Mol. Hum. Reprod. Advance Access originally published online on July 28, 2005
Molecular Human Reproduction 2005 11(7):513-515; doi:10.1093/molehr/gah202
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Survey of the two polymorphisms in DAZL, an autosomal candidate for the azoospermic factor, in Japanese infertile men and implications for male infertility
1Department of Human Genetics and Public Health, Graduate School of Medical Science, University of Tokushima, Tokushima-City, 2Core Research for Evolutional Science and Technology Corporation, Saitama, 3Department of Urology, St. Marianna Medical University School of Medicine, Kawasaki, Japan and 4School of Public Health, Wenzhou Medical College, Wenzhou, Zhejiang province 325035, China
5 To whom correspondence should be addressed at: Department of Human Genetics and Public Health, Graduate School of Medical Science, University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima-City 770-8503, Japan. E-mail: nakahori{at}basic.med.tokushima-u.ac.jp
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Abstract |
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The DAZL (DAZ-like) gene is suggested to be an ancestral gene of the DAZ (deleted in azoospermia) gene on the Y chromosome, which is a strong candidate for the azoospermic factor. Recently, it has been reported that the T54A (Thr54
Ala) polymorphism in exon 3 of the DAZL gene is associated with spermatogenic failure in the Taiwanese population. In this study, to investigate whether this polymorphism is associated with spermatogenic failure in Japanese males, we analysed genomic DNA derived from 234 patients with azoospermia or oligozoospermia and 131 fertile controls. The T54A polymorphism was completely absent in both the patients and the controls. The T12A (Thr12Ala) polymorphism in exon 2 of the DAZL gene was found at a similar frequency in the patients and controls, 15.4% and 13.7%, respectively (P = 0.67). However, the frequency of T12A was higher for the azoospermic (20.5%) than oligozoospermic (9.6%) individuals in infertile men without DAZ deletions, although statistical difference was not so apparent (x2 test: P = 0.037, OR = 2.413, 95% CI = 1.035–5.629; Yate’s x2 test: P = 0.058, OR = 2.319, 95% CI = 0.973–6.166). Our results show that the T54A polymorphism in DAZL has no major role in Japanese males with azoospermia or oligozoospermia. The distribution of the T54A polymorphism may be restricted to the narrow area including Taiwan. Key words: azoospermia/DAZ/DAZL/oligozoospermia/Y chromosome
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Introduction |
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About 10–15% of couples fail to conceive any children, and in nearly half of these couples, it is the male who is infertile (de Kretser, 1997
; McLachlan et al., 1998; Foresta et al., 2002). An important cause of male infertility is spermatogenetic failure such as azoospermia and oligozoospermia. So far, many lines of evidence have unveiled that at least 10% of males with azoospermia or oligozoospermia have deletions encompassing the DAZ (deleted in azoospermia) gene cluster located on the long arm of the Y Chromosome (Nagafuchi et al., 1993; Kobayashi et al., 1994; Reijo et al., 1995; Nakahori et al., 1996). The human DAZ gene family contains at least three members which have RNA-binding domains: DAZ, BOULE and DAZL (DAZ-like). The BOULE gene, which is the ‘grandfather’ of DAZ, has been assigned to 2q33 and is evolutionarily conserved from Drosophila to human (Xu et al., 2001). The DAZL gene, the ‘father’ of DAZ, maps to 3p24 and is known to be important for gametogenesis in different species (Cooke et al., 1996; Yen et al., 1996).
The DAZL gene is homologous to the DAZ gene, with 83% similarity in the coding region. Both genes encode RNA-binding proteins that are expressed only in testis or in testis and ovary, and which are present in both the nucleus and cytoplasm of fetal gonocytes and in spermatogonial nuclei, although the two proteins differ in their intracellular localization (Ruggiu et al., 1997, 2000; Habermann et al., 1998; Brekhman et al., 2000; Reijo et al., 2000). Targeted disruption of the DAZL gene in mice leads to infertility for both males and females (Ruggiu et al., 1997). Intriguingly, spermatogenic failure in mice without Dazl was partially rescued with human DAZ, suggesting a high degree of functional conservation between the DAZ and DAZL genes (Slee et al., 1999). However, there is no direct evidence that the DAZ gene is crucial for spermatogenesis, because deletions in AZFc affect not only the DAZ gene cluster but also other genes expressed in spermatogenic cells (Skaletsky et al., 2003).
Recently, two single-nucleotide polymorphisms (SNPs) in the DAZL gene have been reported (Teng et al., 2002). The first SNP causes the change Thr54Ala (T54A) in the RNA recognition motif and was more frequent in a group of Taiwanese patients with severe oligozoospermia and non-obstructive azoospermia than in controls. The second SNP causes Thr12Ala (T12A) but is not associated with spermatogenic failure (Teng et al., 2002). However, Caucasian populations did not have the T54A polymorphism (Bartoloni et al., 2004; Becherini et al., 2004; Tschanter et al., 2004).
In this study, we evaluated whether two polymorphisms T54A and T12A in the DAZL gene are related with the susceptibility to spermatogenic failure with or without deletion of the DAZ gene cluster in the Japanese population.
