The negative in utero effects of bisphenol A (BPA) on female reproduction are of concern since the ovarian reserve of primordial follicles is constituted during the fetal period. This time-window is difficult to access, particularly in humans. Animal models and explant culture systems are, therefore, vital tools for investigating EDC impacts on primordial germ cells (PGCs). Here, we investigated the effects of BPA on prophase I meiosis in the fetal sheep ovary. We established an in vitro model of early gametogenesis through retinoic acid (RA)-induced differentiation of sheep PGCs that progressed through meiosis. Using this system, we demonstrated that BPA (3 ×10-7 M & 3 ×10-5 M) exposure for 20 days disrupted meiotic initiation and completion in sheep oogonia and induced transcriptomic modifications of exposed explants. After exposure to the lowest concentrations of BPA (3 ×10-7 M), only 2 probes were significantly up-regulated corresponding to NR2F1 and TMEM167A transcripts. In contrast, after exposure to 3 × 10-5 M BPA, 446 probes were deregulated, 225 were down- and 221 were up-regulated following microarray analysis. Gene Ontology (GO) annotations of differentially expressed genes revealed that pathways mainly affected were involved in cell-cycle phase transition, meiosis and spindle assembly. Differences in key gene expression within each pathway were validated by qRT-PCR. This study provides a novel model for direct examination of the molecular pathways of environmental toxicants on early female gametogenesis and novel insights into the mechanisms by which BPA affects meiosis I. BPA exposure could thereby disrupt ovarian reserve formation by inhibiting meiotic progression of oocytes I and consequently by increasing atresia of primordial follicles containing defective oocytes.

Exposure of the fetal testis to numerous individual environmental chemicals (ECs) is frequently associated with dysregulated development, leading to impaired adult reproductive competence. However, 'real-life' exposure involves complex mixtures of ECs. Here we test the consequences, for the male fetus, of exposing pregnant ewes to EC mixtures derived from pastures treated with biosolids fertiliser (processed human sewage). Fetal testes from continuously exposed ewes were either unaffected at day 80 or exhibited a reduced area of testis immunostained for CYP17A1 protein at day 140. Fetal testes from day 140 pregnant ewes that were exposed transiently for 80-day periods during early (0-80 days), mid (30-110 days), or late (60-140 days) pregnancy had fewer Sertoli cells and reduced testicular area stained for CYP17A1. Male fetuses from ewes exposed during late pregnancy also exhibited reduced fetal body, adrenal and testis mass, anogenital distance, and lowered testosterone; collectively indicative of an anti-androgenic effect. Exposure limited to early gestation induced more testis transcriptome changes than observed for continuously exposed day 140 fetuses. These data suggest that a short period of EC exposure does not allow sufficient time for the testis to adapt. Consequently, testicular transcriptomic changes induced during the first 80 days of gestation may equate with phenotypic effects observed at day 140. In contrast, relatively fewer changes in the testis transcriptome in fetuses exposed continuously to ECs throughout gestation are associated with less severe consequences. Unless corrected by or during puberty, these differential effects would predictably have adverse outcomes for adult testicular function and fertility.

L’ambition de l’Atelier de Reflexion Prospective sur l’elevage a ete de repenser la place, les roles de l’elevage et en quoi il doit se transformer pour contribuer pleinement au developpement de systemes agri-alimentaires plus durables ainsi que d’identifier des thematiques de recherches interdisciplinaires permettant d’explorer des fronts de science ou methodologiques porteurs d’avenir pour lever les verrous de connaissances. La reflexion a ete animee par un groupe de 15 chercheurs et a mobilise une centaine de chercheurs. A partir d’une analyse du contexte et des drivers d’evolution des systemes nous proposons un nouveau cadre conceptuel et des voies de progres pour penser l’elevage de demain. L’elevage doit s’inscrire dans le cadre de systemes agri-alimentaires circulaires dans lesquels il doit contribuer, au-dela d’une efficience accrue des moyens de production, a la preservation de la qualite des ressources et a la production d’une alimentation a un prix abordable. Il faut repenser les systemes pour qu’ils soient climato-intelligents et repondent aux enjeux de sante et du bien-etre des animaux et des hommes ; repenser les liens entre elevage, production vegetale et territoire pour maximiser les recyclages et repenser les liens entre elevage, transformation et consommation de ces produits de l’elevage. Les innovations doivent etre basees sur les principes de l’agro-ecologie completes par ceux de l’economie circulaire et par la mobilisation des leviers des (bio)technologies et de l’innovation organisationnelle. Le texte decrit quatre grandes priorites scientifiques avec leurs enjeux de recherche et produit des recommandations pour un plan d’action.

