Distribution of DNA damage in the human sperm nucleus: implications of the architecture of the sperm head.

The sperm nucleus is prone to sustain DNA damage before and after ejaculation. Distribution of the damage is not homogeneous, and the factors determining differential sensitivity among nuclear regions have not yet been characterized. Human sperm chromatin contains three structural domains, two of which are considered the most susceptible to DNA damage: the histone bound domain, harboring developmental related genes, and the domain associated with nuclear matrix proteins. Using a quantitative polymerase chain reaction (qPCR) approach, we analyzed the number of lesions in genes homeobox A3 (HOXA3), homeobox B5 (HOXB 5), sex-determining region Y (SRY)-box 2 (SOX2), ß-GLOBIN, rDNA 18S, and rDNA 28S in human sperm after ultraviolet irradiation (400 µW cm-2, 10 min), H2O2treatment (250 mmol l-1, 20 min), and cryopreservation, which showed differential susceptibility to genetic damage. Differential vulnerability is dependent on the genotoxic agent and independent of the sperm nuclear proteins to which the chromatin is bound and of accessibility to the transcription machinery. Immunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that the highest level of oxidation was observed after H2O2treatment. The distribution of oxidative lesions also differed depending on the genotoxic agent. 8-OHdG did not colocalize either with histone 3 (H3) or with type IIα + ß topoisomerase (TOPO IIα + ß) after H2O2treatment but matched perfectly with peroxiredoxin 6 (PRDX6), which is involved in H2O2metabolism. Our study reveals that the characteristics of the sperm head domains are responsible for access of the genotoxicants and cause differential degree of damage to nuclear areas, whereas chromatin packaging has a very limited relevance. The histone-enriched genes analyzed cannot be used as biomarkers of oxidative DNA damage.

The potential effect of methylseleninic acid (MSA) against γ-irradiation induced testicular damage in rats: Impact on JAK/STAT pathway.

This study suggested that methylseleninic acid (MSA) could respond to the inflammatory signaling associated with ionizing radiation-induced testicular damage. Mature male rats were divided into four groups: negative control, whole body γ-irradiated (IRR) (5 Gy), MSA (0.5 mg/kg, daily for nine consecutive days), and MSA+ IRR groups. MSA increased serum testosterone level and testicular glutathione peroxidase (GPx) as well as decreased the percentage of sperm abnormalities. Radiation prompted inflammatory signaling in the testes through increasing phospho-janus kinase1 (p-JAK1), phospho-signal transducers and activators of transcription 3 (p-STAT3) protein expressions. This induced increment in the inflammatory markers including nuclear factor- kappa B (NF-κB) and interleukin-1beta (IL-1ß) levels. Also, radiation induced elevation of nitric oxide (NO) and malondialdhyde (MDA) levels with consequent reduction in testicular reduced glutathione level (GSH) and superoxide dismutase (SOD) activity. MSA significantly counteracted the radiation effect on testicular nuclear factor erythroid-2-related factor-2 (Nrf2) and suppressor of cytokine signaling (Socs3) protein expressions. In summary, this investigation proposed that MSA preserved spermatogenesis through increasing testosterone levels and GPx activity. Additionally, it diminished testicular inflammation by increasing of Nrf2 and Socs3 levels leading to reducing of p-JAK1, p-STAT3 and NF-κB levels. Histopathological examination results of testicular tissues showed a coincidence with the biochemical analysis.

Radiofrequency radiation (900 MHz)-induced DNA damage and cell cycle arrest in testicular germ cells in swiss albino mice.

Even though there are contradictory reports regarding the cellular and molecular changes induced by mobile phone emitted radiofrequency radiation (RFR), the possibility of any biological effect cannot be ruled out. In view of a widespread and extensive use of mobile phones, this study evaluates alterations in male germ cell transformation kinetics following RFR exposure and after recovery. Swiss albino mice were exposed to RFR (900 MHz) for 4 h and 8 h duration per day for 35 days. One group of animals was terminated after the exposure period, while others were kept for an additional 35 days post-exposure. RFR exposure caused depolarization of mitochondrial membranes resulting in destabilized cellular redox homeostasis. Statistically significant increases in the damage index in germ cells and sperm head defects were noted in RFR-exposed animals. Flow cytometric estimation of germ cell subtypes in mice testis revealed 2.5-fold increases in spermatogonial populations with significant decreases in spermatids. Almost fourfold reduction in spermatogonia to spermatid turnover (1C:2C) and three times reduction in primary spermatocyte to spermatid turnover (1C:4C) was found indicating arrest in the premeiotic stage of spermatogenesis, which resulted in loss of post-meiotic germ cells apparent from testis histology and low sperm count in RFR-exposed animals. Histological alterations such as sloughing of immature germ cells into the seminiferous tubule lumen, epithelium depletion and maturation arrest were also observed. However, all these changes showed recovery to varied degrees following the post-exposure period indicating that the adverse effects of RFR on mice germ cells are detrimental but reversible. To conclude, RFR exposure-induced oxidative stress causes DNA damage in germ cells, which alters cell cycle progression leading to low sperm count in mice.

