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Diana G. Myles - One of the best experts on this subject based on the ideXlab platform.
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analysis of mouse Fertilin in wild type and Fertilin β sperm evidence for c terminal modification α β dimerization and lack of essential role of Fertilin α in sperm egg fusion
Developmental Biology, 2000Co-Authors: Chunghee Cho, Paul Primakoff, Daniel R Branciforte, Diana G. MylesAbstract:Abstract The sperm surface protein Fertilin functions in sperm–egg interaction. On guinea pig and bovine sperm, Fertilin is a heterodimer of α and β subunits. Both subunits are initially synthesized as precursors and then proteolytically processed by removing N-terminal domains. Since the mouse is currently the main mammalian species in which fertilization is studied, in the present report, we analyzed the structure, processing, and expression of Fertilin in mouse. We found that the processing of mouse Fertilin β occurs during epididymal maturation and involves changes in the cytoplasmic tail domain as well as the N-terminal domains. Although we (R. Yuan et al., 1997, J. Cell Biol. 137, 105–112) and others (M. S. Chen et al., 1999, J. Cell Biol. 144, 549–561) have previously reported that mature Fertilin β is 55–57 kDa, here we show that 55 kDa is an unrelated protein in the sperm extract which cross-reacts with an antibody that recognizes precursor, but not mature, Fertilin β. Comparison of Western blots of wild-type and Fertilin β knockout sperm revealed that authentic, mature Fertilin β is 45 kDa. We also obtained direct evidence that mouse Fertilin α and β exist as a heterodimer. In addition, we found that in mice lacking the Fertilin β subunit, Fertilin α is absent from mature sperm. A widely proposed model for sperm–egg fusion suggests that Fertilin α is the sperm component that promotes membrane fusion by undergoing a conformational change that exposes a virus-like, hydrophobic fusion peptide. Because sperm lacking Fertilin α and Fertilin β can fuse with eggs at 50% the wild-type rate, this model is called into question. The results suggest instead that other gamete surface molecules act to promote membrane fusion and that Fertilin's role in gamete fusion is in sperm–egg plasma membrane adhesion.
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Analysis of the Process of Localization of Fertilin to the Sperm Posterior Head Plasma Membrane Domain during Sperm Maturation in the Epididymis
Developmental Biology, 1997Co-Authors: Gary R. Hunnicutt, Dennis E. Koppel, Diana G. MylesAbstract:Abstract Fertilin is a heterodimeric (subunits α and β) sperm plasma membrane protein. Both subunits belong to the ADAM protein family of surface proteins that containa disintegrin anda metalloprotease domain. Fertilin functions in sperm–egg fusion by binding the sperm to the egg plasma membrane via a binding site in the disintegrin domain of Fertilin β. On testicular sperm of guinea pig, Fertilin is distributed on the plasma membrane over the entire sperm head, but is found only on the posterior head once sperm have passed through the epididymis. This redistribution of Fertilin to the posterior head can be partially mimickedin vitroif testicular sperm are briefly treated with trypsin. In this study we used immunofluorescence and digital image analysis to analyze how Fertilin becomes restricted to the posterior head. We found that Fertilin became restricted to the posterior head by migration of anterior head Fertilin molecules into the posterior head domain. Comparison of immunofluorescence patterns and immunoblots of Fertilin from seven regions of the epididymis showed a temporal correlation between the beginning of Fertilin's migration to the posterior head and the proteolytic processing of the full-length Fertilin β precursor (the 85-kDa pro-β form) to a 75-kDa intermediate, pro-β*. Completion of the migration coincided with the further cleavage of pro-β* to the 25- to 28-kDa mature form. Our data suggest that the cleavage of Fertilin pro-β to pro-β* may initiate Fertilin's migration into the posterior head domain and, after localization to that membrane domain, pro-β* is cleaved to mature β. We also report evidence that a common mechanism may be used to change the localization pattern of other sperm surface molecules. Other surface proteins were shown to become localized to either the posterior or the anterior head membrane domains on sperm at the same time Fertilin became localized to the posterior head. These restrictions of surface protein localizations were also shown to immediately precede the development of the sperm's ability to swim and undergo the acrosome reaction, and thus redistribution of surface proteins may be necessary before sperm become functional.
