Role In Sperm-egg Interaction Research Articles

CRISPR/Cas9-mediated genome editing reveals seven testis-enriched transmembrane glycoproteins dispensable for male fertility in mice.

Mammalian fertilization is mediated by multiple sperm acrosomal proteins, many of which are testis-enriched transmembrane glycoproteins expressed during spermiogenesis (e.g., Izumo sperm-egg fusion 1, Sperm acrosome associated 6, and Transmembrane protein 95). We hypothesized that proteins with these features might have a role in sperm-egg interaction and thus carried out an in-silico screen based on multiple public databases. We generated knockout mouse lines lacking seven candidate proteins by the CRISPR/Cas9 system and conducted detailed analyses on the fecundity of the knockout males, as well as their testis appearance and weight, testis and epididymis histology, and sperm motility and morphology. Through the in-silico screen, we identified 4932438H23Rik, A disintegrin and metalloproteinase domain-containing protein 29, SAYSvFN domain-containing protein 1, Sel-1 suppressor of lin-12-like 2 (C. elegans), Testis-expressed protein 2, Transmembrane and immunoglobulin domain-containing 3, and Zinc and ring finger 4. Phenotypic analyses unveiled that the knockout males showed normal testis gross appearance, normal testis and epididymis histology, and normal sperm morphology and motility. Fertility tests further indicated that the knockout male mice could sire pups with normal litter sizes when paired with wild-type females. These findings suggest that these seven proteins are individually dispensable for male reproduction and fertilization. Future studies are warranted to devise advanced in-silico screening approaches that permit effective identification of gamete fusion-required sperm proteins.

P–026 Seminal plasma exosomes: a promising source of biomarkers for fertility evaluation

Abstract Study question Do exosomes from seminal plasma have a role in male fertility? Summary answer Exosomes isolated from seminal plasma have a pivotal role during spermatogenesis and sperm maturation and may represent eligible biomarkers for male fertility/infertility. What is known already During their journey along the male reproductive tract, exosomes contained in seminal fluid are involved in the transfer of several molecules to the maturing sperm. Exosomes are extracellular vesicles (EVs) released by all the cells; they carry a cargo of nucleic acids, proteins and lipids. In the male genital tract, they are released at various levels and their composition differs between men of proven fertility and infertile male patients. Recent studies reported the proteomic profile of exosomes, revealing the presence of several proteins with a well know role in sperm maturation and fertilizing ability acquiring. Study design, size, duration This prospective study consisted of 36 Caucasian men; according to seminal parameters (WHO 2010) they were divided in normozoospermic (N; n = 12), oligoasthenoteratozoospermic (OAT: n = 12) and azoospermic (A; n = 12). Semen samples were collected between October 2020 and January 2021 at the Assisted Reproductive Unit, Siena University Hospital (Italy) after institutional ethical approval and signed written consent from all the participants. Participants/materials, setting, methods Ejaculated sperm were analyzed according to WHO–2010 criteria and divided into the three groups: N, OAT and A. Exosomes were isolated by an in-house modified ExoGAG®-polymer precipitation-based protocol and characterized for size and ultrastructure by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The exosomal proteins were extracted and analyzed by 2D-electrophoresis and the identified profiles were examined by applying bioinformatic tools. The expression of selected genes was evaluated by digital droplets PCR (ddPCR). Main results and the role of chance The present work is readily providing an improvement of the standard ExoGAG® protocol and underlines its advantages over more conventional EVs isolation protocols used to date for recovery from seminal fluid: the number of recovered EVs and their size were finely included in the range of exosomes. This isolation protocol provides samples suitable for proteomic analyses, representing the first 2D-electrophoresis reference map of exosome-pay loaded proteins in N respect to OAT/A groups and providing an innovative and comprehensive functional overview of its proteins. Moreover, the STRING protein-protein interaction analysis revealed the deregulation of specific pathways (e.g. signaling proteins, chromatin packaging and/or remodeling, protein folding and apoptosis) in A and OAT in comparison with N group. Gene expression by ddPCR analysis highlighted that most of the analyzed genes are modulated in according to seminal parameters, in particular: GAPDHS (Glyceraldehyde–3-Phosphate Dehydrogenase, Spermatogenic); SPAM1 (Sperm Adhesion Molecule–1) encoding a members of hyaluronidase family; ADAM2 (ADAM Metallopeptidase Domain–2) that plays an important role in sperm-egg interactions; CRISP1,2,3 (Cysteine Rich Secretory Protein 1,2,3) expressed in the epididymis and secreted into the epididymal lumen; CLGN (Calmegin) encoding a testis-specific chaperone protein and PGK2 (Phosphoglycerate Kinase–2) expressed in the later stages of spermatogenesis. Limitations, reasons for caution This study represents a preliminary experiment. We suggest further comparative studies in larger study cohorts. Wider implications of the findings: This pilot study, demonstrating the unique proteomic and transcriptomic pattern of exosomes in N/OAT/A groups, supports the importance of exosomes in sperm production and maturation. This methodological set-up is expected to open new ways for advancement in the use of exosomes as fertility biomarkers, making possible personalized approaches in ART. Trial registration number Not applicable

