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.

scholarly journals Identification of phosphatidylserine-binding proteins in human white blood cells

1990 ◽  
Vol 269 (3) ◽  
pp. 723-728 ◽  
Author(s):  
M Wolf ◽  
M Baggiolini
Keyword(s):  
Protein Kinase C ◽  
Blood Cells ◽  
Binding Proteins ◽  
White Blood Cells ◽  
Human Neutrophils ◽  
Kinase C ◽  
Sds Page ◽  
Molecular Masses ◽  
Human White Blood Cells ◽  
Monocytes And Lymphocytes

Cytosol and membrane fractions from human neutrophils, monocytes, lymphocytes and platelets were separated by SDS/PAGE, blotted on to nitrocellulose and assayed for selective binding of phosphatidylserine (PS). Two PS-binding proteins with apparent molecular masses of 115 kDa and 100 kDa were identified in the cytosol of neutrophils, monocytes and lymphocytes. Corresponding bands along with other PS-binding proteins were detected in platelets in both cytosol and membrane fractions. These proteins were also found to bind protein kinase C (PKC) provided that PS was present. The 115 kDa and 100 kDa proteins (PS-p115/110) were partially purified from neutrophils and were used for the study of PS and PKC binding. The binding of PS did not require Ca2+ or Mg2+ and was inhibited by phosphatidic acid, by 1-alkyl-2-acetylphosphocholine and, to a lesser extent, by other lipids. The binding of PKC, however, was strictly PS- and Ca2(+)-dependent and seems to occur secondarily to PS binding.

Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 141-149 ◽  
Author(s):  
S. Mortillo ◽  
P.M. Wassarman
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Transmission Electron ◽  
Acrosomal Membrane ◽  
Membrane Compartments ◽  
Sperm Membrane ◽  
Differential Binding ◽  
Inner Acrosomal Membrane ◽  
Low Levels ◽  
Species Specific

Egg zona pellucida glycoproteins mZP3 and mZP2 serve as primary and secondary sperm receptors, respectively, during initial stages of fertilization in mice [Wassarman (1988) A. Rev. Biochem. 57, 415–442]. These receptors interact with complementary egg-binding proteins (EBPs) located on the sperm surface to support species-specific gamete adhesion. Results of whole-mount autoradiographic experiments suggest that purified egg mZP3 and mZP2 bind preferentially to acrosome-intact (AI) and acrosome-reacted (AR) sperm heads, respectively [Bleil and Wassarman (1986) J. Cell Biol. 102, 1363–1371]. Here, we used purified egg mZP2, egg mZP3 and fetuin, which were coupled directly to colloidal gold (‘gold-probes’), to examine binding of these glycoproteins to membrane compartments of AI and AR sperm by transmission electron microscopy. mZP3 gold-probes were found associated primarily with plasma membrane overlying the acrosomal and post-acrosomal regions of AI sperm heads. They were also found associated with plasma membrane overlying the post-acrosomal region of AR sperm heads. mZP2 gold-probes were found associated primarily with inner acrosomal membrane of AR sperm heads, although some gold was associated with outer acrosomal membrane of AI sperm that had holes in plasma membrane overlying the acrosome. Fetuin gold-probes, used to assess background levels of binding, were bound at relatively low levels to plasma membrane and inner acrosomal membrane of AI and AR sperm, respectively. None of the gold-probes exhibited significant binding to sperm tails, or to red blood cells and residual bodies present in sperm preparations. These results provide further evidence that mZP2 and mZP3 bind preferentially to heads of AR and AI sperm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

2012 ◽  
Vol 349 (3) ◽  
pp. 881-895 ◽  
Author(s):  
Marvin Ferrer ◽  
Hilma Rodriguez ◽  
Lindsay Zara ◽  
Yang Yu ◽  
Wei Xu ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Acrosomal Membrane ◽  
Sperm Penetration ◽  
Inner Acrosomal Membrane

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 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.

scholarly journals Ultrastructural localization of guinea pig spermatozoal autoantigens on germinal cells by immunoperoxidase techniques.

