Moderate Dose of Trolox Preventing the Deleterious Effects of Wi-Fi Radiation on Spermatozoa In vitro through Reduction of Oxidative Stress Damage.

BACKGROUND: The worsening of semen quality, due to the application of Wi-Fi, can be ameliorated by Vitamin E. This study aimed to demonstrate whether a moderate dose of trolox, a new Vitamin E, inhibits oxidative damage on sperms in vitro after exposure to Wi-Fi radiation. METHODS: Each of the twenty qualified semen, gathered from June to October 2014 in eugenics clinic, was separated into four aliquots, including sham, Wi-Fi-exposed, Wi-Fi plus 5 mmol/L trolox, and Wi-Fi plus 10 mmol/L trolox groups. At 0 min, all baseline parameters of the 20 samples were measured in sequence. Reactive oxygen species, glutathione, and superoxide dismutase were evaluated in the four aliquots at 45 and 90 min, as were sperm DNA fragments, sperm mitochondrial potential, relative amplification of sperm mitochondrial DNA, sperm vitality, and progressive and immotility sperm. The parameters were analyzed by one-way analysis of variance and Tukey's posttest. RESULTS: Among Wi-Fi plus 5 mmol/L trolox, Wi-Fi-exposed and Wi-Fi plus 10 mmol/L trolox groups, reactive oxygen species levels (45 min: 3.80 ± 0.41 RLU·10-6·ml-1 vs. 7.50 ± 0.35 RLU·10-6·ml-1 vs. 6.70 ± 0.47 RLU·10-6·ml-1, P < 0.001; 90 min: 5.40 ± 0.21 RLU·10-6·ml-1 vs. 10.10 ± 0.31 RLU·10-6·ml-1 vs. 7.00 ± 0.42 RLU·10-6·ml-1, P < 0.001, respectively), percentages of tail DNA (45 min: 16.8 ± 2.0% vs. 31.9 ± 2.5% vs. 61.3 ± 1.6%, P < 0.001; 90 min: 19.7 ± 1.5% vs. 73.7 ± 1.3% vs. 73.1 ± 1.1%, P < 0.001, respectively), 8-hydroxy-2'-deoxyguanosine (45 min: 51.89 ± 1.46 pg/ml vs. 104.89 ± 2.19 pg/ml vs. 106.11 ± 1.81 pg/ml , P = 0.012; 90 min: 79.96 ± 1.73 pg/ml vs. 141.73 ± 2.90 pg/ml vs. 139.06 ± 2.79 pg/ml; P < 0.001), and percentages of immotility sperm (45 min: 27.7 ± 2.7% vs. 41.7 ± 2.2% vs. 41.7 ± 2.5%; 90 min: 29.9 ± 3.3% vs. 58.9 ± 4.0% vs. 63.1 ± 4.0%; all P < 0.001) were lowest, and glutathione peroxidase (45 min: 60.50 ± 1.54 U/ml vs. 37.09 ± 1.77 U/ml vs. 28.18 ± 1.06 U/ml; 90 min: 44.61 ± 1.23 U/ml vs. 16.86 ± 0.93 U/ml vs. 29.94 ± 1.56 U/ml; all P < 0.001), percentages of head DNA (45 min: 83.2 ± 2.0% vs. 68.2 ± 2.5% vs. 38.8 ± 1.6%; 90 min: 80.3 ± 1.5% vs. 26.3 ± 1.3% vs. 26.9 ± 1.1%; all P < 0.001), percentages of sperm vitality (45 min: 89.5 ± 1.6% vs. 70.7 ± 3.1% vs. 57.7 ± 2.4%; 90 min: 80.8 ± 2.2% vs. 40.4 ± 4.0% vs. 34.7 ± 3.9%; all P < 0.001), and progressive sperm (45 min: 69.3 ± 2.7% vs. 55.8 ± 2.2% vs. 55.4 ± 2.5%; 90 min: 67.2 ± 3.3% vs. 38.2 ± 4.0% vs. 33.9 ± 4.0%; all P < 0.001) were highest in Wi-Fi plus 5 mmol/L trolox group at 45 and 90 min, respectively. Other parameters were not affected, while the sham group maintained the baseline. CONCLUSION: This study found that 5 mmol/L trolox protected the Wi-Fi-exposed semen in vitro from the damage of electromagnetic radiation-induced oxidative stress.

