Molecular Human Reproduction, Vol. 5, No. 5, 433-440,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Evidence that a functional fertilin-like ADAM plays a role in human sperm–oolemmal interactions
1 Division of Reproductive Endocrinology, Department of Ob/Gyn, State University of New York at Stony Brook, Stony Brook, NY, USA, and 2 Centro di Fisiopatologia della Riproduzione, Department of Ob/Gyn, Istituto Scientifico San Raffaele, Via Olgettina 60, Milano, Italy
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Abstract |
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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
and ß 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-ß binds to an oolemmal integrin, and it is proposed that the tripeptide FEE (Phe-Glu-Glu) is the integrin recognition sequence in human fertilin-ß. We evaluated whether fertilin ß 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 µM. 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/fertilin/fertilization/integrins/RGD
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Introduction |
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Gamete interactions leading to fertilization can be divided into a series of discrete steps, starting from sperm–zona binding and ending with pronuclear formation. Evidence is accumulating that sperm–oolemmal adhesion and fusion may involve several combined receptor–ligand interactions including members of the family of integrins (Myles, 1993
; Bronson and Fusi, 1996). Integrins are cell surface receptors through which cells attach to extracellular matrices. They also act as co-receptors in many cell–cell interactions. Cells vary their adhesive properties by changing the integrins expressed on their surface (Diamond and Springer, 1994). Apart from functioning in adhesion, a second major property of integrins is their role in signal transduction (Hynes, 1992). Integrins are glycoprotein heterodimers consisting of an chain non-covalently associated with a ß subunit (subunits of molecular weight 95 000–200 000), involved in a variety of cell–matrix and cell–cell adhesion functions (Hemler, 1990). Several of the integrin receptors (ß1, ß3, ß5 subfamilies) recognize as ligands glycoproteins of the extracellular matrix, such as fibronectin, vitronectin, and type I collagen (Ruoslahti and Piershbacher, 1986). Integrins of the ß1 and ß3 subfamilies also play a role in phagocytosis by neutrophils or monocytes, mediated by immunoglobulin G (IgG) Fc receptors (Hemler, 1990). Many integrin–ligand interactions are mediated through the recognition of an RGD (Arg-Gly-Asp) tripeptide sequence (Ruoslahti and Piershbacher, 1986) present in fibronectin, vitronectin, and osteopontin.
Following the observation of Miranda and Tezon (Miranda and Tezon 1992) that human spermatozoa acquire fibronectin during their epididymal passage, we asked whether this molecule might play a role in sperm–egg adhesion, through its binding to an oolemmal integrin. Oligopeptides containing the RGD sequence were shown to competitively inhibit sperm–oolemmal adhesion and oocyte penetration in both heterologous (human–hamster) and homologous (hamster–hamster) gamete interactions, suggesting the involvement of integrins in fertilization (Bronson and Fusi, 1990a,b). Several integrins were detected in hamster oocytes by dot blot analysis, and the oolemma of human oocytes was shown to express 2, 5, 6, v and ß1 integrin chains by means of rosetting of Covasphere coupled with anti-integrin monoclonal antibodies (Fusi et al., 1993).
Conversely, ligands for integrin receptors have been detected on human spermatozoa. The presence of fibronectin has been demonstrated by indirect immunofluorescence on the equatorial segment of permeabilized, ejaculated human spermatozoa (Vuento et al., 1984; Glander et al., 1987). Fibronectin has been identified in extracts of ejaculate human spermatozoa (Vuento et al., 1984; Fusi and Bronson 1992), who also demonstrated the presence of vitronectin on spermatozoa (Fusi et al., 1992). Addition of exogenous vitronectin was shown to promote the adhesion of capacitated human spermatozoa to zona-free hamster eggs in the presence of calcium, and it was postulated that multimeric forms of vitronecton act as a molecular velcro, binding spermatozoa to the egg through ligation of oolemmal integrins (Fusi et al., 1996).
