Hum. Reprod. Advance Access published online on June 10, 2008
Human Reproduction, doi:10.1093/humrep/den191
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Polymorphisms in the human cysteine-rich secretory protein 2 (CRISP2) gene in Australian men
1 Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Clayton, VIC, Australia 2 The Australian Research Council Centre of Excellence for Biotechnology and Development, Monash University, Clayton, VIC, Australia 3 Prince Henry's Institute, Monash Medical Centre, Clayton, VIC, Australia 4 Department of Obstetrics and Gynaecology, University of Melbourne, Carlton, VIC, Australia
5 Correspondence address. Tel +61-3-95947407; Fax: +61-3-95947439; E-mail: moira.obryan{at}med.monash.edu.au
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Abstract |
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BACKGROUND: Cysteine-rich secretory protein 2 (CRISP2) is localized to the human sperm acrosome and tail. It can regulate ryanodine receptors Ca2+ gating and binds to mitogen-activated protein kinase kinase kinase 11 in the acrosome and gametogenetin 1 (GGN1) in the tail.
METHODS AND RESULTS: In order to test the hypothesis that CRISP2 variations contribute to male infertility, we screened coding and flanking intronic regions in 92 infertile men with asthenozoo- and/or teratozoospermia and 176 control men using denaturing HPLC and sequencing. There were 21 polymorphisms identified, including 13 unreported variations. Three SNPs resulted in amino acid substitutions: L59V, M176I and C196R. All were only present in a heterozygous state and found in fertile men. However, the C196R polymorphism was of particular interest as it resulted in the loss of a strictly conserved cysteine involved in intramolecular disulphide bonding. Screening of an additional 637 infertile men identified 23 heterozygous C196R men to give an overall frequency of 3.6%, compared with 3.4% in control men. The functional significance of the C196R polymorphism was defined using a yeast two-hybrid assay. The C196R substitution resulted in the loss of CRISP2–GGN1 binding.
CONCLUSIONS: Although none of the many polymorphisms identified herein showed a significant association with male infertility, functional studies suggested that the C196R polymorphism may compromise CRISP2 function.
Key words: CRISP2/TPX1/GGN/male infertility/single-nucleotide polymorphism
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Introduction |
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Approximately one in six couples experiences some form of infertility and
50% of these cases can be ascribed at least in part to the male partner (de Kretser and Baker, 1999
). A significant proportion of male infertility is accompanied by either inadequate spermatogenesis or defective sperm function. Almost certainly, many contain a genetic component (Rucker et al., 1998; Cram et al., 2001). The establishment of male fertility is a multi-step process and it has been estimated that these processes encompass the coordinated expression and activation of >1500 genes in the testis (Andrews et al., 2000; Reinke et al., 2000). Theoretically, mutations in any one of these genes could lead to infertility. Recently, with the introduction of specific gene targeting techniques and random model organism whole genome mutagenesis programmes, hundreds of candidate genes related to spermatogenesis have been identified (Matzuk and Lamb, 2002; Handel et al., 2006; Kennedy and O'Bryan, 2006; O'Bryan and de Kretser, 2006; Lu et al., 2007). With the exception of a few genes, most have yet to be translated into human medicine (Sun et al., 1999; Miyamoto et al., 2003; Krausz et al., 2006; Dieterich et al., 2007). The difficulties in translation are in part related to reduced family sizes in families carrying fertility-limiting genes, but also to the often frequent reluctance of affected individuals to discuss infertility with family members or to seek treatment. Collectively, this means that traditional mapping studies have had limited success. Polymorphisms or genetic variants in genes involved in spermatogenesis are, however, considered potential risk factors which may contribute to the severity of impaired spermatogenesis (Ferlin et al., 2006; Krausz, 2007; Krausz and Giachini, 2007; Tuttelmann et al., 2007).
Cysteine-rich secretory protein 2 (CRISP2) (previously called TPX1) is a testis-enriched member of the CRISP (O'Bryan et al., 1998), antigen 5 and pathogenesis-related (CAP) superfamily (Schreiber et al., 1997). CRISPs contain 16 absolutely conserved cysteine residues which have been shown by both enzymatic and structural methods to be involved in intramolecular disulphide bonding (Eberspaecher et al., 1995; Guo et al., 2005; Wang et al., 2005; Gibbs et al., 2006). CRISPs are two-domain proteins containing a structurally similar yet evolutionarily diverse N-terminal domain, referred to as the CAP (also Pr-1 or SCP) domain, and a unique C-terminal domain referred to as the cysteine-rich or CRISP domain. The two domains are linked by a hinge region containing two crossed disulphide bonds (Guo et al., 2005; Gibbs et al., 2006).
