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.

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 Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm

1987 ◽  
Vol 104 (4) ◽  
pp. 917-923 ◽  
Author(s):  
AE Cowan ◽  
DG Myles ◽  
DE Koppel
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Domain Boundary ◽  
Lateral Diffusion ◽  
Fold Increase ◽  
Membrane Domains ◽  
Mammalian Sperm ◽  
Acrosomal Membrane ◽  
Percent Recovery ◽  
Lipid Probe

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.

scholarly journals Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

1977 ◽  
Vol 74 (2) ◽  
pp. 561-577 ◽  
Author(s):  
DS Friend ◽  
L Orci ◽  
A Perrelet ◽  
R Yanagimachi
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Phosphatidylcholine Liposomes ◽  
Acrosomal Membrane ◽  
Large Particles ◽  
Fracture Appearance ◽  
Preparatory Stage

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.

scholarly journals Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm.

1986 ◽  
Vol 102 (4) ◽  
pp. 1363-1371 ◽  
Author(s):  
J D Bleil ◽  
P M Wassarman
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Present Report ◽  
Fusion Event ◽  
Acrosomal Membrane ◽  
Specific Receptors ◽  
Species Specific ◽  
Mammalian Eggs ◽  
Silver Grains ◽  
Sperm Receptor

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.

scholarly journals Isoforms of Ankyrin-3 That Lack the NH2-terminal Repeats Associate with Mouse Macrophage Lysosomes

1997 ◽  
Vol 136 (5) ◽  
pp. 1059-1070 ◽  
Author(s):  
Thomas C. Hoock ◽  
Luanne L. Peters ◽  
Samuel E. Lux
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Endocytic Pathway ◽  
Cdna Clones ◽  
Mouse Macrophage ◽  
Regulatory Domains ◽  
Binding Domains ◽  
Mouse Macrophages ◽  
Repeat Domain ◽  
Terminal Repeats

We have recently cloned and characterized ankyrin-3 (also called ankyrinG), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313–330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH2-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29–amino acid segment of the membrane (“repeat”) domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrinR), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3–positive vesicles appear dark on phasecontrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran– containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3–positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

1988 ◽  
Vol 21 (3) ◽  
pp. 297-311 ◽  
Author(s):  
A. Stojanoff ◽  
H. Bourne ◽  
A. G. Andrews ◽  
R. V. Hyne
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Phospholipid Composition ◽  
Acrosomal Membrane

scholarly journals ACROSOMAL DISRUPTION IN SPERM

1974 ◽  
Vol 63 (2) ◽  
pp. 466-479 ◽  
Author(s):  
Daniel S. Friend ◽  
Irene Rudolf
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Direct Consequence ◽  
Thin Sections ◽  
Geometric Patterns ◽  
Acrosomal Membrane ◽  
Principal Piece

"Capacitation" is a physiological event which alters sperm to permit rapid penetration through oocyte investments and fusion between gametes. Acrosomal "reaction," the physiological release of acrosomal contents, occurs after this facilitating process. In this study, acrosomal "disruption" of guinea pig and rat sperm was achieved in vitro by incubating sperm together with the follicular contents of superovulated mice. The samples contained both "reacted" and "disrupted" sperm. Thin sections of affected sperm revealed rupture and vesiculation of the plasma membrane overlying the acrosome, as well as loss of both the outer acrosomal membrane and the acrosomal content. Freeze-fracture revealed disintegration of the characteristic geometric patterns in regions of the acrosomal and plasma membranes thus disrupted and major modifications in particle distribution in the sperm tail. In the guinea pig, strands of 6–8-nm particles, usually confined to the plasma membrane of the midpiece, which overlies mitochondria, also appeared in the principal piece. Likewise, in rat sperm, bands of similarly small particles formed acute angles throughout the membrane of the principal piece. Compared with the membranes of control preparations, these membrane alterations are apparently a direct consequence of incubation with ovarian follicular contents.

scholarly journals A Role for the Disintegrin Domain of Cyritestin, a Sperm Surface Protein Belonging to the ADAM Family, in Mouse Sperm–Egg Plasma Membrane Adhesion and Fusion

1997 ◽  
Vol 137 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Ruiyong Yuan ◽  
Paul Primakoff ◽  
Diana G. Myles
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Active Site ◽  
Surface Protein ◽  
Equatorial Region ◽  
Cdna Clones ◽  
Sperm Surface ◽  
Disintegrin Domain ◽  
Adam Family ◽  
Membrane Adhesion

Sperm–egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell–cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm–egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (α and β subunits). Fertilin α and β are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin β functions in sperm–egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin α, fertilin β, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin β, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm–egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80–90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin β active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin β also strongly inhibited (80–90%) both sperm–egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin β showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin β in sperm–egg plasma membrane adhesion leading to fusion.

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.

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.

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