scholarly journals A role for the migrating sperm surface antigen PH-20 in guinea pig sperm binding to the egg zona pellucida.

1985 ◽  
Vol 101 (6) ◽  
pp. 2239-2244 ◽  
Author(s):  
P Primakoff ◽  
H Hyatt ◽  
D G Myles
Keyword(s):  
Guinea Pig ◽  
Surface Antigen ◽  
Zona Pellucida ◽  
Binding Assays ◽  
Sperm Binding ◽  
Acrosomal Membrane ◽  
Head Surface ◽  
Competition Binding ◽  
Igg1 Subclass ◽  
Different Levels

After the acrosome reaction, the PH-20 surface antigen of guinea pig sperm migrates from its original location on the posterior head surface to a new location on the inner acrosomal membrane (Myles, D.G., and P. Primakoff, 1984, J. Cell Biol., 99:1634-1641). We have isolated three monoclonal antibodies (MAbs) of the IgG1 subclass, PH-20, PH-21, and PH-22, that bind to the PH-20 antigen. The PH-20 MAb strongly inhibited (approximately 90%) sperm binding to the guinea pig egg zona pellucida at saturating antibody concentrations (greater than 20 micrograms/ml). Half-maximal inhibition of sperm binding to the zona was obtained with approximately 2 micrograms/ml PH-20 MAb. The PH-21 MAb at saturating concentration (50 micrograms/ml) partially inhibited (approximately 45%) sperm-zona binding, and the PH-22 MAb (50 micrograms/ml) did not inhibit (0%) sperm-zona binding. Essentially the same amounts of the three MAbs were bound to sperm under the conditions where inhibition (PH-20, PH-21) or no inhibition (PH-22) of sperm-zona binding was observed, which indicates that the different levels of inhibition did not arise from different levels of MAb binding. Competition binding assays with 125I-labeled MAbs showed that PH-21 binding to sperm was not affected by the binding of PH-20 or PH-22. However, that PH-20 and PH-22 blocked each other's binding to sperm suggests that their recognized determinants may be relatively close to one another. The results indicate that the migrating PH-20 antigen has a required function in sperm binding to the zona pellucida and that the PH-20 MAb affects is active site.

Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization

2001 ◽  
Vol 114 (22) ◽  
pp. 4127-4136
Author(s):  
Elizabeth Howes ◽  
John C. Pascall ◽  
Wolfgang Engel ◽  
Roy Jones
Keyword(s):  
Zona Pellucida ◽  
Solid Phase ◽  
Binding Assays ◽  
Vitro Fertilization ◽  
Sperm Binding ◽  
Secondary Receptor ◽  
Specificity Of Binding ◽  
Solid Phase Binding ◽  
Complementary Binding

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

scholarly journals Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization.

1984 ◽  
Vol 99 (5) ◽  
pp. 1634-1641 ◽  
Author(s):  
D G Myles ◽  
P Primakoff
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Guinea Pig ◽  
Surface Antigen ◽  
Acrosome Reaction ◽  
Tail Region ◽  
Head Region ◽  
Surface Molecules ◽  
Acrosomal Membrane ◽  
Sperm Population

We have previously defined distinct localizations of antigens on the surface of the guinea pig sperm using monoclonal antibodies. In the present study we have demonstrated that these antigen localizations are dynamic and can be altered during changes in the functional state of the sperm. Before the sperm is capable of fertilizing the egg, it must undergo capacitation and an exocytic event, the acrosome reaction. Prior to capacitation, the antigen recognized by the monoclonal antibody, PT-1, was restricted to the posterior tail region (principle piece and end piece). After incubation in capacitating media at 37 degrees C for 1 h, 100% of the sperm population showed migration of the PT-1 antigen onto the anterior tail. This redistribution of surface antigen resulted from a migration of the surface molecules originally present on the posterior tail. It did not occur in the presence of metabolic poisons or when tail-beating was prevented. It was temperature-dependent, and did not require exogenous Ca2+. Since the PT-1 antigen is freely diffusing on the posterior tail before migration, the mechanism of redistribution could involve the alteration of a presumptive membrane barrier. In addition, we observed the redistribution of a second surface antigen after the acrosome reaction. The antigen recognized by the monoclonal antibody, PH-20, was localized exclusively in the posterior head region of acrosome-intact sperm. Within 7-10 min of induction of the acrosome reaction with Ca2+ and A23187, 90-100% of the acrosome-reacted sperm population no longer demonstrated binding of the PH-20 antibody on the posterior head, but showed binding instead on the inner acrosomal membrane. This redistribution of the PH-20 antigen also resulted from the migration of pre-existing surface molecules, but did not appear to require energy. The migration of PH-20 antigen was a selective process; other antigens localized to the posterior head region did not leave the posterior head after the acrosome reaction. These rearrangements of cell surface molecules may act to regulate cell surface function during fertilization.

scholarly journals cDNA cloning reveals the molecular structure of a sperm surface protein, PH-20, involved in sperm-egg adhesion and the wide distribution of its gene among mammals.