[Impact of mobile phone radiation on the quality and DNA methylation of human sperm in vitro].

OBJECTIVE: To investigate the influences of mobile phone radiation on the quality and DNA methylation of human sperm in vitro. METHODS: According to the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, we randomly selected 97 male volunteers with normal semen parameters and divided each semen sample from the subjects into two equal parts, one exposed to mobile phone radiation at 1950 M Hz, SAR3. 0 W/kg for 3 hours while the other left untreated as the control. We obtained routine semen parameters as well as the acrosomal reaction ability, apoptosis and DNA methylation of sperm, and compared them between the two groups. RESULTS: Compared with the control, the radiation group showed significantly decreased progressive sperm motility ([36.64 ± 16.93] vs [27.56 ± 16.92]%, P < 0.01) and sperm viability ([63.72 ± 16.35] vs [54.31 ± 17.35]%, P < 0.01) and increased sperm head defects ([69.92 ± 4.46] vs [71.17 ± 4.89]%, P < 0.05), but no significant differences in sperm acrosomal reaction ([66.20 ± 6.75] vs [64.50 ± 3.47]%, P > 0.05). The early apoptosis rate of sperm cells was remarkably higher in the radiation group ([6.89 ± 9.84]%) than in the control ([4.44 ± 5.89]%) (P < 0.05). However, no statistically significant differences were found between the control and radiation groups in the DNA methylation patterns of the paternal imprinting gene H19 ICR ([0.60 ± 0.02] vs [1.40 ± 0.03]%, P > 0.05) or the maternal imprinting gene KvDMR1 ([0.00 ± 0.00] vs [1.80 ± 0.031%, P > 0.05). CONCLUSION: Mobile phone radiation reduces the progressive motility and viability of human sperm and increases sperm head defects and early apoptosis of sperm cells.

Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity.

Interspecific variation in sperm size is enigmatic, but generally assumed to reflect species-specific trade-offs in selection pressures. Among passerine birds, sperm length varies sevenfold, and sperm competition risk seems to drive the evolution of longer sperm. However, little is known about factors favouring short sperm or constraining the evolution of longer sperm. Here, we report a comparative analysis of sperm head abnormalities among 11 species of passerine bird in Chernobyl, presumably resulting from chronic irradiation following the 1986 accident. Frequencies of sperm abnormalities varied between 15.7 and 77.3% among species, more than fourfold higher than in uncontaminated areas. Nonetheless, species ranked similarly in sperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome damage consistent with premature acrosome reaction. Sperm length, but not sperm competition risk explained variation in sperm damage among species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm length and sperm integrity affecting sperm evolution in passerine birds.

[Long-term microwave radiation affects male reproduction in rats].

