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 . -
DAZ1/DAZ2 cluster deletion mediated by gr/gr recombination per se may not be sufficient for spermatogenesis impairment: a study of Chinese normozoospermic men
Yuan Yang1, 3, Cui-Ying Xiao1, 3, Zhou-Cun A1, 3, Si-Zhong Zhang1, 3, Xiang Li2, Si-Xiao Zhang2
1Department of Medical Genetics,
2Department of Urology, West China Hospital, Sichuan University, Chengdu 610041,
China;
3Division of Human Morbid Genomics, National Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041,
China
Abstract
Aim: To explore the possible effect of the deleted in azoospermia
(DAZ) copy cluster deletion on spermatogenesis in
the Chinese population, the deletionof the azoospermia factor c
(AZFc) region was analyzed in 346 normozoospermic
men. Methods: Three DAZ single nucleotide variant loci and seven
AZFc-specific sequence-tagged sites were examined with polymerase chain reaction (PCR)-restriction fragment length polymorphism and routine PCR.
Results: Five (1.4%) of the normozoospermic men were found to have deletion of
gr/gr-DAZ1/DAZ2. None of the men were found to have b2/b4-entire
DAZ deletion. Conclusion: The presence of
gr/gr-DAZ1/DAZ2 deletion in five men with
normozoospermia suggests that this deletion per
se may not be sufficient for spermatogenic impairment in Chinese
men. (Asian J Androl 2006 Mar; 8: 183-187)
Keywords: azoospermia factor c; deleted in azoospermia;gene deletion; normozoospermia
Prof. Si-Zhong Zhang, Department of Medical Genetics, West China Hospital, Sichuan University, Chengdu 610041,
China.
Tel/Fax: +86-28-8516-4009
E-mail: szzhang@mcwcums.com
Received 2005-09-15 Accepted 2005-11-29
1 Introduction
Impaired spermatogenesis is an essential etiology of male infertility. In the last 20 years, several genetic factors
have been correlated to impaired spermatogenesis, in which azoospermia factor
(AZF) microdeletion is the second-most frequent cause after Klinefelter syndrome and accounts for approximately 10% of patients with azoospermia or
oligozoospermia [1-3].
The AZF locus is mapped to the male-specific region of the Y chromosome and is divided into three discrete regions [1,
4], of which the AZFc region is particularly interesting: approximately 80% of
AZF microdeletions occur in this region and
most of them result in entire deleted in azoospermia
(DAZ) gene deletion [5]. The DAZ gene has four copies, most
commonly, and encodes an RNA binding protein exclusively in testicular tissue [6, 7]. Studies have demonstrated that the
DAZ gene plays an important role in spermatogenesis [1, 8].
In recent years, studies on the deletion mechanism of the
AZFc region and the deletion effect of
DAZ gene copies on spermatogenesis have identified that the entire
AZFc deletion, including all DAZ copies, resulting from the
recombination event of two massive repeat units
b2/b4, is responsible for spermatogenic impairment in some populations [6, 9,
10]. In the past 2 years, at least eight
AZFc partial deletion patterns and their clinical significance were reported
[11-13]. Of these, only the DAZ1/DAZ2 deletion caused by gr/gr recombination was suggested to be another important
etiology for spermatogenic impairment as it was observed exclusively in infertile males with a prevalence of 3%
(10/337) in a white study group [14]. However, it is controversial whether
DAZ cluster deletion resulting from the recombination of massive repeat units plays any role in spermatogenesis impairment [11, 15], which suggests that
such study should be performed in larger and more ethnically diverse populations.
Taking into account the possible different distributions of Y chromosome haplogroups among different ethnic
populations, which may result in the various prevalence and phenotype of
AZFc deletion [11, 16], we investigated the
deletion distributions of b2/b4-entire DAZ and
gr/gr-DAZ1/DAZ2 in Chinese men with normozoo-spermia, to
evaluate the genetic effect of the deletions on spermatogenesis.
2 Materials and methods
2.1 Subjects
Three hundred and forty-six Chinese men aged from 23 years to 34 years were recruited from patients attending
the Department of Urology, West China Hospital of Sichuan University (Chengdu, China) from 2003 to 2005. Semen
analyses were performed on all subjects three times and their sperm counts were
>20×106/mL with normal motility and morphology, in accordance with the World Health Organization (WHO) criteria. The subjects were divided into
three groups: 97 with sperm count
(50×106/mL; 186 with sperm count
50-100×106/mL, and 63 with sperm count
>100 and (150×106/mL. The study was approved by the Institutional Ethical Review Board of West China Hospital,
Sichuan University, and signed informed consent forms were obtained from all participants.
2.2 Specific sequence-tagged site (STS) analysis of AZFc
Genomic DNA was extracted from peripheral blood lymphocytes using DNA isolation kits (Takara, Ostu, Japan).
