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PubMed1997

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.

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.

ADAM Proteins↗
PubMed1997

Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit.

Recombinant fertilin subunits produced in a bacterial expression systems were used to test fertilin as an immunocontraceptive antigen in the European rabbit (Oryctolagus cuniculus). Wild female rabbits (n = 40) were immunized with either recombinant rabbit fertilin alpha or beta subunits by the s.c. or intra-Peyer's patch route. High titers of serum anti-fertilin polyclonal IgG antibodies were achieved in all rabbits after repeated boosts, with fertilin-specific IgG but not IgA antibodies detected in vaginal lavages of all animals. The serum IgG antibodies recognized polypeptides in detergent extracts of rabbit sperm with relative molecular masses of 48, 53, and 85 kDa on reducing SDS-PAGE gels and were shown to bind to the head region of methanol-fixed and live caudal rabbit sperm. Preincubation of rabbit sperm with these anti-fertilin IgG antibodies at concentrations of 400 micrograms/ml blocked sperm binding to zona-intact oocytes and inhibited fertilization in vitro by 60-80%. However, despite the levels of circulating and vaginal IgG antibodies achieved, only 4 immunized does failed to become pregnant out of 33 that ovulated. The remaining animals either showed no effect on fertility (n = 29) relative to control animals or failed to ovulate (n = 7). All control animals ovulated and were either fully fertile (n = 15) or were mated to infertile males (n = 4). In addition, proven-fertile male domestic rabbits (n = 3) were immunized s.c. and boosted three times with fertilin beta. Only one animal subsequently showed impaired fertility. These results show that in the rabbit, high levels of circulating sperm-reactive anti-fertilin antibodies and the presence of vaginal IgG does not ensure infertility.

ADAM Proteins↗
PubMed1996

Cloning and expression of recombinant rabbit fertilin.

Fertilin is a sperm surface protein complex which is reported to play an essential role in sperm-egg fusion in mammals. It is comprised of two related subunits, alpha and beta, both of which are glycosylated and have cytoplasmic and extracellular domains. This protein has been reported to play an essential role in sperm-egg fusion in mammals. We report on the cloning and sequencing of the complete cDNA sequences of both subunits from rabbit testis, and the production of recombinant proteins for testing their potential as antigens for use in an immunocontraceptive vaccine to control wild rabbit populations. The cDNAs for rabbit fertilin alpha and beta (Genbank accession numbers, U46069 and U46070) are predicted to encode proteins of 919 and 751 amino acids, respectively, and to show significant levels of homology to fertilin subunits isolated from other species. Analysis of the predicted protein sequences of fertilin alpha but not beta reveals the presence of 21 direct repeats of the hexameric sequence A/PPPPEA at the extreme carboxy terminus, similar to what has been described for a fertilin alpha gene isoform in the monkey. DNA sequences corresponding to the predicted mature alpha and beta fertilin subunits were individually cloned into a bacterial expression system, and the recombinant proteins were used to raise polyclonal antibodies in mice. These antibodies detect components of the native fertilin complex from rabbit sperm.

ADAM Proteins↗
PubMed1996

Molecular cloning of the human fertilin beta subunit.

Fertilin, a heterodimeric sperm surface protein, first identified in guinea pig, has been shown to play an important role in sperm-egg interactions. We report here the complementary DNA and deduced amino acid sequence of human fertilin beta. The human fertilin beta shares significant sequence homology with mouse, guinea pig and monkey fertilin beta and also exhibits similar structural organization. Of particular interest, the mature guinea pig fertilin beta contains an amino-terminal 90 amino acid disintegrin domain. It has been suggested that the integrin recognition sequence (TDE) of the guinea pig fertilin beta disintegrin domain mediates sperm-egg binding. The amino acid sequence at this position in human fertilin beta differs from the mouse, guinea pig and monkey sequence (mouse-QDE; human-FEE; monkey-FDE).

ADAM Proteins↗
PubMed2001

Calmegin is required for fertilin alpha/beta heterodimerization and sperm fertility.

Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607-611). In the present study, we demonstrate that calmegin -/- sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin -/- sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in fertilin beta -/- sperm, we investigated the interaction of calmegin with fertilin beta. Using immunoprecipitation techniques, calmegin was found to bind to sperm membrane proteins, fertilin alpha and beta, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin alpha and beta. In the calmegin -/- mice, a loss of heterodimerization of fertilin alpha and beta was observed and fertilin beta was not detectable in mature sperm. The data not only explain why the calmegin and fertilin beta knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum.

