[Focus on the endocannabinoid system and the reprotoxicity of marijuana in female users]. / Focus sur le système endocannabinoïde et la reprotoxicité du cannabis chez la femme à l'heure du débat sur sa dépénalisation en France.

Among recreative compounds, marijuana is the most used worldwide. Delta9THC binding on brain endocannabinoid receptors drives its psychotropic effects. The endocannabinoid system (ECS) is an endogenous neurohormonal system essential for homeostasis composed of ligands, metabolic enzymes and at least 2 receptors discovered to date. In female reproduction, the ECS regulates the hypothalamic-pituitary axis and many steps of the reproduction process, such as ovulation, tubal transportation and trophoblast implantation. Delta9THC can cross the placental barrier and bind to the fetal endocannabinoid system. In humans, fetal and obstetrical consequences of marijuana use during pregnancy are intrauterine growth restriction and preterm delivery. In the light of legalization projects currently reviewed in several western countries, further research should be conducted to improve knowledge on maternal, fetal and reprotoxic consequences of marijuana use during reproductive age and pregnancy.

Very low concentration of cerium dioxide nanoparticles induce DNA damage, but no loss of vitality, in human spermatozoa.

Cerium dioxide nanoparticles (CeO2NP) are widely used for industrial purposes, as in diesel, paint, wood stain and as potential therapeutic applications. The Organization for Economic Cooperation and Development included CeO2NP in the priority list of nanomaterials requiring urgent evaluation. As metal nanoparticles can cross the blood-testis barrier, CeO2NP could interact with spermatozoa. The genotoxicity of CeO2NP was demonstrated in vitro on human cell lines and mouse gametes. However, the effects of CeO2NP on human spermatozoa DNA remain unknown. We showed significant DNA damage induced in vitro by CeO2NP on human spermatozoa using Comet assay. The genotoxicity was inversely proportional to the concentration (0.01 to 10 mg·L-1). TEM showed no internalization of CeO2NP into the spermatozoa. This study shows for the first time that in vitro exposure to very low concentrations of cerium dioxide nanoparticles can induce significant DNA damage in human spermatozoa. These results add new and important insights regarding the reproductive toxicity of priority nanomaterials, which require urgent evaluation.

Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos.

Our objective was to optimize the CA technique on mammal embryos. MATERIALS AND METHODS: 1000 frozen 2-cell embryos from B6CBA mice were used. Based on a literature review, and after checking post-thaw embryo viability, the main outcome measures included: 1) comparison of the embryo recovery rate between 2 CA protocols (2 agarose layers and 3 agarose layers); 2) comparison of DNA damage by the CA on embryos with (ZP+) and without (ZP-) zona pellucida; and 3) comparison of DNA damage in embryos exposed to 2 genotoxic agents (H2O2 and simulated sunlight irradiation (SSI)). DNA damage was quantified by the % tail DNA. RESULTS: 1) The recovery rate was 3,3% (n=5/150) with the 2 agarose layers protocol and 71,3% (n=266/371) with the 3 agarose layers protocol. 2) DNA damage did not differ statistically significantly between ZP- and ZP+ embryos (12.60±2.53% Tail DNA vs 11.04±1.50 (p=0.583) for the control group and 49.23±4.16 vs 41.13±4.31 (p=0.182) for the H2O2 group); 3) H2O2 and SSI induced a statistically significant increase in DNA damage compared with the control group (41.13±4.31% Tail DNA, 36.33±3.02 and 11.04±1.50 (p<0.0001)). The CA on mammal embryos was optimized by using thawed embryos, by avoiding ZP removal and by the adjunction of a third agarose layer.

Pioglitazone in adult rats reverses immediate postnatal overfeeding-induced metabolic, hormonal, and inflammatory alterations.