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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
Journal of Cell Biology, 1997Co-Authors: Ruiyong Yuan, Paul Primakoff, Diana G. MylesAbstract: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.
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why did the sperm cross the cumulus to get to the oocyte functions of the sperm surface proteins ph 20 and Fertilin in arriving at and fusing with the egg
Biology of Reproduction, 1997Co-Authors: Diana G. Myles, Paul PrimakoffAbstract:The sperm surface has an active role in the events of fertilization. The definition of the sperm surface in both its composition and domain organization begins during spermatogenesis and continues until the moment of sperm-egg fusion. Alterations of the surface proceed as a result of internal programming and environmental cues from both the male and female reproductive tracts, including interactions with the egg itself. We have investigated the sperm surface to understand its domain organization and the ongoing changes in this organization as well as the role of specific surface proteins in fertilization. Much of our research has concentrated on two surface proteins: PH-20 and Fertilin. PH-20 is a single-chain protein, anchored in the membrane via a glycosyl phosphatidylinositol (GPI) anchor. The N-terminal domain of the molecule has a hyaluronidase activity. The hyaluronidase activity of PH-20 on the sperm plasma membrane enables sperm to penetrate the layer of cumulus cells surrounding the oocyte. PH-20 has a second function, unrelated to its hyaluronidase activity, in the binding of acrosome-reacted sperm to the zona pellucida (secondary sperm-zona binding). The Fertilin molecule is an alpha,beta heterodimer whose two subunits are closely related transmembrane proteins. Fertilin beta has a disintegrin domain that has high sequence homology with the snake disintegrins, a known class of soluble integrin ligands. The binding site of the beta disintegrin domain functions to bind sperm to the egg plasma membrane via a mechanism that leads to sperm-egg fusion. The precursor of Fertilin alpha, made in the testis, has an active metalloprotease site that could function in spermatogenesis. This metalloprotease domain is removed by proteolytic processing in the testis. Mature Fertilin alpha on sperm also has a hydrophobic, putative "fusion peptide" that may promote the process of lipid bilayer fusion between sperm and egg plasma membranes. Fertilin alpha and beta are the first identified members of a new gene family of transmembrane proteins, the ADAM family, so called because they contain A Disintegrin And Metalloprotease domain. Many distinct ADAMs have now been found in diverse tissues and species (Drosophila to human) and are proposed to have a variety of functions in development and the adult. In addition to Fertilin, other ADAMs are also present on the sperm plasma membrane and may participate with Fertilin in sperm-egg fusion.
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genomic organization of the mouse Fertilin β gene that encodes an adam family protein active in sperm egg fusion
Developmental Genetics, 1997Co-Authors: Laura Turner, Paul Primakoff, Diana G. MylesAbstract:: The Fertilin alpha and beta genes (Ftna and Ftnb, respectively) encode a sperm surface heterodimer that functions in sperm-egg fusion. They are the first identified members of a large gene family coding for multidomain membrane proteins (ADAMs) that include A Disintegrin And Metalloprotease domain. In this study, we report the isolation and structural organization of the mouse Fertilin beta gene. The gene is present as a single copy and covers a region of approximately 55 kilobases in the genome. The Fertilin beta gene is composed of at least 20 exons interrupted by 19 introns. The sizes of the exons are relatively small and vary from 56 to 193 bases; the sizes of introns vary from 350 bases to 9.4 kilobases. The exon-intron boundaries conform to the GT/AG rule with one exception: GC replaces GT at the 5' splice site in intron 13. Comparison of genomic organization between mouse Fertilin beta and the previously sequenced ADAM family gene, human MDC [Katagiri et al. (1995): Cytogenet Cell Genet 68:39-44] showed 12 conserved exon-intron boundaries. In addition, we analyzed the Fertilin alpha gene, demonstrating that more than one gene is present in the mouse genome.
Paul Primakoff - One of the best experts on this subject based on the ideXlab platform.