1979 ◽  
Vol 27 (4) ◽  
pp. 857-866 ◽  
Author(s):  
P P Le Bouteiller ◽  
F Toullet ◽  
S Righenzi ◽  
G A Voisin
Keyword(s):  
Guinea Pig ◽  
Outer Zone ◽  
Ultrastructural Localization ◽  
Water Soluble ◽  
Acrosomal Membrane ◽  
Specific Antisera ◽  
Additive Combination ◽  
Inner Acrosomal Membrane ◽  
Heterologous Antigens ◽  
Germinal Cells

Three guinea pig spermatozoal autoantigens S, P and T, each one able to induce autoimmune aspermatogenic orchiepididymitis and autoantibodies, were ultrastructurally localized in male germinal cells by immunoperoxidase techniques. Both living and prefixed sectioned cell preparations were treated and examined. Fab antibody fragments were used to study intracellular antigens (whole antibodies were inefficient). Water-soluble S and P autoantigens were found in acrosomal structures in the same sites: proacrosomal and acrosomal granules of the young spermatids, on the head caps of spermatids and acrosomal cap of spermatozoa, along the inner and outer acrosomal membranes and in the outer zone of the acrosomal matrix of the same cells. S was never found in the inner zone of spermatid or spermatozoa acrosomes, while P was present in this inner zone, but only of young spermatids. Water-insoluble T autoantigen was found on the plasmalemma and outer acrosomal membranes of spermatids and spermatozoa, inside the spermatid cytoplasm and, sometimes, on the inner acrosomal membrane of young spermatids. The specificity of the immunological localization for each antigen was confirmed by testing with specific antisera following absorption with homologous and heterologous antigens. No other testicular cell type (including Sertoli cells per se) was found to bear S, P or T autoantigens. When use was made of autoimmune sera obtained through autologous whole spermatozoa, the observed staining was an additive combination of what was observed when using the preceding three immune sera, anti-S, anti-P and anti-T.

scholarly journals Isolation and characterization of galectin-1 binding proteins from human placenta

2000 ◽  
Vol 65 (2) ◽  
pp. 131-140
Author(s):  
Miroslava Jankovic
Keyword(s):  
Human Placenta ◽  
Binding Proteins ◽  
Solid Phase ◽  
Ricinus Communis ◽  
Gel Filtration ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Molecular Masses ◽  
Solid Phase Binding

Galectin-1 binding proteins were isolated from human placenta by affinity chromatography on a column with immobilized endogenous lectin. The molecular masses of the isolated proteins of 170, 67 and 56 kDa were estimated by gel filtration and SDS-PAGE. These proteins were characterized as galactose-containing glycoproteins, based on their reactivity with Ricinus communis agglutinin. In addition, sialylated- lacto-N-fucopentaose II was detected in the 170 kDa protein, using anti CA 19-9 monoclonal antibodies. The interaction of the isolated proteins with human placental galectin-1 was investigated by a solid phase binding assay using asialofetuin as the glycoprotein ligand. The 67 kDa and 56 kDa proteins were found to inhibit galectin-1 binding of asialofetuin, whereas the 170 kDa protein had the opposite effect. It caused an increase in the binding of asialofetuin, suggesting a positive cooperative binding.

The histochemical localization of acrosin in guinea-pig sperm after the acrosome reaction

1978 ◽  
Vol 32 (1) ◽  
pp. 177-184
Author(s):  
D.P. Green ◽  
A.R. Hockaday
Keyword(s):  
Electron Microscopy ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Sperm Head ◽  
Ultrastructural Evidence ◽  
Acrosomal Membrane ◽  
Sperm Penetration ◽  
Inner Acrosomal Membrane ◽  
Dense Marker

The protease acrosin is widely considered to be an essential component of a zona lysin which enables sperm to penetrate the zona pellucida of the egg. Sperm form a characteristic penetration slit little wider than the sperm head itself and this has long suggested that any zona lysin is attached to the sperm surface after an acrosome reaction. This paper provides the first ultrastructural evidence that this is the case. The protein acrosin inhibitor, Kunitz soybean trypsin inhibitor, has been covalently attached to the electron-dense marker, ferritin, and the conjugate incubated with guinea-pig sperm which have undergone an A23187-induced acrosome reaction. Electron microscopy shows that ferritin is distributed unevenly over the outer surface of the newly exposed inner acrosomal membrane but does not extend to the equatorial segment. This is further evidence that acrosin can be considered as a candidate for the role of zona lysin. The mechanism of sperm penetration of the zona is discussed in the light of these observations.

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