP binding.

Role of egg sulfolipidimmobilizing protein 1 on mouse sperm-egg plasma membrane binding.

We have shown that sperm sulfolipidimmobilizing protein 1 (SLIP1, molecular mass of 68 kDa), a sulfogalactosylglycerolipid (SGG)-binding protein, is significant in sperm-zona pellucida (ZP) interaction. The objective of this study was to localize SLIP1 on the egg and determine its role in gamete interaction. Immunofluorescence and immunoprotein A gold electron microscopy localized SLIP1 to the egg plasma membrane. In vitro gamete binding, using zona-free eggs preincubated with antiSLIP1 Fab before coincubation with sperm, showed a significant, dose-dependent decrease in sperm-egg plasma membrane binding. Similar results were obtained when affinity-purified antiSLIP1 IgG was used for egg pretreatment. The significance of egg SLIP1 in sperm-egg plasma membrane binding was further demonstrated by a decrease (36-52%) in in vitro fertilization when zona-intact eggs were pretreated with antiSLIP1 IgG. Since SLIP1 has been shown to bind SGG in vitro, we investigated the possibility that sperm SGG may participate in sperm-egg plasma membrane binding through egg SLIP1. Pretreatment of sperm with antiSGG Fab prior to coincubation with zona-free eggs resulted in a dose-dependent decrease in sperm-egg plasma membrane binding. Collectively, these findings strongly suggest a role for egg SLIP1 in sperm-egg plasma membrane interaction, which may be through its binding to sperm SGG.

A plasma membrane-associated hyaluronidase is localized to the posterior acrosomal region of stallion sperm and is associated with spermatozoal function.

Sperm hyaluronidase has been implicated in sperm penetration of the extracellular matrix of the cumulus oophorus and may play a crucial role in gamete interaction and fertility in mammals. The objectives of this study were to characterize the enzyme activity of equine sperm hyaluronidase and to investigate its cellular distribution. Zymography of stallion sperm plasma membrane extracts was used to identify hyaluronidase activity in protein bands. Affinity-purified polyclonal IgG raised against equine sperm hyaluronidase was used to label fresh and capacitated stallion sperm, followed by indirect immunofluorescence. Equine sperm plasma membrane extracts displayed 3 major protein bands with potent hyaluronidase activity of approximately 54, 59, and 83 kDa. Under reducing conditions, a single protein band was observed at 62 kDa, although the reduced sample exhibited no enzyme activity. The polyclonal IgG labeled the postacrosomal region of stallion sperm and was redistributed over the acrosomal region during in vitro capacitation in a significant percentage of sperm cells. These studies suggest that a specific protein localized to the equine sperm head displays hyaluronidase activity, gets redistributed over the acrosomal region during capacitation, and may be important in fertility in this species.

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization.

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after the sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface.

Vitronectin is an intrinsic protein of human spermatozoa released during the acrosome reaction.

Evidence has been presented that oolemmal integrins and their ligands on spermatozoa may play a role in gamete interactions leading to fertilization. We previously demonstrated that vitronectin (Vn) could be extracted from fresh human spermatozoa and detected in Western blots, and Vn was observed on the surface of living, capacitated sperm by indirect immunofluorescence. In the present experiments, messenger RNA encoding Vn was detected in human testis poly (A+) RNA using Northern analysis, and Vn was localized within the acrosomal region of ejaculated sperm by immunoperoxidase and immunofluorescence staining. During the acrosome reaction, induced in capacitated spermatozoa by lonomycin, Vn was released into the medium in a calcium-dependent manner. Vn appears to be a specific product of intratesticular spermatozoa that is secreted during the acrosome reaction. These findings suggest that Vn is positioned to play a strategic role in gamete interactions leading to fertilization.

The Involvement of Sperm and Egg Glycosidases in Animal Fertilization

A number of glycosidases have been detected in the seminal fluid of the male genital tract of mammals and in sperm and eggs of many invertebrate and vertebrate animals. Although some of these enzymes have been isolated and characterized, the elucidation of their presumed function in the fertilization process proved to be rather difficult. In relatively recent investigations evidence was presented that glycosidases are involved in binding of sperm to the acellular egg-coats of ascidian and mouse eggs, a step that is indispensable for successful fertilization. Furthermore, hyaluronidase and possibly β-N-acetylglucosaminidase from mammalian sperm are claimed to be involved in the penetration of the acellular egg coats, particularly through the cumulus oophorus. The precise mechanism of sperm penetration and whether it can be applied to fertilization in all mammals is still debated. Ascidians and mice are so far the only animals from which evidence exists that egg-bound glycosidases might be involved in the slow block to polyspermy.In summary, good evidence exists that glycosidases play an essential role in gamete interactions, particularly in the interaction of the sperm cell with the acellular egg matrix.

Surface sugar binding components of bovine spermatozoa as evidence by fluorescent neoglycoproteins.

In the present study, the topographical distribution of carbohydrate binding sites on the plasma membrane of bovine epididymal spermatozoa was investigated using 15 different fluorescent neoglycoproteins and asialoglycoproteins. With mannose-bovine serum albumin (BSA)-fluoresceinthiocarbamyl (FTC), mannose-6-phosphate-BSA-FTC, lactose-BSA-FTC, maltose-BSA-FTC, asialolactoferrin-FTC and asialotransferrin-FTC a marked fluorescence was observed in the postacrosomal area. These results further substantiate the concept that carbohydrate binding sites of the spermatozoan plasma membrane and corresponding carbohydrates of the zona pellucida play a significant role in gamete interactions.