CRISP2 protein has been localized to the acrosome and outer dense fibres of the sperm tail (Foster and Gerton, 1996; O'Bryan et al., 2001; Busso et al., 2005; Du et al., 2006), suggesting that it may play a critical role in male fertility. CRISP2 has been implicated in cell adhesion between spermatids and Sertoli cells (Maeda et al., 1998, 1999) and gamete interaction (Busso et al., 2005); however, the mechanisms for these interactions have yet to be revealed. Our recent studies demonstrated that the CRISP domain of mouse CRISP2 can regulate Ca2+ influx through ryanodine receptors (Gibbs et al., 2006) in an analogous manner to several CRISPs isolated from reptile venoms (Yamazaki and Morita, 2004). We have also shown that the CRISP domain of mouse CRISP2 interacts with mitogen-activated protein kinase kinase kinase 11 (MAP3K11) (Gibbs et al., 2007) and the testis-enriched protein gametogenetin 1 (GGN1) (Jamsai et al., 2008). In somatic cells, MAP3K11 is an up-stream kinase in the stress-activated arm of the MAP kinase pathway (Gallo et al., 1994; Rana et al., 1996; Teramoto et al., 1996; Hirai et al., 1997; Davis, 1999) and the NF
B pathway (Hehner et al., 2000). MAP3K11 also has the ability to phosphorylate at least one Golgi protein, Golgin-160 (Cha et al., 2004). GGN1 is a protein of largely unknown function that binds to CRISP2 in the sperm tail via the CRISP domain (Jamsai et al., 2008). For all of these reasons, we hypothesize that mutations or polymorphisms in CRISP2 will be associated with human male infertility.
The human CRISP2 gene contains 10 exons and spans over 21 kb on chromosome 6p21 (http://www.genenames.org/data/hgnc_data.php?hgnc_id=12024; http://www.ncbi.nlm.nih.gov/sites/entrez). Exons 4–10 encode a 243 amino acid protein (Kasahara et al., 1989). On the basis of the conserved expression of CRISP2 across species, we initially assessed the presence and prevalence of human CRISP2 gene single-nucleotide polymorphisms (SNPs) in men with infertility characterized by teratozoospermia (abnormal shape) and/or asthenozoospermia (abnormal motility). This screen identified 21 SNPs including three that resulted in altered amino acid sequences. The C196R SNP was of particular interest and its prevalence was assessed in a larger group of men with a broad range of infertility phenotypes. The functional significance of the C196R SNP was assessed using a yeast two-hybrid assay.
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Materials and Methods |
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Infertile and control men
Infertile male gDNA samples used in this study were taken from the Monash Male Infertility repository (Lynch et al., 2005
). The data set represents a set of non-consecutive men presenting for infertility treatment where female infertility had been extensively ruled out through previous investigations. Clinical data from an assessment by a male fertility specialist physician and from laboratory investigation were stored in the database and included: testis volumes, serum hormone levels for testosterone, follicle stimulating hormone and luteinizing hormone levels and more than one semen analysis as assessed by the World Health Organization (WHO) criteria (World Heath Organization, 1999). Where clinically indicated, a testicular biopsy was performed to determine the histopathology of the testis.
The collection represents patients of particular interest for genetic studies, including those with spermatogenic failure and disorders of sperm motility and morphology. For this study, all men with a definitive diagnosis were excluded. They included men with Y chromosome deletion (including gr/gr), Klinefelter's syndrome or other karyotypic abnormalities, men with cryptorchidism which was not corrected as a young child, men who had undergone cancer therapy or men who had had mumps post-puberty, a congenital absence of the vas deferens, ejaculatory duct abnormalities or complex injury or disease. A total of 729 patients were included in this study, including an initial focus group that contained 92 infertile men with asthenozoospermia (
25% total motility) and/or teratozoospermia (>90% abnormal morphology). An additional 637 infertile men exhibiting a wide range of clinical presentations with semen profiles ranging from azoospermia to normospermic, forming a random selection of infertile male samples from the repository described above, were included in the subsequent exon 9 analysis.