Identification and Characterization of TEX101 in Bovine Epididymal Spermatozoa.

Several studies exhibit the presence of Ricinus Communis Agglutinin I (RCA) binding glycocalyx in mammalian spermatozoa. However, the molecular characterization of RCA binding glycocalyx in sperm membranes and its mechanism of action are poorly understood. The objective of the study was to identify and to characterize RCA binding glycoprotein of the bovine sperm plasma membranes (PM). Lectin blots of caput and cauda sperm PM revealed a 38 kDa polypeptide exhibiting the highest affinity to RCA among the several major RCA binding polypeptides. The 38 kDa RCA binding polypeptide of cauda sperm PM was purified and exhibited a charge train of three distinct spots with isoelectric points (pH 5.3 and 5.8). Proteomic identification yielded ten peptides that matched the sequence of Testis Expressed 101 protein (TEX101). Western blots data revealed that bovine sperm TEX101 is present in both testicular and epididymal sperm PM fractions. The native TEX101 polypeptide contains ~17 kDa N-linked oligosaccharides and the polypeptide is anchored to sperm membrane via a glycosylphosphatidylinositol lipid linkage. Immunofluorescence staining of sperm with anti-TEX101 demonstrated that the polypeptide is localized at the head of cauda sperm. Our biochemical results provide evidence on the presence of TEX101 in bovine epididymal sperm plasma membranes and may have a potential role in sperm-egg interaction.

Construction and Analysis of the Cell Surface’s Protein Network for Human Sperm-Egg Interaction

Sperm-egg interaction is one of the most impressive processes in sexual reproduction, and understanding the molecular mechanism is crucial in solving problems in infertility and failed in vitro fertilization. The main purpose of this study is to map the sperm-egg interaction network between cell-surface proteins and perform an interaction analysis on this new network. We built the first protein interaction network of human sperm-egg binding and fusion proteins that consists of 84 protein nodes and 112 interactions. The gene ontology analysis identified a number of functional clusters that may be involved in the sperm-egg interaction. These include G-protein coupled receptor protein signaling pathway, cellular membrane fusion, and single fertilization. The PPI network showed a highly interconnected network and identified a set of candidate proteins: ADAM-ZP3, ZP3-CLGN, IZUMO1-CD9, and ADAM2-IZUMO1 that may have an important role in sperm-egg interaction. The result showed that the ADAM2 may mediate interaction between two essential factors CD9 and IZUMO1. The KEGG analysis showed 12 statistically significant pathways with 10 proteins associated with cancer, suggesting a common pathway between tumor fusion and sperm-egg fusion. We believe that the availability of this map will assist future researches in the fertilization mechanism and will also facilitate biological interpretation of sperm-egg interaction.

