Previous studies have found that smoking is associated with decreased male fertility via altering the quality of semen. However, the mechanism by which cigarette smoking affects semen quality remains to be fully elucidated. Heavy smoking-induced DNA damage has been reported to correlate with abnormal spermatozoa and male infertility. It has been reported that, in response to DNA damage, activation of the checkpoint kinase 1 (Chk1) facilitates S and G2 checkpoint arrest. The aim of the present study was to investigate the expression levels of Chk1 in sperm cells of smoking and non-smoking men, and to further examine the correlation between DNA fragmentation rates and the expression levels of Chk1 with smoking. The present study was performed on a cohort of 841 smoking men and 287 non-smoking men. In the investigation, sperm concentration, motility, viability, seminal plasma zinc concentration, acrosin activity and sperm DNA fragmentation were examined. The gene and protein expression levels of Chk1 were detected using reverse transcription quantitative-polymerase chain reaction and western blot analyses, respectively. It was observed that the progressive motility of the sperm was significantly decreased in the moderate and heavy smoking groups, whereas no significant changes were observed in the mild smoking group. The sperm in the medium-term smoking group had significantly decreased progressive motility, and the semen concentration, sperm count and progressive motility vitality were markedly decreased in the long-term smoking group. Compared with the non-smoking group, the abnormal head rates in the heavy smoking group and long-term smoking group were significantly increased. The sperm viability and seminal plasma zinc concentration were markedly increased in the smoking group. Increased DNA fragmentation rates were found in the smoking group. The expression of Chk1 was significantly decreased in the smoking group, compared with the non-smoking group. Progressive motility and sperm concentration showed a nonlinear association with the relative mRNA expression of Chk1. However, an inverse association was found between DNA fragmentation rates and the progressive motility and sperm concentration. These data suggested that the decrease of semen quality caused by cigarette smoking was not only correlated with sperm DNA fragmentation rates, but was also correlated with a decline in the expressive level of Chk1. The expression of Chk1 was associated with DNA damage and apoptosis, the reduction of which may lead to decreased sperm repair and increased sperm apoptosis, with a subsequent effect on semen quality.
Infertility is a common disorder affecting some one in seven couples, and subfertility has become a markedly increasing problem in affluent countries, with the most commonly identified cause attributed to 'male factor' (
Studies have demonstrated that Chk1 is a Ser/Thr protein kinase, which controls the G2/M phase transition in response to DNA damage (
The study population consisted of men, who were referred to the Reproductive Medicine Center of Shanxi Women and Infants Hospital (Taiyuan, China) between January 2013 and January 2015. All subjects were of the Han population from Shanxi Province in north China. The study was approved by the ethics committee of the Shanxi Women and Infants Hospital (Taiyuan, China) and the individuals in the relationships provided consent.
The inclusion criteria were as follows: Being the male partner of an infertile couple for a duration of at least 1 year, having regular intercourse, and seeking infertility treatment at the Reproductive Medicine Center, Shanxi Women and Infants Hospital over the study period. A careful history was obtained from each subject to exclude systemic diseases and assess alcohol assumption; careful physical examination was performed, with measurement of testicular size to exclude abnormalities of the external genitalia and cryptorchidism; ultrasonographic examination was performed to exclude varicoceles; microbiological examination and spermioculture were performed to exclude infections; an immunobead binding test was performed to exclude the presence of anti-sperm antibodies; karyotyping was used to exclude any chromosomal abnormality; and genetic examination was performed to exclude Y chromosome microdeletions and cystic fibrosis gene mutations.