Fertilin is a sperm surface protein which also plays an important role in sperm–egg interaction in mammals through its interaction with oolemmal integrins. It is an heterodimer initially identified in guinea pigs that is formed by two distinct subunits, and ß, both glycosylated and structured with an extracellular domain and a cytoplasmic one. There is increasing evidence that fertilin is but one of a conserved family of related cysteine-rich proteins, now designated as ADAM (a disintegrin and a metalloproteinase), that contain metalloproteinase-like and disintegrin-like domains that play important roles in mammalian fertilization (Wolfsberg et al., 1995; Wolfsberg and White, 1996). The fertilin- chain contains a fusion peptide sequence similar to that noted in viral fusion proteins (Huovila et al., 1996). The N-terminal region of the fertilin-ß chain possesses a 93 amino acid disintegrin-like domain (Blobel et al., 1992). Disintegrins are a class of soluble snake venom proteins that act as ligands for certain integrins, and receptor occupation leads to defects in integrin-mediated platelet function (Scarborough et al., 1991). The disintegrin-like domain of fertilin-ß is proposed to interact with an oolemmal integrin (Myles, 1993), enabling the spermatozoan to adhere to the oocyte plasma membrane, leading to a conformational change in the fertilin- subunit that reveals its hydrophobic fusion peptide. The tripeptide TDE derived from guinea pig fertilin-ß, rather than RGD, has been proposed to be an integrin recognition sequence, as is the QDE peptide in mice (Myles et al., 1994; Evans et al., 1995, 1997a; Yuan et al., 1997). Evidence has been presented that the integrin 6ß1 is the receptor for murine sperm-associated fertilin (Almeida et al., 1995; Chen and Sampson, 1999). The human fertilin-ß gene has been cloned (Gupta et al., 1996; Burkin et al., 1997; Vidaeus et al., 1997), and it has been suggested that the amino acid sequence FEE (Phe-Glu-Glu) might be the recognition sequence in human fertilin.
In this study we evaluated the possibility that fertilin-ß plays a role in human fertilization during sperm adhesion to and incorporation by zona-free human eggs. We also studied the relationship between fertilin–oolemmal receptor interactions and gamete interactions involving the RGD integrin recognition sequence. For these purposes we used a linear octapeptide containing the FEE sequence (SFEECDLP) and a scrambled octapeptide with the same amino acids (SFPCEDEL) as a control peptide. G4120, a potent thioether-bridged RGD-containing cyclic peptide which blocks fibronectina and vitronectin receptors (Barker et al., 1992; McDowell and Gadek, 1992), was also used to study the effects of blocking integrins that bind this recognition sequence on gamete interactions as well as its effect on FEE-mediated inhibition of fertilization.
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Oocytes and spermatozoa
Oocytes were obtained by ultrasound-guided transvaginal retrieval after follicle stimulating hormone (FSH) stimulation according to conventional in-vitro fertilization (IVF) stimulation protocols at the Center for Reproductive Medicine of the Istituto Scientifico San Raffaele (Milano, Italy). A number of these oocytes were used for the IVF programme at Ospedale San Raffaele, while the excess ones were used for this study. Only a limited number of embryos can be obtained at this centre, and embryo selection or freezing are not allowed. The use of excess oocytes for this research was approved by the Ethical Committee. After overnight incubation in 5% CO2 in air at 37°C, oocytes were denuded from their cumulus–corona complex using human tubal fluid (HTF)–HEPES-buffered medium (Irvine Scientific, Celbio, Santa Ana, CA, USA) containing 10 IU/ml bovine hyaluronidase (Sigma), through a series of vigorous aspirations using a denuding mouth pipette of 200 and then 150 µm inner diameter (Cook IVF). Fresh HTF–HEPES-buffered medium without hyaluronidase was used to wash the denuded oocytes four to five times. Then, mature metaphase II oocytes were selected for study and freed from their zonae pellucida using HTF–HEPES-buffered medium containing 4 IU/ml protease type VIII, pronase (Sigma) using the same operative method as the previous step but utilizing mouth pipettes with a larger inner diameter (300 µm) to avoid oocyte damage. Again, fresh HTF–HEPES-buffered medium was used for four to five washing steps. Human zona-free oocytes were then placed in Biggers–Whitten–Whittingham (BWW) medium containing 3% bovine serum albumin (BSA), and peptides and/or capacitated spermatozoa at the experimental concentrations were added before incubation at 37°C in humidified atmosphere in the presence of 5% CO2. All the handling procedures and manipulation were conducted on a warm stage.
