Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF

M. Männikkö1, R.-M. Törmälä2, T. Tuuri3, A. Haltia4, H. Martikainen2, L. Ala-Kokko1,5, J.S. Tapanainen2 and J.T. Lakkakorpi2,6,7

1 Collagen Research Unit, Biocenter and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, 2 Department of Obstetrics and Gynecology, University of Oulu, FIN-90014 Oulu, Family Federation of Finland, Infertility Clinics in 3 FIN-20100 Turku, 4 FIN-00101 Helsinki and 6 FIN-90220 Oulu, Finland and 5 Center for Gene Therapy and Department of Medicine, Tulane University, Health Sciences Center, New Orleans, LA 70112, USA

7 To whom correspondence should be addressed at: Family Federation of Finland, Oulu Infertility Clinic, Medipolis, Kiviharjuntie 11, FIN-90220 Oulu, Finland. Email: jouni.lakkakorpi{at}vaestoliitto.fi


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccesful IVF trials. In theory, this may result in gradual reduction of sperm–zona interaction and eventually in total fertilization failure (TFF). METHODS: Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. RESULTS: Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1–87 T->G) and one in exon 6 [c. 894 G->A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). CONCLUSIONS: Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.

Key words: allelic association/genetics/infertility/sequence variation/zona pellucida


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The zona pellucida (ZP) is an extracellular matrix surrounding growing oocytes and early-stage embryos in mammals. Owing to its vital role in reproduction, intensive research has been carried out to resolve its structural and functional characteristics. In humans, this multifunctional structure appears to be composed of at least four known glycoproteins, termed ZP1–4 (Sacco et al., 1981Go; Shabanowitz and O'Rand, 1988Go; Hughes and Barratt, 1999Go; Lefievre et al., 2004Go). During primary follicle growth the production of these proteins is synchronized through at least one common transcription factor, FIG{alpha} (Liang et al., 1997Go). It is hypothesized that both the growing oocyte and surrounding corona radiata cells are responsible for producing these protein components (e.g. Bogner et al., 2004Go), although some variation seems to exist between different mammalian species (Sinowatz et al., 2001Go). Assembly of the ZP appears to require at least ZP2 and ZP3 in mice (Rankin et al., 1996Go; 2001Go), as null mice lacking either of these protein components produce only zonaless eggs and are infertile. Mice lacking ZP1, however, appear to produce a functional zona structure, although the litter size is reduced to half of that observed with normal mice (Rankin et al., 1999Go). According to electron microscopic and biochemical analyses (Bleil and Wassarman, 1980Go; Greve and Wassarman, 1985Go; Wassarman and Mortillo, 1991Go), it is generally accepted that the filaments that make up the main architecture of zona matrix are formed by the joining together of multiple ZP2/ZP3 heterodimers in a head-to-tail fashion. Homodimers of ZP1 (Bleil and Wassarman, 1980Go; Greve and Wassarman, 1985Go) appear to stabilize this structure by cross-linking ZP2/ZP3 multimeres together. For the fourth ZP glycoprotein (ZP4; previously described as ZPB), we can only hypothesize that as a result of its sequence similarity, this protein may share some common features with ZP1.

The prevalent model of the roles of mammalian ZP proteins suggests that ZP3 functions as a primary sperm receptor responsible for sperm binding and induction of the acrosome reaction (Wassarman, 1987Go; 1988Go). The induction of the acrosome reaction may involve multiple interactions between ZP3 moieties and complementary binding sites (receptors) on the sperm surface (Kopf, 1990Go). ZP2 is regarded as a secondary sperm receptor, which mediates the binding of acrosome-reacted spermatozoa to the ZP (Bleil et al., 1988Go; Wassarman, 1988Go; Hinsch et al., 1998Go). ZP1 is thought to maintain the three-dimensional structure of the ZP (Bleil et al., 1988Go; Wassarman, 1988Go). The model described above has mostly been obtained from mouse studies. However, species-specific differences do exist both in the function and number of ZP proteins, for example in porcine and rabbit, ZP1 has been thought to possess sperm binding activity as well (Jones et al., 1992Go; Yurewicz et al., 1993Go; VandeVoort et al., 1995Go; Yamasaki et al., 1995Go; Prasad et al., 1996Go).