Effects of low-dose, long-term formaldehyde exposure on the structure and functions of the ovary in rats.

Formaldehyde (FA) is a ubiquitous environmental pollutant. However, the effects of FA exposure on reproduction are still a matter of scientific controversy. In this study, we assessed the ovarian toxicity of long-term, low-dose FA exposure in rats and explored the potential oxidative stress mechanisms. A total of 30 Sprague-Dawley female rats were randomly allotted to three groups, in which rats were exposed to FA at a dose of 0 mg/m(3) (control), 0.5 mg/m(3) and 2.46 mg/m(3), respectively, by inhalation consecutively for 60 days. The results showed that the ovarian toxicity of FA is dose dependent. Ovarian structure and function in the group of rats exposed to 0.5 mg/m(3) FA showed no obvious difference when compared with those in the control group. However, the activity of superoxide dismutase was significantly decreased, whereas the level of malondialdehyde was significantly increased in ovaries of rats exposed to 2.46 mg/m(3) FA. Moreover, histopathological results demonstrated that the number and size of mature follicles significantly decreased, vascular congestion and interstitial edema in the ovaries of rats exposed to 2.46 mg/m(3) FA. In conclusion, this study may suggest that the FA level of 0.5 mg/m(3) can be considered as a safe level for FA exposure, but long-term FA exposure at a dose of 2.46 mg/m(3) has a harmful effect on ovary by inducing oxidative stress.

Association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in northern Chinese Han males.

X-ray repair cross-complementing group 1 (XRCC1) is a scaffold protein that plays a critical role in DNA base excision repair. To explore the association between XRCC1 single-nucleotide polymorphisms and infertility with idiopathic azoospermia in a northern Chinese Han population, PCR restriction fragment length polymorphism was used to genotype a SNP locus (rs25487) of XRCC1 in 112 patients with idiopathic azoospermia and 156 healthy controls. Furthermore, nucleotide sequences were sequenced. The results showed that, compared with GG genotype, the GA and GA+AA genotypes showed a significant association with an increased risk of idiopathic azoospermia (OR 2.119, 95% CI 1.245-3.606, P=0.005), (OR 2.052, 95% CI 1.227-3.431, P=0.006) respectively. Meanwhile, the A allele frequency was significantly higher in azoospermic patients than that in controls (OR 1.472, 95% CI 1.029-2.105, P=0.034). The substitutions bring about an amino acid alteration: G→A changes the arginine residue into glutamine. In conclusion, the SNP locus rs25487 of XRCC1 could be a marker for genetic susceptibility to idiopathic azoospermia and the A allele might be a risk gene of idiopathic azoospermia in the northern Chinese Han population.

Effects of paternal occupation exposure to formaldehyde on reproductive outcomes.

OBJECTIVE: To investigate whether paternal occupational exposure to formaldehyde (FA) affects the reproductive outcomes. METHODS: Data were collected from 302 male workers occupationally exposed to FA and 305 referent controls through interview questionnaires. Formaldehyde exposure level was measured and calculated for every subject. Different reproductive outcomes were compared for two groups by logistic regression analyses. RESULTS: A significant increased risk of prolonged time to pregnancy (P = 0.034; odds ratio, 2.828; 95% confidence interval, 1.081 to 7.406) and significant elevated risk of spontaneous abortion (P = 0.021; odds ratio, 1.916; 95% confidence interval, 1.103 to 3.329) were observed in wives of male workers occupationally exposed to FA after correction for confounding factors. Moreover, reproductive toxicity due to FA exposure is dose dependent. CONCLUSIONS: This epidemiological study adds some evidence for the hypothesis that paternal FA occupation exposure has adverse effects on reproductive outcomes.

