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From the * Department of Medical Genetics, West China Hospital, Sichuan University and the Division of Human Morbid Genomics, State Key Laboratory of Biotherapy, Chengdu, China.
| Correspondence to: Dr Sizhong Zhang, Professor, Department of Medical Genetics, West China Hospital, Sichuan University, Renmin Nanlu, Section 3 #17 Chengdu, 610041, China (e-mail: szzhang{at}mcwcums.com). |
| Received for publication November 20, 2007; accepted for publication March 13, 2008. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Key words: Male infertility, single nucleotide polymorphism
During spermatogenesis, protein phosphorylation is essential for signaling pathways and regulation of protein activity. Among protein kinases catalyzing phosphorylation, the testis-specific serine/threonine kinase (TSSK) family is a particular one with exclusive or predominant expression in testis and functions in sperm differentiation, capacitation, and fertilization (Bielke et al, 1994; Kueng et al, 1997; Zuercher et al, 2000; Hao et al, 2004; Chen et al, 2005; Xu et al, 2007). To date, 5 members of the TSSK family have been reported, including TSSK1, TSSK2, TSSK3, TSSK4, and TSSK6, and all of the human homologs have been cloned. TSSK1 and TSSK2 are present in the stage of late spermatid to sperm (Kueng et al, 1997; Hao et al, 2004; Xu et al, 2007). Also, TSSK3 is exclusively expressed in human testis (Visconti et al, 2001), and in mature mice, TSSK3 may function in the differentiated Leydig cells (Zuercher et al, 2000). TSSK6, also named small serine/threonine kinase (SSTK), is found predominantly in the elongating spermatids and is involved in postmeiotic chromatin remodeling (Hao et al, 2004; Spiridonov et al, 2005). Male mice with deficient TSSK6 are infertile due to considerable sperm reduction and abnormal motility and morphology of spermatozoa (Spiridonov et al, 2005).
Recently, a new TSSK family member, TSSK4, initially named as TSSK5, has been identified. The TSSK4 gene, mapped to 14q11.2, contains 4 exons and encodes a protein of 328 amino acids with specific expression in testis (Chen et al, 2005; Xu et al, 2007). Further study shows that TSSK4 phosphorylates the cAMP responsive element binding protein (CREB) at Ser-133 and hence facilitates the binding of CREB to the specific cis cAMP responsive element (CRE), which is important for activating transcription of genes related to germ cell differentiation (Don et al, 2002; Chen et al, 2005). By stimulating the CRE/CREB pathway, TSSK4 may be involved in spermatogenesis, probably in the early stages of spermatid differentiation (Don et al, 2002; Chen et al, 2005). Moreover, autosomal aberration analysis in patients with spermatogenic arrest indicated that band 14q11, where TSSK4 is located, is highly linked with infertility, suggesting that certain genes in this region may be associated with impaired spermatogenesis (Guo et al, 2002; Chen et al, 2005).
Therefore, to investigate the possible association between TSSK4 and male infertility, we carried out a mutation screening of this gene in 372 infertile men with azoospermia or severe oligospermia and compared the results with those from 220 controls.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Polymerase Chain Reaction Amplification
Genomic DNA was extracted from peripheral blood lymphocytes using standard
phenol-chloroform procedures. Based on mRNA sequence (GenBank No. NM_174944.2)
and genomic sequence, 4 pairs of primers were designed using PRIMER PREMIER
5.0 (Premier Biosoft International, Palo Alto, California) to amplify all 4
exons including intron/exon boundaries, 5' untranslated region (UTR) and
3' UTR (Table 1).
Polymerase chain reactions (PCR) were carried out in a total volume of 50
µl containing 0.2 µg genomic DNA, 10 pmol of each primer, 10 pmol of
dNTP, and 2 units Taq polymerase (TaKaRa, Shiga, Japan). The PCR profile was:
predenaturation at 94°C for 5 minutes, followed by 35 amplification cycles
comprising denaturation at 94°C for 30 seconds, annealing at a temperature
between 53°C and 57°C for 30 seconds, extension at 72°C for 1
minute, with a final extra extension at 72°C for 10 minutes. Amplicons
were resolved by 1.5% agarose gel electrophoresis to confirm the presence of
specific PCR products.
