1 Institute of Clinical Medicine, School of Medicine, 2 Department of Biochemistry and Center for Cellular and Molecular Biology and 3 Shu-Tien Urological Research Center, National Yang-Ming University, 4 Division of Urology, Department of Surgery, Taipei Veterans General Hospital, 5 Taipei Municipal Jen-Ai Hospital and 6 Graduate Institute of Nutritional Science, Chung Shan Medical University, Taichung, Taiwan, Republic of China
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Abstract |
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Key words: 8-hydroxy-2'-deoxyguanosine/GST M1/polymorphism/sperm/varicocele
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Introduction |
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It has been postulated that human semen contains a significant amount of glutathione S-transferase (GST) and that the enzyme could attenuate the toxicity of ROS to sperm (Mukhtar et al., 1978). The GST gene family produces isoenzyme that is important in protection against oxidative stress (Mann et al., 2000
), and an increase of ROS associated with reduced activity of GST may lead to sperm membrane damage (Gopalakrishnan and Shana, 1998). GST M1 is a member of the GST family, which are proteins involved in the detoxification of several chemical carcinogens by conjugating glutathione or binding them directly. About half of all people from different racial groups lack the enzyme activity of GST, which has a polymorphic expression, and its deficiency is due to homozygous deletion of the gene (Board et al., 1990
). GST M1 () genotype has been reported to be associated with an increased risk of cancer of the lung, colorectum, urinary bladder and stomach (Harada et al., 1987
; Zhong et al., 1991
, 1993
; Bell et al., 1993
; Hirvonen et al., 1993
; Gao and Zhang, 1999
; Hou et al., 2000
). However, its role in varicocele is still unknown.
8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidative products of DNA induced by ROS (Ames, 1998), which can reflect extremely low levels of oxidative DNA damage because it can be detected by high-performance liquid chromatography and an electrochemical detector (HPLC-ECD) system. It has been suggested that sperm DNA damage is closely related to male infertility and that 8-OHdG is a sensitive marker of oxidative DNA damage caused by ROS in human sperm (Shen et al., 1999
,2000
).
Mitochondrial DNA (mtDNA) is a naked and compact DNA molecule, which is rapidly replicated without proofreading. Moreover, mitochondria do not have efficient DNA repair systems. Thus, the mutation rate of mtDNA is 1020-fold higher than that of nuclear DNA (Clayton et al., 1974; Wallace et al., 1987
). We have demonstrated in a previous study that human sperm with lower motility have a higher frequency of occurrence of the 4977 bp deletion of mtDNA (Kao et al., 1995
).
Smoking increases oxidative damage to sperm DNA (Fraga et al., 1996). It has been suggested that varicocele and smoking might affect sperm function (El Mulla et al., 1995
). Our previous studies showed no difference in antioxidant capacity between patients with unilateral and those with bilateral varicocele (Chen et al., 2001
).
To evaluate the role of GST M1 () genotype in patients with varicocele, we conducted this study to evaluate the genetic polymorphism of GST M1 and its association with male infertility and 4977 bp deletion of sperm mtDNA. In addition, sperm function [by computer-assisted semen analysis (CASA)], the levels of 8-OHdG in sperm, and seminal plasma antioxidant capacity were also analysed with respect to GST M1 () genotype, infertility, 4977 bp deletion of sperm mtDNA, smoking, and unilateral or bilateral varicocele.
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Materials and methods |
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GST M1 gene polymorphism
Five millilitres of blood was harvested from each patient. DNA was isolated from peripheral leukocytes by standard procedures using proteinase K digestion and phenol/chloroform extraction. GST M1 gene deletion was detected by published methods (Bell et al., 1993) using a pair of primers for GST M1 gene (G5: 5'-GAACTCCCTGAAAAGCTAAAGC-3'; G6: 5'-GTTGGGCTCAAATATACGGTGG-3') and two primers for ß-globin gene (PC04: 5'-CAACTTCATCCACGTTCACC-3'; GH20: 5'-GAAGAGCCAAGGACAGGTAC-3'). PCR was carried out for 35 cycles in a DNA thermal cycler (PerkinElmer/Cetus) using a thermal profile of denaturation at 94°C for 40 s, annealing at 55°C for 40 s and primer extension at 72°C for 40 s. The PCR products were then separated on a 3% agarose/synergel gel (5:1, w/w) (Diversified Biotech, Newton Center, MA, USA) at 150 V for 1.5 h and stained with 1 µg/ml ethidium bromide at 25°C for 10 min. DNA fragments of the GST M1 gene amplified by PCR were 268 and 215 bp in size, but only the 268 bp band was observed in patients with GST M1 () genotype (Figure 1
).
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Sperm DNA extraction
Total DNA of human sperm was extracted according to the method developed in our laboratory (Kao et al., 1995).
