Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species.

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

Sperm chromatin structure assay parameters measured after density gradient centrifugation are not predictive for the outcome of ART.

BACKGROUND: The sperm chromatin structure assay (SCSA) parameter DNA fragmentation index (DFI) has been shown to predict in vivo and in vitro fertility. So far most SCSA studies have been based on SCSA analysis performed on neat semen. The aim of this study is to assess whether SCSA analysis of sperm prepared by density gradient centrifugation (DGC) could add more information in regard to the prediction of treatment outcome. METHODS: The study included 510 assisted reproductive technique (ART) cycles. SCSA was performed in neat semen and post DGC. SCSA results were expressed in terms of DFI and high DNA stainability (HDS) cell fractions. The outcome parameter was clinical pregnancy (CP). RESULTS: Scatter-plot diagrams demonstrated that for DGC samples, no DFI cut-off values could be set for in vivo or in vitro fertility. In intrauterine insemination, IVF and ICSI groups the mean difference (95% CI) in DFI post DGC between those who achieved CP and those who did not was 0.2% (-1.7 to 2.0%), 0.4% (-1.9 to 2.8%) and 1.3% (-3.1 to 5.9%), respectively, none of these being statistically significant. The corresponding differences for HDS were 0.1% (-1.3 to 1.5%), 0.1% (-0.7 to 0.9%) and 0.6% (-1.6 to 2.7%), respectively (all P-values >0.6). CONCLUSIONS: SCSA performed in semen prepared by DGC cannot predict the outcome of ART.

Xenobiotic activity in serum and sperm chromatin integrity in European and inuit populations.

Lipophilic persistent organic pollutants (POPs) are ubiquitous in the environment and suspected to interfere with hormone activities and reproduction. In previous studies we demonstrated that POP exposure can affect sperm DNA integrity and differences between Inuits and Europeans in sperm DNA integrity and xenobiotic activity were observed. The aim of this study was to investigate possible relations between human sperm chromatin integrity and the xenobiotic serum activity of lipophilic POPs assessed as effects on the estrogen (ER), androgen (AR), and/or aryl hydrocarbon (AhR) receptors. Human sperm chromatin integrity was assessed as DNA fragmentation index (%DFI) and high DNA stainability (%HDS) using the flow cytometric sperm chromatin structure assay (SCSA). Xenobiotic receptor activities were determined using chemically activated luciferase gene expression (CALUX) assay. The study included 53 Greenlandic Inuits and 247 Europeans (Sweden, Warsaw (Poland) and Kharkiv (Ukraine)). A heterogeneous pattern of correlations was found. For Inuits, ER and AhR activities and %DFI were inversely correlated, whereas a positive correlation between AR activity and %DFI was found for Europeans. In contrast, no correlation between receptor activities and %HDS was observed for Inuits but for Europeans positive and negative correlations were observed between ER and AR activities and %HDS, respectively. We suggest that the different patterns of xenobiotic serum activities, in combination with diet associated factors and/or genetics, might be connected to the observed differences in sperm chromatin integrity between the Inuits and Europeans.

Lindane may modulate the female reproductive development through the interaction with ER-beta: an in vivo-in vitro approach.

Lindane (gamma-HCH) is a persistent environmental pollutant that may act as endocrine disrupter, affecting the nervous, immune and reproductive system, possibly through endocrine-mediated mechanisms. Since both estrogen receptors (ER-alpha and -beta) have shown to be target for endocrine disruption, we investigated the role of gamma-HCH on the development of female reproductive system. For an in vivo evaluation of gamma-HCH effects during prenatal period, pregnant CD1 mice were treated p.o. on gestational days 9-16 with 15 mg/kg bw/day of gamma-HCH and vehicle. The in vivo findings in treated F1 pups - in the absence of signs of systemic toxicity - included increase in the absolute and relative and absolute uterus weight revealed on post-natal day 22, earlier vaginal patency and reduced diameters of primary oocytes at fully sexual maturity. No effects on steroid hormone metabolism (aromatase, testosterone catabolism) were observed. Thus, gamma-HCH elicited subtle effects on female reproductive development likely mediated by ER-beta-mediated pathway(s), without a concurrent impairment of steroid hormone metabolism. Furthermore, to verify whether the endocrine interference of gamma-HCH is attributable to stimulation of ER-beta-mediated pathway(s), its effect has been evaluated in vitro on a cell line, LNCaP, expressing only functional ER-beta. In vitro treatments revealed a concentration-related effect on LNCaP cell viability and proliferation. Significantly, the contemporary addition of a pure anti-estrogen, the ER antagonist ICI 182,780, completely reversed gamma-HCH effects indicating an ER-beta-mediated action. Our findings indicate that gamma-HCH may act as endocrine disruptor during the female reproductive system development and ER-beta as a potential target for this compound and other endocrine disrupting chemicals as well.