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Materials and methods |
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Subjects
A total of 234 infertile men with oligozoospermia or non-obstructive azoospermia (age, 23–54 years; median, 35 years) and 131 fertile men (age, 25–50 years; median, 34 years) were subjected to our study. All patients underwent physical examination, semen analysis, checks of FSH, LH and testosterone, karyotyping and molecular tests for Y-chromosome microdeletions. Of the 234 subjects, 141 showed azoospermia, 58 severe oligozoospermia (sperm count <5 x 106/ml) and 35 oligozoospermia (sperm count; 5–20 x 106/ml). All patients exhibited a normal 46, XY karyotype. Microdeletions in AZF regions on the Y chromosome were analysed by multiplex PCR with 9 sequence-tagged sites (STSs) including SRY, AMGY/AMGX, DYZ3, USP9Y, DBY, RBM, DAZ and DYZ1 according to previous reports (Nagafuchi et al., 1993
; Nakahori et al., 1996). This study was approved by the Ethics Committee of The University of Tokushima and St. Marianna Medical University. All participants gave their informed consent.
Detection of polymorphisms
Genomic DNA was extracted from peripheral blood samples with a QIAamp blood kit (Qiagen GmbH, Hilden, Germany). PCRs were carried out to detect the polymorphisms T54A and T12A in the DAZL gene, according to a previous report (Teng et al., 2002). After the reaction, PCR products of exon 3 and exon 2 of the DAZL gene were digested with the restriction enzymes AluI (New England Biolabs, Beverly, USA) and DdeI (BM Biochemica, Mannheim, Germany), respectively. The digested samples were separated with 4% (for exon 3) or 2.5% (for exon 2) agarose gel and visualized with ethidium bromide.
Statistical analysis
Statistical analyses were carried out using Microsoft Excel and SPSS 11.0J (SPSS Inc.).
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Results |
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We aimed to elucidate whether genetic variations of the DAZL gene affect spermatogenic ability in infertile men with or without DAZ deletions in the Japanese population. At the beginning of this study, we found that 14 azoospermic and 10 severely oligozoospermic patients among 234 infertile men had deletions encompassing the DAZ gene cluster on the Y chromosome with a PCR-based method (see Materials and methods).
To determine the frequency of the T54A polymorphism in the DAZL gene, we carried out a PCR-RFLP analysis on 131 fertile and 234 azoospermic or oligozoospermic men. It became clear that no subjects in this study had the polymorphism. This result was consistent with recent reports that the T54A polymorphism was absent in Caucasian populations (Bartoloni et al., 2004; Becherini et al., 2004; Tschanter et al., 2004).
The T12A polymorphism was also analysed with a PCR-RFLP-based method. Each genotype frequency in the infertile patients and the controls was in Hardy-Weinberg equilibrium. A summary of the genotype frequency is shown in Table I. The T12A polymorphism in exon 2 of the DAZL gene was found in both the patients (n = 234) and the controls (n = 131) at a frequency of 15.4% and 13.7%, respectively (P = 0.67). No significant difference in the frequency of each genotype was observed between the patients and the controls (P = 0.77). Moreover, even when infertile men were classified into those with (+) and without (–) DAZ deletions, there was little difference in between them and the controls. However, as summarized in Table II, among the infertile males lacking DAZ deletions, the frequency of the T12A polymorphism was higher in the azoospermic (20.5%) than oligozoospermic (9.6%) individuals, although statistical difference was not so apparent (
2 test: P = 0.037, Yate’s 2 test: P = 0.058).
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Discussion |
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Recently, several independent research groups have reported that Caucasian populations lack the T54A polymorphism in the DAZL gene and that the T12A polymorphism is not associated with male infertility (Bartoloni et al., 2004
; Becherini et al., 2004; Tschanter et al., 2004). In this study, we could not detect the T54A polymorphism in the Japanese population. Hence, it is likely that T54A is found in a very restricted area that includes Taiwan. The T12A polymorphism was found in Japanese men, and its frequency was determined in 234 infertile patients and 131 controls. We demonstrated that the frequency of T12A in the infertile males without DAZ deletions was higher among azoospermic (20.5%) than oligozoospermic (9.6%) individuals, although significant difference was not so apparent. To conclude whether there is an association between T12A and infertility in males without DAZ deletions, more samples should be analysed. At present, the effect this polymorphism has on molecular function is not known. Because T12A is not located in the RNA-binding domain, it probably does not affect the RNA-binding activity of DAZL. T12A may affect the interaction between DAZL and other proteins or the turnover of DAZL.
As it has been reported that some oligozoospermic males have DAZ deletions and infertile sons, genetic background is important for spermatogenic failure even with DAZ deletions (Nakahori et al., 1996; Kuhnert et al., 2004). It would be intriguing to elucidate the frequency of the T12A polymorphism in such oligozoospermic males.
In summary, our data suggest that the T54A variant is not a major cause of infertility in Japanese men. The distribution of the T54A polymorphism may be restricted to the narrow area including Taiwan.
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Acknowledgements |
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This study was supported in part by grants from the Core Research for Evolutional Science and Technology (CREST), 21st Century Center of Excellence (COE) Program and Ministry of Health and Ministry of Health, Labour and Welfare.
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References |
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Submitted on April 20, 2005; resubmitted on May 10, 2005; accepted on June 7, 2005.
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