Antibodies specific to ZP3 have been used to demonstrate that this protein may contain structural alterations that seem to correlate well with failed fertilization, as suggested by Oehninger et al. (1996)Go. In the present study we focused our main interest on a subset of infertile women, termed the total fertilization failure group (TFF), whose medical history did not reveal any known reason for infertility. Their oocytes failed to become fertilized in IVF, but after microinjection (ICSI) the problem was overcome. This may indicate an alteration in gamete recognition. Considering that no visible sign for structural defect in either of the gametes was observed, we focused our attention primarily on genes encoding structural components of ZP. This paper is a preliminary attempt to determine whether single gene variations either alone or in concert with each other could reduce fertilization capability among a subpopulation of infertile couples. To study human ZP1 gene reliably, we also determined its genomic organization by amplifying the full-length complementary DNA (cDNA) from a human ovarian cDNA library, followed by determination of the exon–intron boundaries by comparing the obtained cDNA with the respective human genomic sequence.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study subjects and controls
This study was performed in the Department of Obstetrics and Gynecology at Oulu University Hospital and the Family Federation of Finland (Oulu Clinic) during the years 1999–2003. Study subjects were also partly recruited from Helsinki and Turku clinics at the Family Federation of Finland.

Eighteen infertile couples of Finnish origin, with four or more oocytes produced in a single IVF cycle, were studied. The most important inclusion criterion was that none of the oocytes were fertilized after overnight incubation (following insemination) unless ICSI was performed as an infertility treatment. This group was termed as the TFF group. The age of these women varied from 25 to 36 years (mean 30 years). The minimum of four oocytes as an exclusion criterion was regarded as important to avoid fertilization failure by chance. Furthermore, all the men in this group had to reveal normal sperm parameters (Kruger strict criteria; Kruger et al., 1986Go; 1988Go) either in their native sperm samples and/or after standard sperm washing. This analysis is based on visual observation of sperm count, morphology, motility and sperm antibodies. The values were acceptable when the proportion of fast-moving spermatozoa (category A) was >5%, and this proportion combined with that of much slower, but still progressively forward-moving sperm cells (category B), was >50%. The sperm morphology was within the normal range if the minimal number of normal sperm exceeded 5%. It should be emphasized, however, that structural defects might also exist at the molecular level in sperm proteins, thus making it impossible to completely avoid all those cases with ‘true’ male factor infertility. For this reason, we have excluded only those cases where one or more visible sperm defects were observed, thus accepting that some molecular level defects in sperm architecture may still occasionally exist.

For the comparison, an additional set of 23 infertile couples of Finnish origin was studied. This control group was termed fertilizers in IVF (FIVF). Women in this group had to possess at least four oocytes in a single IVF cycle, but unlike in the TFF group, they had to reveal at least one fertilized oocyte (mean proportion of fertilized oocytes was 61%; range 21–91%). The age variation of the women in this control group was 25–42 years (mean 34 years). As in the TFF group, the spouses in the FIVF group had to reveal normal sperm parameters. The clinical data for the TFF and FIVF groups are presented in Table Ia and b. A second control group, consisting of 68 Finnish women, whose pregnancy had been achieved without assisted reproduction techniques and who had given a birth to at least one healthy baby, was also analysed. This latter control group was termed women with proven fertility (WPF).


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Table Ia. Clinical data for the TFF group

 

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Table Ib. Clinical data for the fertilizers in the FIVF group

 
The recruitment of the all volunteers in both the TFF and FIVF groups was performed only when one of the authors was present as an embryologist in the IVF laboratory to ensure uniform embryo grading with scientific accuracy. A blood sample was then collected from both spouses, the male sample being stored for later use to reveal putative structural defects in the respective sperm genes. This study was evaluated and accepted by the Ethics Committee of Northern Ostrobothnia Hospital District and informed consent was obtained from all participating couples.