Measurement of 8-OHdG content in sperm DNA
8-OHdG level in sperm DNA was measured by HPLCECD (Bioanalytical Systems, West Lafayette, IN, USA) as previously described by (Shigenaga et al., 1994). Deoxyguanosine (dG) and 8-OHdG were used as standards. 8-OHdG levels in sperm DNA are expressed as the number of 8-OHdG molecules per 105 dG.
Detection of mtDNA deletion in sperm
Sperm mtDNA deletions were detected by the method developed in this laboratory (Kao et al., 1998). PCR was carried out for 35 cycles in a DNA thermal cycler (PerkinElmer/Cetus) using a thermal profile of denaturation at 94°C for 40 s, annealing at 55°C for 40 s and primer extension at 72°C for 40 s. PCR products were then separated on a 1.5% agarose/synergel gel (5:1, w/w) (Diversified Biotech) at 150 V for 1.5 h and visualized by staining with 1 µg/ml ethidium bromide at 25°C for 20 min (usually >20 min).
Primer-shift PCR
In order to avoid artefacts in the detection of mtDNA deletions, primer-shift PCR was employed to ascertain that the amplified DNA fragment was not a result of misannealing of primers to the DNA template (Lee et al., 1994). We consistently obtained PCR products of 719, 587, 524 and 392 bp from the 4977 bp-deleted mtDNA by using primer pairs L8150H13845, L8282H13845, L8150H13650 and L8282H13650 respectively.
Measurement of protein thiols of the seminal plasma
Protein thiols of the seminal plasma were measured by a previously described spectrophotometric method by (Boyne and Ellman, 1972). An equal volume of 10% perchloric acid was added to 50 µl of seminal plasma. The pellet was dissolved in 1.4 ml of 50 mmol/l phosphate buffer containing 1% sodium dodecyl sulphate and 50 mmol/l EDTA (pH 7.4). An aliquot of 23 µl of 100 mmol/l 5,5'-dithiolbis-2-nitrobenzoic acid was added and thoroughly mixed. The absorbance of the reaction product at 412 nm was recorded and the protein thiol content was calculated from a standard curve. The value of protein thiol is expressed as nmol/ml seminal plasma.
Measurement of ascorbic acid in the seminal plasma
Ascorbic acid level of the seminal plasma was measured by a spectrophotometric method (Kyaw, 1978). The colour reagent was prepared by mixing 0.3mmol/l sodium tungstate, 1.2mmol/l sodium hydrogen phosphate and 18 mol/l sulphuric acid. Absorbance at 700 nm was read against a reagent blank and plasma ascorbic acid was calculated from a standard curve, and this value is expressed as mg/dl seminal plasma.
Statistical analysis
Data are reported as median with interquartile range because they are not normally distributed. We used the MannWhitney test, KruskalWallis test, Pearson correlation and odds ratio (OR) with a 95% confidence interval for statistical analysis. The software used was SPSS 8.0 for Windows. P < 0.05 was considered to be statistically significant.
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Results |
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The frequencies of GST M1 () genotype were 43.8, 41.9 and 45% for subjects in groups 1, 2 and 3 respectively, and there are no differences between the three groups. However, in group 1 patients with GST M1 () genotype, the frequency of the presence of 4977 bp deletion in sperm mtDNA (+) (54.3%) was significantly higher than that of the absence of 4977 bp deletion in sperm mtDNA () (45.7%, OR: 2.63, P = 0.04), but no significant difference was found for patients in group 2 (OR: 2.95, P = 0.12, Table I). In groups 1 and 2 patients with GST M1 () genotype, the incidence of infertility was higher than that of fertility, but no significant difference was noticed (OR: 2.0, P = 0.19 for group 1 patients and OR: 1.8, P = 0.28 for group 2 patients). The incidence of 4977 bp deletion in sperm mtDNA were 41.3, 17.7 and 1.67% for subjects in groups 1, 2 and 3 respectively. Significantly higher incidence was found in patients of group 1 as compared with those of groups 2 and 3.
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Discussion |
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Ascorbate, urates and thiols in the seminal plasma are major antioxidants for which significantly lower levels have been recorded in infertile compared with fertile men (Lewis et al., 1997). Therefore, we used thiols and ascorbic acid levels to evaluate antioxidant capacity of the seminal plasma. CASA provides an accurate quantitative evaluation of semen quality in patients with varicocele. Our previous study demonstrated that protein thiols and ascorbate in the seminal plasma are good markers for evaluating oxidative stress in patients with varicocele (Chen et al., 2001
). In this study, we found that varicocele and subclinical varicocele patients with GST M1() genotype had lower seminal plasma antioxidant capacity, but only patients with palpable varicoceles and this genotype had lower CASA parameters (except sperm concentration). However, no such difference was found for patients with subclinical varicocele. These results suggest that GST M1 () genotype plays a role in lowering seminal plasma antioxidant capacity, which may result in sperm dysfunction for patients with varicocele.