Semen samples were obtained from five donors, whose semen parameters were normal following the World Health Organization criteria (WHO, 1992) and had normal tests of sperm function (SPA, induced acrosome reaction). Sperm samples were kept at 37°C until liquefaction was completed. A three-step 90/70/50% Percoll (Sigma) gradient, was used to select motile spermatozoa from the semen. Spermatozoa were then stored at 4°C overnight under capacitating conditions using refrigeration buffer Test Yolk medium (Irvine Scientific): BWW/BSA 3% (1:1 v/v). After 18 h spermatozoa were slowly warmed to room temperature, carefully washed three times by centrifugation (300 g) using BWW/BSA 3% and resuspended in experimental incubation medium (BWW/BSA 3%) at 100 000/ml final concentration (Johnson et al., 1995).
Peptides
An FEE containing peptide (P1) and a control peptide (P2) with a scrambled sequence of the same amino acids were synthesized by PRIMM (Istituto Scientifico, San Raffaele, Milan, Italy). The P1 peptide sequence was NH2-SFEECDLP-COOH and the P2 sequence was NH2-SFPCEDEL-COOH. Peptides were furnished as anhydrous powder and were reconstituted with BWW/BSA 3% to a final concentration of 5 mg/ml (stock solution). The stock solutions were aliquoted and stored at –20°C until use. Peptide aliquots were thawed at room temperature 10 min prior to use, and then diluted with preconditioned BWW/BSA 3% to the final concentration used for gamete incubation (0.5–100 µM). G4120 a potent cyclic RGD-containing peptide (molecular weight 668.2), was kindly supplied by Dr R.A.Lazarus (Genentech Inc, South San Francisco, CA). It was resuspended at 1 mg /ml concentration and stored as the other peptides. The non-RGD containing GRGES peptide (Sigma) was used as a control.
Experiment I: inhibitory effects of SFEECDLP on sperm–egg interaction
Zona-free human eggs were coincubated in parallel with capacitated human spermatozoa at 100 000/ml final concentration in 300 µl BWW/BSA 3% medium in the presence of different concentrations (0.5–500 µM) of the FEE containing peptide SFEECDLP (P1), a non-FEE-containing octapeptide SFPCEDEL (P2), and in the absence of any oligopeptide for 90 min at 37°C in humidified atmosphere in the presence of 5% CO2. At the end of incubation, eggs were carefully washed 3–4 times with fresh BWW/BSA 3% medium and freed from unbound and loosely bound spermatozoa by serial aspiration through a finely drawn pipette. Eggs were then stained by short-term (5–15 s) exposure to 1 mM Acridine Orange–3% dimethyl sulphoxide (DMSO) in BWW/BSA 3% and prepared as whole mounts under 22x35 mm coverlips (Bronson et al., 1981). At x400 magnification, adherent spermatozoa appeared as bright green or orange heads against the dark field under UV illumination. Penetrated spermatozoa exhibited expanded green heads against the orange background of the ooplasm. In these experiments, both incubation and scoring were performed blinded to avoid bias, and the person who performed the scoring did not know the nature of the peptide utilized nor the concentrations of peptide.
Experiment II: SFEECDLP is not oocyte-toxic
After denuding the cumulus–corona complex and freeing oocytes from the zona pellucida, they were co-incubated with spermatozoa at a final concentration of 100 000/ml for 90 min at 37°C in a 300 µl drop of BWW/BSA 3% medium containing either peptide P1 or P2 at each of two different concentrations (1 and 5 µM) shown to be inhibitory in experiment I. A third group of oocytes that had been pre-incubated for 30 min with peptide P1 was washed 4–5 times in fresh BWW/BSA 3% medium and inseminated in parallel with capacitated spermatozoa and incubated in medium free from peptides for 90 min at 37°C (P1 preincubation). Spermatozoa adhesion and penetration were assessed as described in experiment I.