ADAM Proteins↗
PubMed2000

Identification of key functional amino acids of the mouse fertilin beta (ADAM2) disintegrin loop for cell-cell adhesion during fertilization.

Fertilin beta (also known as ADAM2) is a cell adhesion molecule on the surface of mammalian sperm that participates in sperm-egg membrane binding. Fertilin beta 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 beta lacks an RGD tripeptide and instead has the consensus sequence X(D/E)ECD (QDECD in mouse fertilin beta) in its putative disintegrin loop, and there is controversy over which amino acids comprise the active site of the fertilin beta 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 beta 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 beta 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.

ADAM Proteins↗
PubMed1997

Cloning and sequence analysis of rat fertilin alpha and beta--developmental expression, processing and immunolocalization.

Fertilin alpha and beta 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 alpha and beta 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 alpha and beta was determined in pre-pubertal and mature rat testes. Fertilin alpha mRNA was present at all stages of development, suggesting that it is not exclusively expressed in post-meiotic germ cells. In contrast, fertilin beta mRNA was first identified in day 19 testes, coincident with the presence of pachytene spermatocytes. Polyclonal antisera raised against a 28-residue 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 beta 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.

ADAM Proteins↗
PubMed2002

Expression of fertilin and CD9 in bovine trophoblast and endometrium during implantation.

The superficial placentation of cattle involves the development of fetal binucleate cells that arise from the chorion and migrate between adjacent cell tight junctions to fuse with maternal epithelium. Thus, the temporal and spatial patterns of expression of the cell migration, adhesion, and fusion molecules fertilin and CD9 were investigated in bovine trophoblast and endometrium. Bovine fertilin alpha and fertilin beta messenger RNA sequences were amplified by reverse transcriptase-polymerase chain reaction in testis (positive control), peri-implantation (Days 18, 19, and 21), and postimplantation (Days 35-40) trophoblast RNA, but not in caruncular endometrium (Day 40). Northern blot analysis indicated that the transcript hybridizing to fertilin alpha in trophoblast RNA was approximately 4.0 kilobases (kb), whereas in testis, 2 transcripts of approximately 3.3 and 3.8 kb were indicated. The transcript hybridizing to the fertilin beta probe was also larger in trophoblast than in testis ( approximately 3.8 vs. 2.4 kb, respectively). In situ hybridization revealed that fertilin beta mRNA was expressed by trophoblast cells, including binucleate cells. Immunohistochemical study of CD9, a member of the transmembrane-4-superfamily which is thought to be involved in sperm-egg fusion, showed that CD9 was present on the apical surface of uterine epithelium and in a subpopulation of binucleate cells of the trophoblast. Immunoprecipitation followed by Western blot analysis showed association between CD9 and integrin alpha3 in endometrium. The results support the hypothesis that fertilin and CD9 are involved in bovine binucleate cell migration and fusion.

ADAM Proteins↗
PubMed1999

Evidence that a functional fertilin-like ADAM plays a role in human sperm-oolemmal interactions.

Fertilin is a protein initially identified in guinea pig spermatozoa; it is the prototype of a larger family of conserved, proteins designated as a disintegrin and a metalloproteinase (ADAM). These heterodimers which consist of alpha and beta subunits, containing metalloproteinase-like and disintegrin-like domains, appear to play a role in mammalian fertilization. Peptides derived from the disintegrin domains of two ADAMs, fertilin and cyritestin, interfere with gamete adhesion and sperm-egg membrane fusion in non-human species. It has been suggested that fertilin-beta binds to an oolemmal integrin, and it is proposed that the tripeptide FEE (Phe-Glu-Glu) is the integrin recognition sequence in human fertilin-beta. We evaluated whether fertilin beta plays a role in human fertilization by studying the effects of a linear octapeptide containing the FEE sequence, SFEECDLP, and a scrambled octapeptide with the same amino acids, SFPCEDEL, on the incorporation of human spermatozoa by human zona-free eggs. The effects of G4120, a potent RGD-containing (Arg-Gly-Asp) thioether-bridged cyclic peptide which blocks both fibronectin and vitronectin receptors, and the relationship between FEE- and RGD-receptor interactions on sperm-egg interactions were also studied. The FEE-containing peptide, but not the scrampled peptide, inhibited sperm adhesion to oocytes and their penetration, over the range 1-5 microM. The inhibition induced by SFEECDLP was reversible and occurred only in the presence of peptide itself. The G4120 peptide exhibited 10-fold less inhibitory effects on sperm adhesion and penetration than did SFEECDLP. When combined, SFEECDLP and G4120 exhibited strong inhibition of both adhesion and penetration at concentrations that individually had been ineffective, suggesting co-operation between the two receptor-ligand interactions during fertilization. We propose that a fertilin-like molecule is functionally active on human spermatozoa and that its interaction with an oolemmal integrin receptor plays a role in fertilization in humans.