A brief medical history was obtained, primarily by informal interview with the patient, or from the patient's clinical notes or a self-reported questionnaire. According to the standardization of the World Health Organization (WHO) on smoking and associated literature (
A semen sample was obtained from all subjects via masturbation following 2–7 days of abstinence for routine sperm counts, according to the WHO (2010) criteria (sperm concentration, motility, morphology and viability) (
Sperm viability was assessed within 30 min of ejaculation. Analysis was performed using eosin Y staining (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), for which 1 g of eosin was dissolved with 1 g fresh sperm. The percentage of viable sperm, indicated by an unstained sperm head, and non-viable sperm, indicated by staining of the sperm head, was assessed by counting a minimum of 200 spermatozoa. Replicate counts of 200 sperm on each of two slides were performed using a using a CX31 microscope (Olympus Corporation, Tokyo, Japan), which were then repeated if >5% difference was found (
The spermatozoa in each group were analyzed for acrosin activity using a Human Spermatozoa Acrosin Activity Quantitative Assay kit (Huakang Biotech, Shenzhen, China), according to manufacturer's protocol. At 24°C, the quantity of substrate hydrolyzing 1.0
The seminal fluid in each group was analyzed for zinc using a Seminal Plasma Zinc Quantitative Assay kit (Huakang Biotech), according to the manufacturer's protocol. The seminal fluid (1 ml) was centrifuged for 5 min at 1,000 × g at 5 min. The supernatant was transferred into a test tube for use in seminal plasma analysis. The sediment was washed with 1 ml physiological saline solution, mixed on an XH-B vortex-type mixer (Jiangsu Kangjian Medical Apparatus Co., Ltd., Taizhou, China) for 30 sec, and centrifuged again, as previously. The supernatant was discarded and the sediment was used for zinc determination, rather than using 200
The analysis of DNA fragmentation was performed in fresh semen using fluorescence staining with a kit supplied by (Huakang Biotech), based on the fluorescence emission from individual sperm, which were stained with acridine orange (AO). AO molecules are intercalated into double-stranded DNA, and green fluorescence is emitted from the sperm nuclei. The DNA in sperm with immature nuclei are readily denatured into single strands and, following AO molecule aggregation in the nuclei, the color of the fluorescence becomes orange-red. The cell suspension was pipetted onto a glass slide and observed under a BX51 fluorescence microscope (Olympus Corporation) with a 480–490 nm filter. The percentages of green (normal DNA integrity) and orange-red (abnormal DNA integrity) spermatozoa in each sample of 200 spermatozoa were calculated by the same examiner. The integrity of sperm nuclear DNA was considered abnormal when the percentage of denaturation (orange-red spermatozoa on AO staining) was >34%.
RT-qPCR analysis was used to assess the transcriptional expression of tumor-associated genes, including Chk1. Total RNAs were extracted from the spermatozoa using a Total RNA Purification kit (Nanjing KeyGen Biotech Co., Ltd.), the concentration of the RNA was determined using a NanoDrop 1000 (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and reverse transcribed using an Transcriptor First Strand cDNA synthesis kit (Nanjing KeyGen Biotech Co., Ltd.). The reaction was performed in the following conditions: 30°C for 10 min; 42°C for 30 min; 99°C for 5 min; and 5°C for 5 min. qPCR was conducted in a CFX-96 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a One Step SYBR PrimeScript RT-PCR kit (Takara Bio., Inc.), as described. The specific primers (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were as follows: Chk1, forward 5′-ATATGAAGCGTGCCGTAGACT-3′ and reverse 5′-TGCCTATGTCTGGCTCTATTCTG-3′; GAPDH, forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. qPCR was performed as follows: Initial denaturation at 98°C for 5 min; 40 cycles of 95°C for 10 sec, and 72°C for 15 sec. The 2−ΔΔCq method was used for quantification, calculated according to the manufacturer's protocol, with results expressed as the mean ± standard deviation.
Spermatozoa were prepared from each group for western blot analysis to determine the expression level of the tumor-associated protein, Chk1. The seminal fluid (1 ml) was centrifuged for 5 min at 1,000 × g. The precipitate was washed with 1 ml physiological saline solution, mixed on a vortex-type mixer for 30 sec, and centrifuged again, as above. For analysis of cellular protein levels, spermatozoa cells were rinsed twice with ice-cold phosphate-buffered saline and then lysed in ice-cold lysis buffer [containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ETDA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, and 10
All data were analyzed using SPSS 17.0 (SPSS, Inc. Chicago, IL, USA). Normally distributed data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. To assess the normality of the distribution, a Shapiro-Wilk test was performed. One-way analysis of variance was used for comparison among multiple groups if the variance was homogeneous, whereas non-normally distributed variables were analyzed using a Mann-Whitney U test or Kruskal-Wallis variance analysis, as appropriate.
