Molecular Human Reproduction, Vol. 7, No. 4, 313-317,
April 2001
© 2001 European Society of Human Reproduction and Embryology
Commentary |
Do fertilin ß 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
Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
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Abstract |
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 Top Abstract A role for integrins...Evidence in support of...ConclusionsAcknowledgementsReferences |
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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 ß 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 ß and cyritestin in sperm–oolemma interactions, this lack of specificity indicates the need for considerable caution when interpreting results obtained using this approach.
ADAM protein/disintegrin/integrin/IVF/MDC protein/sperm-oocyte binding
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A role for integrins in sperm–oocyte binding |
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TopAbstractA role for integrins...Evidence in support of...ConclusionsAcknowledgementsReferences |
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Over the past 10 years, integrins have become increasingly implicated in the complex series of sperm–oocyte interactions which lead to fertilization (Bronson and Fusi, 1990a
,b); both human and hamster oocytes have long been known to express oolemmal integrins (Fusi et al., 1993). Hence, the discovery that the 
and ß subunits of the guinea-pig sperm surface fertilin complex each contained a disintegrin-like domain, similar to the integrin-binding disintegrins associated with some snake venom haemorrhagic proteins, naturally led Blobel and co-workers to propose a model in which fertilin ß played a role in oolemma binding (Blobel et al., 1992). However, whilst many snake venom disintegrins contain an RGD (arg-gly-asp) integrin-binding tripeptide sequence, the disintegrin-like domain of the guinea-pig fertilin ß subunit (and all subsequently sequenced species orthologues) contained an ECD (glu-cys-asp) motif at a similar position (see Figure 1). However, this did not preclude a role for fertilin ß in integrin binding since ECD is the alternative tripeptide in a number of the non-RGD-containing snake venom disintegrin domains; it was this observation which first led us to propose that ECD may act as a possible oolemma-integrin-binding ligand (Perry et al., 1995). Indeed, ECD-containing peptides have been shown to play a similar role to RGD-containing peptides in inhibiting platelet aggregation and clotting (see McLane et al., 1998), albeit with alterations in the affinity (Jia et al., 1997) or specificity (Shimokawa et al., 1997) of integrin binding.
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Fertilin ß is a member of a large family of integral membrane proteins, the MDC (or ADAM) family which, in addition to a disintegrin-like domain, also possess a prodomain, a metalloproteinase-like domain and a cysteine-rich domain, reminiscent of a number of snake venom reprolysins. Several MDC proteins are abundantly-expressed in the male reproductive tract and a number have been shown to be present on the surface of spermatogenic cells (Heinlein et al., 1994
; Wolfsberg et al., 1995; Frayne et al., 1997, 1998a, Frayne et al., b; McLaughlin et al., 1997). All possess a disintegrin-like domain containing a conserved XCD putative integrin-binding motif (where X represents one of a limited, but as yet undefined, subset of amino acid residues). Of those identified in the reproductive tract, fertilin ß, cyritestin (also known as tMDC I), tMDC II and tMDC III are exclusively expressed by spermatogenic cells and are therefore potential oolemma-binding candidates. However, most functional studies to date have concentrated on fertilin ß and cyritestin. |
Evidence in support of a role for MDC proteins in oolemma-binding |
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TopAbstractA role for integrins...Evidence in support of...ConclusionsAcknowledgementsReferences |
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The advent of IVF assay systems has provided an invaluable tool for studying gamete interactions. One commonly adopted approach is based on the axiom that sperm–oocyte interactions can be significantly decreased in vitro when eggs are pre-incubated with a peptide mimic based on a sperm protein implicated in oocyte binding; this experimental approach is analogous to the inhibition of integrin-mediated somatic cell adhesion by RGD-containing peptides (Ruoslahti, 1996
). Indeed the greatest body of evidence to support a role for fertilin ß and cyritestin in oolemma binding stems from the ability of short synthetic peptides, based on their disintegrin-like domains, to inhibit sperm–oocyte binding and fusion in IVF systems (e.g. Myles et al., 1994; Almeida et al., 1995; Evans et al., 1995; Yuan et al., 1997; Linder and Heinlein, 1997; Pyluck et al., 1997; Chen and Sampson, 1999). However, there is considerable variation and inconsistency in the literature regarding both the efficacy of peptide mimics used in such IVF assays and the identification of the residues important for oolemma-binding, leading to a number of conflicting reports. Indeed some of the earlier studies suggested the involvement of an alternative integrin-binding tripeptide motif (Myles et al., 1994; Almeida et al., 1995; Evans et al., 1995). Such problems can be largely attributed to a number of technical factors. First, there are significant variations in experimental protocols between research groups. For example, the method of zona removal and sperm/oocyte ratios are critical factors in zona-free oocyte assays (Evans, 1999). Second, there are differences in the way that results are reported, making direct comparisons difficult, as some workers present effects on sperm–oocyte fusion only and not sperm–oocyte binding (Myles et al., 1994; Linder and Heinlein, 1997; Pyluck et al., 1997; Chen and Sampson, 1999) or do not measure fertilization or fusion rates (Almeida et al., 1995). Thirdly, synthetic peptides of different lengths may behave in different ways. Very short peptides (up to 6 residues) are likely to be relatively rigid with very few molecules adopting the type of conformation associated with the integrin-binding tripeptide motif found at the tip of the loop of native snake venom disintegrins; this is arguably one reason why very high peptide concentrations are needed to elicit a significant effect. In contrast, longer peptides (e.g. greater than 12 residues) are likely to be more flexible with a greater proportion of molecules adopting a favourable binding conformation. Finally, the amino acid composition of synthetic peptides, particularly in terms of net overall charge and degree of hydrophobicity, may well influence their properties. In this respect it is important that test and control peptides have identical amino acid compositions and identically positioned cysteine residues wherever possible; several published studies have attempted to compare results using peptides with very different amino acid compositions and this may account in part for some of the problems encountered.