Experiment III: reversibility of SFEECDLP effects on sperm–egg interaction
Human capacitated spermatozoa and human zona-free eggs were co-incubated in the presence of peptides at 1 µM and 5 µM concentrations, at 37°C for 90 min, as in experiment I. Sperm adhesion and penetration were assessed as previously described. Eggs co-incubated with spermatozoa in the presence of the FEE-containing peptide P1 were then carefully washed 4x in 50–75 µM drops of BWW/BSA 3% and re-exposed to spermatozoa from the same donor in the presence of the scrambled control peptide P2. After 90 min incubation at 37°C, sperm adhesion and penetration was evaluated.
Experiment IV: the effect of G4120, a cyclic RGD-containing peptide that blocks RGD-sensitive integrins, on sperm–egg interaction
The G4120 peptide or a control peptide GRGES that did not possess an RGD sequence was added to the sperm–egg medium at different concentrations (0.5–500 µM). The incubation medium, incubation times and scoring were the same as previously indicated.
Experiment V: effects of SFEECDLP and G4120 on sperm–egg interaction are additive
The effects of G4120 and SFEECDLP (P1) on gamete interactions were studied in parallel, in the presence of each peptide alone and in combination at 0.5 µM. At this concentration, the number of oolemmal adhering to, and penetrating, spermatozoa in the presence of P1 or G4120 had previously been shown not to be significantly different than that observed in the presence of the control peptides P2 or GRGES respectively. Sperm adhesion and penetration were assessed as previously described after 90 min of incubation at 37°C in the presence of 100 000/ml final concentration of capacitated spermatozoa.
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Results |
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The FEE containing peptide SFEECDLP (P1) inhibited the adhesion of human spermatozoa to human zona-free eggs in a concentration-dependent manner, starting from 1 µM (P1: 8.3 ± 2.1 versus P2: 21.9 ± 5.3; mean ± SD) and reaching a plateau (2.2 ± 1.1) at 5 µM (Figure 1a
). The inhibition was not complete, but the number of spermatozoa adherent to oocytes when incubated in the presence of P1 was markedly lower than that observed in the presence of the scrambled peptide SFPCEDEL (P2) and in the peptide-free controls (21.4 ± 5.6), and the difference was statistically significant (P < 0.001). At lower concentrations (0.1 or 0.5 µM), there was no difference between the two peptides. No difference was observed in the number of spermatozoa adherent to the oolemma when gametes were incubated in the absence of peptides when compared with the peptide P2, except at 500 µM P2 (6.3 ± 5.4). Peptide P1 also inhibited sperm penetration of oocytes, when compared with results in the presence of either P2 or those in the absence of peptide, and the phenomenon had the same characteristics as sperm adhesion. No differences were observed in sperm penetration at 0.5 µM peptide concentration, while inhibition started at 1 µM concentration, reaching a plateau at 5 µM (Figure 1b).
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The inhibitory effects of SFEECDLP (P1) on adhesion and penetration occurred only in the presence of peptide. As observed in experiment II, co-incubation of spermatozoa and eggs in the presence of the FEE-containing peptide P1 resulted in a lower number of adherent and penetrated spermatozoa than in the presence of the scrambled peptide P2, but pre-exposure of the eggs to P1 had no effect on subsequent gamete interaction, indicating that the inhibitory effects observed were not due to non-specific egg toxicity (Figure 2a,b
). These findings were also confirmed by the results of experiment III, in which the interaction of spermatozoa and eggs was observed in the presence of peptide P1, the scrambled peptide P2, or in the presence of P2 following the prior exposure of these same oocytes to inhibitory concentrations of the peptide P1. While sperm–oolemmal adhesion and penetration were impaired in the presence of P1, when these eggs were washed out of P1 and reinseminated in the presence of P2 the number of adherent and penetrating spermatozoa was similar to that in the group of eggs exposed to P2 without prior exposure to peptide P1 (Figure 3a,b). These results indicate that the inhibition induced by the SFEECDLP is reversible and occurs only in the presence of the peptide itself.
