Editor-in-Chief
Prof. Yi-Fei WANG,
Asian J Androl 2006; 8 (2): 213-218 |
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This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you. - .Original Article . - Associations of homologous RNA-binding motif gene on the X chromosome (RBMX) and its like sequence on chromosome 9 (RBMXL9) with non-obstructive azoospermia Akira Tsujimura1, Kazutoshi Fujita1, Kazuhiko Komori1, Phanu Tanjapatkul1, Yasushi Miyagawa1, Shingo Takada1, Kiyomi Matsumiya2, Masaharu Sada3, Yoshihiko Katsuyama4, Masao Ota5, Akihiko Okuyama1 1Department of Urology, Graduate School of Medicine, Osaka University, Suita 565-0871, Japan Abstract Aim: To investigate the associations of autosomal and X-chromosome homologs of the RNA-binding-motif (RNA-binding-motif on the Y chromosome, RBMY) gene with non-obstructive azoospermia (NOA), as genetic factors for NOA may map to chromosomes other than the Y chromosome. Methods: Genomic DNA was extracted using a salting-out procedure after treatment of peripheral blood leukocytes with proteinase K from Japanese patients with NOA (n=67) and normal fertile volunteers (n=105). The DNA were analyzed for RBMX by expressed sequence tag (EST) deletion and for the like sequence on chromosome 9 (RBMXL9) by microsatellite polymorphism. Results: We examined six ESTs in and around RBMX and found a deletion of SHGC31764 in one patient with NOA and a deletion of DXS7491 in one other patient with NOA. No deletions were detected in control subjects. The association study with nine microsatellite markers near RBMXL9 revealed that D9S319 was less prevalent in patients than in control subjects, whereas D9S1853 was detected more frequently in patients than that in control subjects. Conclusion: We provide evidence that deletions in or around RBMX may be involved in NOA. In addition, analyses of markers in the vicinity of RBMXL9 on chromosome 9 suggest the possibility that variants of this gene may be associated with NOA. Although further studies are necessary, this is the first report of the association between RBMX and RBMXL9 with NOA. (Asian J Androl 2006 Mar; 8: 213-218) Keywords: spermatogenesis; homologous RBMY; polymorphism; microsatellite marker; azoospermia; Y chromosome Dr Akira Tsujimura, Department of Urology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita,
Osaka 565-0871, Japan. 1 Introduction Approximately 15%-20% of infertile men exhibit azoospermia, which may be caused by failure of spermatogenesis or obstruction of the seminal tract [1]. Congenital dysfunction in spermatogenesis, referred to as non-obstructive azoospermia (NOA), might, in many cases, be the result of genomic abnormalities. Many men who have NOA of presumed genetic origin might possess some spermatozoa by testicular sperm extraction (TESE). Furthermore, a microdissection TESE procedure that uses direct visualization with an operating microscope to target the large whitish tubules that presumably contain the greatest number of spermatogenetically active germ cells was recently reported [2-6]. We previously reported that the spermatozoa retrieval rate was improved by up to 42.9% using microdissection TESE in patients with NOA [6]. These technical improvements and expanded indications for TESE with intracytoplasmic sperm injection are advantageous for patients with NOA. However, such success also means that the genetic abnormalities in NOA can be transmitted to the next generation. Therefore, it is important to understand the genetic basis of NOA. Microdeletions of the Y chromosome at the azoospermia factor (AZF) locus have been suggested as a major cause of NOA. The AZF locus was recently shown to contain four loci, AZFa, b, c and d [7]. Strong candidate genes for AZF belong to two gene families that encode testis-specific RNA-binding proteins: the RNA-binding-motif (RBMY) gene family from the AZFb region [8]; and the deleted in azoospermia (DAZ) gene from the AZFc region [9]. Although numerous studies have reported a relation between deletions of AZF, including RBMY and DAZ, and azoospermia and severe oligospermia [10-12], fewer than 10% of cases of NOA involve these deletions. Therefore, additional genetic factors that cause NOA are thought to be located on the X chromosome or aouto-somes. The DAZ gene in the AZF region has been reported to have a homolog, which may be one of the genes responsible for NOA, on chromosome 3 in humans [13]. In contrast, there are no data supporting a relation between the RBMY-related gene and NOA, although a homolog of the RBMY gene on Xq26 (RBMX) and a homologous RBMX-like sequence on chromosome 9 (RBMXL9), which is specifically expressed in testis, were recently described [14, 15]. In the present study, we examined the associations of expressed sequence tags (ESTs) in and near RBMX and polymorphic microsatellite markers near RBMXL9 in NOA patients and fertile control subjects. 