Indices of methylation in sperm DNA from fertile men differ between distinct geographical regions.

STUDY QUESTION: Which are the main determinants, if any, of sperm DNA methylation levels? SUMMARY ANSWER: Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. WHAT IS KNOWN ALREADY: DNA methylation level, assessed on repetitive sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts. STUDY DESIGN, SIZE, DURATION: A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from Greenland, Caucasians from Warsaw (Poland) and Kharkiv (Ukraine). Semen samples were collected between May 2002 and February 2004 and aliquots were immediately frozen. PARTICIPANTS/MATERIALS, SETTING, METHODS: We estimated sperm DNA global methylation level (DGML) in two ways. First DNA methylation in repetitive DNA sequences (LINE-1, Satα and Alu) was quantified by PCR pyrosequencing after bisulfite conversion and second by flow cytometry (FCM) using fluorescently labeled monoclonal antibodies anti-5-methylcytosine. We analyzed whether personal characteristics and habits, body mass index, semen quality parameters, sperm chromatin integrity, biomarkers of accessory gland function and the plasma concentration of reproductive hormones were associated with sperm DNA methylation levels in men. Associations were evaluated by analysis of variance and linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The geographical location emerged as the main determinant when using the methylation level in repetitive sequences. FCM DGML results were not associated with those from repetitive sequence analysis. No other consistent associations between methylation markers and the assessed variables were identified across countries. LIMITATIONS, REASONS FOR CAUTION: The methods used are only surrogates of the actual sperm methylome and the methylation levels at individual specific loci were not explored. WIDER IMPLICATIONS OF THE FINDINGS: Sperm DGML is relatively independent from semen quality parameters and is a new candidate biomarker for epidemiological studies of the impact of environmental contaminants on male fertility. STUDY FUNDING/COMPETING INTERESTS: The study is part of the project CLEAR (Climate change, Environmental contaminants and Reproductive health) supported by the European Commission 7th framework program, contract no: FP7-ENV-2008-1-226217. No competing interest is declared.

Sperm chromatin integrity in DDT-exposed young men living in a malaria area in the Limpopo Province, South Africa.

BACKGROUND: There is mounting evidence that deteriorated semen quality may be associated with increased serum concentration of 1,1,1-trichloro-2,2-bis(chlorodiphenyl)ethane (DDT) and its metabolites. The problem is exacerbated in situations where DDT is the only resource available to control malaria mosquitoes and DDT metabolite plasma concentration can reach 1000-fold the level found in other populations. There are limited and contradictory epidemiological data on whether DDT/dichlorodiphenyl-dichloroethylene (DDE) can also damage sperm DNA. Therefore, there is a need to investigate the possible adverse effects on human sperm genetic integrity in a sufficiently large study population with adequate exposure contrasts, especially in the high exposure range. METHODS: We conducted a cross-sectional study, recruiting 209 young males from three communities in an endemic malaria area where DDT is sprayed annually. Blood plasma p,p'-DDT and its metabolite p,p'-DDE levels were measured and expressed as lipid adjusted p,p'-DDT and p,p'-DDE values. The sperm chromatin structure assay and Aniline Blue test were used to assess sperm DNA/chromatin integrity. RESULTS: The lipid adjusted p,p'-DDT mean (+/-SD) and median concentrations were 109.2 (+/-106.6) and 83.9 microg/g, respectively; and the lipid adjusted p,p'-DDE mean (+/-SD) and median concentrations were 246.2 (+/-218.5) and 177.8 microg/g, respectively. The results point to a weak association between DDT/DDE plasma concentration and the incidence of sperm with chromatin defects. CONCLUSIONS: The results suggest that non-occupational environmental DDT exposure may have a negative impact on sperm chromatin integrity in young South African males.

Use of hematopoietic cytokines to accelerate the recovery of the immune system in irradiated mice.

In our previous studies aimed at designing appropriate strategies to accelerate recovery of the immune system after irradiation, we found that the hematopoietic cytokine recombinant murine (rmu) interleukin (IL)-3 was able to induce differentiation and growth of thymocytes and splenic T and B lymphocytes in mice exposed to x-rays (200-500 cGy). The recovery, however, was complete at 7 days only after a dose of 200 cGy, whereas 2, 3, and 4 weeks were necessary to achieve full recovery after 300, 400, and 500 cGy, respectively. These studies were extended to investigate the effects of another hematopoietic cytokine, recombinant human (rhu) IL-11, a bone marrow stromal-derived cytokine, administered together with IL-3 to irradiated mice. The synergistic effect of the two cytokines was evident when relatively small doses of rhu IL-11 were injected with an optimal dose of rmu IL-3.