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The cyclic RGD peptide (G4120) alone was not effective in blocking sperm adhesion at 0.5 µM concentration, when compared with the control peptide GRGES, but exhibited an inhibitory effect starting at higher concentrations (Figure 4
). The inhibition of sperm–oolemmal adhesion increased at 10 µM and reached a plateau at 50 µM. Comparison of the results obtained utilizing the FEE-containing peptide P1 versus G4120 revealed that P1 had an effect at lower concentrations than the RGD-containing one. Under the same experimental conditions, the interference in sperm–egg interaction using SFEECDLP (P1) reached a plateau at 5 µM, while utilizing the RGD-containing peptide, the same effect was observed at a concentration 10 times higher (50 µM). While both the cyclic RGD peptide G4120 and P1 peptide were individually without effect on sperm–oolemmal interactions at 0.5 µM, their combination, when studied in parallel, resulted in significant inhibition of both sperm adhesion and penetration (Figure 5).
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Discussion |
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This is the first work to demonstrate in a homologous system using human gametes that a peptide derived from the disintegrin loop of human fertilin-ß (SFEECDLP), as well as a peptide containing the RGD recognition motif, at µmol concentrations, each inhibit the binding of spermatozoa to the oolemma of zona-free eggs. Previously, we had shown that GdRGDSP, a cyclized peptide that blocks fibronectin and vitronectin receptors, inhibited the binding of capacitated human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, at 0.5–10 µM (Fusi et al., 1996
). The effect of the addition of both FEE- and RGD-containing peptides to the culture medium was additive at a concentration (0.5 µM) that individually did not inhibit sperm–oolemmal adherence, suggesting the existence of co-ordinated receptor–ligand interactions during sperm–oocyte interactions. Similar receptor co-operativity has been noted during phagocytosis of target particles by leukocytes (Greenberg, 1995).
We have postulated that recognition of the spermatozoon adhering to the oolemma, and its incorporation into the oocyte, bears some similarity to those processes that occur during phagocytosis (Bronson, 1998). Phagocytosis-promoting receptors (integrins, Fc
receptors and complement receptors) have been identified on the oolemma of mammalian eggs. That RGD-containing sperm-associated proteins such as vitronectin and fibronectin might play a role in this process is supported by studies of the role of invasin in the entry of Yersinia into cells. Yersinia enterocolitica and Y.pseudotuberulosis are enteropathogens that infect Peyer's patches and cause gastroenteritis and mesenteric lymphadenitis. Following their attachment to the cell surface, the entry of enteropathogenic Yersinia into cultured mammalian cells involves a process morphologically similar to internalization of complement-coated particles by phagocytes, and these organisms reside in membrane-bound phagosomes. The bacterial protein invasin mediates the entry of Yersinia into cultured cells. Invasin is a 986 amino acid membrane protein that binds to five different members of the ß1 integrin family (Falkow et al., 1992). Escherichia coli transfected with the gene for invasin and latex beads coated with purified invasin derivatives are each internalized by cultured mammalian cells in a similar manner to Yersinia. In addition, the tripeptide sequence Arg-Gly-Asp (RGD) has been shown to inhibit binding to invasin (Isberg et al., 1995). Attachment and entry of coxsackie virus A9 to GMK cells is also dependent on an RGD sequence in the viral capsid protein. Antibodies specific for the v and/or ß3 integrin subunits protect GMK cells from A9 infection (Roivanen et al., 1994). Human adenovirus type 2 enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins vß3 and vß5 via an RGD amino acid sequence (Wickham et al., 1993). Membrane permeabilization appears to be mediated through the vß5 integrin (Nemerow et al., 1994).
The multi-functional molecule vitronectin, which has been detected in human spermatozoa, is a strong candidate to play a role similar to invasin. While a major secretory product of the liver, it also appears to be an intrinsic protein of human spermatozoa. Message for vitronectin has been detected by Northern blotting analysis in human testis poly-A mRNA (Fusi et al., 1994) and in spermatocytes by in-vitro reverse transcription–polymerase chain reaction (RT–PCR) (Nuovo et al., 1995). It contains an RGD recognition sequence that could act as a ligand for oolemmal integrins, as well as a cryptic heparin binding site. The integrin chains v and ß3, which together constitute the vß3 receptor for vitronectin, have been detected on the oolemma of human oocytes. Vitronectin is detected on the surface of capacitated spermatozoa (Fusi et al., 1992) and is released into the medium during a calcium ionophore-induced acrosome reaction (Fusi et al., 1996).