Testicular and epididymal ADAMs: expression and function during fertilization

The disintegrin and metalloprotease domain-containing protein (ADAM) family of multidomain membrane proteins comprises at least 34 members in mammals. More than half of these proteins are expressed specifically or predominantly in mammalian testes and epididymis, implying their prominence in male reproduction. These reproductive ADAMs can be classified into three phylogenetic groups; designated I, II, and III. Each group displays remarkably contrasting features. Group I contains 11 ADAMs expressed in the testis. The genes that encode these proteins lack introns in their coding sequences and most of the proteins are processed into prodomain-lacking forms in mature sperm. Five ADAMs--encoded by genes with multiple exons and introns--belong to phylogenetic group II. These ADAMs are also expressed in testicular germ cells, but both prodomains and metalloprotease domains are lacking in mature sperm. Two phylogenetic group III ADAMs are synthesized in the epididymis; one of which is secreted and transferred to the sperm surface. Some of these sperm ADAMs are assembled into potentially functional complexes, including ADAM1B-ADAM2, ADAM2-ADAM3-ADAM4, ADAM2-ADAM3-ADAM5, and ADAM2-ADAM3-ADAM6. It has been suggested that ADAM2 and ADAM3 have roles in sperm-egg interactions. Mouse knockout studies have revealed that the ADAM2-ADAM3 complex is critical for in vivo sperm migratory function in the female reproductive tract.

Dicalcin Inhibits Fertilization through Its Binding to a Glycoprotein in the Egg Envelope in Xenopus laevis

Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca(2+)-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca(2+)-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization.

Stabilization of membrane‐associated α‐l‐fucosidase by the human sperm equatorial segment

Previous reports from this laboratory documented the existence of two novel isoforms of alpha-L-fucosidase in human semen and showed that membrane-associated alpha-L-fucosidase is cryptically held within the acrosomal compartment and enriched within the sperm equatorial segment. The occurrence of these novel isoforms is provocative. Sperm proteins potentially involved in sperm-egg interactions must maintain their functional integrity as they travel through the female reproductive tract. The goal of this project was to investigate the stability of membrane-associated alpha-L-fucosidase in human sperm. Whole seminal plasma and Percoll purified sperm cell populations were incubated for 72 h at 37 degrees C, with 5% CO(2) or ambient air. At various times during prolonged incubation, sperm cells were permeabilized with 0.1% Triton X-100 and enzyme assays using the fluorogenic substrate 4-MU-fuc were performed to evaluate the stability of both the seminal plasma and membrane-associated alpha-L-fucosidase. Here, we report seminal plasma alpha-L-fucosidase activity rapidly decreased within 24 h. Conversely, alpha-L-fucosidase activity from Percoll purified sperm cell populations persisted up to 72 h. Data from these experiments support the notions that (i) membrane-associated alpha-L-fucosidase is stable for extended periods of time, consistent with a possible role in sperm-egg interaction and (ii) membrane domains and compartmentalization within the human sperm are key to preserving protein integrity.

Loss of Zona Pellucida Binding Proteins in the Acrosomal Matrix Disrupts Acrosome Biogenesis and Sperm Morphogenesis

Zona pellucida binding protein 1 (ZPBP1), a spermatid and spermatozoon protein that localizes to the acrosome, was originally identified in pigs and named for its binding to the oocyte zona pellucida. In an in silico search for germ cell-specific genes, Zpbp1 and its novel paralog, Zpbp2, were discovered and confirmed to be expressed only in the testes in both mice and humans. To study the in vivo functions of both ZPBP proteins, we disrupted Zpbp1 and Zpbp2 in mice. Males lacking ZPBP1 were sterile, with abnormal round-headed sperm morphology and no forward sperm motility. Ultrastructural studies demonstrated that absence of ZPBP1 prevents proper acrosome compaction, resulting in acrosome fragmentation and disruption of the Sertoli-spermatid junctions. Males null for ZPBP2 were subfertile, demonstrated aberrant acrosomal membrane invaginations, and produced dysmorphic sperm with reduced ability to penetrate zona pellucida. Molecular phylogenetic analysis of ZPBPs from amphibians, birds, and mammals suggests that these paralogous genes coevolved to play cooperative roles during spermiogenesis. Whereas ZPBP1 was discovered for an in vitro role in sperm-egg interactions, we have shown that both ZPBP proteins play an earlier structural role during spermiogenesis.