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analysis of mouse Fertilin in wild type and Fertilin β sperm evidence for c terminal modification α β dimerization and lack of essential role of Fertilin α in sperm egg fusion
Developmental Biology, 2000Co-Authors: Chunghee Cho, Paul Primakoff, Daniel R Branciforte, Diana G. MylesAbstract:Abstract The sperm surface protein Fertilin functions in sperm–egg interaction. On guinea pig and bovine sperm, Fertilin is a heterodimer of α and β subunits. Both subunits are initially synthesized as precursors and then proteolytically processed by removing N-terminal domains. Since the mouse is currently the main mammalian species in which fertilization is studied, in the present report, we analyzed the structure, processing, and expression of Fertilin in mouse. We found that the processing of mouse Fertilin β occurs during epididymal maturation and involves changes in the cytoplasmic tail domain as well as the N-terminal domains. Although we (R. Yuan et al., 1997, J. Cell Biol. 137, 105–112) and others (M. S. Chen et al., 1999, J. Cell Biol. 144, 549–561) have previously reported that mature Fertilin β is 55–57 kDa, here we show that 55 kDa is an unrelated protein in the sperm extract which cross-reacts with an antibody that recognizes precursor, but not mature, Fertilin β. Comparison of Western blots of wild-type and Fertilin β knockout sperm revealed that authentic, mature Fertilin β is 45 kDa. We also obtained direct evidence that mouse Fertilin α and β exist as a heterodimer. In addition, we found that in mice lacking the Fertilin β subunit, Fertilin α is absent from mature sperm. A widely proposed model for sperm–egg fusion suggests that Fertilin α is the sperm component that promotes membrane fusion by undergoing a conformational change that exposes a virus-like, hydrophobic fusion peptide. Because sperm lacking Fertilin α and Fertilin β can fuse with eggs at 50% the wild-type rate, this model is called into question. The results suggest instead that other gamete surface molecules act to promote membrane fusion and that Fertilin's role in gamete fusion is in sperm–egg plasma membrane adhesion.
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fertilization defects in sperm from mice lacking Fertilin β
Science, 1998Co-Authors: Chunghee Cho, Paul Primakoff, Donna O Bunch, Jean Emmanuel Faure, Eugenia H Goulding, E M Eddy, Diana C MylesAbstract:Fertilin, a member of the ADAM family, is found on the plasma membrane of mammalian sperm. Sperm from mice lacking Fertilin beta were shown to be deficient in sperm-egg membrane adhesion, sperm-egg fusion, migration from the uterus into the oviduct, and binding to the egg zona pellucida. Egg activation was unaffected. The results are consistent with a direct role of Fertilin in sperm-egg plasma membrane interaction. Fertilin could also have a direct role in sperm-zona binding or oviduct migration; alternatively, the effects on these functions could result from the absence of Fertilin activity during spermatogenesis.
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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
Journal of Cell Biology, 1997Co-Authors: Ruiyong Yuan, Paul Primakoff, Diana G. MylesAbstract: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.
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why did the sperm cross the cumulus to get to the oocyte functions of the sperm surface proteins ph 20 and Fertilin in arriving at and fusing with the egg
Biology of Reproduction, 1997Co-Authors: Diana G. Myles, Paul PrimakoffAbstract:The sperm surface has an active role in the events of fertilization. The definition of the sperm surface in both its composition and domain organization begins during spermatogenesis and continues until the moment of sperm-egg fusion. Alterations of the surface proceed as a result of internal programming and environmental cues from both the male and female reproductive tracts, including interactions with the egg itself. We have investigated the sperm surface to understand its domain organization and the ongoing changes in this organization as well as the role of specific surface proteins in fertilization. Much of our research has concentrated on two surface proteins: PH-20 and Fertilin. PH-20 is a single-chain protein, anchored in the membrane via a glycosyl phosphatidylinositol (GPI) anchor. The N-terminal domain of the molecule has a hyaluronidase activity. The hyaluronidase activity of PH-20 on the sperm plasma membrane enables sperm to penetrate the layer of cumulus cells surrounding the oocyte. PH-20 has a second function, unrelated to its hyaluronidase activity, in the binding of acrosome-reacted sperm to the zona pellucida (secondary sperm-zona binding). The Fertilin molecule is an alpha,beta heterodimer whose two subunits are closely related transmembrane proteins. Fertilin beta has a disintegrin domain that has high sequence homology with the snake disintegrins, a known class of soluble integrin ligands. The binding site of the beta disintegrin domain functions to bind sperm to the egg plasma membrane via a mechanism that leads to sperm-egg fusion. The precursor of Fertilin alpha, made in the testis, has an active metalloprotease site that could function in spermatogenesis. This metalloprotease domain is removed by proteolytic processing in the testis. Mature Fertilin alpha on sperm also has a hydrophobic, putative "fusion peptide" that may promote the process of lipid bilayer fusion between sperm and egg plasma membranes. Fertilin alpha and beta are the first identified members of a new gene family of transmembrane proteins, the ADAM family, so called because they contain A Disintegrin And Metalloprotease domain. Many distinct ADAMs have now been found in diverse tissues and species (Drosophila to human) and are proposed to have a variety of functions in development and the adult. In addition to Fertilin, other ADAMs are also present on the sperm plasma membrane and may participate with Fertilin in sperm-egg fusion.