Background: The increased incidence of diseases, including metabolic syndrome and infertility, may be related to exposure to the mixture of chemicals, which are ubiquitous in the modern environment (environmental chemicals, ECs). Xeno-detoxification occurs within the liver which is also the source of many plasma proteins and growth factors and plays an important role in the regulation of homeostasis. Objectives: The objective of this study was to investigate the effects of ECs on aspects of liver function, in a well characterized ovine model of exposure to a real-life EC mixture. Methods: Four groups of sheep (n = 10-12/sex/treatment) were maintained long-term on control or sewage sludge-fertilized pastures: from conception to culling at 19 months of age in females and from conception to 7 months of age and thereafter in control plots until culling at 19 months of age in males. Environmental chemicals were measured in sheep livers and RNA and protein extracts were assessed for exposure markers. Liver proteins were resolved using 2D differential in-gel electrophoresis and differentially expressed protein spots were identified by liquid chromatography/tandem mass spectroscopy. Results: Higher levels of polycyclic aromatic hydrocarbons (PAHs) and lower levels of polychlorinated biphenyls (PCBs) in the livers of control males compared to control females indicated sexually dimorphic EC body burdens. Increased levels of the PAHs Benzo[a] anthracene and chrysene and reduced levels of PCB 153 and PCB 180 were observed in the livers of continuously exposed females. EC exposure affected xenobiotic and detoxification responses and the liver proteome in both sexes and included major plasma-secreted and blood proteins, and metabolic enzymes whose pathway analysis predicted dysregulation of cancer-related pathways and altered lipid dynamics. The latter were confirmed by a reduction in total lipids in female livers and up-regulation of cancerrelated transcript markers in male livers respectively by sewage sludge exposure. Conclusions: Our results demonstrate that chronic exposure to ECs causes major physiological changes in the liver, likely to affect multiple systems in the body and which may predispose individuals to increased disease risks.

The ambition of the foresight on livestock farming was to rethink the place, the roles of livestock and how it should be transformed to fully contribute to the development of more sustainable agri-food systems. It was also to identify cross-disciplinary research themes that explore promising fronts of science or methodologies to solve knowledge locks. The foresight was managed by a group of 15 researchers and mobilized a hundred researchers. Based on an analysis of the context and the drivers for the evolution of the systems, we propose a new conceptual framework to think about innovations. Livestock farming must be part of circular agri-food systems in which it must contribute, beyond an increased efficiency of production factors, to the preservation of the quality of the resources and to the production of a food at an affordable price. Systems need to be rethought to be climate-smart and to respond to the health and welfare challenges of animals and people, rethink the linkages between livestock, crop production and territories to maximize recycling and rethink livestock linkages, processing and consumption of livestock products. Innovations should be based on the principles of agro-ecology supplemented by those of the circular economy and mobilizing levers of (bio) technologies and organizational innovation. The text describes 4 major scientific priorities with their research issues and produces recommendations for an action plan.

L’ambition de l’Atelier de Réflexion Prospective sur l’élevage a été de repenser la place, les rôles de l’élevage et en quoi il doit se transformer pour contribuer pleinement au développement de systèmes agri-alimentaires plus durables ainsi que d’identifier des thématiques de recherches interdisciplinaires permettant d’explorer des fronts de science ou méthodologiques porteurs d’avenir pour lever les verrous de connaissances. La réflexion a été animée par un groupe de 15 chercheurs et en a mobilisé une centaine. À partir d’une analyse du contexte et des drivers d’évolution des systèmes nous proposons un nouveau cadre conceptuel et des voies de progrès pour penser l’élevage de demain. L’élevage doit s’inscrire dans le cadre de systèmes agri-alimentaires circulaires dans lesquels il doit contribuer, au-delà d’une efficience accrue des moyens de production, à la préservation de la qualité des ressources et à la production d’une alimentation à un prix abordable. Il faut repenser les systèmes pour qu’ils soient climato-intelligents et répondent aux enjeux de santé et du bien-être des animaux et des Hommes ; repenser les liens entre élevage, production végétale et territoire pour maximiser les recyclages et repenser les liens entre élevage, transformation et consommation des produits de l’élevage. Les innovations doivent être basées sur les principes de l’agroécologie complétés par ceux de l’économie circulaire et par la mobilisation des leviers des (bio)technologies et de l’innovation organisationnelle. Le texte décrit quatre grandes priorités scientifiques avec leurs enjeux de recherche et produit des recommandations pour un plan d’action.