P-575: Seminal fibronectin in certain cases of male infertility

ObjectiveFibronectin is a dimeric filament forming glycoprotein. It is a secretory product of accessory sex glands. fibronectin is a major compound of the human seminal gel and also pointed to its potential role in sperm trapping in it. It may play a major role in sperm-egg interaction. The objective was to study seminal fibronectin level in certain cases of male infertility in relation to other variables.DesignA prospective studyMaterials and methodsA study on 30 infertile men grouped into three groups with asthenozoospermia (9 cases), asthenoteratozoospermia (10 cases), and oligoasthenoteratozoospermia (11 cases). Ten fertile normozoospermic men were selected as control group. Thorough history and full clinical examination were done with complete semen analysis using a simple computer-assisted (Autosperm) method. Seminal plasma fibronectin was measured by Radial Immune Diffusion plate. Serum testosterone and F.S.H. levels were detected using enzyme immunoassay.ResultsHigh significant increase of seminal plasma fibronectin levels were detected among different infertile groups compared to control one. Significant negative correlation was found between fibronectin level and grade (A, B and A+B) motility. There was a significant negative correlation between fibronectin level and sperm velocity, linear velocity and linearity index. Fibronectin was negatively correlated with serum testosterone level while seminal WBCs count was positively correlated significantly.ConclusionDecrease sperm motility is found in association with increase concentration of fibronectin in seminal plasma. This may add a diagnostic tool for patients with unexplained infertility and would provide an important lead to develop new therapeutic strategies directed towards the mechanisms of male infertility. ObjectiveFibronectin is a dimeric filament forming glycoprotein. It is a secretory product of accessory sex glands. fibronectin is a major compound of the human seminal gel and also pointed to its potential role in sperm trapping in it. It may play a major role in sperm-egg interaction. The objective was to study seminal fibronectin level in certain cases of male infertility in relation to other variables. Fibronectin is a dimeric filament forming glycoprotein. It is a secretory product of accessory sex glands. fibronectin is a major compound of the human seminal gel and also pointed to its potential role in sperm trapping in it. It may play a major role in sperm-egg interaction. The objective was to study seminal fibronectin level in certain cases of male infertility in relation to other variables. DesignA prospective study A prospective study Materials and methodsA study on 30 infertile men grouped into three groups with asthenozoospermia (9 cases), asthenoteratozoospermia (10 cases), and oligoasthenoteratozoospermia (11 cases). Ten fertile normozoospermic men were selected as control group. Thorough history and full clinical examination were done with complete semen analysis using a simple computer-assisted (Autosperm) method. Seminal plasma fibronectin was measured by Radial Immune Diffusion plate. Serum testosterone and F.S.H. levels were detected using enzyme immunoassay. A study on 30 infertile men grouped into three groups with asthenozoospermia (9 cases), asthenoteratozoospermia (10 cases), and oligoasthenoteratozoospermia (11 cases). Ten fertile normozoospermic men were selected as control group. Thorough history and full clinical examination were done with complete semen analysis using a simple computer-assisted (Autosperm) method. Seminal plasma fibronectin was measured by Radial Immune Diffusion plate. Serum testosterone and F.S.H. levels were detected using enzyme immunoassay. ResultsHigh significant increase of seminal plasma fibronectin levels were detected among different infertile groups compared to control one. Significant negative correlation was found between fibronectin level and grade (A, B and A+B) motility. There was a significant negative correlation between fibronectin level and sperm velocity, linear velocity and linearity index. Fibronectin was negatively correlated with serum testosterone level while seminal WBCs count was positively correlated significantly. High significant increase of seminal plasma fibronectin levels were detected among different infertile groups compared to control one. Significant negative correlation was found between fibronectin level and grade (A, B and A+B) motility. There was a significant negative correlation between fibronectin level and sperm velocity, linear velocity and linearity index. Fibronectin was negatively correlated with serum testosterone level while seminal WBCs count was positively correlated significantly. ConclusionDecrease sperm motility is found in association with increase concentration of fibronectin in seminal plasma. This may add a diagnostic tool for patients with unexplained infertility and would provide an important lead to develop new therapeutic strategies directed towards the mechanisms of male infertility. Decrease sperm motility is found in association with increase concentration of fibronectin in seminal plasma. This may add a diagnostic tool for patients with unexplained infertility and would provide an important lead to develop new therapeutic strategies directed towards the mechanisms of male infertility.