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genomic organization of the mouse Fertilin β gene that encodes an adam family protein active in sperm egg fusion
Developmental Genetics, 1997Co-Authors: Laura Turner, Paul Primakoff, Diana G. MylesAbstract:: The Fertilin alpha and beta genes (Ftna and Ftnb, respectively) encode a sperm surface heterodimer that functions in sperm-egg fusion. They are the first identified members of a large gene family coding for multidomain membrane proteins (ADAMs) that include A Disintegrin And Metalloprotease domain. In this study, we report the isolation and structural organization of the mouse Fertilin beta gene. The gene is present as a single copy and covers a region of approximately 55 kilobases in the genome. The Fertilin beta gene is composed of at least 20 exons interrupted by 19 introns. The sizes of the exons are relatively small and vary from 56 to 193 bases; the sizes of introns vary from 350 bases to 9.4 kilobases. The exon-intron boundaries conform to the GT/AG rule with one exception: GC replaces GT at the 5' splice site in intron 13. Comparison of genomic organization between mouse Fertilin beta and the previously sequenced ADAM family gene, human MDC [Katagiri et al. (1995): Cytogenet Cell Genet 68:39-44] showed 12 conserved exon-intron boundaries. In addition, we analyzed the Fertilin alpha gene, demonstrating that more than one gene is present in the mouse genome.
Janice Perry Evans - One of the best experts on this subject based on the ideXlab platform.
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analysis of the roles of rgd binding integrins α4 α9 integrins α6 integrins and cd9 in the interaction of the Fertilin β adam2 disintegrin domain with the mouse egg membrane
Biology of Reproduction, 2002Co-Authors: Xiaoling Zhu, Janice Perry EvansAbstract:Abstract Fertilin β (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin β utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize Fertilin β binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the α5/α8/αv/αIIb or RGD-binding subfamily (α5β1, α8β1, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, and αIIbβ3) and the α4/α9 subfamily (α4β1, α9β1, and α4β7). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for Fertilin β binding. Peptides with the sequence MLDG, which perturb α4/α9 integrin-mediated interactions, signific...
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analysis of Fertilin α adam1 mediated sperm egg cell adhesion during fertilization and identification of an adhesion mediating sequence in the disintegrin like domain
Journal of Biological Chemistry, 2001Co-Authors: Grace E Wong, Xiaoling Zhu, Chrissy E Prater, Janice Perry EvansAbstract:Abstract Fertilin α (also known as ADAM1) is a member of the ADAM (A disintegrin and A metalloprotease domain) family of proteins. In this study, we examine the mechanism of mouse Fertilin α's in adhesion of sperm to the egg plasma membrane during fertilization. We find that recombinant forms of Fertilin α corresponding to either the disintegrin-like domain or the cysteine-rich domain and the EGF-like repeat can perturb sperm-egg binding, suggesting that both of these domains can participate in Fertilin α-mediated adhesion events. In further examination of the Fertilin α disintegrin-like domain, we find that a subdomain of disintegrin-like domain with the sequence DLEECDCG outside the putative disintegrin loop but with homology to the Fertilin β disintegrin loop can inhibit the binding of both sperm and recombinant Fertilin α to eggs, suggesting that this is an adhesion-mediating motif of the Fertilin α disintegrin-like domain. This sequence also inhibits the binding of recombinant Fertilin β to eggs and thus is the first peptide sequence found to block two different sperm ligands. Finally, a monoclonal antibody to the tetraspanin protein CD9, KMC.8, inhibited the binding of recombinant Fertilin α to eggs in one type of binding assay, suggesting that, under certain conditions, Fertilin α may interact with a KMC.8-sensitive binding site on the egg plasma membrane.