Verifying exon–intron boundaries of the human ZP1 gene
The exon–intron boundaries of the human ZP1 gene were determined by amplifying the full-length cDNA, using a Human Ovary Marathon Ready cDNA kit (Clontech, Palo Alto, CA, USA) and an ExpandTM Long Template PCR system (Roche, Mannheim, Germany). In the first PCR cycle a specific primer for ZP1 cDNA based on the predicted sequence and the AP1 primer from the Marathon Ready kit were used. Nested PCR was performed with ZP1 cDNA-specific primers. Primer sequences and locations in the genomic sequence (GenBank accession no. AC004126) are indicated in Table II. The amplification reactions were carried out in a volume of 50 µl containing 5 µl of human ovary cDNA (Clontech), 5 pmol of each primer, 1.75 mmol/l MgCl2, 0.2 mmol/l dNTPs and 3.5 U Expand Long Enzyme mix (Roche). The conditions, after initial denaturation at 94°C for 2 min, were 25 cycles of 30 s at 94°C, 30 s at 60°C and 2 min at 68°C, followed by final extension at 68°C for 4 min. Nested PCR was performed under identical conditions with the exception that 5 µl of the first PCR product was used as a template. The PCR products were purified from 1.2% agarose gel and sequenced (ABI PRISMTM 377 Sequencer, Applied Biosystems, Foster City, CA, USA.). The obtained cDNA sequence was compared with the genomic sequence to determine the exon-intron boundaries of the human ZP1 gene.


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Table II. Primers used for ZP1 cDNA (all 5' to 3')

 
Conformation-sensitive gel electrophoresis analysis and sequencing
Genomic DNA extracted from EDTA-treated blood samples was used for mutation screening by conformation-sensitive gel electrophoresis (CSGE) (Körkkö et al., 1998Go). The sequences corresponding to 12 exons of ZP1 (AC004126), 19 exons of ZP2 (NT_010393), eight exons of ZP3 (NT_007933), 12 exons of ZP4 (NT_004836) and exon-flanking sequences were amplified by PCR (GenBank accession numbers indicated in parentheses). The sizes of the PCR products were designed to vary between 200 and 500 bp. The amplification reactions were carried out in a volume of 23 µl containing 20–40 ng of genomic DNA, 5–10 pmol of each primer, 1.5 mmol/l MgCl2, 0.2 mmol/l dNTPs and 0.6 U AmpliTaq Gold DNA polymerase (Applied Biosystems Roche, Brenchburg, NJ, USA). After initial denaturation at 95°C for 10 min, each of the 35 cycles consisted of the following steps: 30 s at 95°C, 30 s at 54–65°C and 30 s at 72°C, followed by final extension at 72°C for 8 min. The PCR products were denatured at 95°C for 5 min, followed by annealing at 68°C for 30 min in order to generate heteroduplexes for CSGE. A 3-µl aliquot of the reaction mixture was analysed on a 1.2% agarose gel to evaluate the quantity and quality of the PCR product. Approximately 20 ng of the product was used for heteroduplex analysis by CSGE as described previously, with the exception that gels were stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR, USA) (Körkkö et al., 1998Go). PCR products containing heteroduplexes were sequenced using a DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and an ABI PRISMTM 377 Sequencer to detect the sequence variations. The genotypes of the homozygotes were determined either by digestion or sequencing. For ZP3, exons 6, 7 and 8 were analysed by direct sequencing of the PCR product. The nomenclature of sequence variations is based on instructions by den Dunnen and Antonarakis (2001)Go. These instructions are also available online at http://www.archive.uwcm.ac.uk/uwcm/mg/docs/mut_nom.html.

Allele frequencies
Allele frequencies were determined for three sequence variations found in the ZP3 gene, c. 1–87 T->G, c. 535–17 C->A and c. 894 G->A (p. K298) that were found as heterozygotes more often in TFF subjects than in the other two groups. Digestion with a restriction enzyme was performed on c. 1–87 T->G with PvuII and on c. 894 G->A (p. K298) with StuI. Sequencing by means of the ABI PRISMTM 377 Sequencer was used for c. 535–17 C->A.

Statistical analysis
Fisher's exact test was used to analyse the statistical significance of the observed allele and genotype frequencies. Calculations were performed by means of GraphPad Calculations Inc. 2002 software.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Verifying exon–intron boundaries of the human ZP1 gene
To carry out a reliable search for mutations in the human ZP1 gene from the DNA of infertile women, we verified the predicted cDNA for ZP1 by amplifying the full-length cDNA from a human ovary cDNA library (Clontech), and determined the exon–intron boundaries by comparing the obtained cDNA sequence with the genomic sequence (AC004126). Two PCR products were obtained with primers 1 and 2 (Table II; Figure 1A). The first product (1629 bp) contained ZP1 exons 1–11, and the second (1332 bp), exons 1, 2, 4–11 and intron 8 (69 bp). Separate PCRs were performed to obtain the 5' and 3' ends of the cDNA. Primers 3 and 4 were used to amplify the 5' end. A 318-bp product containing ZP1 exons 1 and 2 was obtained. A PCR product of 320 bp with ZP1 exons 10–12 was obtained for the 3' end with primers 5 and 6. The results of these amplifications indicated that the ZP1 cDNA from human ovary is 1952 bp in size and consists of 12 exons. The resulting protein contains 638 amino acids. The results thus confirmed the predicted ZP1 cDNA (Hughes and Barratt, 1999Go). Interestingly, exon 3 was missing from the second PCR product, which seems to cause a translation termination codon in exon 4. Theoretically, this may result in formation of a truncated protein of only 109 amino acids. The structures of the two ZP1 gene variants are shown in Figure 1B.