Immediate postnatal overfeeding in rats, obtained by reducing the litter size, results in early-onset obesity. Such experimental paradigm programs overweight, insulin resistance, dyslipidemia, increased adipose glucocorticoid metabolism [up-regulation of glucocorticoid receptor (GR) and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1)], and overexpression of proinflammatory cytokines in mesenteric adipose tissue (MAT) in adulthood. We studied the effects of pioglitazone, a PPARγ agonist, treatment on the above-mentioned overfeeding-induced alterations. Nine-month-old rats normofed or overfed during the immediate postnatal period were given pioglitazone (3 mg/kg/day) for 6 weeks. Pioglitazone stimulated weight gain and induced a redistribution of adipose tissue toward epididymal location with enhanced plasma adiponectin. Treatment normalized postnatal overfeeding-induced metabolic alterations (increased fasting insulinemia and free fatty acids) and mesenteric overexpression of GR, 11ß-HSD11, CD 68, and proinflammatory cytokines mRNAs, including plasminogen-activator inhibitor type 1. Mesenteric GR mRNA levels correlated positively with mesenteric proinflammatory cytokines mRNA concentrations. In vitro incubation of MAT obtained from overfed rats demonstrated that pioglitazone induced a down-regulation of GR gene expression and normalized glucocorticoid-induced stimulation of 11ß-HSD1 and plasminogen-activator inhibitor type 1 mRNAs. Our data show for the first time that the metabolic, endocrine, and inflammatory alterations induced by early-onset postnatal obesity can be reversed by pioglitazone at the adulthood. They demonstrate that pioglitazone, in addition to its well-established effect on adipose tissue redistribution and adiponectin secretion, reverses programing-induced adipose GR, 11ß-HSD1, and proinflammatory cytokines overexpression, possibly through a GR-dependent mechanism.

A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus-oocyte complexes.

Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original-CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus-oocyte complexes (COC-CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC-CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC-CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC-CA is less time-consuming and simplifies the protocol of the original-CA, because COC-CA is easier to perform without the washing-out procedure.

In vivo exposure to benzo(a)pyrene induces significant DNA damage in mouse oocytes and cumulus cells.

STUDY QUESTION: Does in vivo exposure to benzo(a)pyrene (BaP) induce DNA damage in oocytes and cumulus cells (CCs) in mice? SUMMARY ANSWER: Significant increases in DNA strand breaks in oocytes and CCs and in BaP-induced DNA adducts in CCs were detected in exposed mice compared with controls. WHAT IS KNOWN ALREADY: BaP has well-known mutagenic and carcinogenic effects on somatic cells, and is also registered as potential reproductive toxicant by several environmental protection agencies. It has been shown to cause a significant increase in DNA adducts in ovarian tissues; however, to our knowledge, the genotoxic effects of BaP on oocytes and CCs have not been studied to date. STUDY DESIGN, SIZE, DURATION: Female CD1 mice were exposed to BaP via the oral administration of a single dose of 13 mg/kg body weight (bw); matched controls were exposed to the vehicle only (soya oil). A total of 15 groups of 6 mice (exposed or controls) were sacrificed 2, 4, 6, 15 or 22 days after BaP exposure, and after collection of oviducts, the oocyte-CC complexes (COC) were released. PARTICIPANTS/MATERIALS, SETTING, METHODS: The alkaline comet assay was used to quantify the DNA breaks in oocytes and CCs; DNA damage was expressed as the Olive Tail Moment (OTM). Immunofluorescent staining was used to quantify BaP-induced DNA adducts in CCs. Fluorescence was expressed as the average grey value (AGVA; arbitrary units). The differences between the exposed and control groups were assessed using analysis of variance (ANOVA) and the non-parametric Mann-Whitney test. MAIN RESULTS AND THE ROLE OF CHANCE: Higher levels of DNA damage were observed in the oocytes and CCs of BaP-exposed mice than in those of vehicle controls. Significant increases in OTM (mean ± SE) were detected in (i) oocytes from females exposed for 4 (10.5 ± 0.9 versus 3.1 ± 0.4, P < 0.0001) or 6 days before collection (15.6 ± 2.0 versus 3.6 ± 0.9, P < 0.0001) and (ii) CCs from females exposed 2 (6.4 ± 0.6 versus 2.1 ± 0.2, P < 0.0001), 4 (7.8 ± 0.4 versus 2.4 ± 0.1, P < 0.0001) or 6 days before collection (7.3 ± 0.3 versus 3.2 ± 0.5, P < 0.0001) compared with controls. A significant increase in benzo(a)pyrene-7,8-9,10 diol epoxide (BPDE)-DNA adducts and higher AGVA (mean ± SE) scores were observed in CCs from females exposed 2 (6.1 ± 0.3 versus 3.6 ± 0.5, P < 0.0001), 4 (7.5 ± 0.1 versus 3.4 ± 0.1, P < 0.0001) or 6 days before collection (11.6 ± 0.4 versus 3.7 ± 0.1, P < 0.0001) compared with control mice. LIMITATIONS, REASONS FOR CAUTION: Mice were given one treatment via the oral route because this dose and mode of administration have been shown to induce detectable BPDE-DNA adduct levels in mouse organs and sperm cells. Additional data are needed to assess DNA damage in oocytes and CCs after chronic exposure to BaP in vivo. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study examining the in vivo genotoxicity of BaP in oocytes and CCs. We observed significant DNA damage in the oocytes and CCs of mice after acute BaP exposure. BPDE-DNA adducts result directly from BaP metabolism while DNA breaks could result mainly from BPDE-DNA adduct excision and repair and/or through direct genotoxicity from increased reactive oxygen species. These results add new and important insights regarding the recently suggested toxicity of chronic BaP exposure in the ovary. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant (93-CPQ 2012-05) from the DIRRECTE, Provence Alpes Côte d'Azur, France. None of the authors have any conflict of interest to declare.