1990 ◽  
Vol 111 (6) ◽  
pp. 2939-2949 ◽  
Author(s):  
W F Lathrop ◽  
E P Carmichael ◽  
D G Myles ◽  
P Primakoff
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Zona Pellucida ◽  
Genomic Dna ◽  
Surface Protein ◽  
Wide Distribution ◽  
Cdna Clones ◽  
Southern Blots ◽  
Binding Domains ◽  
Acrosomal Membrane

Sperm binding to the egg zona pellucida in mammals is a cell-cell adhesion process that is generally species specific. The guinea pig sperm protein PH-20 has a required function in sperm adhesion to the zona pellucida of guinea pig eggs. PH-20 is located on both the sperm plasma membrane and acrosomal membrane. We report here the isolation and sequence of a full-length cDNA for PH-20 (available from EMBL/GenBank/DDBJ under accession number X56332). The derived amino acid sequence shows a mature protein of 468 amino acids containing six N-linked glycosylation sites and twelve cysteines, eight of which are tightly clustered near the COOH terminus. The sequence indicates PH-20 is a novel protein with no relationship to the mouse sperm adhesion protein galactosyl transferase and no significant homology with other known proteins. The two PH-20 populations, plasma membrane and acrosomal membrane, could arise because one form of PH-20 is encoded and differentially targeted at different spermatogenic stages. Alternatively, two different forms of PH-20 could be encoded. Our evidence thus far reveals only one sequence coding for PH-20: Southern blots of guinea pig genomic DNA indicated there is a single PH-20 gene, Northern blots showed a single size PH-20 message (approximately 2.2 kb), and no sequence variants were found among the sequenced cDNA clones. Cross-species Southern blots reveal the presence of a homologue of the PH-20 gene in mouse, rat, hamster, rabbit, bovine, monkey, and human genomic DNA, showing the PH-20 gene is conserved among mammals. Since genes for zona glycoproteins are also conserved among mammals, the general features of sperm and zona proteins involved in mammalian sperm-egg adhesion may have been evolutionarily maintained. Species specificity may result from limited changes in these molecules, either in their binding domains or in other regions that affect the ability of the binding domains to interact.

scholarly journals Role of guinea-pig sperm autoantigens in sperm binding to the zona pellucida and oocyte penetration

Reproduction ◽  
1986 ◽  
Vol 77 (2) ◽  
pp. 347-353 ◽  
Author(s):  
G. M. de Vera ◽  
B. M.-L. Guienne ◽  
M. De Almeida ◽  
G. A. Voisin
Keyword(s):  
Guinea Pig ◽  
Zona Pellucida ◽  
Sperm Binding

Midazolam Selectively Potentiates the A2A- but not A1-receptor– mediated Effects of Adenosine

2000 ◽  
Vol 92 (2) ◽  
pp. 567-567 ◽  
Author(s):  
Christoph N. Seubert ◽  
Timothy E. Morey ◽  
Anatoly E. Martynyuk ◽  
Roy F. Cucchiara ◽  
Donn M. Dennis
Keyword(s):  
Guinea Pig ◽  
Dependent Manner ◽  
Nucleoside Transporter ◽  
Binding Assays ◽  
A2a Adenosine Receptor ◽  
Concentration Dependent Manner ◽  
Isolated Hearts ◽  
Mediated Effects ◽  
Competition Binding ◽  
A1 Receptor