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Denaturing High-Performance Liquid Chromatography Analysis and DNA Sequencing
Mutation screening of TSSK4 was carried out using denaturing
high-performance liquid chromatography (DHPLC) on an automated WAVE system
(Transgenomic, Omaha, Nebraska). WAVEMAKER 4.1 software (Transgenomic) was
used to determine the optimal melting temperature for tested fragments. Prior
to DHPLC, the PCR products were denatured at 94°C for 5 minutes and cooled
at room temperature for 45 minutes. Then, 6 µL products were injected into
a high-throughput DNASep column and eluted with a linear acetonitrile gradient
of 2% per minute at a flow rate of 0.9 mL/min. The elution profiles of
heterozygous fragments were represented as aberrant shaped peaks, while the
homozygous fragments were represented as single peaks. After DHPLC,
heterozygous fragments were reamplified and purified with QIAquick PCR
Purification Kit (Qiagen, Hilden, Germany) and sent for direct sequencing in
both directions using an ABI 3100 DNA Sequencer (Applied Biosystems, Foster
City, California).
Genotyping and Statistical Analysis
All participants were genotyped for identified variations including single
nucleotide polymorphisms (SNPs) by PCR-restriction fragment length
polymorphism (PCR-RFLP) analysis with corresponding restriction endonucleases
(Dde I, Bsa HI, Tsp 45I, Dpn II; NEB,
Beverly, Massachusetts). After endonuclease digestion, the products were
electrophoresed on a 3% agarose gel and observed with a Gel Doc1000 system
(Bio-Rad, Hercules, California).
Hardy-Weinberg equilibrium (HWE) was tested using an HWE program.
Differences in genotypic and allelic frequencies between infertile patients
and controls were assessed by 
2 test using SPSS 11.0 software
(SPSS Inc, Chicago, Illinois).
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Results |
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The genotypic distributions of the 2 SNPs were in HWE, and their genotype
and allele frequencies are shown in Table
2. The 2 SNPs were found in both azoospermic and oligospermic
patients without apparent differences in either genotype or allele frequencies
between them (data not shown). As shown in
Table 2, although no
significant difference was observed among the 3 genotypes of c.679G>A, the
frequency of allele A in controls was significantly higher than that in
patients (3% vs 1.2%, P = .031) with an odds ratio (OR) of 0.400 (95%
confidence interval [CI]: 0.170–0.944). At c.987+108G>A, only 2
genotypes, GG and GA, were identified in both groups. The frequencies of
genotype GA and allele A decreased significantly in controls compared with
patients (genotype GA: 15% vs 5.5%, P < .001; allele A: 7.5% vs
2.7%, P = .001), giving an OR of 3.057 (95% CI: 1.600–5.842)
and 2.89 (95% CI: 1.531–5.453),
respectively.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Four novel variations of TSSK4 were identified. Although c.765C>A did not change the amino acid sequence, the results from bioinformatics analysis with ESEfinder 3.0 (Exonic Splicing Enhancer [ESE]; Cartegni et al, 2003) showed that the substitution of C by A at c.765C>A would result in disappearance of the ESE sequence GGTCACTT, which could be recognized and bound by the splicing factor SC35, an important member of the serine/arginine-rich (SR) protein family that plays a essential role in pre-mRNA splicing (Cartegni et al, 2003; Sanford et al, 2005). Thus, compared with allele C, allele A may decrease the activity of pre-mRNA splicing and reduce the expression of TSSK4. However, since c.765C>A is found in a limited number of infertile men (5/372), whether it is related to impaired spermatogenesis still needs to be clarified by functional studies in more patients. Another rare variation, c.-155C>G, should not be considered a disease-causing mutation for the time being because its possible influence on gene function has not been studied.
At c.679G>A, a significant difference in allele A frequency was observed between patients with oligospermia/azoospermia and controls, suggesting that this SNP may be associated with impaired spermatogenesis. This SNP changes an amino acid, but no effect on pre-mRNA splicing could be predicted, so it is not clear whether this polymorphism is a functional one or only a genetic marker in linkage disequilibrium with other loci responsible for spematogenic impairment. The statistical analysis also showed that allele A of SNP c.987+ 108G>A was associated with an increased risk for male infertility, since the frequencies of allele A (OR: 2.89) and genotype GA (OR: 3.057) were significantly higher in patients than in controls. In addition, some researchers have suggested that SNPs in the UTRs may impact mRNA stability, translation efficiency, and gene expression (Miller et al, 2002; Kamiyama et al, 2007; Nief et al, 2007; Wang et al, 2007). Therefore, to further reveal the possible role of TSSK4 in male infertility, more deep functional study of c.987+108G>A on gene expression is necessary.
In conclusion, as the first mutational analysis of TSSK4 in infertile men with azoospermia or severe oligospermia, our investigation provided some preliminary results and suggestions for in-depth studies of this gene on male infertility in the future.
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Footnotes |
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These authors contributed equally to this article and share
coauthorship. 
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