The Mouse t Complex Gene Tsga2, Encoding Polypeptides Located in the Sperm Tail and Anterior Acrosome, Maps to a Locus Associated with Sperm Motility and Sperm-Egg Interaction Abnormalities1

Previous studies of sperm from mice heterozygous for a t haplotype (t) and heterospecific combinations of the t complex identified two tightly linked genetic factors responsible for t/t male sterility related to expression of the flagellar waveform aberration, curlicue. Dnahc8, an axonemal dynein heavy chain gene, is a strong candidate for the proximal factor, Ccua, but the identity of the distal factor, Ccub, is unknown. In the present study, we employ motility assays of sperm from males heterozygous for t and novel heterospecific combinations of the t complex to demonstrate that Ccub is a composite of at least two synergic elements, Ccub1, positioned within a genomic interval spanning approximately 0.6 Mb immediately distal to Dnahc8, and Ccub2, situated in a region approximately 4-7 Mb distal to Ccub1. We also show that Tsga2, a testis-restricted gene, fulfills many of the prerequisites required to make it a strong candidate for Ccub1. These include: 1) its location within the aforementioned genomic interval; 2) a highly reduced level of testis expression by its heterospecific allele relative to the level of expression of its t allele; 3) determination that TSGA2(t) carries numerous nonsynonymous mutations in residues otherwise highly conserved in all known orthologous proteins; 4) the detection of major TSGA2 polypeptides in sperm protein extracts; and 5) the apparent distribution of these polypeptides in major sperm tail structures. Surprisingly, these TSGA2 isoforms appear to localize in the vicinity of the anterior acrosome, as well, suggesting that Tsga2 may also play a role in sperm-egg interaction. Finally, our results indicate that a TSGA2 polypeptide with apparent similarities to the smaller of the two sperm isoforms is expressed by epididymal cells.

SAMP14, a Novel, Acrosomal Membrane-associated, Glycosylphosphatidylinositol-anchored Member of the Ly-6/Urokinase-type Plasminogen Activator Receptor Superfamily with a Role in Sperm-Egg Interaction

We report a new member of the Ly-6/urokinase-type plasminogen activator receptor (uPAR) superfamily of receptors, SAMP14, which is retained on the inner acrosomal membrane of the human spermatozoan following the acrosome reaction and may play a role in fertilization. The SAMP14 sequence predicted a glycosylphosphatidylinositol (GPI)-anchored protein with a signal peptide, a transmembrane domain near the carboxyl terminus, and a putative transamidase cleavage site in the proprotein. Attachment of SAMP14 to the membrane by a lipid anchor was confirmed by its sensitivity to phosphatidylinositol phospholipase C. SAMP14 has a single functional domain similar to the Ly-6 and urokinase plasminogen activator receptor superfamily of proteins, and the gene mapped to 19q13.33, near the PLAUR locus for uPAR at 19q13.2. Northern and dot blotting showed that SAMP14 expression was testis-specific. Indirect immunofluorescence and immunoelectron microscopy with antisera to purified recombinant SAMP14 localized the protein to outer and inner acrosomal membranes as well as the acrosomal matrix of ejaculated human sperm. Acrosome-reacted sperm demonstrated SAMP14 immunofluorescence, indicating its retention on the inner acrosomal membrane following the acrosome reaction. However, SAMP14 localized to the entire sperm when unwashed swim-up sperm from the ejaculate were stained, indicating that some SAMP14 is loosely associated with the plasma membrane. Antibodies against recombinant SAMP14 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that SAMP14 may have a role in sperm-egg interaction. SAMP14 represents a GPI-anchored putative receptor in the Ly-6/uPAR family that is exposed on the inner acrosomal membrane after the acrosome reaction.