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Fertilin β and other ADAMs as integrin ligands: insights into cell adhesion and fertilization
BioEssays, 2001Co-Authors: Janice Perry EvansAbstract:One of the most important cell-cell interactions is that of the sperm with the egg. This interaction, which begins with cell adhesion and culminates with membrane fusion, is mediated by multiple molecules on the gametes. One of the best-characterized of these molecules is Fertilin beta, a ligand on mammalian sperm and one of the first ADAMs (A Disintegrin and A Metalloprotease domain) to be identified. Fertilin beta (also known as ADAM2) participates in sperm-egg membrane binding, and it has long been hypothesized that this function is achieved through the interaction of the disintegrin domain of Fertilin beta with an integrin on the egg surface. There are now approximately 30 members of the ADAM family and, to date, five different ADAMs (Fertilin beta, ADAM9, ADAM12, ADAM15, ADAM23) have been described to interact with integrins (specifically alpha(6)beta(1), alpha(v)beta(3), alpha(9)beta(1), alpha(v)beta(5), and/or alpha(5)beta(1)). This field will be discussed with respect to what is known about specific ADAMs and the integrins with which they interact, and what the implications are for sperm-egg interactions and for integrin function. These data will also be discussed in the context of recent knockout studies, which show that eggs lacking the alpha(6) integrin subunit can be fertilized, and eggs lacking the integrin-associated tetraspanin protein CD9 fail to fertilize. Key issues in cell adhesion that pertain to gametes and fertilization will also be highlighted.
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identification of key functional amino acids of the mouse Fertilin β adam2 disintegrin loop for cell cell adhesion during fertilization
Journal of Biological Chemistry, 2000Co-Authors: Xiaoling Zhu, Niharika P Bansal, Janice Perry EvansAbstract:Abstract Fertilin β (also known as ADAM2) is a cell adhesion molecule on the surface of mammalian sperm that participates in sperm-egg membrane binding. Fertilin β is a member of the molecular family known as ADAMs or MDCs. These proteins have a disintegrin domain with homology to integrin ligands found in snake venoms; several of these snake proteins have an RGD tripeptide presented on an extended “disintegrin loop.” However, Fertilin β lacks an RGD tripeptide and instead has the consensus sequenceX(D/E)ECD (QDECD in mouse Fertilin β) in its putative disintegrin loop, and there is controversy over which amino acids comprise the active site of the Fertilin β disintegrin loop. We have used point-mutated versions of the sequence AQDECDVT and two bioassays to identify the key functional amino acids of this sequence from the mouse Fertilin β disintegrin domain. Amino acid substitutions for the terminal aspartic acid residue of the QDECD sequence result in dramatically reduced activities in the two assays for protein function, implicating the terminal aspartic acid residue as critical for protein function. Substitutions for the glutamic acid and the cysteine residues in the QDECD sequence result in slight reductions in activity, whereas substitution of the first aspartic acid has virtually no effect. These data suggest that the conserved ECD sequence of the mouse Fertilin β disintegrin loop, especially the terminal D residue, contributes more to the protein's activity than does the QDE sequence that aligns with the RGD tripeptide in other disintegrins.
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characterization of the binding of recombinant mouse sperm Fertilin β subunit to mouse eggs evidence for adhesive activity via an egg β1integrin mediated interaction
Developmental Biology, 1997Co-Authors: Janice Perry Evans, Gregory S. Kopf, Richard M. SchultzAbstract:Abstract The sperm protein Fertilin (also known as PH-30) is a candidate for mediating the interactions between sperm and egg plasma membranes. Fertilin is a heterodimer. The β subunit, which has a region with homology to the family of integrin ligands known as disintegrins, has been hypothesized to be involved in the binding of sperm to the egg surface. To investigate this hypothesis and determine what role Fertilin β plays in fertilization, we have expressed the putative extracellular domain of mouse Fertilin β in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant Fertilin β-EC) and used two assays to characterize its binding to mouse eggs. Immunocytochemistry was used to examine the localization of recombinant Fertilin β-EC binding. A luminometric assay was also developed to quantify levels of binding of recombinant Fertilin β-EC to single eggs. We find that recombinant Fertilin β-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that Fertilin β has adhesive properties. Peptides corresponding to the disintegrin domain of Fertilin β reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of Fertilin β by binding sites on the egg. Treatment of zona pellucida-free eggs with chymotrypsin reduces the ability of the eggs to support the binding of recombinant Fertilin β-EC, implicating an egg surface protein as a binding site for recombinant Fertilin β-EC. Binding of recombinant Fertilin β-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mMCa2+, Mg2+, or Mn2+. Furthermore, eggs incubated in recombinant Fertilin β-EC prior toin vitrofertilization show reduced levels of sperm binding. Finally, we have examined the possible role of integrins on eggs as receptors for Fertilin β, since an anti-α6integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E. A. C. Almeidaet al.(1995)Cell81, 1095–1104). We find that: (a) an increased amount of GoH3 epitope on the egg surface does not correlate with an increased ability of the eggs to bind sperm or recombinant Fertilin β-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant Fertilin β-EC binding; and (c) recombinant Fertilin β-EC binding is reduced in the presence of anti-β1integrin antibodies. These results suggest that a β1-containing integrin participates in the binding of recombinant Fertilin β-EC to mouse eggs.