All men were clinically infertile and gave informed consent. The study was approved by the Human Research and Ethics Committees of Southern Health (Monash Medical Centre) and the Royal Women's Hospital (Melbourne) and by the Monash University Standing Committee.
The control group was composed of the following groups of men: 52 men of proven fertility who had not undergone physical examination, 42 previously fertile men presenting with obstructive azoospermia after vasectomy and 82 healthy young men with normal semen and hormone profiles that are consistent with fertility, but the majority of whom were of unproven fertility. Specifically, 13 of the 82 men in this group had fathered children (Sikaris et al., 2005). All subjects gave their informed consent and the study was approved by the Human Research and Ethics Committees of Southern Health (Monash Medical Centre), the Royal Women's Hospital (Melbourne) and Concorde Hospital (Sydney).
Denaturing high performance liquid chromatography
Primer sequences and PCR conditions are shown in Table I. Primer sets were designed to amplify all 10 exons of the CRISP2 gene and 50 bp of intronic sequence flanking the exons using a web-based program, Primer3 (http://frodo.wi.mit.edu). Approximately 50 ng of total genomic DNA samples from 92 asthenozoo- and/or teratozoospermic men and 176 control men were used as templates for PCR amplification in 30 µl reactions using Fisher Biotec Taq polymerase F2 (Fisher Biotec Subiaco, Western Australia, 1 unit/30 µl reaction). Reactions were subjected to an initial denaturation of 94°C for 5 min, followed by 35 cycles of 94°C for 30 s; annealing at the temperature given in Table I for 45 s and extension at 72°C for 30 s. A final extension of 72°C for 7 min was adopted. Following PCR amplification, post-PCR re-annealing was performed to ensure equimolar quantities of homo- and heteroduplex products. This was achieved by mixing patient PCR products and fertile men control PCR products (of known sequence) at 1:1 ratio and incubating them at 94°C for 4 min followed by melt profile cooling from 94°C to 65°C over a 30 min time interval. Samples were run on a Varian Pro-Star Helix System (Walnut Creek, USA). Pre-equilibration was performed prior to sample loading using 100 mM TEAA, pH7.0, 0.1 mM EDTA (Varian Buffer Pak A) as a mobile phase. Elution was achieved by an increasing gradient of 25% (v/v) acetonitrile delivered in 100 mM TEAA, pH 7.0, 0.1 mM EDTA.
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Restriction fragment length polymorphism
Restriction fragment length polymorphism (RFLP) analysis was performed on an additional 637 infertile patients to investigate the frequency of the C196R polymorphism. PCR amplifications were performed using primers: Tpxex9mutF (5'-TTTGATGACTGATACTGCCCTTA-3') and Tpxex9mutR (5'-TTACTGCAT AGTCCTTTGTCCC-3') under the conditions described above. Aliquots of 10 µl PCR product were subjected to NciI restriction digestion in 20 µl reaction volumes using NEB buffer 4 and 4 units of NciI. Digestions were performed for a minimum of 2 h at 37°C and subsequently analysed on 3% TBE agarose gels. A heterozygous C196R gDNA sample was included as a positive control. The Tpxex9mutF and Tpxex9mutR primers yielded a PCR product of 164 bp. Upon NciI digestion, heterozygous samples yielded three fragments of 164, 142 and 22 bp. Two fragments of 142 and 22 bp were expected for a homozygous C196R sample. Any apparent SNPs or individuals demonstrating an unusual profile were sequenced to confirm the presence of the SNP.
Data analysis
Sequencing data was analysed with Sequencher version 4.2 (Gene Codes Corporation, MI, USA). SNP data were compared with previously described SNP databases and the NCBI reference sequence (NC_000006
[GenBank]
.10) using Ensembl Geneseqview (ENSG00000124490). The gene reference is given as Homo sapiens chromosome 6-ref assembly-complement NC_000006
[GenBank]
.10:g (49767431.49789858). When reporting positions of SNPs in the text, only the number inside the bracket has been shown for brevity. SNP frequency comparisons between infertile male and fertile male groups were performing using 
2 tests (two-tailed) within Graph Pad PRISM 4 Quick Calcs (San Diego, CA, USA) with P-values <0.05 being statistically significant. Denaturing high performance liquid chromatography (DHPLC) products were compared and analysed using Star Reviewer Software (Varian).