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Sperm viability was analyzed using eosin Y staining. As shown in
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Analyses using RT-qPCR (
The present study found a non-linear association between the relative mRNA expression of Chk1, and the progressive motility and sperm concentration (P<0.05). However, DNA fragmentation rates were inversely association (P<0.05) with progressive motility and sperm concentration (
Cigarette smoking is a recognized health hazard, and the highest prevalence of smokers is in young men of reproductive age (
In the present study, it was observed that the progressive motility of the sperm in the moderate and heavy smoking groups were significantly decreased, compared with the non-smokers, whereas no significant changes were observed in the mild smoking group. No significant differences were observed in the routine semen parameters of the short-term smoking group, compared with the non-smoking group. However, the sperm in the medium-term smoking group had significantly decreased progressive motility, and the long-term smoking group had decreased semen concentration, sperm count and progressive motility, compared with the non-smoking group. Compared with the non-smoking group, no significant changes were found in the sperm morphology in the mild smoking group or moderate smoking group. This was also the case for the short-term smoking group and medium-term smoking group. However, the abnormal head rates in the heavy smoking group and long-term smoking group showed significant increases, compared with the non-smoking group. Compared with the non-smoking group, the smoking group exhibited a significant increase in sperm viability. The seminal plasma zinc concentration decreased significantly in the smoking group, compared with non-smoking group, and the smoking group had significantly increased DNA fragmentation rates, compared with the non-smoking group. Similar results were obtained for spermatozoa acrosin activity between the smoking group and non-smoking group. These data are consistent with the results of previous studies (
It has been reported that, in response to DNA damage, the activation of the Chk1 facilitates S and G2 checkpoint arrest (
In the present study, the expression of Chk1 was significantly decreased in the smoking group, compared with the non-smoking group. There was a nonlinear association between the relative mRNA expression of Chk1 and the progressive motility and sperm concentration. However, an inverse association was found between DNA fragmentation rates and the progressive motility and sperm concentration. These data suggested that the decrease of semen quality caused by cigarette smoking was not only correlated with sperm DNA fragmentation indices, but was also correlated with a decline in the expression of Chk1. The expression of Chk1 was correlated with sperm DNA damage and apoptosis, and its reduction may lead to decreased sperm repair and increased sperm apoptosis, with a subsequent effect on semen quality.
In conclusion, the results obtained in the present study provide useful information regarding the expression of Chk1 in sperm cells of smoking and non-smoking men, and the association between DNA fragmentation rates and the expression levels of Chk1 with smoking. They may also offer information for the prevention and treatment of male infertility as a result of smoking.
The present study was supported by the Research Fund of National Health and Family Planning Commission of China (grant no. RFNHFPCC, 201402004).
Comparison of sperm viability between smokers and non-smokers. (A) Sperm viability was analyzed using eosin Y staining. The (a) non-viable sperm, indicated by a stained head, and (b) viable sperm indicated by an unstained head, were examined using microscopy (magnification, ×20). (B) Sperm viability in the smoking group was significantly decreased, compared with that in the non-smoking group. (P<0.05). Data are presented as the mean ± standard deviation.
Comparison of plasma zinc concentration, spermatozoa acrosin activity and DNA fragmentation rates between the non-smoking and smoking groups. (A) Seminal plasma zinc concentration decreased significantly in the smoking group, compared with the non-smoking group (P<0.05). (B) Spermatozoa acrosin activity decreased significantly in the smoking group, compared with the non-smoking group (P<0.05). (C) DNA fragmentation rates were analyzed using acridine orange staining. The (a) green (normal DNA integrity) and (b) orange-red (abnormal DNA integrity) spermatozoa were examined using a BX51 Olympus fluorescence microscope with a 480–490 nm filter (magnification, ×20). (D) A significant increase in DNA fragmentation rate was observed in the smoking group, compared with the non-smoking group (P<0.05). Data are presented as the mean ± standard deviation.
Expression levels of Chk1 in the sperm of smokers and non-smokers. (A) Expression levels of Chk1 were analyzed using reverse transcription-quantitative-polymerase chain reaction analysis, which demonstrated that the mRNA expression of Chk1 was significantly decreased in the smoking group, compared with the non-smoking group (P<0.05). Data are presented as the mean ± standard deviation. (B) Western blot analysis and (C) quantification showed that the protein expression levels of Chk1 were significantly decreased in the smoking group, compared with the non-smoking group (P<0.05). Chk1, checkpoint kinase 1.
Comparison of routine semen parameters between non-smokers and smokers grouped according to daily cigarette consumption.
