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Adverse temporal trends in human semen quality and cryptorchidism in infants have been associated with exposure to environmental chemicals (ECs) during development. Here we report that a population of breeding dogs exhibit a 26 year (1988-2014) decline in sperm quality and a concurrent increased incidence of cryptorchidism in male offspring (1995-2014). A decline in the number of males born relative to the number of females was also observed. ECs, including diethylhexyl phthalate (DEHP) and polychlorinated biphenyl 153 (PCB153), were detected in adult dog testes and commercial dog foods at concentrations reported to perturb reproductive function in other species. Testicular concentrations of DEHP and PCB153 perturbed sperm viability, motility and DNA integrity in vitro but did not affect LH stimulated testosterone secretion from adult testis explants. The direct effects of chemicals on sperm may therefore contribute to the decline in canine semen quality that parallels that reported in the human.

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Background Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown. Methods In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses. Results In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1). Conclusions Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism. Electronic supplementary material The online version of this article (doi:10.1186/s12916-014-0251-x) contains supplementary material, which is available to authorized users.

Prophase I meiosis and follicle formation are two crucial events of the ovarian development, determining for the future female fertility. Disruption of these initial steps of folliculogenesis results in incomplete sexual development and may contribute to gonadal dysgenesis, infertility or ovarian cancer. Several environmental chemicals are known to affect these developmental processes in rodents following in utero exposure or in vitro culture. However, some mechanisms governing these processes differ between species and reproductive toxicity studies in rodent models correlate poorly with the human. For this reason, sheep are an alternative animal model with many relevant similarities to human ovarian development and to human toxicokinetics. The use of low doses of environmental pollutants close to those found in human biological fluids is crucial in order to mimic the real-life exposure conditions of human gonads. Our aims were: (i) to develop a long-term in vitro ovary organotypic culture allowing both meiosis initiation/progress and follicle morphogenesis/differentiation, and (ii) to evaluate the effects of MEHP (10-6M and 10-8M), PCB 101-118 (10 6M; 10 7M), MEHP + PCB101-118 and BPA (3.10-5M; 3.10-7M), on folliculogenesis and ovary gene expression by microarray analysis. Foetal ovaries were collected at 48dpc, 90dpc and 110dpc then cultured for 21 days in presence of growth/differentiation factors. After each period of culture, per each condition, ovarian fragments were split between histological examination and transcriptome analysis. We have established a foetal ovary culture system in sheep, able to: (i) initiate and maintain meiosis in oogonia, (ii) allow follicle transition and differentiation. Using this culture system, we have shown that in vitro exposure to environmental doses of MEHP and PCB 101-118, produced very few alterations in the sheep foetal ovary transcriptome. MEHP + PCB101-118 and BPA (3.10-7M) exposure at low doses induced less than 10 transcripts differentially expressed at any developmental stage. The dysregulated transcripts were specific for each stage of ovarian development. In contrast, BPA (3.10-5M) exposure resulted in alteration of a large number of transcripts, especially during prophase I meiosis. In conclusion, while BPA (3.10-5M) exposure induced more in vitro changes to foetal ovarian transcripts that other pollutants tested, none at environmental doses (10-7M and 10-8M) produced significant alterations both in transcriptome and ovarian morphology. This research was supported by EU grant (FP7; REEF #212885).

Our previous studies of sewage sludge exposures, and the literature, highlight polychlorinated bisphenols (PCB) and diethylhexyl phthalate (DEHP) as biologically active at environmental concentrations. We have demonstrated in sheep that a real-life cocktail of environmental chemicals, containing these chemicals, preferentially accumulate in the fetus and affects the development of the ovary.We therefore investigated the effects of dietary intake of DEHP, the two PCBs and a mixture of both, on sheep female reproductive development in vivo and in vitro. Ovaries were collected from day 140 foetal sheep foetuses exposed in-utero to DEHP and/or PCBs (congeners 101+118) and also control (vehicle). 54 ovaries distributed in four groups were included in this study and were analysed by histology and by transcriptomic analysis with a dedicated sheep microarray.There were no overall significant effects of any of the exposures on fetal ovarian follicle dynamics at day 140 compared with control fetuses. In DEHP-exposed ovaries, a total of 136 genes were differentially expressed between controls and exposed fetuses (FDR 5% and a fold-change >1.5). A greater number of genes were down-regulated (106/136) as compared to those that were up-regulated (30/136). A much smaller number of genes were significantly affected by PCB exposure; only 4 genes were differentially expressed. Surprisingly, the mixture DEHP+PCBs produced fewer changes than DEHP alone: 24 genes were differentially expressed (9 up, 13 down).In parallel, we have developed a long-term (20 days) organotypic culture system to investigate the histological and molecular effects of MEHP (the main metabolite of DEHP) and PCB101 & 118 on early ovarian development. In these culture experiments, MEHP and PCB 101 & 118 at 10-6 M or a combination of both and MEHP and PCB 101-118 at 10-8 M or combination of both were tested. Three stages of ovary development were investigated: meiosis (48-70dpc in sheep), primary follicle formation (90-110dpc) and follicle differentiation (110-130dpc). Very few genes were differentially expressed between controls and PCB, MEHP and mixture exposures. There were no significant differences in morphology or in oocyte or follicle numbers.In conclusion, a larger number of effects of exposure were observed in vivo than in vitro for the same compounds. This also suggests further reasons for caution in assessing developmental consequences of exposure to ECs in vitro.