Len Hall - One of the best experts on this subject based on the ideXlab platform.
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do Fertilin β and cyritestin play a major role in mammalian sperm oolemma interactions a critical re evaluation of the use of peptide mimics in identifying specific oocyte recognition proteins
Molecular Human Reproduction, 2001Co-Authors: Eileen A Mclaughlin, Jan Frayne, Graham Bloomerg, Len HallAbstract:Integrins have been proposed to play a role in mammalian sperm-oocyte interactions for many years. To a large extent this hypothesis stems from the ability of short synthetic peptides, based on the disintegrin-like domains of two sperm surface integral membrane proteins, Fertilin beta and cyritestin, to inhibit sperm--oocyte binding and fusion in vitro. Here we argue that such peptide mimics lack specificity in these simple IVF assay systems. Hence, whilst not precluding a role for Fertilin beta and cyritestin in sperm-oolemma interactions, this lack of specificity indicates the need for considerable caution when interpreting results obtained using this approach.
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mammalian sperm egg recognition does Fertilin β have a major role to play
BioEssays, 1999Co-Authors: Jan Frayne, Len HallAbstract:The advent of simple in vitro fertilisation techniques has provided the reproductive biologist with an invaluable system for assaying sperm fertilising ability. In particular, they provide a useful way of identifying and characterising gamete-specific proteins that play a role in sperm-egg interactions, and in recent years, a growing number of sperm surface proteins have been identified that appear to be involved in these processes. Fertilin beta was one of the first sperm membrane proteins to be implicated in egg interactions and it has been proposed that this is mediated by means of binding of its disintegrin-like domain to cognate integrin receptors on the egg plasma membrane. A recent paper in Science by Cho and colleagues [Cho et al. 1998. Fertilisation defects in sperm from mice lacking Fertilin beta. Science 281: 1857-1859 (Ref. 1)] provides preliminary data on a Fertilin beta knockout mouse. Whilst Fertilin beta null males had greatly reduced fertility, somewhat surprisingly, this could be largely attributed to causes other than impaired binding to the egg plasma membrane.
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sequence analysis of a variety of primate Fertilin α genes evidence for non functional genes in the gorilla and man
Molecular Reproduction and Development, 1998Co-Authors: Jennifer A Jury, Jan Frayne, Len HallAbstract:The sperm surface Fertilin complex was first described in the guinea pig where it was found as a heterodimer of alpha and beta subunits, both of which were proposed to play a role in sperm-oolemma recognition and plasma membrane fusion during fertilisation. Whilst the beta subunit is apparently testis-specific, the finding of low levels of Fertilin alpha in nonreproductive tissues has cast some doubt on a unique role in fertilisation. Moreover, the absence of a functional Fertilin alpha gene in the human would imply that this gene product is not absolutely essential for fertilisation, although it could play a facilitatory role. We now describe the organisation and sequence of the Fertilin alpha genes in a range of primates, including the great apes, and find that the gorilla gene, like that of the human, is non-functional.