Correlation of Sperm Associated Antigen 11 (SPAG11) and its Isoforms with Varicocele in Rats.

BACKGROUND: We undertook this study to investigate the variation relationship of sperm associated antigen 11 (Spag11) mRNA expression and SPAG11E protein in the epididymis and spermatozoa of experimental left varicocele (ELV) rats. These findings could contribute to the understanding of the role of epididymal proteins in sperm functions and the mechanism of male infertility induced by varicocele. METHODS: The ELV model was established in adolescent male Sprague-Dawley rats. Four weeks after the operation, tissue distribution and changes in the expressions of Spag11 mRNA and SPAG11E protein caused by ELV in the whole of left epididymis and spermatozoa were studied using quantitative reverse transcription-polymerase chain reaction (RT-QPCR), immunohistochemistry and immunofluorescence. Significant differences were identified using one-way ANOVA followed by Student-Newman-Keuls test. Significance level (p) was fixed at 0.05. RESULTS: The expected product of Spag11 was 96 bp that amplified by RT-QPCR was detected in the epididymal tissue and spermatozoa. SPAG11E protein was confined mainly to the supranuclear region of the principal cells and the stereocilium of the epididymal epithelium, it was concentrated on the acrosome and the tail of spermatozoa except the terminal piece. Statistical analyses of the images and the data indicated that Spag11 mRNA and SPAG11E protein expressions in the left epididymis and spermatozoa of ELV rats presented a considerable decrease (p<0.001) compared with that of the corresponding control group. CONCLUSION: The expressions of Spag11 mRNA and SPAG11E protein declined markedly in ELV rats, which suggest that SPAG11E may not only play an important role in sperm maturation, but it may also be influenced by varicocele.

Association study of HLA-B alleles with idiopathic male infertility in Han population of China.

PURPOSE: To investigate the distributions of HLA-B alleles and estimate their associations with idiopathic male infertility in Chinese Han population. METHODS: Polymerase chain reaction-sequence-based typing (PCR-SBT) method was used for DNA typing at HLA-B locus in 109 patients with idiopathic male infertility and 152 healthy controls in male Han population of Shaanxi Province, situated in northwestern China. RESULTS: In total, we detected 45 HLA-B alleles in idiopathic infertile patients, 48 HLA-B alleles in control subjects. However, no significant differences of these allelic frequencies were found between the infertile patients and the controls. CONCLUSION: HLA-B gene was unlikely a major risk factor of idiopathic male infertility in this sample population. As different populations have different HLA polymorphisms, investigation of the relationship of other HLA genes and idiopathic male infertility with larger sample size, is warranted in the future.

[Experimental varicocele affects VEGF and Flt-1 protein expressions in the testis and epididymis of adolescent rats].

OBJECTIVE: To investigate the effects of experimental left varicocele (ELV) on the vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase-1 (Flt-1) proteins in the testis and epididymis of adolescent rats, and to find out the correlation of the two proteins with varicocele-induced male infertility. METHODS: We established the ELV model in adolescent male SD rats, and detected the expressions of VEGF and Flt-1 proteins in the testis and epididymis by immunohistochemistry at 2 and 4 weeks after surgery. RESULTS: Cell- and region-specific expressions of VEGF and Flt-1 were observed in the testis and epididymis of the ELV and control groups. Statistical analysis showed that, in comparison with the corresponding control groups, the 2- and 4-week ELV groups exhibited a notable increase in the VEGF protein expression in the hibateral testis and epididymis (P < 0.01, P < 0.05); the Flt-1 expression was obviously upregulated in the hibateral testis and epididymis of the 2-week ELV group (P < 0.01, P < 0.01), but remarkably reduced in the hibateral testis and left epididymis of the 4-week ELV group (P < 0.01, P < 0.05), with no statistic difference in the right epididymis (P > 0.05). CONCLUSION: ELV can cause changes in the expressions of VEGF and Flt-1 proteins in the testis and epididymis of adolescent rats, and consequently affect spermatogenesis and spermiotelcosis, which may be one of the causes of varicocele-induced male infertility or subfertility.