Sperm–oolemmal adhesion and fusion also involves the sperm glycoprotein fertilin, which appears to bind to an oolemmal integrin, in this manner promoting sperm–egg adhesion and gamete membrane fusion (Myles, 1993). Fertilin is an integral membrane heterodimer initially identified through the use of monoclonal antisperm antibodies raised against guinea pig spermatozoa (Primakoff et al., 1987). Fertilin- and fertilin-ß are now recognized to be members of a larger family of multidomain intergral membrane proteins designated ADAMs, all of which posses a metalloproteinase-like domain, a disintegrin domain, and a cysteine-rich domain (Wolfsberg et al., 1995; Wolfsberg and White, 1996.) Antibodies to fertilin partially inhibit fertilization in vitro in the rabbit (Hardy et al., 1997). The gene for fertilin-ß has been sequenced, and it appears to be conserved between species, although the putative tripeptide recognition sequence of the disintegrin binding domain varies between species. Members of this family have been identified in monkey Macaca fasicularis epididymis (Perry et al., 1992) and testis (Perry et al., 1994), suggesting its wider role in reproduction. The cDNA cloning, deduced amino acid sequence, tissue specificity and chromosomal mapping of human fertilin-ß have been reported (Gupta et al., 1996; Burkin et al., 1997; Vidaeus et al. 1997). Its amino acid sequence shares 90%, 56% and 55% identity respectively, to monkey, guinea pig and mouse fertilin-ß homologues.
It has been proposed that a phenylalanine–glutamate– glutamate (FEE) tripeptide at positions 449–45 within the disintegrin-like domain of human fertilin-ß, which is homologous to fertilin-ß tripeptides of other species, could act as an integrin recognition site (Videus et al., 1997). There is some dispute, however, regarding the precise fertilin equivalent of the RGD integrin-binding motif. Gichuhi et al. (Gichuhi et al., 1997) have suggested that the tripeptide ECD located two residues following the FEE tripeptide plays this role, as it is conserved across species and is probably the most common alternative tripeptide in non-RGD-containing snake venom disintegrins. The effects of short peptides containing either the RGD or ECD motif present in human fertilin-ß on the binding of human spermatozoa to zona-free hamster eggs has been studied (Gichuhi et al., 1997). In these experiments, sperm binding and penetration of zona-free hamster oocytes were inhibited in parallel, broadly in line with results of Bronson and Fusi (Bronson and Fusi 1990). The hexapeptide DECDLP inhibited both binding to and penetration of zona-free hamster eggs in a concentration-dependent manner, as did an RGD-containing linear peptide GRGDLP, with near complete inhibition at a peptide concentration of 300 µM. In contrast, an irrelvant hexapeptide (GERSTY) produced no significant inhibition of binding or penetration at concentrations up to 150 µM. These results, utilizing a heterologous assay, strongly suggested that receptors for fertilin and an RGD-containing sperm-associated protein play a role in the recognition of human spermatozoa by the oolemma. Further evidence that the ECD tripeptide could act as an integrin recognition sequence comes from the study of Yuan et al. (Yuan et al., 1997), who found that the ECD-containing peptide AQDECDVT inhibited the fusion of mouse spermatozoa with oocytes in vitro and from Evans et al. (Evans et al., 1997a), who showed that CAQDEC inhibited the binding of recombinant murine fertilin-ß to eggs. In addition, it has recently been demonstrated in mice, (Chen and Sampson, 1999) using photoaffinity labelling, that the DECD binds to the oolemmal integrin 6ß1. The present experiments, however, do not allow us to distinguish the relative importance of the FEE and ECD tripeptides in receptor recognition for human gametes, as the octapeptide used (SFEECDLP) in studying the interaction of homologous human gametes contains both amino acid sequences derived from the disintegrin domain of human fertilin-ß.