Seven AZFc-specific STS were used to detect entire and partial
AZFc deletions, of which sY1161/sY1191/sY1291 and
sY1206/sY1201 were co-amplified separately, and sY254/sY255 was amplified according to the European Academy of
Andrology and the European Molecular Genetics Quality Network (EAA/EMQN) best practice guidelines [5]. All
polymerase chain reaction (PCR) products were separated with 2% agarose gel and visualized by the ethidium
bromide staining method. The b2/b4-entire
DAZ deletion was identified by the positive result of sY1201 and the negative
results of sY254, sY255, sY1191, sY1291 and sY1206 [9]. The gr/gr deletion was identified by the negative result of
sY1291 and the positive results of the other STS [12]. The primer sequences and PCR conditions were as previously
described [9, 12].
2.3 Single nucleotide variant (SNV) analysis of the DAZ gene
The partial deletion of DAZ gene copies was
detected in SNV sites of the DAZ gene, including SNVII, SNVV and
sY581, by PCR-restriction fragment length polymor-phism, in which the
MboI digestion fragment of SNVII distinguishes
DAZ1 from other DAZ copies, the
DraI digestion fragment of SNVV distinguishes
DAZ1/DAZ2 from DAZ3/DAZ4, and the
Sau3A digestion fragment of sY581 distinguishes
DAZ1/DAZ4 from DAZ2/DAZ3. The digestion
products of SNVII and SNVV/sY581 were separated with 2% agarose gel and 8% polyacrylamide gel and visualized
by ethidium bromide and silver staining methods. The
DAZ1/DAZ2 deletion was identified by the presence of
pair-fragments of 195bp/49bp and 122bp/60bp with the
SNVV-DraI and SNVII-MboI tests, respectively, together with
integrated restriction fragments of
sY581-Sau3A. The primer sequences and PCR conditions were described in Ferlin
et al. in 2004 [17]. The GenBank accession numbers are G73166 for SNVII, G63908 for SNVV, and G63906 for
sY581.
3 Results
With specific STS analysis of AZFc, eight of 346 men showed partial
AZFc deletions (2.3%), and all of them were the
gr/gr deletion (Figure 1). The b2/b4-entire
DAZ deletion was not found in any subject according to the positive results of
sY254 and sY255 (Figure 2).
In the SNVanalysis of the DAZ gene, 21 men were found to have lost sequence family variants in the SNV loci,
12 of which were identified as presenting two fragments of 195bp and 49bp with
SNVV-DraI, and three fragments of 489bp, 122bp and 60bp with
SNVII-MboI, together with integrated restriction fragments of
sY581-Sau3A, indicative of the deletion of
DAZ1/DAZ2 (Figure 3) and the prevalence was 3.5% (12/346). Nine out of 21 males
were identified by the three fragments of 122bp, 73bp and 49bp with
SNVV-DraI, and four fragments of 489bp, 182bp, 122bp and 60bp with
SNVII-MboI, in addition to the integrated restriction fragments of
sY581-Sau3A, suggesting that the deletion frequency of
DAZ3/DAZ4 was 2.6% (9/346).
Taking specific AZFc STS and SNV analysis together, we found that, of eight men with gr/gr recombination, five
showed the doublet deletion of DAZ1/DAZ2 and three showed the
DAZ3/DAZ4 deletion. These deletion patterns in
the three groups of subjects categorized by their sperm counts are shown in Table 1.
4 Discussion
In the present study, b2/b4-entire DAZ deletion was not detected in any of the 346 subjects. This result, in
addition to the approximately 10% of entire
DAZ deletion frequency reported in a previous study in Chinese infertile
men with azoospermia or oligozoospermia [18], suggests that the b2/b4-entire
DAZ deletion may be a major AZFc abnormality responsible for impaired spermatogenesis in Chinese men, which is in agreement with reports in other
populations [9].
One study has suggested that the
gr/gr-DAZ1/DAZ2 deletion represents an important genetic defect leading to
impaired spermatogenesis in white men [14]. However, as shown in Table 1, the
gr/gr-DAZ1/DAZ2 deletion was observed in 1.4% (5/346) of normozoo-spermic Chinese men with no significant differencse found in frequencies
between two groups of semen sperm counts,
(20-50)×106/mL and
(50-100)×106/mL, suggesting that the
gr/gr-DAZ1/DAZ2 deletion on its own may be insufficient to result in spermatogenic impairment in Chinese men. This is in
contrast to previous studies in which
gr/gr-DAZ1/DAZ2 deletion was only found in patients with impaired
spermatogenesis, but not in the 263 normal men examined [14]. The reason for the different results in different
studies could be manifold, such as environmental factors, genetic modification, as well as Y chromosome haplogroups
in different ethnic populations [19]. As for
gr/gr-DAZ3/DAZ4 deletion, it occurs with a frequency of 0.9% (3/346),
which is similar to that reported in white men [14].