Deoxyribonucleic bases are susceptible to oxidative stress and a high level of oxidative DNA damage of the sperm may be involved in male infertility (Hideya et al., 1997). It has been reported that the 8-OHdG contents of sperm DNA in smokers were higher than those in non-smokers (Shen et al., 1998
). In this study, we found that the level of sperm 8-OHdG was significantly higher in patients with varicocele and subclinical varicocele, and GST M1 () genotype, 4977 bp deletion of mtDNA, smoking and infertility. However, no difference was found between control patients with GST M1 () genotype and those without. Besides, there is a negative correlation between sperm function and the level of 8-OHdG of sperm DNA for all the patients. Moreover, the level of sperm 8-OHdG was the highest in patients with varicocele followed by those with subclinical varicocele and normal controls. This confirms that sperm 8-OHdG is a good marker for evaluating oxidative stress in patients with varicocele.
It has been demonstrated that mtDNA mutations play an important role in some pathophysiological conditions of human spermatozoa, and that multiple mtDNA deletions occur more frequently in the sperm with low motility and infertile patients (Kao et al. 1995, 1998
). In this study, patients with varicocele had a higher frequency of 4977 bp deletion of sperm mtDNA and infertility than patients with subclinical varicocele and normal controls. Also, patients with varicocele and subclinical varicocele associated with 4977 bp deletion in sperm mtDNA had higher levels of sperm 8-OHdG and lower antioxidant capacity of the seminal plasma than those without the 4977 bp deletion of mtDNA.
Genetic factors that could mediate the pathogenesis of male infertility are mostly unclear and increased frequencies of cytochrome P4501A1 gene polymorphisms have been found in infertile men (Fritsche et al., 1998; Schuppe et al., 2000
). Asthenozoospermia is associated with mtDNA halogroups in Caucasians and it was suggested that genetic alterations of mtDNA can induce sperm dysfunction (Ruiz-Pesini et al., 2000
). One study found that long androgen receptor CAG alleles are associated with male infertility and defective spermatogenesis (Mifsud et al., 2001
). GST M1 is located at human chromosome 1p13, which displays three alleles: GST M1a, GST M1b and GST M1 null () (Board, 1981a
,b
; Suzuki et al., 1987
). GST plays an important role in biotransformation and detoxification of many xenobiotics. GST activity is widely distributed in hepatic and extrahepatic tissues including ovary, testes, and serum. It has been demonstrated that GST might have a relevant protective role during spermatogenesis (Castellon, 1999
). It has been suggested that polymorphism of the GST M1 gene might be an important factor in determining the susceptibility of a patient to develop alcohol-induced disorders of human spermatogenesis (Pajarinen et al., 1996
). Mu and theta class GST play an important role in the detoxification of products of oxidative damage, such as lipid hydroperoxides, alkenals and DNA hydroperoxides, as well as some carcinogens including methyl halides and benzo(a)pyrene epoxides (Smith et al., 1995
). However, Gopalakrishnan and Shaha (1998) argued that the role of GST in the antioxidant defence mechanism of sperm is still not clear although increased ROS production by the cell was noticed when GST activity was suppressed (Gopalakrishnan and Shaha, 1998
). Therefore, in this study we used antioxidant capacity of seminal plasma and the level of sperm 8-OHdG to evaluate the effect of GST M1 () genotype instead of GST enzyme because patients with GST M1 () genotype had deficiency of this enzyme.
We demonstrated for the first time that there is an association between GST M1 gene polymorphism and 4977 bp deletion of sperm mtDNA for patients with varicocele. In group 1 patients with GST M1 () genotype, the frequency of the presence of the 4977 bp deletion in sperm mtDNA (54.3%) was significantly higher than that of the absence of the 4977 bp deletion in sperm mtDNA (45.7%, OR: 2.63, P = 0.04). Besides, significantly higher 8-OHdG content in sperm DNA and lower protein thiols and ascorbic acid in seminal plasma were found in patients of groups 1 and 2 with GST M1 () genotype than those with GST M1 (+) genotype. These results suggest that the sperm of varicocele patients with GST M1() genotype are more vulnerable to oxidative damage. Therefore, we should pay more attention to oxidative stress-related pathological manifestations in varicocele-bearing patients with GST M1 () genotype.
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Acknowledgements |
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Notes |
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Submitted on May 16, 2001; resubmitted on August 14, 2001
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References |
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Submitted on May 16, 2001; resubmitted on August 14, 2001; accepted on November 2, 2001.