Immunohistochemistry
In order to define the sites of CRISP2 production in the human testis Bouin's-fixed human testis was immuno-stained using an avidin–biotin amplified technique with the T4 (amino acids 34–49 within the CAP domain of the human CRISP2 protein) and T8 (amino acids 222–236 within the CRISP domain) peptide antisera at a dilution of 1 in 1000 as described previously (O'Bryan et al., 2001). The specificity of staining was assured by pre-absorption with the immunizing peptide as described previously (O'Bryan et al., 2001). Human testis tissue was obtained with consent from a healthy donor (Kennedy et al., 2004).
In situ hybridization
The expression of CRISP2 mRNA within the normal human testis was determined by in situ hybridization using DIG-labelled antisense cRNA probes as previously described (O'Bryan et al., 1998). DIG-riboprobes were prepared from a partial CRISP2 cDNA corresponding to exon 10 (ZBB2/pGEM-T clone). This plasmid was generated by RT–PCR from human testis total RNA using primers tpxBB 5'-TTTATGCAAGTAAAAACTCAGGTAGTAGG-3' and tpxZ 5'-GATCTCCTAAGTAACTGTGATTCCTTGA-3'. The resultant 502 bp product was gel purified and sub-cloned into pGEM-T as outlined by the supplier (Promega Corporation). Using a BLAST analysis of the RefSeq database, no significant homology was seen between this sequence and any other sequence. Labelled sense and antisense cRNA was synthesized by incubation of either NotI or NcoI linearized ZBB2/pGEM-T plasmid template with DIG-labelled UTP (Boehringer Mannheim) in the presence of SP6 or T7 RNA polymerase, appropriate for the respective restriction enzyme employed. In addition to the inclusion of a sense control, the antisense probe was hybridized with a section of human epididymis tissue which expresses CRISP1 and CRISP3, but not CRISP2. The latter assessed the specific possibility of cross-reactivity between CRISP family members. No cross-reactivity was observed (data not shown).
Cloning of the human CRISP2 and human GGN1 cDNAs into yeast expression vectors
Full-length human CRISP2 cDNA was amplified from total human testis cDNA using primers: hfCRISP2Fw 5'-CACCTGCAAGAAAGAGCACA-3' and hfCRISP2Rev 5'-GCAGTC TTGCACAATGCTCA-3'. An 841 bp fragment was blunt-end cloned into the pPCR-ScriptSK cloning vector (Stratagene). Cysteine position 196 (C196) was converted to arginine (R196) using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The mature human CRISP2 cDNA coding region (amino acids 21–243) containing either C196 or R196 was cloned in the NcoI and EcoRI sites of the pAS2-1 bait vector (BD Clontech, Palo Alto, CA, USA) to make a fusion protein between the yeast GAL4 DNA binding domain and CRISP2.
Full-length human GGN1 cDNA was also amplified from total human testis cDNA using primers: pACT2hfGGN1NcoIFw:5'-gatgccatggGTATGGGGAACTTGCAGTCG-3' and pACT2hfGGN1EcoRIRev: 5'-gcggaattcGTCAGTTGGAATGGGTGGCCT-3'. Upper case bases represent GGN1 specific sequences, underlined lower case bases represent restriction enzyme cutting sites (NcoI and EcoRI) added onto primers to facilitate cloning and the remaining lower case bases are extra bases added to extend PCR fragments at each end to facilitate efficient restriction digestion. A 1986 bp fragment containing full-length GGN1 (652 amino acids) and flanking NcoI and EcoRI sites was subsequently cloned into pACT2 prey vector (BD Clontech) to make a fusion protein between the yeast GAL4 DNA activation domain and GGN1. Sequencing analysis of both human CRISP2 and GGN1 full-length inserts after sub-cloning into yeast expression vectors was performed using BigDyeTerminator DNA sequencing Kit (Applied Biosystems, Foster City, CA, USA).