Background Inhibition of adenosine metabolism offers a unique approach to harness the cardioprotective properties of adenosine in a site- and event-specific manner. Benzodiazepines inhibit adenosine metabolism by blocking nucleoside transporter. Therefore, the authors studied the binding affinities of structurally different benzodiazepines to nucleoside transporter and benzodiazepine-induced potentiation of A1-adenosine (negative dromotropy) and A2A-adenosine (coronary vasodilation) receptor-mediated effects. Methods In membranes from porcine striatum and guinea pig ventricle, competition binding assays to displace [3H]nitrobenzylmercaptopurine riboside ([3H]NBMPR) from nucleoside transporter were performed using alprazolam, chlorodiazepoxide, diazepam, flurazepam, and midazolam. The augmentation by the most potent benzodiazepine of A1- and A2A-adenosine receptor-mediated responses, elicited by exogenous administration of adenosine or brief periods of global hypoxia, was subsequently studied in guinea pig Langendorff-perfused hearts. Results All benzodiazepines completely displaced [3H]NBMPR in a concentration-dependent manner with Hill coefficients not significantly different from unity in both striatal and ventricular membranes. Midazolam was the most potent inhibitor of nucleoside transporter (ventricle:pKi = 5.22+/-0.41, Ki = 6 microM). In isolated hearts, midazolam (5, 10, 20 microM) significantly augmented coronary flow in a concentration-dependent manner in the presence of adenosine (30 nM), an effect reversed by ZM 241385, a selective A2A-receptor antagonist. In contrast, midazolam did not increase the effect of adenosine (30 nM) on atrioventricular conduction. Similarly, midazolam potentiated A2A- but not A1-receptor-mediated effects of endogenous adenosine released during hypoxia. Conclusions Structurally distinct benzodiazepines inhibit nucleoside transporter to different degrees. Midazolam selectively augments A2A- but not A1-receptor-mediated effects of adenosine by inhibiting nucleoside transporter.

Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation

2013 ◽  
Vol 25 (1) ◽  
pp. 26 ◽  
Author(s):  
B. M. Gadella
Keyword(s):  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Molecular Level ◽  
Mammalian Species ◽  
Perivitelline Space ◽  
Dynamic Regulation ◽  
Sperm Binding ◽  
Cumulus Oocyte Complex ◽  
Acrosomal Membrane ◽  
Matrix Properties

Recent findings have refined our thinking on sperm interactions with the cumulus–oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona–cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm–ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.

scholarly journals Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

2012 ◽  
Vol 197 (7) ◽  
pp. 897-905 ◽  
Author(s):  
Boris Baibakov ◽  
Nathan A. Boggs ◽  
Belinda Yauger ◽  
Galina Baibakov ◽  
Jurrien Dean
Keyword(s):  
Zona Pellucida ◽  
Human Sperm ◽  
Binding Assays ◽  
Terminal Domain ◽  
Sperm Binding ◽  
Gamete Recognition ◽  
Recombinant Peptides ◽  
Sperm Penetration ◽  
Mouse Eggs

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1–3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm–egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy.

Identification of zona- and fucoidan-binding proteins in guinea-pig spermatozoa and mechanism of recognition

Development ◽  
1990 ◽  
Vol 109 (1) ◽  
pp. 41-50
Author(s):  
R. Jones ◽  
R.M. Williams
Keyword(s):  
Guinea Pig ◽  
Zona Pellucida ◽  
Binding Proteins ◽  
Sulphated Polysaccharides ◽  
Acrosomal Membrane ◽  
Sds Page ◽  
Molecular Masses ◽  
Low Mass ◽  
Inner Acrosomal Membrane ◽  
Mechanism Of Recognition

Binding of guinea-pig spermatozoa to the zona pellucida of homologous eggs has been reported to involve ‘receptors’ on the inner acrosomal membrane (Huang et al. 1981). These receptors can be blocked by sulphated polysaccharides such as fucoidan (Huang and Yanagimachi, 1984). The aims of the present investigation were to identify these putative zona receptors using 125I-fucoidan as a probe and examine their mechanism of recognition. Results show that 125I-fucoidan binds to several proteins extracted from guinea-pig spermatozoa with molecular masses of 95, 60, 48, 34, 30 and 18–20 × 10(3) (K) on SDS-PAGE. The 48K, 34K and 30K components represent proacrosin and two forms of acrosin, respectively. 125I-zona pellucida glycoproteins also bound strongly to the 48K, 34K and 30K sperm proteins. The other high and low mass binding proteins were not positively identified but cytochemical experiments with fluoresceinamine-fucoidan and FITC-soybean trypsin inhibitor indicate that they are intraacrosomal. The mechanism of binding of 125I-fucoidan to proacrosin/acrosin (and also the 95K, 60K and 18K-20K components) involves multiple sulphate groups on the polysaccharide in a specific orientation to allow them to interact with basic residues on the protein. It is suggested that guinea-pig spermatozoa retain sufficient proacrosin/acrosin bound to the inner acrosomal membrane after the acrosome reaction to mediate binding to the zona pellucida and that functionally proacrosin is analogous to sea urchin binding.

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

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