Testicular cancer and sperm DNA damage: short- and long-term effects of antineoplastic treatment.

The aim of this study was to investigate sperm DNA damage induced by chemo- and radiotherapy in patients with testicular cancer to provide data on the extent and persistence of nuclear damage that might affect individual reproductive potential. We evaluated pre- and post-antineoplastic treatment sperm DNA integrity, expressed as DNA Fragmentation Index (DFI), in a large caseload of testicular cancer patients by sperm chromatin structure assay. The mean total DFI for all patients at T0 was 18.0 ± 12.5%. Sperm chromatin profile was markedly impaired at T3 (27.7 ± 17.4%) and T6 (23.2 ± 15.3%), improving considerably at T12 and T24 (14.0 ± 8.9% and 14.4 ± 10.3%). After chemotherapy, we found a marked increase in DFI at T3 and T6 and a significant reduction at T12 and T24 in comparison with the baseline. In contrast, DFI increased at T3 and T6 after radiotherapy but the subsequent reduction was far less marked, reaching baseline values at T12 and T24. Finally, post-treatment DNA damage was not age or histotype dependent, but was more marked in the advanced stage of cancer. In this study, we showed that the chromatin profile may be affected in the months immediately following the end of the treatment, improving after 12-24 months. Our results thus indicate that post-treatment DNA damage is influenced both by the type and intensity of the therapy and by the pathological and clinical stage of the disease.

Differential effects of gonadectomy on thymic stromal cells in promoting T cell differentiation in mice.

Twenty-six week-old BDF1 mice were gonadectomized and grafted with thymus from irradiated (8.5 Gy) newborn, 6-week-old, or 26-week-old mice. One month later, grafted thymuses were recovered and examined in terms of thymocyte numbers, subpopulations and proliferative responses to Concananavlin A (Con A). The growth of the irradiated thymus was significantly higher in gonadectomized (Gx) than in sham-operated (Sham) mice and the magnitude of thymic growth was apparently age-dependent, as it was greater for newborns than for older mice. Con A response of thymocytes was also significantly higher in Gx mice than in Sham mice, and the magnitude of the response declined with advancing age of the thymus donors. Flow cytometric analysis revealed that a significant increase in the percentage of CD4+CD8- was observed in thymus grafts showing high Con A responses. However, this effect of Gx on the thymus graft was dependent on age of the thymus donor. Namely, newborn thymus grafts could grow equally well in both Gx and Sham recipients, whereas thymus grafts from 6- and 26-week-old mice could grow well only in Gx, but not in Sham recipients. The number of thymocytes was comparable in thymus grafts from 6- and 26-week-old mice, but the proliferative response to Con A was higher in the former than in the latter graft. Collectively, Gx appeared to promote immigration of thymocyte precursors into the thymus and to enhance proliferation and differentiation of thymocytes towards CD4+CD8- T cells, in an age-related manner.

Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation.

To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.

A laser assisted deposition technique suitable for the fabrication of biosensors and molecular electronic devices.

The recently developed technique of laser induced plasma deposition is used to obtain layers of different protein molecules on a substrate. The biological activity of the deposits is checked and micropatterning of active antibodies is achieved.

Sperm chromatin damage impairs human fertility. The Danish First Pregnancy Planner Study Team.

OBJECTIVE: To examine the relationship between sperm chromatin defects, evaluated by the flow cytometric (FCM) sperm chromatin structure assay (SCSA), and the probability of a pregnancy in a menstrual cycle (fecundability). DESIGN: Follow-up study. SETTING: The Section of Toxicology and Biomedical Sciences, ENEA Casaccia, Rome, Italy, and the Department of Occupational Medicine, Aarhus University Hospital, Aarhus, Denmark. PATIENT(S): Two hundred fifteen Danish first pregnancy planners with no previous knowledge of their fertility capability. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen samples were collected at enrollment to measure semen volume, sperm concentration, motility, and morphology (by microscopy), as well as chromatin susceptibility to in situ, acid-induced partial denaturation by the FCM SCSA. Time to pregnancy was evaluated during a 2-year follow-up period. Demographic, medical, reproductive, occupational, and lifestyle data were collected by questionnaire. Fecundability was correlated with SCSA-derived parameters. RESULT(S): Fecundability declines as a function of the percentage of sperm with abnormal chromatin and becomes small when aberrant cells are >40%. CONCLUSION(S): Optimal sperm chromatin packaging seems necessary for full expression of the male fertility potential. The SCSA emerged as a predictor of the probability to conceive in this population-based study.