Expression and nutritional regulation of the (pro)renin receptor in rat visceral adipose tissue.

BACKGROUND: Early life nutritional environment plays an important role in the development of visceral adipose tissue and interacts with nutritional regulations in adulthood, leading to metabolic dysregulations. AIM: We hypothesized that the renin-angiotensin system may play a role in the programming-induced development of visceral adipose tissue. MATERIAL AND METHODS: We studied, using a model of programming of overweight and glucose intolerance, obtained by post-natal overfeeding with consecutive highfat diet, the status of plasma renin activity and mesenteric adipose renin-angiotensin system, including the recently identified (pro)renin receptor, in adult rats. RESULTS: Post-natal overfeeding or high-fat feeding lead to overweight with increased visceral fat mass and adipocytes surface. When both paradigms were associated, adipocytes surface showed a disproportionate increase. A strong immunoreactivity for (pro)renin receptor was found in stromal cells. Plasma renin activity increased in programmed animals whereas (pro)renin receptor expressing cells density was stimulated by high-fat diet. There was a positive, linear relationship between plasma renin activity and (pro)renin receptor expressing cells density and adipocytes surface. CONCLUSIONS: Our experiments demonstrate that association of post-natal overfeeding and high-fat diet increased plasma renin activity and adipose (pro)renin receptor expression. Such phenomenon could explain, at least in part, the associated disproportionate adipocyte hypertrophy and its accompanying increased glucose intolerance.

[Sex chromosomes and meiosis]. / Chromosomes sexuels et méiose.

Sex chromosome behaviour fundamentally differs between male and female meiosis. In oocyte, X chromosomes synapse giving a XX bivalent which is not recognizable in their morphology and behaviour from autosomal bivalents. In human male, X and Y chromosomes differ from one another in their morphology and their genetic content, leading to a limited pairing and preventing genetic recombination, excepted in homologous region PAR1. During pachytene stage of the first meiotic prophase, X and Y chromosomes undergo a progressive condensation and form a transcriptionally silenced peripheral XY body. The condensation of the XY bivalent during pachytene stage led us to describe four pachytene substages and to localize the pachytene checkpoint between substages 2 and 3. We also defined the pachytene index (PI=P1+P2/P1+P2+P3+P4) which is always less than 0.50 in normal meiosis. XY body undergoes decondensation at diplotene stage, but transcriptional inactivation of the two sex chromosomes or Meiotic Sex Chromosome Inactivation (MSCI) persists through to the end of spermatogenesis. Sex chromosome inactivation involves several proteins, some of them were now identified. Two isoforms of the HP1 protein, HP1beta and HP1gamma, are involved in the facultative heterochromatinization of the XY body, but the initiation of this process involves the phosphorylation of the protein H2AX by the kinase ATR whose recruitment depends on BRCA1. Extensive researches on the inactivation of the sex chromosomes during male meiosis will allow to a better understanding of some male infertilities.