This work was supported by the European Community 's Seventh Framework Programme (FP7/2007-2013); absent

This work was supported by the EC7th Framwork Programme (FP7/2007-2013) grant agrement n° 212885; absent

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Animals and humans are chronically exposed to endocrine disrupting chemicals (EDCs) that are ubiquitous in the environment. There are strong circumstantial links between environmental EDC exposure and both declining human/wildlife reproductive health and the increasing incidence of reproductive system abnormalities. The verification of such links, however, is difficult and requires animal models exposed to 'real life', environmentally relevant concentrations/mixtures of environmental contaminants (ECs), particularly in utero, when sensitivity to EC exposure is high. The present study aimed to determine whether the foetal sheep reproductive neuroendocrine axis, particularly gondotrophin-releasing hormone (GnRH) and galaninergic systems, were affected by maternal exposure to a complex mixture of chemicals, applied to pasture, in the form of sewage sludge. Sewage sludge contains high concentrations of a spectrum of EDCs and other pollutants, relative to environmental concentrations, but is frequently recycled to land as a fertiliser. We found that foetuses exposed to the EDC mixture in utero through their mothers had lower GnRH mRNA expression in the hypothalamus and lower GnRH receptor (GnRHR) and galanin receptor (GALR) mRNA expression in the hypothalamus and pituitary gland. Strikingly, this, treatment had no significant effect on maternal GnRH or GnRHR mRNA expression, although GALR mRNA expression within the maternal hypothalamus and pituitary gland was reduced. The present study clearly demonstrates that the developing foetal neuroendocrine axis is sensitive to real-world mixtures of environmental chemicals. Given the important role of GnRH and GnRHR in the regulation of reproductive function, its known role programming role in utero, and the role of galanin in the regulation of many physiological/neuroendocrine systems, in utero changes in the activity of these systems are likely to have long-term consequences in adulthood and represent a novel pathway through which EC mixtures could perturb normal reproductive function.

National audience; Fetal liver tissue concentrations of selected pollutant classes were determined in sheep fetuses and their dams, at 110 days of gestation (term = 145d) following exposure to pasture treated with either inorganic fertiliser (Control; C) or with sewage sludge (Treated; T). In flock 1, ewes were maintained on either T or C pastures throughout their breeding lives. Ewes of flock 2 were mated 2 weeks later and exposed to the same T pastures either, throughout life up to the time of experimental mating when they were transferred to control pasture (TC), or only from mating until slaughter at 110days gestation having been on untreated pastures before mating (CT). Neither maternal nor fetal tissue concentrations of DEHP, PCB, PBDE and PAH was affected by sludge exposure in any group. At 110 days gestation, mean DEHP concentrations were similar in adult and fetal tissues. Concentrations of PCB congeners 28, 52, 101, 118, 138, 153 and 180 were generally higher in maternal tissues (P

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International audience; Environmental exposure to endocrine disrupting compounds (EDCs) during development could adversely affect reproductive function via effects on the hypothalamo-pituitary-gonadal axis (HPG). The HPG axis is regulated by many steroid sensitive inputs, including the KiSS-1 gene product kisspeptin. We have previously shown that KiSS-1 mRNA and protein are expressed within gonadotrophs in the foetal ovine pituitary and that KiSS-1 mRNA expression is decreased following gestational exposure to EDCs. The current study aimed to determine whether KiSS-1 mRNA expression is also changed in pituitary glands of adult animals exposed to EDCs in-utero. Tissue was obtained from 12 month old male and female sheep born to mothers pastured on fields fertilised with inorganic fertiliser (C) or sewage sludge (T) throughout pregnancy (n=12 per group). mRNA was extracted, reverse transcribed and KiSS-1 mRNA expression quantified by QrtPCR (relative to β-actin). Data were analysed using two-way ANOVA. Results - KiSS-1 mRNA expression was significantly (P

International audience