In summary, we confirmed that the b2/b4-entire
DAZ deletion might be the essential genetic abnormality for
impaired spermatogenesis in Chinese men. However, the
gr/gr-DAZ1/DAZ2 deletion was identified in Chinese
normozoospermic men, which suggests that the
gr/gr-DAZ1/DAZ2 deletion per se is not an essential cause of
spermatogenic failure in the Chinese population. Further study on partial
AZFc deletion should be performed in infertile
Chinese men to compare the gr/gr-DAZ1/DAZ2 deletion frequency, so as to reveal the genetic effect of the cluster
deletion and to explore novel deletion patterns contributing to spermatogenic impairment.
Acknowledgment
This research was supported by the National High Technology Research and Development Program (863 Program,
China, grant numbers 2002BA711A08 and 2004AA216090), National Natural Science Foundation of China (grant
number 30371491), Foundation of Population and Family Planning Committee, Sichuan Province, China (grant
number 200149) and the China Medical Board Foundation.
References
1 Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F,
et al. Human Y chromosome azoospermia factors
(AZF) mapped to different subregions in Yq11. Hum Mol Genet 1996; 5: 933-43.
2 El Awady MK, El Shater SF, Ragaa E, Atef K, Shaheen IM, Megiud NA. Molecular study on Y chromosome microdeletions in
Egyptian males with idiopathic infertility. Asian J Androl 2004; 6: 53-7.
3 Chiang HS, Yeh SD, Wu CC, Huang BC, Tsai HJ, Fang CL. Clinical and pathological correlation of the microdeletion of Y chromosome
for the 30 patients with azoospermia and severe oligoasthenospermia. Asian J Androl 2004; 6: 369-75.
4 Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG,
etal. The male-specific region of the human Y
chromosome is a mosaic of discrete sequence classes. Nature 2003; 423: 825-37.
5 Simoni M, Bakker E, Krausz, C. EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions. State
of the art 2004. Int J Androl. 2004; 27: 240-9.
6 Saxena R, de Vries JW, Repping S, Alagappan RK, Skaletsky H, Brown LG,
et al. Four DAZ genes in two clusters found in the
AZFc region of the human Y chromosome. Genomics 2000; 67: 256-67.
7 Reijo R, Lee TY, Salo P, Alagappan R, Brown LG, Rosenberg M,
et al. Diverse spermatogenic defects in humans caused by Y
chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet 1995; 10: 383-93.
8 Slee R, Grimes B, Speed RM, Taggart M, Maguire SM, Ross A,
et al. A human DAZ transgene confers partial rescue of the mouse
DAZ1 null phenotype. Proc Natl Acad Sci USA 1999; 96: 8040-5.
9 Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH,
et al. The AZFc region of the Y chromosome
features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet 2001; 29: 279-86.
10 Repping S, Skaletsky H, Lange J, Silber S, Van Der Veen F, Oates RD,
et al. Recombination between palindromes P5 and P1 on the
human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet 2002; 71: 906-22.
11 Hucklenbroich K, Gromoll J, Heinrich M, Hohoff C, Nieschlag E, Simoni M. Partial deletions in the
AZFc region of the Y chromosome occur in men with impaired as well as normal spermatogenesis. Hum Reprod 2005; 20: 191-7.
12 Repping S, Skaletsky H, Brown L, van Daalen, SK, Korver CM, Pyntikova T,
et al. Polymorphism for a 1.6-Mb deletion of the human
Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 2003; 35: 247-51.
13 de Llanos M, Ballesca JL, Gazquez C, Margarit E, Oliva R. High frequency of gr/gr chromosome Y deletions in consecutive oligospermic
ICSI candidates. Hum Reprod 2005; 20: 216-20.
14 Ferlin A, Tessari A, Ganz F, Marchina E, Barlati S, Garolla A,
et al. Association of partial AZFc region deletions with spermatogenic
impairment and male infertility. J Med Genet 2005; 42: 209-13.
15 Machev N, Saut N, Longepied G, Terriou P, Navarro A, Levy N,
et al. Sequence family variant loss from the
AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility. J Med Genet 2004; 41: 814-25.
16 Fernandes S, Paracchini S, Meyer LH, Floridia G, Tyler-Smith C, Vogt PH. A large
AZFc deletion removes DAZ3/DAZ4 and nearby
genes from men in Y haplogroup N. Am J Hum Genet 2004; 74: 180-7.
17 Ferlin A, Bettella A, Tessari A, Salata E, Dallapiccola B, Foresta C. Analysis of the
DAZ gene family in cryptorchidism and idiopathic
male infertility. Fertile Steril 2004; 81: 1013-8.
18 Yang Y, Xiao CY, Zhang SZ, Zhang SX, Huang MK, Lin L. High risk genetic factor in Chinese patients with idiopathic male infertility:
deletion of DAZ gene copy on Y chromosome. Chin Med J 2004; 117: 1092-4.
19 Vogt PH. AZF deletions and Y chromosomal haplogroups: history and update based on sequence. Hum Reprod Update 2005; 11:
319-36.
|