2 Materials and methods 2.1 Subjects The study subjects were 67 infertile Japanese men with NOA who had undergone microdissection TESE. The remaining subjects were 105 fertile men who had fathered children delivered at affiliated maternity clinics and who served as the control subjects. All study participants provided informed consent. The 67 infertile patients were diagnosed with NOA on the basis of a complete history, physical examination and endocrinologic profile. NOA was confirmed by a Johnsen score of less than 7 in a histological specimen from the testis obtained after the participant was enrolled in the study. The mean patient age was (33.7±5.0) years, and preoperative serum concentrations were (7.3±5.1)mIU/mL luteinizing hormone, (24.7±14.6)mIU/mL follicle-stimulating hormone, (3.5 ± 1.4)ng/mL total testosterone, (13.0±3.7)pg/mL free testosterone, (10.5±9.7)ng/mL prolactin, (25.1±13.0)pg/mL estradiol, and (39.0±45.6) pg/mL inhibin B. We excluded any patients with Klinefelter syndrome or obstructive azoospermia from the study. 2.2 Genomic DNA extraction Genomic DNA was extracted with a salting-out procedure after treatment of peripheral blood leukocytes with proteinase K, as described by Tsujimura et al. [16]. 2.3 Detection of deletion of ESTs in and around RBMX We performed polymerase chain reaction (PCR) to detect deletions of X-chromosome ESTs, W11365, DXS7491, G42696, SHGC31764, D11S2422 and RH45175, which are all located in and around the RBMX gene (Figure 1). The PCR primers for each EST are shown in Table 1. After initial denaturation for 12min at 95°C, amplification was carried out in an automated thermal cycler for 35 cycles at 95°C for 30s, 58°C for 60s and 72°C for 60s, with a final extension of 72°C for 10min for W11365 and DXS7491. The annealing temperature was changed to 55°C for D11S2422 and60°C for G42696, SHGC31764 and RH45175. After electrophoretic separation on 2.0% agarose gels, PCR products were visualized with ethidium bromide staining and ultraviolet transillumination. 2.4 Genotyping microsatellites around RBMXL9 To determine the number of repeats of nine polymorphic microsatellite loci near BBMXL9 (Figure 2), we synthesized unilateral primers by labeling the 5' ends with a fluorescent reagent, 6-FAM, HEX, or TET (PE Biosystems, Foster City, CA, USA). The PCR primers for amplifying D9S161, D9S263, D9S270, D9S1868, D9S319, D9S1853, D9S205, D9S1118, and D9S1845 are shown in Table 2. After initial denaturation for 12min at 95°C, amplification was carried out in an automated thermal cycler for 35 cycles of 95°C for 30s, 58°C for 45s and 72°C for 60s, followed by a final extension of 72°C for 10min for D9S161, D9S263, D9S270, D9S1868, D9S319 and D9S1853. The annealing temperature was changed to 55°C for D9S205,52°C for D9S1118 and 60°C for D9S1845. The PCR products were denatured for 5min at 100°C, mixed with formamide-containing stop buffer and loaded together with a size standard (GS500 TAMRA; PE Biosystems, Foster City, CA, USA) on a 4% polyacrylamide denaturing gel containing 8mol/L urea. The gels were run in a Model 377 Automated DNA Sequencer (PE Biosystems, Foster City, CA, USA). Fragment sizes were determined automatically with GeneScan software (PE Biosystems, Foster City, CA, USA). 2.5 Statistical analysis Significance of differences between patients with NOA and fertile control subjects in the distribution of microsatellite markers around RBMXL9 was assessed by the c2 method with continuity correction and by Fisher’s exact probability test. P was corrected by multiplication by the number of alleles observed at each locus. P (0.05 was considered statistically significant. 3 Results 3.1 Deletion of ESTs in and around RBMX Of the six ESTs in and around RBMX, a deletion of SHGC31764 was found in one patient with NOA, and a deletion of DXS7491 was found in one other patient with NOA. No deletions were detected in control subjects (Table 3). The clinical characteristics of patients with deletions of ESTs did not differ substantially from those of the other NOA patients (Table 4). 3.2 Genotyping of microsatellite markers around RBMXL9 Association analysis of susceptibility to NOA with nine polymorphic markers near RBMXL9 revealed the presence of two markers with significantly low P values (D9S319: c2=4.59, P=0.03; D9S1853: c2=5.28, P=0.02; Table 5). Interestingly, the frequency of D9S319 was lower in NOA patients than in control subjects (relative risk, 0.51), whereas the frequency of D9S1853 was higher in patients than in control subjects (relative risk, 3.85). 