A re-evaluation of sperm protein 17 (Sp17) indicates a regulatory role in an A-kinase anchoring protein complex, rather than a unique role in sperm-zona pellucida binding.

Sp17 was originally proposed to be a sperm-specific protein and thought to play a role in sperm-egg interactions by binding to the zona pellucida via two conserved heparin-binding motifs. However, more recent data indicate that it may be expressed more widely, both in tumours and in normal somatic tissues. The complete nucleotide and deduced amino acid sequences of human Sp17 transcripts are reported and it is shown that they are expressed in a wide range of tissues, albeit at a much lower expression than in the testis. On the basis of the extremely high sequence conservation throughout the N-terminal half of Sp17, and the presence within this region of an A-kinase anchoring protein (AKAP)-binding motif, it is postulated that the proposed role of Sp17 in zona pellucida binding is unlikely to be its principal function.

Localization of N‐linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm-egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N-acetyllactosamine regions of the carbohydrate chains are removed by endo-beta-Galactosidase digestion. Endo-beta-Galactosidase-digested ZPB retains stronger sperm-binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N-linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm-ligand active high-mannose-type chain and the acidic complex-type chains in bovine ZPA. Three N-glycopeptides of ZPA, containing an N-glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo-beta-Galactosidase-digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N-linked chains obtained from each N-glycopeptide were characterized by two-dimensional sugar mapping analysis, while considering the structures of the N-linked chains of the zona protein mixture reported previously. Acidic complex-type chains were found at all three N-glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm-ligand active chain at Asn527 in the ZP domain of ZPA.

Density and distribution of anionic sites on boar ejaculated and epididymal spermatozoa.

Current knowledge implies that spermatozoa successively acquire negative surface charges as they migrate through the epididymis. Until recently, however, techniques used were not amenable to statistical analysis. In the present study, a novel approach allowing numerical assessment of negative charge labelling was used in order to determine the density and distribution of anionic sites on ejaculated and maturing spermatozoa collected from six regions of the boar epididymis. Labelling was assessed quantitatively for the three morphologically distinct membrane domains on the sperm head. Statistical analysis revealed that labelling density was highest on efferent duct spermatozoa, declined up to the proximal corpus and then increased again. Densities of anionic sites on distal corpus, proximal cauda and ejaculated sperm cells were similar but significantly below the values obtained for efferent duct spermatozoa. All three sperm membrane domains underwent parallel changes. However, the overall density of negative charges on the postacrosomal segment was significantly higher as compared to the acrosomal plasma membrane. These alterations reflect sperm surface modifications through removal and addition of anionic groups. Since charge interactions are considered to play a pivotal role in sperm-egg interactions, these processes should be viewed as an integral part of sperm maturation.

Identification of perivitelline N-linked glycans as mediators of sperm-egg interaction in chickens

This study demonstrates that carbohydrates play an essential role in sperm-egg interactions in birds. Sperm-egg interaction was measured in vitro as the ability of spermatozoa to hydrolyse a small hole in the inner perivitelline layer, the equivalent of the mammalian zona pellucida. Preincubation with Triticum vulgaris lectin (WGA) and succinyl-WGA (S-WGA) at 10 microgram ml(-1) resulted in complete inhibition of sperm-egg interaction, whereas at the same concentration a range of other lectins (Canavalia ensiformis (Con A), Arachis hypogea (PNA), Ulex europaeus II (UEA II), Solanum tuberosum (STA), Tetragonolobus purpureas (LTA) and Pisum sativum (PSA)) were unable to inhibit sperm egg interaction significantly, although fluorescein-labelled derivatives of these lectins were found to stain the inner perivitelline layer. Significant inhibition of sperm-egg interaction was achieved by the addition of N-acetyl-D-glucosamine and fucoidin to the assay mixture; however, D-glucose, D-galactose, D-fucose and L-fucose had no significant effect on sperm-egg interaction. Pretreatment of the inner perivitelline layer with N-glycanase significantly reduced sperm-egg interaction, whereas treatment with O-glycanase had no effect. These results demonstrate that N-linked glycans play an essential role in sperm-egg interaction in chickens.

