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
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Abstract |
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Key words: allelic association/genetics/infertility/sequence variation/zona pellucida
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Introduction |
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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, 1987; 1988
). The induction of the acrosome reaction may involve multiple interactions between ZP3 moieties and complementary binding sites (receptors) on the sperm surface (Kopf, 1990
). ZP2 is regarded as a secondary sperm receptor, which mediates the binding of acrosome-reacted spermatozoa to the ZP (Bleil et al., 1988
; Wassarman, 1988
; Hinsch et al., 1998
). ZP1 is thought to maintain the three-dimensional structure of the ZP (Bleil et al., 1988
; Wassarman, 1988
). 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., 1992
; Yurewicz et al., 1993
; VandeVoort et al., 1995
; Yamasaki et al., 1995
; Prasad et al., 1996
).
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). 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 exonintron boundaries by comparing the obtained cDNA with the respective human genomic sequence.
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Materials and methods |
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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., 1986; 1988
) 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 2191%). The age variation of the women in this control group was 2542 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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Verifying exonintron boundaries of the human ZP1 gene
The exonintron 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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Allele frequencies
Allele frequencies were determined for three sequence variations found in the ZP3 gene, c. 187 TG, c. 53517 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. 187 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. 53517 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.
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Results |
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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. 187 TG 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. 187 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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Discussion |
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A novel hypothesis concerning molecular level mechanisms underlying oocytesperm interaction has been proposed, involving multiple low-affinity binding sites (Castle, 2002). 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 (ZP14). 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)
, 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., 1998
; 2003
).
One of the major findings concerning a sequence variation that directly alters the respective amino acid was found at position c. 91 GA (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., 2000
). Considering that besides the ZP, ZP3 proteins are also present in the perivitelline membrane of the oocyte (Green, 1997
), it is reasonable to believe that this sequence is of importance at least in spermoocyte 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., 2001
; Swanson and Vacquier, 2002
). 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., 1991). 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 helixloophelix transcription factors (e.g. FIG
) and regulates the co-expression of all ZP proteins during zona formation (Liang et al., 1997
). Interestingly, we found statistically significant sequence variation (c. 187 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., 2003), 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 structurefunction 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on March 29, 2004; resubmitted on September 24, 2004; accepted on February 10, 2005.
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