OBJECTIVE: To investigate the effect of long-term microwave radiation on male reproduction in rats. METHODS: A total of 100 male Wistar rats were exposed to microwave radiation with average power density of 0, 2.5, 5 and 10 mW/cm2 for 4 weeks, 5 times a week and 6 minutes per time. Changes in serum testosterone, testicular index, histology and ultrastructure, and the percentage of teratospermia in the epididymis were observed dynamically at 6 h, 7 d, 14 d, 28 d and 60 d after the exposure. RESULTS: There was a significant decrease in serum testosterone concentration at 28 d after microwave radiation at 2.5, 5 and 10 mW/cm2 ([10.20 +/- 4.31] ng/ml, [5.56 +/- 3.47] ng/ml and [7.53 +/- 4.54] ng/ml) and at 60 d at 10 mW/cm2 ( [15.95 +/- 9.54] ng/ml), as compared with the control group ([23.35 +/- 8.06] ng/ml and [31.40 +/- 9.56] ng/ml) (P < 0.05 or P < 0.01). No significant changes were found in the testis index at 6 h -60 d after microwave radiation at the three doses, but different degrees of degeneration, necrosis and shedding of spermatogenic cells, thinning of spermatogenic epithelia, and decrease or deletion of spermatozoa were observed, and more obvious at 28 d and 60 d. Swelling and cavitation of mitochondria in all spermatogenic cells, agglutination and margin translocation of nuclear chromatin in the spermatogonial and Leydig cells were seen at 7 d and 60 d after 5 mW/cm2 microwave radiation. The rate of teratospermia of the epididymis was increased, more obviously at 7 d after 2.5, 5 mW/cm2, 60 d after 5 mW/cm2, and 7 d, 28 d and 60 d after 10 mW/cm2 microwave radiation (P < 0.05 or P < 0.01). CONCLUSION: Long-term microwave radiation may cause injury to male reproduction, which is positively correlated with the radiation dose, and has an obvious late effect.

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa.

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 µm² vs. 18.8 ± 1.4 µm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

[Biological effects of phytoecdysteroids and chronic low-dose irradiation].

The influence of serpistene in dose of 5 and 50 mg/kg on chronic low-dose gamma-irradiation (22.6 cGy) effects on cytogenetic (abnormal sperm cell, marrow bone micronucleus) and function and morphology (thyroid and adrenal glands) parameters of mice was estimated. The serpistene modifies effects of gamma-irradiation depends on the administration regime and a dose of the substance. The most expressive radioprotective effect on endocrine organs after serpistene prophylactic administration was found. The prophylactic dose was 5 mg/kg for adrenal gland and both doses--for thyroid gland. The most expressive radioprotective effect on marrow bone cells after serpistene therapeutic administration in a dose of 5 mg/kg was found. The most expressive antimutagenic effect on somatic and germinal cells of prophylactic and therapeutic administration in a dose of 50 mg/kg was found.

Preliminary study on the induction of sperm head abnormalities in mice, Mus musculus, exposed to radiofrequency radiations from global system for mobile communication base stations.

The exposure of male mice to radiofrequency radiations from mobile phone (GSM) base stations at a workplace complex and residential quarters caused 39.78 and 46.03%, respectively, in sperm head abnormalities compared to 2.13% in control group. Statistical analysis of sperm head abnormality score showed that there was a significant (p < 0.05) difference in occurrence of sperm head abnormalities in test animals. The major abnormalities observed were knobbed hook, pin-head and banana-shaped sperm head. The occurrence of the sperm head abnormalities was also found to be dose dependent. The implications of the observed increase occurrence of sperm head abnormalities on the reproductive health of humans living in close proximity to GSM base stations were discussed.

Effects of low dose radiation and vitamin C treatment on chloroquine-induced genotoxicity in mice.

Chloroquine (CHQ) is a commonly used antimalarial agent. We evaluated the genotoxic potential of CHQ using chromosome aberration (CA), micronucleus (MN), and sperm head abnormality (SA) assays in vivo in Swiss albino mice. The interaction between a low dose of radiation and CHQ, as well as the effect of vitamin C on CHQ-induced genotoxicity, was also evaluated. It was observed that CHQ induced CA, as well as MN, in the bone marrow cells under certain treatment conditions. Further, CHQ induced significant increase in the frequency of SA both at 24 hr and 21 days of the treatment. In the present study vitamin C pretreatment apparently reduced the frequency of CA, MN, and SA induced by CHQ. In the combination studies with radiation and CHQ, we found that exposure to low doses of radiation (0.5 Gy) either prior to or following CHQ treatment, in the dose ranges tested, has little or no synergistic effect in the mutagenic evaluations in somatic cells. However, radiation exposure along with CHQ treatment resulted in significant increase in the frequency of SA as compared to the groups receiving CHQ alone at 21 days of the treatment. In summary, CHQ has the potential to induce genotoxicity in mammalian cells. Further, germ cells may be relatively more sensitive as compared to the somatic cells.

Rat testis as a radiobiological in vivo model for radionuclides.