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Figure 1. (A) Amplification of the human ZP1 cDNA from human ovary cDNA library. Lane 1, 1 kb DNA ladder; lane 2, two PCR products (1629 and 1332 bp) with primers from exon 1 to exon 11 of ZP1. (B) Schematic presentation of the two cDNAs obtained for human ZP1. Exons are indicated by black rectangles, and introns and flanking sequences by diagonal lines. The longer cDNA with 12 exons is shown on top and the shorter cDNA with 10 exons in the middle. The scale bar (in kb) is shown at the bottom. *The position of the predicted stop colon.

 
CSGE analysis and sequencing
Eighteen subjects (TFFs) and two control groups consisting of 23 FIVFs and 68 WPFs were investigated for mutations in the four ZP genes. Samples that had heteroduplexes in CSGE analysis were sequenced and the genotypes of the homozygotes were determined either by digestion or sequencing, Stu I, Pvu II. (Amersham Biosciences AB, Uppsala, Sweden). Altogether, 20 sequence variations were detected in ZP genes: four in both ZP1 and ZP2, eight in ZP3 and four in ZP4 (Table III). Human ZP3 is located at chromosome region 7q11.23 and is not a single copy gene. The region contains a polymorphic locus that could give rise to a probably non-functional polypeptide, POM-ZP3 (van Duin et al., 1993Go). The 3' end of the POM-ZP3 transcript is 99% identical to that of ZP3 and it appears to have arisen from duplication of the last four exons (exon 5–8) of ZP3 (Kipersztok et al., 1995Go). Since intronic regions are also highly homologous, it was impossible to amplify these two genes separately by PCR. Exon 5 of ZP3 is identical to the corresponding region in POM-ZP3 and was analysed by CSGE. One sequence variation (c. 741–22 C->T) was observed in this region in seven of 18 TFFs, in six of 23 FIVFs and in 23 of 68 WPFs. The sequence for the region containing exons 6, 7 and 8 differs between ZP3 (NM_007155) and POM-ZP3 (U10099) genes in six positions: c. 923 + 6 has AGG in ZP3 that is lacking in POM-ZP3, POM-ZP3 has an additional G at c. 1293 and there are four single nucleotide differences between ZP3 and POM-ZP3 (c. 1013 A/G, c. 1018 A/G, c. 1034 G/C and c. 1114 G/T, ZP3 and POM-ZP3, respectively). The observation of these differences in the sequences indicated that both ZP3 and POM-ZP3 were amplified in PCR. The variation c. 894 G->A (p. K298) was seen in samples where, on the basis of the above sequence differences, only ZP3 was amplified, suggesting that it is a sequence variation of ZP3. In addition, a CC to TT change in exon 7 of ZP3 was observed, converting the codon CCG for proline-315 to the codon TTG for leucine. Because of the high degree of homology between ZP3 and POM-ZP3 it is not possible to say whether the variation is in the ZP3 or the POM-ZP3 gene, but it was found in 18/24 WPFs (not all 68 were analysed), thus suggesting that it is not unique to the study subjects.


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Table III. Observed sequence variations in ZP1, ZP2, ZP3 and ZP4

 
Of the 20 sequence variations found in ZP genes, four were statistically significant P = 0.05, one in ZP1 and three in ZP3 (Table III). The most interesting and statistically significant sequence variations were found from ZP3 at two locations (c. 1–87 T->G and c.->535–17 C->A). They both were present more readily in the TFF group than in the WPF (P-values = 0.016 and 0.020). The third sequence variation in ZP3 [c. 894 G->A (p. K298)] was found more frequent in the TFF group when compared with the FIVF and WPF groups (P-values = 0.0001 and 0.041). In turn, the sequence variation in ZP1 [c. 471 T->G (p. I158 T)] was observed to be statistically less frequent in FIVF than both in TFF and WPF (P-values = 0.013 and 0.035).