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cloning and sequence analysis of rat Fertilin alpha and beta developmental expression processing and immunolocalization
Molecular Human Reproduction, 1997Co-Authors: Eileen A Mclaughlin, Roy Jones, Jan Frayne, H L Barker, J A Jury, W C L Ford, Len HallAbstract:Fertilin a and b are members of the MDC (metalloproteinase-like, disintegrin-like, cysteine-rich) protein family and are expressed on the sperm surface where they have been proposed to play a role in mammalian fertilization. Inhibition of sperm‐oocyte binding and sperm‐oocyte fusion make Fertilin an attractive target for the development of an immunocontraceptive vaccine. Full-length cDNAs encoding a and b Fertilin subunits were isolated from a rat testis cDNA library and sequenced. Using reverse transcription‐polymerase chain reaction (RT‐PCR), the developmental expression of Fertilin a and b was determined in pre-pubertal and mature rat testes. Fertilin a mRNA was present at all stages of development, suggesting that it is not exclusively expressed in post-meiotic germ cells. In contrast, Fertilin b mRNA was first identified in day 19 testes, coincident with the presence of pachytene spermatocytes. Polyclonal antisera raised against a 28residue peptide (corresponding to part of the disintegrin domain) and two recombinant fusion proteins identified a 90 kDa protein in testicular sperm extracts and a 60 kDa protein in caput and cauda epididymidal sperm extracts, the predicted sizes for rat Fertilin b precursor and mature protein respectively. Indirect immunofluorescence using the anti-peptide antisera stained the acrosomal cap of permeabilized testicular, caput and caudal spermatozoa and elongating spermatids in testicular sections.
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evidence that peptides derived from the disintegrin domain of primate Fertilin and containing the ecd motif block the binding of human spermatozoa to the zona free hamster oocyte
International Journal of Andrology, 1997Co-Authors: P M Gichuhi, W C L Ford, Len HallAbstract:The binding of human spermatozoa to, and penetration of, zona-free hamster oocytes were significantly inhibited in the presence of (i) a 28-residue peptide corresponding to part of the disintegrin-like domain of monkey Fertilin beta, or (ii) a hexapeptide containing the ECD motif found in the disintegrin-like domain of monkey and human Fertilin beta, or (iii) a hexapeptide containing an RGD motif, implicated in integrin recognition. Polyclonal antibodies raised against the 28-residue peptide were used in conjunction with immunobeads to demonstrate that this peptide bound to the oolemma of zona-free hamster oocytes. These data support the view that the ECD motif is involved in the recognition of the oolemma receptor by human Fertilin.
Gregory S. Kopf - One of the best experts on this subject based on the ideXlab platform.
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Roles of the disintegrin domains of mouse Fertilins alpha and beta in fertilization
1998Co-Authors: Janice P. Evans, Richard M. Schultz, Gregory S. KopfAbstract:Fertilin is a heterodimer of a and b subunits, both of which are members of the ADAM (A Disintegrin and A Metalloprotease do-main)/MDC (Metalloprotease-Disintegrin-Cysteine-rich) family of proteins. We have previously demonstrated that recombinant forms of the putative extracellular domains of mouse Fertilin a and Fertilin b bind to mouse eggs and inhibit sperm-egg membrane binding. In this study, we examined the roles of the disintegrin domains of Fertilins a and b by producing recombinant forms of Fertilins a and b that included the disintegrin domains (aDCE and bDCE) or that were truncated so that they lack the disintegrin domains (aCE and bCE) and tested the abilities of these proteins to bind to eggs and to inhibit sperm-egg binding. Fertilin bDCE was able to inhibit sperm-egg binding, but Fertilin bCE was relatively ineffective, in-dicating that the disintegrin domain of Fertilin b is required for interactions with egg binding sites and/or for proper protein fold-ing. Fertilins aDCE and aCE both inhibited sperm-egg interactions, but Fertilin aDCE tended to be more effective. Thus, the presence of the disintegrin domain in Fertilin aDCE apparently enhanced the ability of this recombinant protein to inhibit sperm-egg binding, either by interacting with egg binding sites or by improving the efficiency of protein folding. These data also indicate that the other domains of the Fertilin a extracellular region (cysteine-rich and/or epidermal growth factor-like repeat) have the ability to block sperm binding and suggest that these domains of Fertilin a may participate in sperm-egg adhesion