4 Discussion Mammalian spermatogenesis is a developmental process in which male germ cells undergo meiosis and complex morphological changes to form mature sperm. Many genes affecting male fertility have been identified in mice, and these genes are located both on autosomes and sex chromosomes. However, the search for human genes involved in spermatogenesis has so far focused only on the long arm of the Y chromosome. The relation between NOA and genes located on the X chromosome or on autosomes has not been clarified. We previously reported that a gene associated with NOA may be located on human chromosome 6 (6p21.3) near the DRB1 and the DQB1 loci [16, 17]. We also reported that a single nucleotide polymorphism in the protamine-2 gene on human chromosome 16 (16p13.3) was present in 1 of 153 patients with NOA and absent in 270 fertile control subjects [18]. Furthermore, it was recently reported that a 1 bp deletion of the synapto-neal complex protein 3 (SYCP3) gene located on human chromosome 12 (12q23.3) was present in 2 of 19 patients with NOA [19]. These findings indicate that the genes associated with spermatogenesis in humans are located on chromosomes other than the Y chromosome [20]. The RBMX/RBMY gene family comprises a complex set of genes with multiple copies in the human genome. RBMY-like sequences were found on chromosomes 1, 4, 6, 9 and 11 as well as on the X chromosome by fluorescence in situ hybridization [15]. Of these RBMY-like sequences, the one on chromosome 9 is expressed specifically in testis and, to a lesser extent, in brain. The possibility that RBMXL9 represents a novel gene involved in testicular function has been reported [14]. Thus, we examined the association of RBMXL9 with NOA using nine polymorphic microsatellite markers located near RBMXL9 in patients with NOA and in normal fertile volunteers. We found that 2 of 9 of these markers (D9S319 and D9S1853) displayed strong associations with NOA. Interestingly, D9S319 was found less frequently in patients than in control subjects, whereas D9S1853 was detected more frequently in patients than in control subjects. Because D9S20, which is the nearest microsatellite marker to RBMXL9, did not show significant association with NOA, RBMXL9 can be excluded as a candidate gene for NOA. However, it is possible that one or more gene(s) responsible for idiopathic azoospermia are localized in the narrow segment between D9S319 and D9S1853. The possibility that a marker within this segment may affect the function of RBMXL9 in spermatogenesis remains. We report associations between genetic variations in RBMX in Japanese patients with NOA. Several ESTs in or near RBMX were deleted in a subset of patients with NOA. Although we did not find an association between RBMXL9 and NOA, two markers near this gene were associated with NOA. The study described here is the first to investigate the possibility that RBMX or RBMXL9 is responsible for NOA. Finally, we emphasize the importance of understanding the genes on the X and autosomal chromosomes that underline NOA. Acknowledgment We are grateful to Mayuka Omune, Mariko Oki, Sachiko Tanabe, Chizu Nakamura and Tomomi Enomoto for their technical assistances and useful discussions. References 1 Matsumiya K, Namiki M, Takahara S, Kondoh N, Takada S, Kiyohara H, et al. Clinical study of azoospermia. Int J Androl 1994; 17: 140-2. 2 Amer M, Ateyah A, Hany R, Zohdy W. Prospective comparative study between microsurgical and conventional testicular sperm extraction in non-obstructive azoospermia: follow-up by serial ultrasound examinations. Hum Reprod 2000; 15: 653-6. 3 Schlegel PN, Li PS. Microdissection TESE: sperm retrieval in non-obstructive azoospermia. Hum Reprod Update 1998; 4: 439. 4 Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999; 14: 131-5. 5 Silber SJ. Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia. Hum Reprod 2000; 15: 2278-84. 6 Tsujimura A, Matsumiya K, Miyagawa Y, Tohda A, Miura H, Nishimura K, et al. Conventional multiple or microdissection testicular sperm extraction: a comparative study. Hum Reprod 2002; 17: 2924-9. 7 Friel A, Houghton JA, Maher M, Smith T, Noel S, Nolan A, et al. Molecular detection of Y chromosome microdeletions: an Irish study. 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Susceptibility gene for non-obstructive azoospermia located near HLA-DR and -DQ loci in the HLA class II region. Hum Genet 2002; 110: 192-7. 18 Tanaka H, Miyagawa Y, Tsujimura A, Matsumiya K, Okuyama A, Nishimune Y. Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations. Mol Hum Reprod 2003; 9: 69-73. 19 Miyamoto T, Hasuike S, Yogev L, Maduro MR, Ishikawa M, Westphal H, et al. Azoospermia in patients heterozygous for a mutation in SYCP3. Lancet 2003; 362: 1714-9. 20 Truong BN, Moses EK, Armes JE, Venter DJ, Baker HW. Searching for candidate genes for male infertility. Asian J Androl 2003; 5: 137-47. |
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