The existence of possible allelic association in sequence variations found in ZP3 was analysed by determining the allele and genotype frequencies in the study and the two other groups (Table IV). Statistical analysis suggested that the T/G genotype and the G allele of c. 1–87 T->G variation was present more often in TFF group than in the combined group of FIVFs and WPFs, with P-values of 0.0174 and 0.0218, respectively. A similar observation was made concerning the c. 894 G->A (p. K298) variation, with P-values of 0.0055 and 0.0098 for G/A genotype and A allele, respectively. Homozygotes G/G for c. 1–87 T->G and A/A for c. 894 G->A (p. K298) were not observed. Analysing the co-segregation of the two variations using the SNPHAP program (http://www-gene.cimr.cam.ac.uk/clayton/software/) did not reveal any statistically significant haplotype that would be more common in the TFF groups than in the combined group of FIVFs and WPFs (data not shown).


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Table IV. Genotype and allelic frequencies (f) for three sequence variations in ZP3

 
To further elucidate the possible involvement of the sequence variations in complete fertilization failure found, we also calculated the mean number of cumulative sequence variations per person in all the three groups (Table V). Table V aims to give a general view of whether some of the zona proteins possess a higher number of sequence variations in the TFF group than in the two control groups, and whether this reflects a possible cause of fertilization failure. Although this calculation may give us only a rough estimation, it was interesting to find that ZP3 and ZP1 contained on average the highest number of sequence variations. Indeed, when the values were compared between the groups, TFFs had about 1.5x higher mean number of sequence variations in ZP3 and ZP1 than FIVFs and WPFs. We did not find, however, any differences in ZP2 and ZP4.


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Table V. Mean number of sequence variations per person in different ZP genes

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Binding of spermatozoa to the ZP appears to be a complex phenomenon, requiring distinct proteins with varying degrees of carbohydrate and peptide motifs. Proteins participating in this interaction have been widely investigated and many candidate genes have been proposed. As the molecular structure of the ZP is well characterized, and because it has a pivotal role in activating sperm during the early stages of gamete interaction, the four ZP proteins were here subjected for further characterization for putative sequence variations to gain more insight into molecular events in unexplained infertility.

A novel hypothesis concerning molecular level mechanisms underlying oocyte–sperm interaction has been proposed, involving multiple low-affinity binding sites (Castle, 2002Go). This hypothesis is easy to accept, as this may be a natural way to prevent complete fertilization failure arising from a putative single de novo nucleotide change in one or several important genes. For this reason it was not surprising to discover that no single point mutation observed solely explains fertilization failure in IVF among TFFs. Instead, we found various sequence variations throughout the four known human ZP genes (ZP1–4). It was interesting to learn that the TFF group exhibited an ~1.5-fold increase in sequence variations within genes encoding not only ZP3 but also ZP1, when comparing the values with those of the FIVF and WPF groups (Table V). Although the cumulative effect of the four sequence variations found in the ZP1 is currently difficult to predict, it is tempting to speculate that ZP1 plays a more significant role in human fertilization. Indeed, several pieces of evidence now argue for a more profound role of ZP1 action. These include, among others, the results obtained by VandeVoort et al. (1995)Go, who showed that polyclonal antibodies generated against recombinant rabbit ZP1 inhibit monkey sperm from binding to ZP of the same species. Accordingly, using knockout technology and subsequent replacement of ZP3 and ZP2 either separately or together, Rankin and co-workers came to the conclusion that ZP2 and ZP3 proteins might not be sufficient to support human sperm binding and that an additional human zona protein(s) may be required (Rankin et al., 1998Go; 2003Go).