Summary. Declining fertility and perturbations in reproductive development in a variety of species have been linked to exposure to endocrine disrupting chemicals (EDCs), which are ubiquitous in the environment. Ruminants are largely exposed to such chemicals in sewage sludge fertiliser widely used in animal production systems. This has been investigated experimentally through the deliberate exposure of pregnant ewes to sewage sludge fertilised pastures or control pastures treated with inorganic fertiliser containing no detectable EDCs. Perturbations in the developing fetal hypothalamic–pituitary–gonadal axis have been observed in both male and female fetuses. A sub-population of rams exposed both pre- and post-natally exhibited adversely altered testis development. Periods of developmental sensitivity to EDCs have also been identified. An in vitro culture system for ovine fetal ovaries has shown that mixtures have a greater effect than individual chemicals. The dog provides a sentinel model of human dietary exposure to chemicals in ruminant-derived food products. Evidence of altered fertility and testicular cancer, along with EDC detection in dog testes and pet foods, support this concept. Established rodent models of EDC exposure provide a means to investigate mechanisms and transgenerational effects. Overall, monitoring and in vitro studies carried out in sentinel species and on human tissues, combined with in vivo mechanistic studies carried out in ruminants and rodents, provides a clear picture of the global impact of EDCs on reproductive development. In particular, the ovine model of sewage sludge exposure provides the only currently available "real life" model of exposure to a cocktail of EDCs.

We have shown that continuous maternal exposure to the complex mixture of environmental chemicals (ECs) found in human biosolids (sewage sludge), disrupts mRNA expression of genes crucial for development and long-term regulation of hypothalamic-pituitary gonadal (HPG) function in sheep. The present study investigated whether exposure to ECs only during preconceptional period or only during pregnancy perturbed key regulatory genes within the hypothalamus and pituitary gland and whether these effects were different from chronic (life-long) exposure to biosolid ECs. The findings demonstrate that the timing and duration of maternal EC exposure influences the subsequent effects on the foetal neuroendocrine system in a sex-specific manner. Maternal exposure prior to conception, or during pregnancy only, altered the expression of key foetal neuroendocrine regulatory systems such as gonadotrophin-releasing hormone and kisspeptin to a greater extent than when maternal exposure was 'life-long'. Furthermore, hypothalamic gene expression was affected to a greater extent in males than in females and, following EC exposure, male foetuses expressed more 'female-like' mRNA levels for some key neuroendocrine genes. This is the first study to show that 'real-life' maternal exposure to low levels of a complex cocktail of chemicals prior to conception can subsequently affect the developing foetal neuroendocrine system. These findings demonstrate that the developing neuroendocrine system is sensitive to EC mixtures in a sex-dimorphic manner likely to predispose to reproductive dysfunction in later life.

The development of fetal ovarian follicles is a critical determinant of adult female reproductive competence. Prolonged exposure to environmental chemicals (ECs) can perturb this process with detrimental consequences for offspring. Here we report on the exposure of pregnant ewes to an environmental mixture of ECs derived from pastures fertilized with sewage sludge (biosolids): a common global agricultural practice. Exposure of pregnant ewes to ECs over 80 day periods during early, mid or late gestation reduced the proportion of healthy early stage fetal follicles comprising the ovarian reserve. Mid and late gestation EC exposures had the most marked effects, disturbing maternal and fetal liver chemical profiles, masculinising fetal anogenital distance and greatly increasing the number of altered fetal ovarian genes and proteins. In conclusion, differential temporal sensitivity of the fetus and its ovaries to EC mixtures has implications for adult ovarian function following adverse exposures during pregnancy.