Diepoxybutane cytotoxicity on mouse germ cells is enhanced by in vivo glutathione depletion: a flow cytometric approach.

Diepoxybutane is one of the key metabolites of butadiene, a compound of high environmental and occupational concern. The effects of diepoxybutane on mouse reproductive cells have been previously characterized by flow cytometry demonstrating a specific, dose-dependent cytotoxicity for differentiating spermatogonia. It is known that butadiene epoxides, deriving from butadiene bioactivation by cytochrome P450-monooxygenase systems, can be enzymatically conjugated to glutathione by glutathione S-transferases. In this paper, we tested the hypothesis whether a pretreatment with phorone, a well-known intracellular glutathione depleter, would enhance the germ cell cytotoxicity of diepoxybutane. Results were consistent with an active role played in vivo by the glutathione-detoxifying system, as diepoxybutane cytotoxicity was increased after chemically induced reduction of glutathione concentration.

Analysis of DNA oxidative damage related to cell proliferation.

In vivo and in vitro cell populations exhibit a different sensitivity and a heterogeneous response to many genotoxic agents. Several studies have been carried out to evaluate the possibility that the different sensitivity of the cells is related to their proliferative status. In this study, the sensitivity of proliferating (P) and quiescent (Q) C3H10T1/2 cells to oxidative damage and their repair capability has been investigated by single cell gel electrophoresis (SCGE) and micronucleus test. Furthermore the possibility to simultaneously detect DNA damage and cell cycle position has been evaluated. Our results showed a dose-related increase of DNA damage in exponential and plateau phase cells treated with hydrogen peroxide (doses ranging between 2.5 and 100 microM). DNA damage was almost completely repaired within 2 h after treatment in both culture conditions. The percentage of cells in the various phases of the cell cycle has been determined by comet assay and by flow cytometry, and a good agreement between the results of the two techniques was found. Untreated exponentially growing cells in G1 phase showed a lower tail moment than S and G2/M cells. The same cell cycle dependence was evidenced in cells treated with low doses of H(2)O(2), while, at the higher doses, all cells showed a similar level of damage. These results confirm the sensitivity of the Comet Assay in assessing DNA damage, and support its usefulness in evaluating cell cycle-related differential sensitivity to genotoxic agents.

Reproductive toxicity of 1,3-butadiene in the mouse: cytogenetic analysis of chromosome aberrations in first-cleavage embryos and flow cytometric evaluation of spermatogonial cell killing.

Reproductive effects of 1,3 butadiene inhalation have been evaluated in male mice by reduction of post-meiotic germ cells, alteration of sperm chromatin structure and transmission of chromosome aberrations to one-cell embryos. Animals were exposed for 5 consecutive days for 6 h per day to butadiene concentrations of 130, 500 or 1300 ppm. The testicular fraction of post-meiotic germ cells was measured by flow cytometric analysis on the basis of their DNA content. Round spermatids were discriminated from mature, elongated spermatids by their different degree of chromatin condensation. Butadiene-induced cytotoxic effects on differentiating spermatogonia were shown by a concentration-dependent decrease of round spermatids occurring 21 days after chemical exposure, confirmed by a similar decrease of elongated spermatids measured in testes sampled 7 days later. Statistically significant effects were seen already at 130 ppm. An incomplete repopulation of the elongated spermatid compartment observed 35 days after exposure to 1300 ppm suggested that, at the highest concentration tested, butadiene toxicity extended to stem cells. Alterations of sperm chromatin were revealed by its increased sensitivity to acidic denaturation in situ. The percentage of abnormal sperm was significantly increased after butadiene exposure of differentiating spermatogonia and spermatocytes. This suggested the induction of persistent effects interfering with chromatin remodelling during spermiogenesis. Chromosome-type structural aberrations were significantly elevated in first-cleavage embryos conceived by males mated during the first and second week after the end of exposure. The lowest effective tested concentration was 500 ppm, the same reported for dominant lethal induction under identical exposure conditions. As in the dominant lethal assay, the effect of this dose was confined to exposed sperm, while both sperm and late spermatids were affected by the inhalation of 1300 ppm. A quantitative comparison between the effects induced by intraperitoneal injections of diepoxybutane or butadiene inhalations suggested that other reactive intermediates, in addition to diepoxybutane, might contribute to mediate butadiene-induced reproductive toxicity.