ADAM Proteins↗
PubMed1997

Genomic organization of the mouse fertilin beta gene that encodes an ADAM family protein active in sperm-egg fusion.

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.

ADAM Proteins↗
PubMed2004

Possible function of the ADAM1a/ADAM2 Fertilin complex in the appearance of ADAM3 on the sperm surface.

In mouse, two different isoforms of ADAM1 (fertilin alpha), ADAM1a and ADAM1b, are produced in the testis. ADAM1a is localized within the endoplasmic reticulum of testicular germ cells, whereas epididymal sperm contain only ADAM1b on the plasma membrane. In this study, we show that the loss of ADAM1a results in the male infertility because of the severely impaired ability of sperm to migrate from the uterus into the oviduct through the uterotubal junction. However, epididymal sperm of ADAM1a-deficient mice were capable of fertilizing cumulus-intact, zona pellucida-intact eggs in vitro despite the delayed dispersal of cumulus cells and the reduced adhesion/binding to the zona pellucida. Among testis (sperm)-specific proteins examined, only the level of ADAM3 (cyritestin) was strongly reduced in ADAM1a-deficient mouse sperm. Moreover, the appearance of ADAM3 on the sperm surface was dependent on the formation of a fertilin protein complex between ADAM1a and ADAM2 (fertilin beta) in testicular germ cells, although no direct interaction between the fertilin complex and ADAM3 was found. These results suggest that ADAM1a/ADAM2 fertilin may be implicated in the selective transport of specific sperm proteins including ADAM3 from the endoplasmic reticulum of testicular germ cells onto the cell surface. These proteins then can participate in sperm migration into the oviduct, the dispersal of cumulus cells, and sperm binding to the zona pellucida.

ADAM Proteins↗
PubMed1998

Fertilization defects in sperm from mice lacking fertilin beta.

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.

ADAM Proteins↗
PubMed1998

Sequence analysis of a variety of primate fertilin alpha genes: evidence for non-functional genes in the gorilla and man.

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.

ADAM Proteins↗
PubMed1999

Mammalian sperm-egg recognition: does fertilin beta have a major role to play?

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.

ADAM Proteins↗
PubMed2001

Fertilin beta and other ADAMs as integrin ligands: insights into cell adhesion and fertilization.

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.

ADAM Proteins↗
PubMed1995

Surface expression of the pre-beta subunit of fertilin is regulated at a post-translational level in guinea pig spermatids.

During spermiogenesis in the guinea pig, the spermatid plasma membrane becomes sequentially segregated into three domains of distinct composition. We have previously shown that plasma membrane proteins appear on the cell surface in a temporally regulated manner such that proteins localized to the same domain reach the surface membrane at the same time in sperm development. Fertilin is a cell surface protein restricted to the whole head of testicular sperm; like other proteins restricted to this membrane domain, it does not appear on the cell surface until late (steps 11-13) in spermiogenesis. Using confocal microscopy of immunofluorescently labeled testicular sections, we demonstrate that the pre-beta subunit of fertilin is present in pachytene spermatocytes. It is initially observed in long, strand-like structures that likely represent the endoplasmic reticulum; it later appears in a punctate distribution in the cytoplasm of early spermatids prior to its appearance on the surface membrane in late elongating spermatids. Immunoblotting experiments confirm the presence of the fertilin pre-beta subunit in spermatocytes and early spermatids at the same apparent molecular weight as in later stages. These results suggest that the appearance of fertilin pre-beta subunit on the spermatid surface is regulated by a post-translational mechanism.

ADAM Proteins↗
PubMed1998

Structural properties of the putative fusion peptide of fertilin, a protein active in sperm-egg fusion, upon interaction with the lipid bilayer.

We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.

ADAM Proteins↗
PubMed1997

The human fertilin alpha gene is non-functional: implications for its proposed role in fertilization.

In the guinea-pig, the alpha subunit of the fertilin complex, a heterodimeric surface membrane glycoprotein found on the head region of spermatozoa, has previously been proposed to mediate membrane fusion with the oolemma plasma membrane during fertilization. Here we describe experiments which indicate that the only fertilin alpha-like gene in humans is an expressed, but nonfunctional, pseudogene, possibly derived by genetic recombination between the two fertilin alpha genes found in some primates. This finding clearly raises questions about the importance and/or role of fertilin alpha in mammalian fertilization.

ADAM Proteins↗