Topaz1 (Testis and Ovary-specific PAZ domain gene 1) is a germ cell specific gene highly conserved in vertebrates. The putative protein TOPAZ1 contains a PAZ domain, specifically found in PIWI, Argonaute and Zwille proteins. Consequently, Topaz1 is supposed to have a role during gametogenesis and may be involved in the piRNA pathway and contribute to silencing of transposable elements and maintenance of genome integrity. Here we report Topaz1 inactivation in mouse. Female fertility was not affected, but male sterility appeared exclusively in homozygous mutants in accordance with the high expression of Topaz1 in male germ cells. Pachytene Topaz1 – deficient spermatocytes progress through meiosis without either derepression of retrotransposons or MSCI dysfunction, but become arrested before the post-meiotic round spermatid stage with extensive apoptosis. Consequently, an absence of spermatids and spermatozoa was observed in Topaz1−/− testis. Histological analysis also revealed that disturbances of spermatogenesis take place between post natal days 15 and 20, during the first wave of male meiosis and before the generation of haploid germ cells. Transcriptomic analysis at these two stages showed that TOPAZ1 influences the expression of one hundred transcripts, most of which are up-regulated in mutant testis at post natal day 20. Our results also showed that 10% of these transcripts are long non-coding RNA. This suggests that a highly regulated balance of lncRNAs seems to be essential during spermatogenesis for induction of appropriate male gamete production.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

In mammals, implantation is associated with major changes in gene profiles in the female reproductive tract. Molecular signatures of the endometrium have also been shown to vary according to the ability of the embryo to develop to term. Nevertheless, analysing endometrial gene patterns during implantation is incompatible with the maintenance of pregnancy. Therefore early determination of pregnancy issue requires a noninvasive method. Peripheral blood mononuclear cells (PBMC) could represent such an alternative but their reaction to the presence of an implanting embryo has to be investigated. The aim of this study was to investigate gene expression profiles of endometrial caruncular tissue (CAR) and PBMC collected from pregnant ewes (n = 4) and nonpregnant ewes inseminated with inactivated sperm (n = 4) at Day 15 after oestrus. Differentially expressed genes (DEG) were identified using an ovine custom-designed array derived from the ovine 15K Agilent array (Ruscanu et al. 2013 J. Virol. 87, 9333–9343). Data were normalized by Loess and analysed by a linear model in the Limma R package. P-values were corrected using the Benjamini and Hochberg procedure. Comparing pregnancy versus nonpregnancy led to the identification of 2826 DEG in CAR (P < 0.05) and 396 DEG in PBMC (P < 0.10; 265 DEG common with CAR). Ingenuity Pathway Analysis (IPA; Ingenuity Inc., Mountain View, CA, USA) analysis of the 396 PBMC-related DEG revealed 72 overrepresented biological functions (P < 0.001). Among the 15 most overrepresented functions, 13 were common between CAR and PBMC and were mostly related to the immune system, as “infectious disease”, “cell-to-cell signalling and interaction”, “immunological disease”, “immune cell trafficking and inflammatory response”. Using the downstream effect analysis (DEA) of IPA, we identified 163 functions predicted to be increased and 8 functions predicted to be decreased for the CAR DEG dataset, whereas 12 functions were predicted to be increased and 40 functions predicted to be decreased for the PBMC DEG dataset. Interferon (IFN) signalling was strongly present in both datasets, with 44% of PBMC DEG and 29% of CAR DEG found to be related to IFN type I response according to the Interferome database (www.interferome.org). A selection of 12 DEG was validated by qRT-PCR in CAR, intercaruncular areas, and PBMC using 8 pregnant and various groups of nonpregnant ewes (n = 7–9/group). Our data show that PBMC transcriptome is influenced by early pregnancy in sheep, including a major impact of IFN type I such as IFN tau, the signal of maternal recognition of pregnancy. Identifying relevant circulating biomarkers reflecting the quality of the embryo will require further investigation. The authors thank UCEA team (INRA), B. Jost (IGBMC) and F. Moreews (Sigenae).