Acrylamide-induced chromosomal damage in male mouse germ cells detected by cytogenetic analysis of one-cell zygotes.

Within a project coordinated by the Commission of the European Communities for the detection of germ cell mutagens, the cytogenetic analysis of first-cleavage metaphases was carried out to detect chromosomal damage induced by acrylamide (AA) in meiotic and postmeiotic stages of mouse spermatogenesis. Male mice were intraperitoneally injected with single acute doses of 75 or 125 mg/kg or treated with five daily injections of 50 mg/kg and mated either 7 or 28 days after the end of treatment. Chromosomal aberrations were scored in C-banded metaphases prepared from one-cell zygotes by a mass harvest technique. AA treatment of late spermatids-spermatozoa resulted in significant increases of structural aberrations at all doses tested. The data could be fitted to a curvilinear regression and a doubling dose of 23 mg/kg was calculated. The large majority of observed aberrations were of the chromosome type, including dicentrics, rings and translocations, in agreement with a mechanism of chromosomal damage mediated through the alkylation of DNA-associated protamines. Even though the frequency of aberrations 28 days after treatment was not significantly higher than the control value, the presence of multiple rearrangements in two cells suggested that AA might also have a minor effect on spermatocytes. The results of the cytogenetic analysis of first cleavage metaphases agreed well both qualitatively and quantitatively with the outcome of dominant lethal and heritable translocation assays. AA-induced cytotoxicity was monitored by flow cytometric DNA content analysis of testicular cells. By this method, a dose-dependent depletion of mature spermatids after treatment of spermatogonia and a toxic effect upon primary spermatocytes were detected.

Murine recombinant interleukin-3 induces recovery of T and B cells in irradiated mice.

Injection of murine recombinant interleukin-3 (IL-3) into (C57BL/10 x DBA/2)F1 mice, sublethally irradiated with 300 cGy and killed 14 days later, induced in the thymus recovery of the cell number and mitotic responsiveness to concanavalin A (Con A), as well as an increase in number of double-negative CD4-CD8-, double-positive CD4+ CD8+, and single-positive CD4+CD8- and CD4-CD8+ cells. Also in the spleen, the cell count and mitotic responsiveness to Con A and lipopolysaccharide were increased to normal levels by IL-3 treatment. If the assays were performed 21 or 28 days after irradiation, IL-3 treatment was able to restore thymus and spleen cell counts as well as T- and B-cell mitotic responsiveness, even when mice were exposed to 400 or 500 cGy, respectively. These results altogether indicate that IL-3 induces differentiation and growth of thymocytes and recovery of T- and B-cell functions in mice exposed to sublethal irradiation.

Effect of recombinant IL-3 on lymphocyte populations in irradiated mice.

Administration of murine recombinant interleukin 3 (IL-3) to sublethally irradiated mice induced in the thymus recovery of the cell count and mitotic responsiveness to Con A, as well as a decrease in CD4-CD8- cells concomitant with an increase in single positive cells. Also in the spleen, the cell count and mitotic responsiveness to Con A and lipopolysaccharide (LPS) were recovered by IL-3 treatment. These findings show that IL-3 induces differentiation and growth of thymocytes and recovery of T and B cell functions in sublethally irradiated mice.

Flow cytometric assessment of trophosphamide toxicity on mouse spermatogenesis.

The effects of trophosphamide on mouse reproductive cells have been investigated by flow cytometric analysis of testicular cell populations and alterations of sperm chromatin structure. Mice were treated with single intraperitoneal injections of TP, the doses ranging between 50 and 150 mg/kg, and were killed after 7, 14, 21, 28, 35, or 49 days. Dose-dependent reductions of tetraploid cells, round spermatids, and elongated spermatids were detected at 7, 21, and 28 days, respectively, reflecting cytotoxic damage to the differentiating spermatogonia compartment. The dose necessary to reduce the number of differentiating spermatogonia to half the control value was approximately 70 mg/kg. Stem cells were not affected by this treatment, and the normal spermatogenic process was restored after 7 weeks. In addition, cauda epididymal sperm were analyzed by the sperm chromatin structure assay, a flow cytometric measurement of the susceptibility of the sperm nuclear DNA to in situ acid denaturation; a statistically significant increase of sperm with altered chromatin structure was detected after a TP treatment of 150 mg/kg. Together with previous findings published in the literature, where the same doses induced heritable genetic damage, this study demonstrates a marked adverse cytotoxic effect of TP on the male reproductive integrity. All this information should be taken into consideration when TP is used in chemotherapeutic regimens.