Identification of perivitelline N-linked glycans as mediators of sperm-egg interaction in chickens

This study demonstrates that carbohydrates play an essential role in sperm-egg interactions in birds. Sperm-egg interaction was measured in vitro as the ability of spermatozoa to hydrolyse a small hole in the inner perivitelline layer, the equivalent of the mammalian zona pellucida. Preincubation with Triticum vulgaris lectin (WGA) and succinyl-WGA (S-WGA) at 10 microgram ml(-1) resulted in complete inhibition of sperm-egg interaction, whereas at the same concentration a range of other lectins (Canavalia ensiformis (Con A), Arachis hypogea (PNA), Ulex europaeus II (UEA II), Solanum tuberosum (STA), Tetragonolobus purpureas (LTA) and Pisum sativum (PSA)) were unable to inhibit sperm egg interaction significantly, although fluorescein-labelled derivatives of these lectins were found to stain the inner perivitelline layer. Significant inhibition of sperm-egg interaction was achieved by the addition of N-acetyl-D-glucosamine and fucoidin to the assay mixture; however, D-glucose, D-galactose, D-fucose and L-fucose had no significant effect on sperm-egg interaction. Pretreatment of the inner perivitelline layer with N-glycanase significantly reduced sperm-egg interaction, whereas treatment with O-glycanase had no effect. These results demonstrate that N-linked glycans play an essential role in sperm-egg interaction in chickens.

Role of sperm sulfogalactosylglycerolipid in mouse sperm-zona pellucida binding.

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of mammalian male germ cells. Like other sulfoglycolipids, SGG is believed to be involved in cell-cell/extracellular matrix adhesion. Specifically, we investigated whether sperm SGG played a role in sperm-egg interaction. Initially, we produced an affinity-purified, rabbit polyclonal immunoglobulin (Ig) G antibody that specifically recognized SGG (anti-SGG). Indirect immunofluorescence using anti-SGG IgG localized SGG to the convex and concave ridges and the postacrosome of the mouse sperm head. Pretreatment of sperm with anti-SGG IgG/Fab inhibited sperm-zona pellucida (ZP) binding in vitro in a concentration-dependent manner (to a maximum of 62%). This inhibition was observed at the level of primary binding. Sperm treated with anti-SGG IgG underwent the spontaneous and ZP-induced acrosome reaction at the same rate as control sperm treated with preimmune rabbit serum IgG. Fluorescently labeled SGG liposomes were shown to associate specifically with the egg ZP, whereas fluorescently labeled liposomes of galactosylglycerolipid (SGG's parental lipid) and phosphatidylserine (negatively charged like SGG) did not. Furthermore, coincubation of SGG liposomes with sperm and isolated ZP inhibited sperm-ZP binding in a concentration-dependent manner. These results strongly suggest an involvement of sperm SGG in direct binding to the ZP.