Yeast two-hybrid assay
A yeast two-hybrid assay to assess the interaction between human CRISP2 protein and its interacting partner GGN1 was performed as described previously (Gibbs et al., 2007). Briefly, C196-CRISP2/pAS2-1 or R196-CRISP2/pAS2-1 bait vector was co-transfected with the GGN1/pACT2 prey vector into reporter Saccharomyces cerevisiae strain AH109. Cells were plated on nutrient-deficient media (leucine–tryptophan–histidine dropout) to verify the interaction, according to the manufacturer's instructions (BD Clontech). Positive clones were subsequently grown on high stringency leucine–tryptophan–histidine–adenine dropout media.
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CRISP2 expression in the human testis
Consistent with the distribution of CRISP2 in rodents (O'Bryan et al., 2001
; Busso et al., 2007), CRISP2 was localized solely to germ cells in the human testis; specifically to spermatids and the developing and mature acrosome and tail (Fig. 1A and B). The specificity of the T4 staining was confirmed by pre-absorption with the immunizing peptide (Fig. 1A, inset). Identical results were obtained using the T8 antiserum (data not shown). The expression of CRISP2 within the testis was further confirmed using in situ hybridization methods. Consistent with previously published data in the rat indicating that like many testis transcripts, CRISP2 mRNA undergoes a translational delay (O'Bryan et al., 1998; Maeda et al., 1999; O'Bryan et al., 2001); low levels of CRISP2 mRNA were observed in the later stages of spermatocyte development. Expression was significantly up-regulated in round through to elongated spermatids (Fig. 1C).
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Polymorphisms in CRISP2 and male infertility
On the basis of the localization of CRISP2 protein, we postulated that inactivating mutations would lead to male infertility characterized by abnormal sperm morphology and/or motility. As such, we sought to define the type and frequency of polymorphisms in the CRISP2 gene. Using PCR amplification with primers flanking each exon followed by DHPLC and direct DNA sequencing, we screened the nucleotide sequence variations in a total of 10 exons and adjacent intron/exon boundaries in the CRISP2 gene of 92 infertile men with astheno- and/or teratoszoopermia. Results were compared with sequences from 176 control men. Exons 1–8 were successfully screened by DHPLC, whereas exon 2 and the extremely polymorphic exons 9–10 were screened by sequencing.
As shown in Table II and Fig. 2A, the human CRISP2 gene displayed a relatively high degree of heterogeneity. The majority of polymorphisms were in introns or UTR regions and included 13 which had not previously been reported or detailed in publicly available databases. Previously unreported polymorphisms within the non-coding region of the gene included: an insertion of a G at position 49789331 (98 bp upstream of non-coding exon 1); a T>C substitution at position 49789171 (within non-coding exon 1); two in intron 3–4: one of which was the replacement of a 7 bp sequence (TAGAGAC) with a completely different 11 bp sequence (GTGTTAGAAGG) starting at position 49786818 and the second of which was a deletion of 4 bp (GGCT) starting at position 49786790; two in intron 4–5: a C>A substitution at position 49776702 and an A>T substitution at position 49776495; three in intron 7–8: an A>T substitution at position 49773968, a G>A substitution at position 49773936 and a T>A substitution at position 49773689; and a T>G substitution at position 49771478 in intron 9–10. A 2 test without Yates correction revealed that the frequency of none of these SNPs varied significantly between infertile and fertile men.
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Two novel SNPs and a recently reported SNP affecting protein-coding sequences were detected: a C>G substitution at position 49776348 in exon 5 which would result in a conservative L59V change at the protein level; a G>A substitution at position 49771584 in exon 9 which would result in a conservative M176I change at the protein level and a T>C substitution at 49771526 in exon 9 which would result in a non-conservative C196R change at the protein level, respectively (Table II). All three SNPs were only ever found in a heterozygous state. The L59V and M176I polymorphisms were only seen in control men and were not pursued further. The C196R polymorphism was seen in 3 out of 92 infertile men (3.3%) and in 6 out of 175 control men (3.4%) (Table III). These data indicate that none of the protein-coding SNPs resulted in a dominant form of male infertility.