The radiobiological effect of intracellularly localised radionuclides emitting low energy electrons (Auger electrons) has received much attention. Most in vivo studies reported have been performed in the mouse testis. We have investigated the rat testis as an in vivo radiobiological model, with sperm-head survival, testis weight loss and also alteration in the blood plasma hormone levels of FSH and LH as radiobiological endpoints. Validation of the rat testis model was evaluated by using mean absorbed doses of up to 10 Gy from intratesticularly (i.t.) injected (111)In oxine or local X-ray irradiation. Biokinetics of the i.t. injected radionuclide was analysed by scintillation camera imaging and used in the absorbed dose estimation. By the analysis of the autoradiographs, the activity distribution was revealed. Cell fractionation showed (111)In to be mainly associated with the cell nuclei. External irradiations were monitored by thermoluminescence dosimeters. The sperm-head survival was the most sensitive radiobiological parameter correlated to the mean absorbed dose, with a D(37) of 2.3 Gy for (111)In oxine and 1.3 Gy for X rays. The levels of plasma pituitary gonadal hormones FSH and LH were elevated for absorbed doses >7.7 Gy. This investigation shows that the radiobiological model based on the rat testis has several advantages compared with the previously commonly used mouse testis model. The model is appropriate for further investigations of basic phenomena such as radiation geometry, intracellular kinetics and heterogeneity, crucial for an understanding of the biological effect of low-energy electrons.

[The estimation of molecular and cytogenetic effects in mice exposed to chronic low dose gamma-radiation]. / Otsenka molekuliarnykh i tsitogeneticheskukh éffektov khronicheskogo vozdeistviia nizkointensivnogo gamma-izlucheniia u myshei.

Molecular and cytogenetic parameters were estimated in male CBA/lac mice exposed to chronic low dose-rate gamma-radiation (62 cGy/year) for 40, 80, 120, 210, and 365 days. After 40 days of exposure (6.7 cGy), spleen lymphocyte susceptibility to hydrogen peroxide was shown to increase. However, beginning from the day 120 of the treatment (20.4 cGy), the opposite effect was observed. An increase in number of the DNA-protein crosslinks was recorded in spleen lymphocytes only on day 40 of the experiment. The number of DNA breaks increased significantly beginning from day 120 of the experiment, as shown by the DNA-comet method. On the day 210 of irradiation, the frequency of abnormal sperm heads in the mice significantly increased. The number of normochromatic micronucleated erythrocytes of the peripheral blood remained unchanged.

[The effect of chronic exposure to cadmium and gamma radiation at low doses on the genetic structures in mice]. / Vliianie khronicheskogo vozdeistviia kadmiia i gamma-izlucheniia v malykh dozakh na geneticheskie struktury myshei.

The DNA-protein cross-links (DPC) in mouse thymocytes and spleen lymphocytes, the number of abnormal sperm heads (ASH) and the number of micronuclei (MN) in normochromatic erythrocytes (NCE) of peripheral blood were studied in mice exposed to long-term low-intensity gamma radiation (0.072 cGy/days) and/or cadmium with drinking water (0.01 mg Cd2+/l) for 20, 40 and 80 days. The dependence of DPC level on the total dose (exposure time) of gamma radiation and/or cadmium is nonlinear. The maximal level of DPC in cells of lymphoid organs upon exposure to gamma radiation or cadmium was recorded on the 40-th day, and under combined exposure on the 20-th day of the experiment. The long-term exposure to cadmium or gamma radiation causes an increase in the ASH frequency. The increase in frequencies of MN in NCE and reciprocal translocations in spermatocytes was not found.

Induction of micronuclei in bone marrow and sperm head abnormalities after combined exposure of mice to low doses of X-rays and acrylamide.

People come into contact with chemical and physical agents which are present in the environment and in workplaces. We investigated the effects of combined exposures to low doses of X-rays (0.05-0.25 Gy) and acrylamide (AA; 75 mg/kg bw) in the somatic and germ cells of outbred male mice by using a bone-marrow micronucleus test and a sperm morphology test. Combined treatment of germ cells to 0.25 Gy of X-rays + 75 mg/kg bw of acrylamide enhanced the effect induced by each agent given alone. The results confirmed the sensitivity to damage of spermatozoa and late spermatids, which can be demonstrated by sperm head abnormalities and reduced fertility. The sensitivity of somatic cells to acrylamide alone was similar to that of germ cells. Combined exposure to 0.05 Gy + 75 mg/kg bw of AA induced micronuclei in polychromatic erythrocytes of bone marrow although each dose did not produce a mutagenic effect. Teratogenesis Carcinog. Mutagen. 20:133-140, 2000.