Our results support in part those of Evans et al. (Evans et al., 1995), who compared in a murine system the effects of a linear RGD-containing oligopeptide versus a peptide that contained the tripeptide QDE sequence of the cell recognition region of mouse fertilin-ß disintegrin domain on the fertilization of zona-free mouse eggs in vitro. While RGD containing peptides only partially inhibited sperm–egg adhesion, the tripeptide QDE decreased binding and fusion by ~70%. The authors postulated that the inhibitory effects of RGD-containing oligopeptides previously observed in a heterologous assay utilizing human spermatozoa and zona-free hamster eggs (Bronson and Fusi, 1990) might have been related to the unique nature of the Syrian hamster oocyte, which may be penetrated by spermatozoa of many heteologous species. The latter hypothesis does not appear to be the case, however, given the results of the present experiments.
We have addressed the question of differences in gamete receptor–ligand interactions between species, by studying the effects on receptor blocking oligopeptides on the interaction of human spermatozoa with zona-free human eggs. G4120, a thioether-bridged cyclized peptide known to block RGD-sensitive integrins inhibited both sperm oolemmal adhesion as well as sperm penetration, although at higher concentrations than did an FEE-containing octapeptide (SFEECDLP). In contrast, a peptide containing similar amino acids but in scrambled form (SFPCEDEL) did not inhibit gamete interactions, when compared with the no peptide control. In addition, the effects of the two active peptides were additive. These data indicate that the interaction of human spermatozoa and eggs, at the level of the oolemma, requires the recognition of at least two distinct sperm-associated ligands containing different amino acid recognition sequences. They suggest that integrins which recognize fertilin and RGD containing sperm-associated proteins, such as vitronectin, each play a role in gamete interactions leading to fertilization and that they may cooperate in gamete interactions leading to fertilization. It is unclear at this time, however, whether the same or different oolemmal receptors recognize both human fertilin and vitronectin.
The fact that the inhibition of sperm adhesion or penetration mediated through the addition of SFEECDLP occurs only in the presence of the peptide provides additional information. First of all, the inhibition mediated by FEE is not due to non-specific effects of the peptide, but is related to the occupancy of the receptor. Second, the fertilin-receptor interaction is reversible and does not lead to a stable modification of the receptor itself or to its disappearance. Third, the times of receptor block are brief and the receptor can easily be functionally recovered.
Our findings are significant as they present circumstantial evidence that a functionally active fertilin-like moiety is expressed on human spermatozoa. This possibility had recently been called into question (Jury et al., 1997), who have provided evidence that the fertilin- gene is not expressed in humans nor a wide range of primates (Jury et al., 1998) but is a non-functional pseudogene. Cytritestin, another member of the ADAM family, has been shown to be expressed in spermatocytes of rodents and proposed to play a role as a binding protein (Yuan et al., 1997), may also be non-functional in humans. Although Adham et al. (Adham et al., 1998) have reported that the human genome contains two cyritestin genes and that internal deletions do not cause a frame shift in the C-terminal coding region, Frayne and Hall (Frayne and Hall 1998) have presented evidence to the contrary. They have proposed that the human cyritestin gene is non-functional due to the presence of a variety of deletions, insertions, and in-frame termination codons. The absence of cyritestin expression was further supported by their inability to detect its presence on Western blots of human testis and human sperm extracts probed with macaque and human anti-cyritestin antisera, while fertilin-ß was detected in these preparations.
While these results would indicate that the fertilin-/ß (ADAM1/2) heterodimer first identified in guinea pig spermatozoa is unlikely to be expressed on human spermatozoa, the finding that a fertilin-ß derived peptide containing putative integrin recognition sequences competitively inhibits human sperm–egg adhesion and fusion in vitro strongly suggests that fertilin-ß is expressed on the human sperm surface, in association with a fertilin- substitute. Van Huijsduijen et al. (Van Huijsduijen et al., 1998) have recently reported the cDNA cloning of two new, closely related ADAM family members (ADAM 20, 21), which are expressed exclusively in human testis. Their encoded products show good sequence homology with fertilin- and contain potential fusion peptide sequences. It is possible that ADAM 20, ADAM 21, or another member of this widely growing family may form a heterdimer with fertilin-ß (ADAM2), leading to the expression of a functional molecule on human spermatozoa.
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3 To whom correspondence should be addressed at: Department of Obstetrics & Gynecology, Health Sciences Center, SUNY, Stony Brook, New York 11794–8091, USA
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References |
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Submitted on April 24, 1998; accepted on February 12, 1999.
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