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The C196R polymorphism was of particular interest as it would result in the loss of a strictly conserved cysteine which is involved in formation of one of the two crossed disulphide bonds in the hinge region which joins the CAP and CRISP domains in CRISPs (Guo et al., 2005
; Gibbs et al., 2006). Loss of this bond would likely result in hyper-flexibility of the hinge and thus the potential for deleterious effects on protein activity or interactions with binding partners. In order to assess the possibility that homozygous C196R polymorphisms are a cause of human male infertility, an additional 637 patients were screened by RFLP analysis. As can be seen in Fig. 2C, digestion of PCR products amplified from exon 9 of the CRISP2 gene with NciI resulted in a differential banding pattern depending on the presence or absence of the T>C polymorphism. In the presence of the T base at position 18333, this assay generated a single band of 164 bp. In the presence of the C base, two bands of 142 and 22 bp were generated. Heterozygous patients contained three bands at 164, 142 and 22 bp. Using agarose gel electrophoresis, only the 164 and 142 bp products were visible. However, these were sufficient to resolve all three genotypes. All potential C196R heterozygous or homozygous samples were confirmed by sequencing. As illustrated in Table III, 23 additional men carrying heterozygous C196R encoding sequences were found out of 637. The overall frequency of C196R heterozygous individuals in the infertile Australian male population was 3.6% (26/729). This frequency was not statistically different from that observed in the control group (6/175, 3.4%) using a 2 test without Yates correction (P = 0.9293). No homozygous men were found. In order to further assess the possibility of an association between the C196R polymorphism and a particular aspect of sperm function, patients were further stratified on the basis of sperm density, percentage motile sperm or percentage normal morphology as defined using WHO criteria. No significant association was found (Supplementary Table 1), showing that this polymorphism in a heterozygous state (in isolation) is not a critical determinant of sperm function.
Consistent with this conclusion were the normal semen analyses for both of the control men, carrying the C196R polymorphism, who had undergone a semen analysis: CC26 had a sperm count of 70 x 106/ml, 64% total motility and 17% abnormal forms; CC78 had a sperm count of 21.9 x 106/ml, 67% total motility and 29% abnormal forms. The semen profiles of all men carrying the C196R polymorphism can be found in Supplementary Table 2.
Potential functional relevance of C196R polymorphism in CRIPS2 protein
In previous studies, we have shown that the ion channel regulatory region (ICR), a sub-region within the CRISP domain of mouse CRISP2, binds to the COOH end of GGN1 in the sperm tail (Jamsai et al., 2008). To test the hypothesis that loss of the cysteine at amino acid position 196 (located within the adjacent hinge region) would interfere with binding partner interactions, we developed a yeast two-hybrid assay to test the effect of the C196R polymorphism on GGN1 binding. Our data confirmed that human CRISP2 carrying the C196R polymorphism lost the ability to interact with human GGN1 protein.
We have previously shown that CRISP2–GGN1 interaction occurs between the ICR of CRISP2 and the COOH end of GGN1 (Jamsai et al., 2008). The modification of the hinge region through introduction of the C196R mutation, however, ablates GGN1 binding to the ICR in the full-length CRISP2. These data suggest that there are additional sequences outside the ICR within the CRISP2 structure which affect GGN1 binding. Our assessment is that the C196R mutation will have two possible effects, notably: that the removal of a conserved disulphide bond increases the degree of flexibility in the hinge resulting in steric hindrance between the CRISP2 and GGN1 primary or secondary binding sites and that the introduction of a charged amino acid significantly affects a secondary protein interaction site thereby destabilizing interaction with the ICR.
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Discussion |
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CRISP2 is a highly variable gene in the human male population; it is expressed predominantly in the testis and is produced in the haploid germ cell compartment where it becomes localized to the sperm acrosome and tail. This study represents the first analysis of CRISP polymorphisms in a human population of fertile and infertile men. We have identified 21 separate CRISP2 polymorphisms including 13 which had not previously been identified and three of which resulted in changes in the amino acid sequence. Importantly, the C196R polymorphism resulted in the loss of an absolutely conserved cysteine involved in disulphide bonding within the hinge region linking the CAP and CRISP domains of CRISP2. Although none of the identified SNPs appears to be independently causative for male infertility in this population, functional data suggest that in the homozygous state the C196R polymorphism would interfere with protein function including the ability to bind to GGN1 in the sperm tail. The C196R polymorphism occurs in a heterozygous state in
3.6% of the Australian male population.