One of the major findings concerning a sequence variation that directly alters the respective amino acid was found at position c. 91 G->A (p. G31R) of ZP3. This sequence variation is interesting, since it removes the last G residue in the LWLL - - G amino acid sequence, which, in turn, has been found to be one of the structures binding to the equatorial segment and the post-acrosomal sheath of human spermatozoa (Eidne et al., 2000Go). Considering that besides the ZP, ZP3 proteins are also present in the perivitelline membrane of the oocyte (Green, 1997Go), it is reasonable to believe that this sequence is of importance at least in sperm–oocyte recognition and fusion. Additional evidence to support the significance of this sequence comes from studies by Swanson and co-workers, who carried out phylogeny-based analysis of ZP2 and ZP3 (Swanson et al., 2001Go; Swanson and Vacquier, 2002Go). Interestingly, these authors were able to identify several sites, including the LWLL - - G sequence, likely to be under positive Darwinian selection and therefore important for speciation. Therefore, an interesting possibility exists as to whether any of the elements in speciation process are involved, thus explaining at least partly the background of unexplained infertility. As loss of the LWLL - - G sequence was also observed in both control groups, we can only conclude, however, that this sequence variation cannot solely explain the complete fertilization failure in TFFs, suggesting that additional factors are involved.

The regulatory regions of both human and mouse ZP3 contain five conserved elements, termed I, IIA, IIB, II and IV (Millar et al., 1991Go). Millar et al. suggested that element IV is both necessary and sufficient for transcription from the ZP3 promoter. This element contains the nucleotide sequence CANNTG, which binds basic helix–loop–helix transcription factors (e.g. FIG{alpha}) and regulates the co-expression of all ZP proteins during zona formation (Liang et al., 1997Go). Interestingly, we found statistically significant sequence variation (c. 1–87 T->G) in element IIA. Since this sequence variation destroys a putative CANNTG sequence and was more frequently found among TFFs than control groups, it is possible that element IIA also has a role in controlling human ZP3 transcription. A subgroup of additional sequence variations with statistical significance were also detected, although their effect on the respective zona protein is difficult to predict. These include c. 894 G->A (p. K298) in ZP3, which does not directly affect the amino acid content. The second variation, c. 471 T->G (p. I 158 T), was found in ZP1, and replaces isoleucine with threonine at position 158. Interestingly, this latter sequence variation was less common in the FIVF group than in the TFF and WPF groups. Considering that additional sequence variations may be present in the remaining locations that has not yet characterized, it is possible that a given subset of these variations (or even single variations alone) within each ZP gene may modify the respective primary mRNA transcript in such a manner that affects, for instance, post-transcriptional processes and subsequently the resulting protein product.

In conclusion, our study on ZP genes of infertile women revealed a high degree of sequence variation among genes studied. We noted that the TFF group had on average 1.5x more sequence variations in ZP3 and ZP1 compared with the two control groups. As each infertile (or alternatively fertile) couple builds up a unique set of complementary structures that has to act in synchrony, all pairs of interacting molecules, in either the egg or the sperm, has to possess correct architecture to function properly. Whether a given set of these sequence variations ultimately modifies, in addition to protein backbone, important glycosylation sites of functionally significant ZP proteins remains to be resolved. Technically this task is challenging, as the most significant (especially O-linked) sugar moieties seem to be located within the last four exons of the ZP3 gene (e.g. Dell et al., 2003Go), which, in turn, are duplicated to another gene locus (POM-ZP3) during the course of evolution. In addition, the respective proteins from sperm surface must be characterized thoroughly. If the multiple low-affinity binding site theory turns out hold true in human, it will be interesting to see then whether increasing amount of given sequence variations will gradually reduce the recognition and/or binding capacity of the two gametes, and as a consequence, eventually leading to TFF. Understanding the structure–function relationships more precisely in the two human gametes will guide us in the near future to be able to select individually the most suitable infertility treatment for each infertile couple.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to express gratitude to Minta Lumme, Satu Koljonen, Anna-Maija Asukas, Raija Tolkkinen, Raili Valtanen, Irma Sundqvist and Maarit Kinnunen for their excellent technical assistance. We also thank our collaborators Sirpa Mäkinen and Tarja-Leena Penttilä, Family Federation of Finland, for collecting patient data and sending blood samples. The staff of the Pharmacy of Haukipudas, Department of Obstetrics and Gynaecology and Department of Medical Microbiology at the Oulu University Hospital, and the Family Federation of Finland in Oulu are gratefully acknowledged for providing WPF blood samples. This study was supported by grants from the Academy of Finland, the Sigrid Jusélius Foundation and Oulu University Hospital.


    Notes
 
M.Männikkö and R.-M.Törmälä equally contributed to this work.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 29, 2004; resubmitted on September 24, 2004; accepted on February 10, 2005.





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