Abstract
Proper regulation of meiosis is essential for normal spermatogenesis and abnormalities may be associated with infertility, as shown in both animal knockout studies and studies identifying anomalies in key proteins, such as SCP3 and MLH1. Disruptions of meiosis are associated with azoospermia or severe oligozoospermia, and may increase the incidence of sperm aneuploidy in some men. Based on its function and animal studies, REC8, a key component of the meiotic cohesion complex, has been identified as a candidate male infertility gene. In this study, we have evaluated sequence variation in the REC8 gene of severely infertile men of European descent with azoospermia or severe oligozoospermia compared to a fertile control population. The direct sequencing of these populations revealed nine polymorphic sites, four within intron/exon boarders, four within coding exons and one in the three prime untranslated region. These sites did not show significantly different allelic frequencies in the study populations compared to fertile controls. This indicates that polymorphisms of the Rec 8 gene are not a common cause of infertility in this population. Additional studies are warranted in patients with defined meiotic disruption.
| Abbreviations | ||
| SC | = | synaptonemal complex |
| UGRP | = | Utah Genetic Reference Project |
| SNP | = | single nucleotide polymorphism |
Communication
During the past decade, gene-targeting studies have identified a growing list of male infertility candidate genes. Among this list are a number of genes important for the regulation of meiosis [Christensen and Carrell [Citation2002]; Carrell et al. [Citation2006]; Sanderson et al. [Citation2008]]. One gene of particular interest is REC8, a key component of the meiotic cohesion complex. During meiosis, the mitotic RAD21/SCC1 cohesion subunit is replaced by the meiosis-specific REC8 isoform in budding and fission yeast, and multiple meiotic dysfunctions occur when the gene is disrupted [Molnar et al. [Citation1995]; Klein et al. [Citation1999]].
A recent report demonstrates that meiotic errors also occur when REC8 is disrupted in mice. Specifically, meiosis is disrupted during prophase I, resulting in male and female infertility, and synaptonemal complex-like (SC) structures form aberrantly between sister chromatids rather than homologous chromosomes [Xu et al. [Citation2005]]. Based on these findings, the purpose of this study was to determine if mutations in human REC8 are responsible for some cases of severe male factor infertility.
All aspects of this prospective study were approved through the institutional review board. After informed consent, DNA was collected at the Andrology Laboratory at the University of Utah from the first study group, comprised of severely oligozoospermic (spermatozoa of less than 5 M/ml) or azoospermic patients presenting with primary infertility and with no known genetic risks for infertility. Additional DNA for the second study group, fertile controls, was obtained from the Utah Genetic Reference Project (UGRP). Both groups were evaluated by direct sequencing for mutations in the REC8 gene. The predominant ethnic background of both groups was Western European (>95%). Individuals previously diagnosed with any condition or treatments linked with infertility (e.g., cystic fibrosis, Klinefelter's, varicocele, chemotherapy, etc.) were not included.
In total, 96 infertile males (48 azoospermic, 48 oligozoospermic) were screened for alterations of the REC8 gene. An additional 96 men of known paternity (grandfathers and fathers from the UGRP repository), though semen analysis parameters were unknown, were also screened as a fertile control group. Four sets of primers were designed to amplify the nineteen coding exons and flanking intronic sequence of REC8. All reactions were optimized to give clean, ample quantities of DNA and additional internal primers were designed to allow sequencing in both the forward and reverse directions. Sequencing was conducted on an ABI 3700 capillary sequencer. Sequence trace files generated by the ABI 3700 were subsequently assembled using Phrap program software and evaluated for alterations using both the Phred and Consed sequence analysis programs. In addition to software analysis, sequence trace files were visually examined by a trained technician to confirm putative polymorphisms.
A total of nine sequence variations were identified in the groups screened. Of these nine, four were in the coding regions, four in the intronic sequence and one in the 3′ untranslated region. The four exonic variations () consisted of two synonymous changes, and two that resulted in an amino acid change. The synonymous changes, in exons 4 and 13 (H94H and P332P, dbSNPs rs11547058 and rs1885711, respectively), have been identified previously as part of the HapMap project and were common in both our fertile controls and infertile patients. The two nonsynonymous changes, in exons 2 and 15, R31C and M414T, respectively, were each found in individual infertile patients. The R31C change was also previously identified through the HapMap project (dbSNP rs34075659).
Allelic Frequency of the Four Exonic Sequence Variations.
The first amino acid variation caused by a C/T transition in exon 2 results in the poorly conserved conversion of a positively charged arginine residue to a polar, uncharged cysteine residue. The change occurs within the conserved C-terminal region of the Rad21, Rec8 and Scc1 family eukaryotic cohesions [Lee and Orr-Weaver [Citation2001]]. Though we identified the variant as a heterozygous change in a single azoospermic patient, the SNP has a reported allele frequency of 0.5 in European populations. The second identified variant, a T/C transition resulting in the conservative substitution of methionine for threonine (M414T) has not been previously identified. This alteration was also identified as a heterozygous change in a single azoospermic patient.
Our data suggests that mutations in the REC8 gene are not a common cause of azoospermia or severe oligozoospermia, although polymorphisms identified in this study could be associated with polymorphisms of multigenic effects in some patients. This study did not evaluate men with elevated aneuploidy or observed recombination defects, and the incidence of polymorphisms may be different when evaluating that phenotype. Therefore, REC8 may still be considered as an infertility candidate gene in men demonstrating abnormal SC formation in immunofluorescence studies of meiotic recombination [Sanderson et al. [Citation2008]].
Acknowledgments
Control samples obtained from the UGRP were collected with support from a Public Health Services research grant to the Huntsman General Clinical Research Center, number M01-RR00064, from the National Center for Research Resources. The UGRP was also supported by generous gifts from the W.M. Keck and Delores Dore Eccles Foundations. We'd like to extend our sincere thanks to all family members who participated in the UGRP and the patients who consented to DNA collection for the infertile panel.
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