Increasing evidence supports the idea that reproductive efficiency may be “programmed” during prenatal or early postnatal life. Developmental programming is the concept that an “insult” during any developmental stage can result in programming of organ/organismal function in the short or long term. Insults that result in developmental programming include external factors such as (1) malnutrition pre-mating, during pregnancy, or during infancy; and (2) exposure to environmental factors such as social stress, high temperature-humidity, smoke, herbicides, pesticides, or phytosteroids. Intrinsic factors that result in developmental programming include (1) maternal age, (2) multiple fetuses, and (3) maternal and embryonic genetic background. Based on large epidemiological studies primarily in humans, as well as controlled studies in animal models including livestock, developmental programming affects the function of various organ systems in the offspring, resulting in altered growth, body composition, metabolism, and behavior. The organ systems that have been shown to be affected include, among others, adipose tissue, brain, cardiovascular system, endocrine system, gastrointestinal tract, kidney, muscle, and reproductive system, including the ovary, utero-placenta, testis, and hypothalamic-pituitary-gonadal axis. In addition, the effects of developmental programming have been shown to be transmissible across generations. The 2015 Triennial Reproduction Symposium will focus on how developmental processes and systems can affect reproductive success in males and females in livestock and other species.

The origin of sex reversal in XX goats homozygous for the polled intersex syndrome (PIS) mutation was unclear because of the complexity of the mutation that affects the transcription of both FOXL2 and several long noncoding RNAs (IncRNAs) [1, 2]. Accumulating evidence suggested that FOXL2 could be the sole gene of the PIS locus responsible for XX sex reversal, the IncRNAs being involved in transcriptional regulation of FOXL2 [2]. In this study, using zinc-finger nuclease-directed mutagenesis, we generated several fetuses, of which one XX individual bears biallelic mutations of FOXL2. Our analysis demonstrates that FOXL2 loss of function dissociated from loss of IncRNA expression is sufficient to cause an XX female-to-male sex reversal in the goat model and, as in the mouse model, an agenesis of eyelids. Both developmental defects were reproduced in two newborn animals cloned from the XX FOXL2(-/-) fibroblasts. These results therefore identify FOXL2 as a bona fide female sex-determining gene in the goat. They also highlight a stage-dependent role of FOXL2 in the ovary, different between goats and mice, being important for fetal development in the former but for postnatal maintenance in the latter.

Biosolids (processed human sewage sludge), which contain low individual concentrations of an array of contaminants including heavy metals and organic pollutants such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB), and polychlorinated dibenzodioxins/polychlorinated dibenzofurans known to cause physiological disturbances, are increasingly being used as an agricultural fertilizer. This could pose a health threat to both humans and domestic and wild animal species. This review summarizes results of a unique model, used to determine the effects of exposure to mixtures of environmentally relevant concentrations of pollutants, in sheep grazed on biosolids-treated pastures. Pasture treatment results in nonsignificant increases in environmental chemical (EC) concentrations in soil. Whereas EC concentrations were increased in some tissues of both ewes and their fetuses, concentrations were low and variable and deemed to pose little risk to consumer health. Investigation of the effects of gestational EC exposure on fetal development has highlighted a number of issues. The results indicate that gestational EC exposure can adversely affect gonadal development (males and females) and that these effects can impact testicular morphology, ovarian follicle numbers and health, and the transcriptome and proteome in adult animals. In addition, EC exposure can be associated with altered expression of GnRH, GnRH receptors, galanin receptors, and kisspeptin mRNA within the hypothalamus and pituitary gland, gonadotroph populations within the pituitary gland, and regional aberrations in thyroid morphology. In most cases, these anatomical and functional differences do not result in altered peripheral hormone concentrations or reproductive function (e.g., lambing rate), indicating physiological compensation under the conditions tested. Physiological compensation is also suggested from studies that indicate that EC effects may be greater when exposure occurs either before or during gestation compared with EC exposure throughout life. With regard to human and animal health, this body of work questions the concept of safe individual concentration of EC when EC exposure typically occurs as complex mixtures. It suggests that developmental EC exposure may affect many different physiological systems, with some sex-specific differences in EC sensitivity, and that EC effects may be masked under favorable physiological conditions.