Within mammals, there are 3–4 CRISPs depending on species (three in humans), all of which are either incorporated into sperm during their production in the testis or during epididymal maturation (Gibbs and O'Bryan, 2007). CRISP2 is a testis-enriched protein which is incorporated in the developing acrosome and accessory structures of the sperm tail (O'Bryan et al., 2001). CRISP1 and CRISP4 are produced predominantly in the epididymal lumen wherein they are associated with sperm membranes (Jalkanen et al., 2005; Nolan et al., 2006). CRISP3 is also expressed in the epididymis, but is more widely expressed, including in the salivary gland and B-cells (Haendler et al., 1993; Pfisterer et al., 1996; Laine et al., 2007) and is up-regulated in several forms of pathology including prostate cancer and pancreatitis (Liao et al., 2003; Bjartell et al., 2006). In addition to the reproductive tract of mammals, CRISPs are also found in the venom of many species of poisonous snakes (Yamazaki and Morita, 2004; Fry et al., 2006), poisonous lizards (Morrissette et al., 1995), in the hatching gland of Xenopus laevis (Schambony et al., 2003) and buccal gland of the parasitic lamprey (Ito et al., 2007).
All CRISPs contain 16 absolutely conserved cysteines which form intramolecular disulphide bonds to form two discrete domains, the CAP and CRISP domains, which are linked by a hinge region (Guo et al., 2005; Shikamoto et al., 2005). The molecular function of CRISPs as a whole, and the two domains separately, is only now starting to be revealed. CRISP domains, as typified by the mouse CRISP2 CRISP domain, can regulate Ca2+ through RyR channels (Gibbs et al., 2006). By inference, it is also the CRISP domains in reptile venoms and lamprey buccal gland secretions which differentially regulate a range of ion channels (Ito et al., 2007). Further, the CRISP domain of CRISP2 has recently been shown to interact with several binding partners including MAP3K11 (Gibbs et al., 2007) and GGN1 (Jamsai et al., 2008). The function of the CAP domain, which is also found in non-CRISP proteins in species from a wide range of phyla, remains virtually unknown, although it is associated with an antifungal activity in plants (Stintzi et al., 1993; Niderman et al., 1995) and a recent study on a cone snail CAP, Tex31, suggests CAPs may act as site-specific proteases (Milne et al., 2003). The human CRISP3 has also been shown to interact with β-microseminoprotein (MSP) (Udby et al., 2005) and to bind to 
1B-glycoprotein (A1BG) in the seminal plasma (Udby et al., 2004). The data presented herein suggest that the spatial orientation of the CAP and CRISP domains relative to one another is important for such molecular interactions.
Although none of the polymorphisms identified herein results in a dominant form of infertility, evidence supports the idea that SNPs involved in complex traits, like infertility, can sustain amino acid changes in less-critical areas of the protein. A number of the complex trait-associated SNPs, spread over several genes, may then combine to generate a phenotype (Thomas and Kejariwal, 2004), or, in rare instances, occur in a homozygous state which results in infertility in its own right. Viewed from this perspective, some of the identified CRISP2 SNPs may play a role in male infertility when combined with SNPs in other genes.
However, a complementary analysis of CRISP SNPs in the Hanoverian warmblood horse has recently been published, wherein the authors report the identification of a non-synonymous E208K polymorphism which was significantly associated with decreased male fertility in a heterozygous state (Hamann et al., 2007). Collectively, these data support the notion the CRISPs are regulators of male fertility. Clearly, however, additional studies assessing phenotype–genotype correlations in different populations and functional analysis in animal models are required to fully grasp the function of CRISPs and the necessity for multiple CRISPs in sperm development and function.
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Supplementary Data |
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Supplementary data are available at http://humrep.oxfordjournals.org/.
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The research was supported by grants from the National Health and Medical Research Council (NHMRC) and the Australian Research Council (ARC). D.J., M.K.O.B. and R.I.M.L. are the recipients of NHMRC Fellowships. The Monash Male Infertility Repository was funded by Andrology Australia, Monash IVF, The Victorian State Government, Monash University and Prince Henry's Institute.
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Submitted on October 10, 2007; resubmitted on March 25, 2008; accepted on April 16, 2008.
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