The expression of Usp26 gene in mouse testis and brain.

Deubiquitinating enzymes (DUBs) play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 (USP26) is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription (RT)-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids (steps 9-16), Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial cells, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.

[Effects of progesterone on intracellular free Ca2+ concentration in the spermatozoa of fertile men and patients with unexplained infertility].

OBJECTIVE: To investigate the difference in the responsiveness of intracellular free Ca2+ concentration ([Ca2+]i) to progesterone in the spermatozoa of normal fertile men and patients with unexplained infertility. METHODS: Nine normal fertile men and 10 patients with unexplained infertility were selected in this study. After swim-up separation of the motile fraction and 2-hour in vitro capacitation, the spermatozoa were loaded with the fluorescent calcium indicator Fluo-3/AM (8.85 micromol/L) for 40 minutes away from the light, and then the sperm suspension was mixed with equal amount of 20% gelatin to immobilize the spermatozoa. The basal intracellular free [Ca2+]i and that induced by 10 micromol/L progesterone in the individual sperm were assessed by laser scanning confocal microscopy. RESULTS: The infertile patients had a significantly lower basal level of [Ca2+]i in the capacitated sperm than the fertile men (P < 0.01). The sperm from the normal controls responded to progesterone by exhibiting a rapid but transient rise in [Ca2+]i, with the peak level significantly higher than the basal level (P < 0.05), while those from the infertile patients by showing a slight increase, with no significant difference between the peak and basal levels (P > 0.05). Both the peak of the progesterone-induced [Ca2+]i and its increase amplitude expressed as the difference between the peak and basal levels were significantly higher in the normal fertile group than in the infertile patients (P < 0.01). CONCLUSION: The responsiveness of [Ca2+]i to progesterone is reduced in the spermatozoa of patients with unexplained infertility, which suggests a functional defect in the non-genomic sperm membrane progesterone receptor responsible for calcium influx.

Androgen receptor: a new player associated with apoptosis and proliferation of pancreatic beta-cell in type 1 diabetes mellitus.

Androgen receptor (AR) mediates a wide range of cellular processes, such as proliferation, differentiation and apoptosis. Here we sought to identify whether AR was located in pancreatic beta-cells and investigate its functions in type 1 diabetes induced by multiple low doses of streptozotocin. Double/triple immunofluorescence, Western blot and semi-quantitative RT-PCR were carried out to determine variances of AR expression in beta-cells and correlation between AR and apoptosis/proliferation of beta-cells with progress of diabetes. In addition, in vitro primary beta-cells from control mice were cultured for 3 days or 6 days with compound stimulation in order to further identify effect of AR on beta-cell apoptosis and proliferation. AR expression in beta-cells peaked in control and 1-day diabetic mice, gradually and significantly decreased, even disappeared in diabetic mice with progress of diabetes. TUNEL-positive beta-cells were concomitant with overexpression of AR, and Ki67-positive beta-cells showed extremely weak, even negative AR staining. In vitro, AR could mediate beta-cell apoptosis, and AR antagonist flutamide contributed to beta-cell proliferation. In conclusion, AR is abundantly expressed in pancreatic beta-cell cytoplasm of control mice. With progress of type 1 diabetes, decrement of AR expression in diabetic mice contributes to prohibit beta-cells from apoptosis, and is strongly associated with beta-cell proliferation.

[Expressions of VEGF and Flt-1 in the testis, epididymis and epididymal sperm of adolescent rats].