IL-11 synergizes with IL-3 in promoting the recovery of the immune system after irradiation.

In our previous studies aiming at the design of appropriate strategies to accelerate the recovery of the immune system after irradiation, we found that recombinant murine (rmu) IL-3 treatment induces differentiation and growth of thymocytes and splenic T and B lymphocytes in mice exposed to X-rays (200-500 cGy). These studies were extended to investigate the effects of recombinant human (rhu) IL-11. Results indicate that rhuIL-11 is able to restore thymus and spleen cell numbers as well as T and B cell mitotic responsiveness in mice exposed to 200 cGy but not to 300 cGy. However, recovery of thymus and spleen cell numbers and functions could be accelerated also in mice exposed to higher dose if rhuIL-11 was given with rmuIL-3. Recovery was complete as soon as 7 days after irradiation. A large dose of both cytokines was explored and the synergistic effect of the two cytokines was evident when a relatively small dose of rhuIL-11 was injected with graded doses of rmuIL-3. The recovery of the immune system in irradiated mice injected with these cytokines was independent from Bcl-2 expression, suggesting that elimination of damaged cells by apoptosis is unaffected by hematopoietic cytokines.

Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay.

The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which exploits the metachromatic properties of Acridine Orange to monitor the susceptibility of sperm chromatin DNA to in-situ acid denaturation. SCSA was used to study the chromatin structure variations of human spermatozoa in semen, both before and after swim-up and after cryopreservation. Semen samples were provided by 19 healthy normozoospermic subjects attending pre-marriage checks. Each sample was divided into three aliquots: the first aliquot was evaluated without further treatment, the second underwent swim-up, and the third was stored according to standard cryopreservation techniques in liquid nitrogen at -196 degrees C. Samples were also analysed by light and fluorescence microscopy (after Acridine Orange staining to evaluate the number of green fluorescent sperm heads), and by computer-assisted semen analysis. The results showed that post-rise spermatozoa represent a subpopulation characterized by a general improvement of the morphological (reduction of the percentage of abnormal forms and heads, increase of the green head sperm percentage) and kinetic parameters. This subpopulation also exhibited improved chromatin structure properties, confirming that these cells have the best structural and functional characteristics, indicative of optimal fertilizing ability. On the other hand, overall sperm quality deteriorates after cryopreservation. When thawed spermatozoa underwent an additional swim-up round, a general improvement of nuclear maturity was seen in the post-rise spermatozoa.

The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios.

The impact of demographic, lifestyle, and seminal factors on the sperm chromatin structure assay (SCSA) parameters was evaluated in a population of 277 healthy Danish men. This cohort was established within the framework of a European Concerted Action on occupational hazards to male reproductive capability in order to examine the possible reproductive effects of exposure to styrene or pesticides. The SCSA measures the susceptibility of sperm DNA to in-situ acid-induced denaturation, by multiparameter flow cytometric analysis after staining with the DNA-specific fluorescent dye acridine orange. The green versus red bivariate cytogram patterns were quite variable among donors, showing a wide heterogeneity of sperm DNA denaturability. Nevertheless, in those cases where we had the possibility to measure two semen samples from the same donor, the cytogram pattern remained stable over time (0.64 < r < 0.78). Analysis of variance demonstrated that the SCSA results can be influenced by the age of the donor (P < 0.0001), smoking habits (P < 0.05), the presence of leukocytes and immature germ forms in the ejaculate (P < 0.0001), and the duration of sexual abstinence (P < 0.0001). Furthermore, the relationship between the SCSA data and sperm concentration, morphology, and vitality was weak (-0.22 < r < -0.46). Therefore, the SCSA provides independent and complementary measurements of semen quality and is thus a useful tool for epidemiological studies, but the effects of some confounders should be accounted for in the survey design and analysis.