Despite these inconsistencies, in most reported studies short fertilin ß- or cyritestin-based peptide mimics have been found to cause a very substantial inhibition of sperm–oocyte interactions, implying that both of these proteins are indispensable. Yet recent gene `knockout' experiments in mice would not appear to support this conclusion. Whilst fertilin ß `knockout' mice (Cho et al., 1998; Frayne and Hall, 1999) exhibited drastically reduced fertility and cyritestin (Shamsadin et al., 1999) `knockout' males were infertile, in each case much of the reduction in fertility was attributed to causes other than reduced oolemma binding. For example, both types of `knockout' spermatozoa exhibited an apparent reduction in zona pellucida binding and fertilin ß-null spermatozoa also showed a reduction in migration from the uterus to the oviduct. Indeed cyritestin-null spermatozoa were able to bind normally to the oolemma of zona-free eggs in vitro, implying that cyritestin is not essential for sperm–oocyte interactions at the level of the oolemma.
We believe that this apparent dilemma can be explained by the promiscuous nature of short, disintegrin-based MDC peptide mimics when used in IVF assays. Implicit in the way that many of these studies have been presented, and the interpretation of the results obtained from them, is the belief that a given synthetic peptide will only mimic the MDC protein upon which it is based. For example, inhibition of IVF by a fertilin ß-based synthetic peptide is taken to support a role for fertilin ß in fertilization in vivo. For such an interpretation to be valid, these peptide mimics clearly need to possess the necessary sequence specificity assumed to be inherent in their native counterparts. Without this specificity each peptide mimic could inhibit a range of different MDC-integrin interactions in complex IVF assays, thereby yielding little, if any, information on the role of individual MDC proteins in vivo. We believe that this is precisely what happens and have found that in principle any peptide containing an XCD motif (where X represents one of a limited, but as yet undefined, subset of amino acid residues) is able to inhibit mouse sperm–oocyte binding and fusion in a zona-free oocyte assay, indicating that the flanking sequences do not confer any sequence specificity in these simple in-vitro assays. For example, CRLAQD-ECDVTEYC (corresponding to the putative disintegrin-binding loop of mouse fertilin ß) and CQALDRECDTYVEC (a scrambled version of the same peptide, but maintaining the ECD motif) were equally effective at inhibiting sperm– oocyte interactions in vitro (Table I and Figure 2). Similarly, CRKSKDQCDFPEFC (corresponding to the putative disintegrin-binding loop of mouse cyritestin) and CKFD- SKQCDPFREC (a scrambled version of the same peptide, but maintaining the QCD motif) were also equally effective (Table I and Figure 2). These findings are in contrast to data reported by Yuan and colleagues (Yuan et al., 1997) which indicated that a fertilin ß peptide resulted in only a slight inhibition (13%) of sperm–oocyte binding, substantially less than that of a comparable cyritestin-based peptide. In our studies, which utilized highly-purified peptides, no statistically significant differences were obtained between the fertilin ß peptide and the cyritestin peptide in their abilities to inhibit sperm binding at any of the peptide concentrations tested (Figure 2Ai and Aii). Indeed, at the higher peptide concentrations tested, the levels of inhibition obtained were very similar for both peptides.