[Morphological changes in mice sperm head after exposure to radiation and to cyclophosphamide]. / Zmiany morfologiczne mysich plemników po skojarzonym dzialaniu promieniowania X i cyklofosfamidu.

Male mice Sfis:Pzh were exposed to X-rays, cyclophosphamide or combination of both agents. Each of agent was given in low (0.25 Gy, 25 mg/kg bw CP) or high (1.00 Gy, 100 mg/kg bw CP) doses. Germ cells were exposed to agents as spermatogonia. After 35 days sperm abnormalities test was performed. Exposure to one of agents only, did not enhance statistically significant frequency of morphologically abnormal spermatozoa. Combined treatment of spermatogonia to both agents in low as well in high doses induce clear biological effects, but only combination of high doses (1.00 Gy + 100 mg/kg bw CP) induce statistically significant effect. Results obtained in this study confirmed, that ability of different agents to induce sperm-shape abnormalities is related to its ability to induce mutations in germ cells.

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm.

Morphologic and morphometric sperm characteristics of mouse epididymal extracts from animals exposed to static magnetic fields were evaluated. For this purpose, animals were exposed for 35 days to a field of 0.7 T generated by a commercial permanent magnet for either 1 or 24 h per day. The values of morphometric parameters were obtained using the morphometric module of the Sperm Class Analyzer computerized image analysis system, and percentages of abnormalities were calculated. The size of sperm heads was unaffected by exposure to static magnetic fields. Lack of hook was a sperm head abnormality found significantly more frequently in animals exposed continually than in nonexposed animals, showing a possible alteration to the spermatogenic process after exposure to static magnetic fields. The percentage of sperm with coiled tails or of sperm with abnormal midpiece or tail was not altered by exposure.

Transferrin-dependent uptake and dosimetry of Auger-emitting diagnostic radionuclides in human spermatozoa.

UNLABELLED: Localization of Auger-emitting radionuclides within spermatozoa could lead to the induction of transmissible genetic damage. We have quantified in vitro uptake of the widely used diagnostic Auger-emitters, (111)In and 99mTc, by ejaculated human spermatozoa and investigated the role of transferrin in their cellular localization. The resultant dose to sperm heads, including cellular dosimetry for Auger emissions, has been calculated for each radionuclide and compared with that achieved using conventional macrodosimetry. METHODS: Freshly isolated human spermatozoa were incubated in a physiological salt solution containing (111)In-chloride, 99mTc-pertechnetate or the transferrin-binding isotope 59Fe-citrate as a positive control. Cellular uptake mechanisms were investigated with transferrin competition and temperature dependence studies. The percentage uptake of each radionuclide was determined, and the dose to individual sperm heads was calculated using both conventional macrodosimetric methods and by consideration of radionuclide localization and energy deposition at the cellular level, including Auger electron emissions from (111)In and 99mTc. RESULTS: On in vitro incubation, human spermatozoa were found to accumulate (111)In and 59Fe but not 99mTc. Cell uptake of (111)In and 59Fe was transferrin-mediated; however, an alternative transferrin-independent uptake pathway was also present for (111)In. The dose to sperm heads from (111)In, calculated using measured uptake and cellular dosimetry, was found to be larger than that calculated using conventional dosimetry by a factor of more than 100. In contrast, conventional dosimetry was adequate for 99mTc and 59Fe. CONCLUSION: Isolated human spermatozoa appear to accumulate transferrin-binding isotopes, such as the Auger-emitter (111)In. If this uptake mechanism operates in the male reproductive tract, the resultant high dose to the sperm head could indicate that contraception may be advisable after large diagnostic doses of (111)In and, possibly, other transferrin-binding radionuclides. Such precautions could prevent transmission of any genetic damage from irradiated spermatozoa.

[Genetic disorders in house mice after the accident at Chernobyl power plant]. / Geneticheskie narusheniia u domovykh myshei posle avarii na Chernobyl'skoi AES.

Genetic effects were studied in house mice caught from 1986 to 1994 in regions polluted by radionuclides as a result of the Chernobyl disaster. The dose rates of gamma-radiation on the soil surface ranged from 0.02 to 200 mR/h. The frequency of reciprocal translocations in mouse spermatocytes was relatively low, but increased with the dose rate. Embryo mortality was increased only in the progeny of male mice caught in 1987 in the area with maximum contamination. The frequency of mice heterozygous for recessive lethal mutations decreased with the time after the accident.