OBJECTIVE: To study the expressions of the vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase-1 (Flt-1) in the testis, epididymis and epididymal sperm of adolescent rats and explore the functions of both the proteins in the male reproductive system. METHODS: The expressions of VEGF and Flt-1 were detected in 20 adolescent SD rats, immunohistochemical staining used for both the testis and the epididymis and immunofluorescent staining for sperm. RESULTS: VEGF and Flt-1 proteins were specifically present in the testis, epididymis and sperm. In the testis, VEGF immunoreactive particles were localized in the cytoplasm of spermatogenic cells, the developing acrosome of spermatids, Sertoli cells and Leydig cells, while Flt-1 expressed mainly in the developing acrosome of spermatids and Leydig cells. In the epididymis, the cell-specific and region-specific expressions of VEGF and Flt-1 proteins were observed in the principal cells of epididymal epithelia, VEGF in the whole epididymis, while Flt-1 only in the caput and cauda segments. Both VEGF and Flt-1 were localized in the acrosome of the sperm head as well as in the neck, middle and principal segments of the sperm tail. CONCLUSION: The specific expression patterns of VEGF and Flt-1 in the rat testis, epididymis and sperm indicate that they may independently or collectively affect spermatogenesis and spermiotelcosis in either an autocrinological or a

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility.

AIM: To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 (USP26) gene and its involvement in idiopathic male infertility in China. METHODS: Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. RESULTS: Of 41 infertile men, 9 (22.0%, P = 0.01) had changes in USP26 gene on the X chromosome. A compound mutation (364insACA; 460G right triple arrow A) was detected in 8 patients (19.5%, P = 0.01) and a 1044T right triple arrow A substitution was found in 1 patient (2.4%, P > 0.05). All three variations led to changes in the coding amino acids. Two substitutions predict some changes: 460G right triple arrow A changes a valine into an isoleucine, and 1044T right triple arrow A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. CONCLUSION: The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

[Radioligand binding assay of progesterone receptors on normal fertile human sperm membrane].

OBJECTIVE: To investigate the progesterone-binding site on the normal fertile human sperm membrane after 2 hours of in vitro capacitation. METHODS: Viable spermatozoa were selected by a swim-up method. After 2 hours of in vitro capacitation, multipoint saturation binding experiments were performed. Sperm suspension and increasing concentrations of progesterone-11alpha-glucuronide-[125I] iodotyramine (125I-P) were added to 7 total binding tubes respectively, and equal amounts of sperm suspension and 125I-P were added to another 7 corresponding non-specific binding tubes in the presence of 10 micromol/L progesterone. After incubation for 1 hour at 4 degrees C, the radioactivity of both the tubes and the pellets after centrifugation was measured respectively. The equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) were calculated using the mathematical model of single site multi-point saturation method of Scatchard function and least-squares regression. RESULTS: Kd was (0.61 +/- 0.04) nmol/L and Bmax was (830 +/- 344) sites/cell. The significance test of the regression equation indicated that r = -0.980, P < 0.01. CONCLUSION: There is a high affinity and low capacity binding site for the progesterone (progesterone receptor) on the normal fertile human sperm membrane.

[Investigation of progesterone receptor on human sperm plasma membrane].

OBJECTIVES: To investigate the localization and positive percentage of progesterone receptor (PR) on the human sperm surface. METHODS: After in vitro capacitation, progesterone binding sites on the sperm were quantitatively analyzed by fluorescence microscopy and flow cytometry using fluorescein isothiocyanate-labeled bovine serum albumin-progesterone complex (P-BSA-FITC). RESULTS: The spermatozoa stained by P-BSA-FITC mainly showed two labeling patterns, with the green fluorescence on the whole acrosomal region or the equatorial acrosomal region only and the stainless postacrosomal and tail regions. The percentage of progesterone-binding sperm was (30.2 +/- 2.4)%. CONCLUSIONS: There is selective expression of PR on the human sperm acrosome surface.