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). (ii) Peptide based on mouse cyritestin (•), or a scrambled cyritestin peptide containing a QCD motif (In contrast, a scrambled cyritestin peptide which lacked the QCD motif (CRQDKSKCFDPFEC), or in which the QCD was replaced by RGD (CKFDSKRGDPFREC), had no effect on sperm–oocyte interactions (Table I
and Figure 2), confirming the importance of an XCD motif [a conclusion supported by recent site-directed mutagenesis experiments (Bigler et al., 2000; Zhu et al., 2000)] and indicating that an XCD tripeptide could not be substituted by the alternative RGD integrin-binding ligand. Hence, whilst studies with MDC-based peptide mimics fully support the proposed role for integrins in sperm–oocyte interactions, they do not permit any conclusions to be drawn with respect to the specific MDC protein(s) which may be involved in these processes.
Such a finding is perhaps not that surprising by analogy to somatic cell integrin ligands. For example, in the matrix glycoprotein, fibronectin, a prototype integrin ligand about which a great deal is known from detailed structure-function studies, only two short peptide sequences are recognized by cells: an RGD tripeptide motif and a `synergy' site located some distance away (Humphries and Newham, 1998). If MDC proteins bind to integrins in a similar manner, with the XCD motif mimicking the RGD motif, then any putative `synergy' sequence required for specificity is likely to be some distance away on the primary sequence, beyond the limits of the short peptides commonly used in IVF assays.
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Conclusions |
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TopAbstractA role for integrins...Evidence in support of...ConclusionsAcknowledgementsReferences |
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We have previously postulated that a number of sperm-associated MDC proteins are likely to play a role in oolemma-binding and that these proteins may act co-operatively (Hall and Frayne, 1999
). In other words, we propose that the concerted effects of several MDC proteins will lead to maximum `fertilizing ability' but individual proteins are not indispensable, implying a degree of functional redundancy. Such a model is supported by our work on the human orthologues of MDC genes implicated in fertility, several of which we have shown to be non-functional in man (Jury et al., 1997; 1998; Frayne and Hall, 1998; Frayne et al., 1999). In particular, human tMDC I/cyritestin transcripts contain a variety of deletions, insertions and in-frame termination codons, precluding the synthesis of a functional protein; a finding which is further supported by the lack of immunoreactivity on Western blots of human testis and sperm protein extracts probed with macaque and human anti-tMDC I antisera (Frayne and Hall, 1998). However, our findings are in contrast to an earlier report (Adham et al., 1998) which suggested the presence of a functional cyritestin gene (cyrn1) and a second, closely related gene (cyrn2) in the human; the former we have argued contains significant sequencing errors (see Frayne and Hall, 1998) and the latter, of which there is only data for one exon, appears to be a non-transcribed genomic sequence, probably a pseudogene. Other non-functional human MDC genes include fertilin and tMDC II (Jury et al., 1997; 1998; Frayne et al., 1999). Our proposed model of MDC functional redundancy and co-operativity would also be consistent with the preliminary data reported for the mouse fertilin ß and cyritestin `knockouts' (see above), where neither of these MDC proteins was found to be essential for sperm–oolemma interactions; indeed oolemma-binding by cyritestin-null sperm was apparently unaffected.
Furthermore, the ability of a single peptide mimic to drastically reduce sperm–oocyte interactions in vitro, which at first sight would appear to be inconsistent with our model and with the mouse `knockout' data (particularly for cyritestin where sperm–oolemma binding was unaffected), is easily explained by the lack of sequence specificity which we have shown to be inherent in such peptides, with a single peptide being able to block most, if not all, gamete-associated disintegrin-integrin interactions. Hence, although current data is still consistent with a role for fertilin ß and cyritestin in rodent sperm–oolemma interactions, it is reasonable to consider the possibility that other, sperm-surface MDC proteins, may be equally important.
In the light of the limitations associated with the use of peptide mimics, confirmation of a role for fertilin ß and cyritestin in sperm–oocyte interactions, characterization of the specific disintegrin residues involved in binding and identification of the cognate, oolemma-associated integrins would be greatly facilitated by the availability of correctly folded recombinant MDC proteins for in-vitro binding studies. Unfortunately, this is fraught with problems, mainly associated with their large size, cysteine-rich nature (with associated requirement for correct disulphide bond formation) and post-translational processing. Whilst there have been a number of recent reports using recombinantly expressed MDC proteins (or domains derived from them), it is important to bear in mind that unless such proteins are correctly folded (which could be difficult to establish), they may simply behave as `long peptides' with lack of specificity problems similar to those associated with short peptide mimics.
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Acknowledgements |
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TopAbstractA role for integrins...Evidence in support of...ConclusionsAcknowledgementsReferences |
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The data presented in this report was supported by the Wellcome Trust (UK).
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Notes |
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1 To whom correspondence should be addressed. E-mail: L.Hall{at}bris.ac.uk
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References |
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Submitted on October 17, 2000; accepted on February 1, 2001.
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