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characterization of the binding of recombinant mouse sperm Fertilin β subunit to mouse eggs evidence for adhesive activity via an egg β1integrin mediated interaction
Developmental Biology, 1997Co-Authors: Janice Perry Evans, Gregory S. Kopf, Richard M. SchultzAbstract:Abstract The sperm protein Fertilin (also known as PH-30) is a candidate for mediating the interactions between sperm and egg plasma membranes. Fertilin is a heterodimer. The β subunit, which has a region with homology to the family of integrin ligands known as disintegrins, has been hypothesized to be involved in the binding of sperm to the egg surface. To investigate this hypothesis and determine what role Fertilin β plays in fertilization, we have expressed the putative extracellular domain of mouse Fertilin β in bacteria as a fusion protein with maltose-binding protein (hereafter referred to as recombinant Fertilin β-EC) and used two assays to characterize its binding to mouse eggs. Immunocytochemistry was used to examine the localization of recombinant Fertilin β-EC binding. A luminometric assay was also developed to quantify levels of binding of recombinant Fertilin β-EC to single eggs. We find that recombinant Fertilin β-EC binds to the region of the plasma membrane of the egg to which sperm bind, thus providing the first direct evidence that Fertilin β has adhesive properties. Peptides corresponding to the disintegrin domain of Fertilin β reduce its binding to eggs, suggesting that this domain is at least partially involved in the recognition of Fertilin β by binding sites on the egg. Treatment of zona pellucida-free eggs with chymotrypsin reduces the ability of the eggs to support the binding of recombinant Fertilin β-EC, implicating an egg surface protein as a binding site for recombinant Fertilin β-EC. Binding of recombinant Fertilin β-EC to eggs is also reduced in the absence of divalent cations and is supported by 2.0 mMCa2+, Mg2+, or Mn2+. Furthermore, eggs incubated in recombinant Fertilin β-EC prior toin vitrofertilization show reduced levels of sperm binding. Finally, we have examined the possible role of integrins on eggs as receptors for Fertilin β, since an anti-α6integrin subunit monoclonal antibody, GoH3, has been shown to inhibit sperm binding (E. A. C. Almeidaet al.(1995)Cell81, 1095–1104). We find that: (a) an increased amount of GoH3 epitope on the egg surface does not correlate with an increased ability of the eggs to bind sperm or recombinant Fertilin β-EC; (b) the GoH3 antibody has virtually no inhibitory effect on recombinant Fertilin β-EC binding; and (c) recombinant Fertilin β-EC binding is reduced in the presence of anti-β1integrin antibodies. These results suggest that a β1-containing integrin participates in the binding of recombinant Fertilin β-EC to mouse eggs.
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characterization of the binding of recombinant mouse sperm Fertilin α subunit to mouse eggs evidence for function as a cell adhesion molecule in sperm egg binding
Developmental Biology, 1997Co-Authors: Janice Perry Evans, Richard M. Schultz, Gregory S. KopfAbstract:Fertilin (previously known as PH-30) is a sperm protein that is a candidate molecule for mediating the binding and fusion of the sperm and egg plasma membranes. Fertilin is a heterodimer, with a beta subunit that has a region of homology to the disintegrin family of integrin ligands and an alpha subunit that has a region of homology to viral fusion peptides. It has been hypothesized that Fertilin beta and alpha subunits mediate the interactions between sperm and egg plasma membranes, namely, binding and fusion, respectively. To address this hypothesis and to examine specifically the role of Fertilin alpha in fertilization, we have expressed the predicted extracellular domain of mouse Fertilin alpha as a bacterial fusion protein with maltose-binding protein. This fusion protein (hereafter referred to as recombinant Fertilin alpha-EC) binds to the microvillar region of zona pellucida (ZP)-free eggs, the region of the membrane to which sperm bind. This binding is reduced in the absence of divalent cations and is supported by Ca2+, Mg2+, or Mn2+. Eggs that have been treated with chymotrypsin bind less recombinant Fertilin alpha-EC than do untreated eggs, suggesting that a chymotrypsin-sensitive binding site for recombinant Fertilin alpha-EC is present on egg surfaces. Binding to eggs is also affected by the method used to remove the ZP. Finally, recombinant Fertilin alpha-EC inhibits the binding of sperm to eggs during in vitro fertilization of ZP-free eggs. These data are the first evidence to suggest that Fertilin alpha can function as a cell adhesion molecule during fertilization, mediating the binding of sperm and egg plasma membranes.