Functional processing of fertilin: evidence for a critical role of proteolysis in sperm maturation and activation

Fertilin is a sperm surface protein with an essential role in fertilization. It is required for the migration of spermatozoa through the oviduct, for binding to the zona pellucida, and for efficient binding to the egg plasma membrane. Fertilin consists of two subunits, fertilin alpha and beta, both of which belong to the metalloprotease-disintegrin protein family (ADAMs). Fertilin alpha and beta are made as larger precursors that are processed proteolytically at different stages of sperm maturation in the testis and epididymis. Fertilin alpha is processed first, most likely by a pro-protein convertase in the secretory pathway of testicular cells. Fertilin beta is processed later, while spermatozoa are in transit through the epididymis. The processing of fertilin beta in the epididymis correlates with the acquisition of fertilization competence in spermatozoa, exposes an epitope that has a role in sperm-egg interactions, and triggers the relocalization of fertilin from the whole sperm head to the posterior head. These findings indicate that the proteolytic processing of fertilin and perhaps also other sperm proteins plays an important role in sperm maturation and activation in the epididymis. Further evaluation of the functional significance of proteolysis for sperm maturation should lead to new and exciting insights into the mechanism of sperm maturation, and may also uncover the cause of certain types of male infertility. The identification of the responsible proteases could provide novel targets for contraceptive drugs.

Functional processing of fertilin: evidence for a critical role of proteolysis in sperm maturation and activation

Fertilin is a sperm surface protein with an essential role in fertilization. It is required for the migration of spermatozoa through the oviduct, for binding to the zona pellucida, and for efficient binding to the egg plasma membrane. Fertilin consists of two subunits, fertilin alpha and beta, both of which belong to the metalloprotease-disintegrin protein family (ADAMs). Fertilin alpha and beta are made as larger precursors that are processed proteolytically at different stages of sperm maturation in the testis and epididymis. Fertilin alpha is processed first, most likely by a pro-protein convertase in the secretory pathway of testicular cells. Fertilin beta is processed later, while spermatozoa are in transit through the epididymis. The processing of fertilin beta in the epididymis correlates with the acquisition of fertilization competence in spermatozoa, exposes an epitope that has a role in sperm-egg interactions, and triggers the relocalization of fertilin from the whole sperm head to the posterior head. These findings indicate that the proteolytic processing of fertilin and perhaps also other sperm proteins plays an important role in sperm maturation and activation in the epididymis. Further evaluation of the functional significance of proteolysis for sperm maturation should lead to new and exciting insights into the mechanism of sperm maturation, and may also uncover the cause of certain types of male infertility. The identification of the responsible proteases could provide novel targets for contraceptive drugs.

Mannose-binding molecules of rat spermatozoa and sperm-egg interaction.

We have previously reported the occurrence and partial characterisation of an alpha-D-mannosidase activity on plasma membranes of rat, mouse, hamster and human spermatozoa. A soluble isoform of the rat sperm surface mannosidase was purified and polyclonal antibody raised. Since several reports have suggested that mannosyl residues on the rat, mouse and human zona pellucida may be involved in sperm-zona binding, studies were undertaken to examine the receptor-like role of mannose-binding molecules on rat spermatozoa. Sprague-Dawley rats (25-30-days old) were superovulated and eggs collected from the oviduct were treated with 0.3% hyaluronidase to remove the cumulus cells. Spermatozoa, collected from the cauda epididymis were capacitated for 5 h at 37 degrees C in 5% CO2 in air. The sperm-zona binding assay was performed in the presence of increasing concentrations of several sugars as well as preimmune and immune (anti-mannosidase or anti-mannose binding protein) IgG. Data from these studies show that: (1) significantly fewer sperm bound per egg in the presence of competitive inhibitors of mannosidase; (2) among the sugars examined, D-mannose was the most potent inhibitor causing 70% reduction in the number of sperm bound per egg; (3) anti-mannosidase or anti-mannose binding protein (but not preimmune) IgG showed a dose-dependent reduction in the number of sperm bound per egg; (4) anti-mannosidase IgG (but not anti-mannose binding protein IgG) showed a dose-dependent inhibition of sperm surface mannosidase activity; (5) the competitive inhibitors of mannosidase or the immune IgG had no effect on sperm motility or the sperm acrosome reaction. These result suggest that mannose-binding molecule(s) such as alpha-D-mannosidase or mannose-binding protein on the spermatozoa may recognise mannosyl residues on zona pellucida, and play a receptor-like role in sperm-egg interaction in the rat.