Parameter | Non-smoking | Smoking | Daily cigarette consumption
| ||
---|---|---|---|---|---|
Mild | Moderate | Heavy | |||
Cases (n) | 298 | 920 | 256 | 365 | 299 |
Semen volume (ml) | 3.63±1.48 | 3.44±1.23 | 3.53±1.18 | 3.40±1.21 | 3.47±1.23 |
Sperm concentration (×106/ml) | 45.38±24.83 | 41.57±21.93 | 44.41±21.42 | 42.62±25.26 | 40.13±24.73 |
Sperm count (×106) | 49.28±31.29 | 44.62±31.94 | 47.17±29.96 | 45.84±31.24 | 43.18±32.61 |
Progressive motility (%) | 27.97±10.66 | 18.26±11.48 | 26.42±12.63 | 19.58±11.24 | 15.21±9.17 |
Data are presented as the mean ± standard deviation. Mild, ≤9 cigarettes/day; moderate, 10–19 cigarettes/day; heavy, ≥20 cigarettes/day.
Comparison of routine semen parameters between non-smokers and smokers grouped according to the duration of smoking.
Parameter | Non-smoking | Short-term | Duration of smoking
| |
---|---|---|---|---|
Medium-term | Long-term | |||
Cases (n) | 298 | 268 | 282 | 370 |
Semen volume (ml) | 3.63±1.48 | 3.57±1.24 | 3.43±1.42 | 2.12±1.11 |
Sperm concentration (×106/ml) | 45.38±24.83 | 43.65±20.39 | 41.59±21.11 | 28.85±22.22 |
Sperm count (×106) | 49.28±31.29 | 48.47±21.56 | 44.54±22.39 | 37.67±16.22 |
Progressive motility (%) | 27.97±10.66 | 25.67±11.18 | 18.69±12.24 | 13.28±11.43 |
Data are presented as the mean ± standard deviation. Short-term, ≤5 years; medium-term, 5–10 years; long-term, ≥10 years.
Comparison of sperm morphology between non-smokers and smokers grouped according to daily cigarette consumption.
Parameter | Non-smoking | Smoking | Daily cigarette consumption
| ||
---|---|---|---|---|---|
Mild | Moderate | Heavy | |||
Cases | 298 | 920 | 256 | 365 | 299 |
Normal sperm | 7.22±1.49 | 6.43±1.33 | 7.13±1.38 | 6.97±1.55 | 6.13±1.07 |
Abnormal head | 82.51±11.66 | 88.38±15.11 | 86.36±13.10 | 86.02±10.16 | 98.22±18.54 |
Abnormal body | 41.38±8.58 | 49.32±14.43 | 47.73±12.89 | 46.52±14.81 | 52.37±14.23 |
Abnormal tail | 6.23±7.19 | 11.64±12.77 | 9.38±8.64 | 10.57±10.32 | 12.39±14.71 |
Data are presented as the mean ± standard deviation. Mild, ≤9 cigarettes/day; moderate, 10–19 cigarettes/day; heavy, ≥20 cigarettes/day.
Comparison of sperm morphology between non-smokers and smokers grouped according to duration of smoking.
Parameter | Non-smoking | Duration of smoking
| ||
---|---|---|---|---|
Short-term | Medium-term | Long-term | ||
Cases (n) | 298 | 268 | 282 | 370 |
Normal sperm | 7.22±1.38 | 7.42±1.23 | 6.29±1.26 | 5.29±1.27 |
Abnormal head | 82.51±10.62 | 80.91±14.25 | 88.27±12.77 | 99.43±16.28 |
Abnormal body | 41.38±9.48 | 47.88±8.76 | 51.93±10.36 | 52.50±9.71 |
Abnormal tail | 6.23±7.44 | 11.59±8.61 | 9.37±11.27 | 11.31±12.19 |
Data are presented as the mean ± standard deviation. Short-term, ≤5 years; medium-term, 5–10 years; long-term, ≥10 years.
Association between DNA fragmentation rates and the expression of Chk1 with semen parameters in all participants.
Parameter | Mean ± SD | Relative expression of Chk1
|
DNA fragmentation rate
| ||
---|---|---|---|---|---|
r-value | P-value | r-value | P-value | ||
Relative expression of Chk1 | 1.43±0.28 | – | – | – | – |
DNA fragmentation rate | 0.58±0.01 | – | – | – | – |
Progressive motility (%) | 22.16±9.32 | 0.042 | 0.027 |
0.042 | 0.027 |
Sperm concentration (×106/ml) | 39.27±15.22 | 0.047 | 0.026 |
0.037 | 0.012 |
Statistically significant result. Chk1, checkpoint kinase 1; SD, standard deviation.