Exposure to PCBs and p,p'-DDE and human sperm chromatin integrity.

Persistent organochlorine pollutants (POPs) such as polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (p,p'-DDE), the major metabolite of dichlorodiphenyltrichloroethane (DDT), are stable lipophilic compounds widely found in the environment and in the general population. They can enter the food chain, and their negative impact on male reproduction is currently under active scrutiny. To explore the hypothesis that environmental exposure to these compounds is associated with altered sperm chromatin structure integrity in human sperm, we conducted a study of 176 Swedish fishermen (with low and high consumption of fatty fish, a very important exposure source of POPs). We determined serum levels of 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and p,p'-DDE, and we used the sperm chromatin structure assay (SCSA) to assess sperm DNA/chromatin integrity. When CB-153 serum levels (individual dose range, 39-1,460 ng/g lipid) were categorized into equally sized quintiles, we found an association with the DNA fragmentation index (%DFI). A significantly lower %DFI was found in the lowest CB-153 quintile (< 113 ng/g lipid) compared with the other quintiles; there was a similar tendency, although not statistically significant, between %DFI and p,p'-DDE. These results suggest that POP exposure may have a slight negative impact on human sperm chromatin integrity.

Flow cytometry applications in the evaluation of sperm quality: semen analysis, sperm function and DNA integrity.

Flow cytometry (FCM) has been extensively used to study mammalian sperm in the areas of reproductive toxicology (to monitor effects from environmental, occupational and therapeutic exposures), veterinary science (to preselect the gender of offspring by sorting X- and Y-chromosome-bearing sperm) and clinical andrology (to assess individual fertility potential). Using FCM, a variety of sperm features can now be rapidly measured on a cell-by-cell basis such as sperm count, viability, acrosomal integrity, mitochondrial function and DNA integrity; the last one is involved in postfertilization failure and embryo toxicity. It is foreseen that only a multiplex approach, which includes FCM assays together with the new genomics/proteomics methods, could increase the predictive power of fertility status and help identify susceptible subpopulations of men at risk for infertility, spontaneous abortions and birth defects.

Lonidamine transiently affects spermatogenesis in pubertal CD1 mice.

Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a well-known antispermatogenic drug, was studied for the first time in pubertal mice to assess its possible effects on spermatogenesis. Male CD1 mice were orally treated on Postnatal Day (PND) 28 with a single dose of LND (100 mg/kg body weight) and sacrificed on PND30, PND42, PND74 and PND123. On PND30 (48 h after dosing), severe testicular effects were evidenced in the treated animals: (a) reduction of the testicular sperm head concentration (approximately 50% of the control value); (b) changes in the spermatogenic cell type distribution (mild decrease of the elongated spermatids and S-phase cells fractions); and (c) morphological alterations of the Sertoli cell cytoplasm and germ cell exfoliation. These changes were recovered in adulthood, on PND74 and PND123. However, no effect on sperm chromatin structure was detected on the epididymal sperm of mature mice by sperm chromatin structure assay, suggesting that LND did not interfere with the process of chromatin reorganization and DNA packaging.

Long-term effects of lonidamine on mouse testes.

Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is a well-known antispermatogenic drug. The aim of this study was to identify its possible long-term sequelae on the reproductive system of mice as compared with rats, where most data have been obtained until now. Sexually mature CD1 male mice were administered a single dose of LND (200 mg/kg bw by gavage) and killed 24 and 48 h, 6 days and 2, 4 and 8 weeks after the treatment. Testes were collected, weighed and (1) fixed in Bouin's solution for histological analysis or (2) reduced to monocellular suspensions and ethanol fixed to undergo flow cytometry (FCM) DNA content analysis. No effect on body weight and/or food consumption was observed in the treated group in comparison with the control group. Testicular weight was significantly reduced 24 h after the treatment. Reduced seminiferous epithelium with a progressive lack of intercellular cohesion and marked depletion of spermatids, infiltration of granulocytes, desquamation into the tubular lumen and increased intertubular spaces were present by 24 h after the treatment and persisted to a marked degree at 48 h, 6 days and 2 and 4 weeks up to a marked degeneration of tubular structures with absence of spermatogenesis. The same effects, albeit with a moderate severity, were still present 8 weeks after the treatment. As also detected by FCM, primary spermatocytes appeared to be the main cellular target. Sertoli and Leydig cells were remarkably spared. The histological findings are consistent with those previously observed in rats and point out that testicular damage may persist for several weeks after a single-dose administration. Findings are discussed in comparison with testicular toxicity elicited by other xenobiotics.

Histological and histomorphometric alterations in thyroid and adrenals of CD rat pups exposed in utero to methyl thiophanate.

Pregnant CD rats were treated with an initial dose of 0, 310 or 560 mg/kg bw per day of the fungicide methyl thiophanate (MT) on gestational days 10-14, corresponding to formation of thyroid and adrenal primordia; newborns were sacrificed on postnatal days (PNDs) 10 and 23. No apparent maternal toxicity and no effects on litter size, viability or weight gain were present. Delayed ear pinna detachment and eye opening were present at top dose level. Thyroid histology showed increased irregular nuclei and/or mitoses (PND 10-both doses), cells with necrotic or hydropic changes (PND 23-top dose). The adrenal cortex showed increased karyomegaly and hydropic degeneration (PND 23-both doses). Thyroid histomorphometry showed reduced follicular density, moderately increased follicular cell height and number of nuclei/follicle (PND 10-top dose and PND 23-both doses), suggesting retarded follicular maturation. The adrenal cortex relative area was slightly decreased (PND 10-top dose and PND 23-both doses).MT may act as weak endocrine disrupter, suggesting that attention should be paid to delayed endocrine alterations elicited by agrochemicals.

Long-lasting effects of lindane on mouse spermatogenesis induced by in utero exposure.

Long-lasting effects on mouse spermatogenesis induced by prenatal exposure to the insecticide lindane have been investigated by conventional reproductive endpoints complemented by the flow cytometric (FCM) DNA content analysis of testis cells and by the Sperm Chromatin Structure Assay (SCSA). Two lindane dose levels, 15 and 25 mg/kg bw, and diethylstilboestrol (DES, 10 microg/kg bw) as positive control, were administered daily by gavage to pregnant CD1 mice on gestation days (GD) 9-16. Reproductive endpoints were evaluated on F1 male mice on postnatal day (PND) 60; additionally, animals treated with lindane 25 mg/kg per day and DES were examined on PND 100 to evaluate the possible reversibility of the effects. On PND 60, lindane and DES caused a reduction in the sperm head count and concentration, with recovery in older lindane 25 mg/kg per day animals (PND 100). By contrast, the DES group exhibited a greater reduction in the sperm head count on PND 100 than on PND 60. Changes in biochemical parameters in the testes, lactate dehydrogenase-C(4) (LDH-C(4)), and sorbitol dehydrogenase (SDH) activities, were also observed in adult treated F1 mice. Furthermore on PND 60, the FCM analysis revealed changes in the pattern of testicular germ cell distribution, especially in the haploid subcompartment, in the lindane 25 mg/kg per day group. A dose-dependent increase in chromatin abnormalities of the epididymal sperm was also shown by SCSA. These changes recovered on PND 100. Preliminary qualitative examination did not reveal any significant difference in the structure of testicular tissue; however, there were suggestions of a moderate increase in number and size of Leydig cells in both DES- and lindane-treated animals. The partial reversibility of these effects and the lack of structural modification of the testicular tissue as evidenced by histopathologic assessment suggest a functional impairment of sperm production and maturation, possibly associated with changes induced by lindane on factors affecting intratesticular steroidogenesis.