Timing of maternal exposure and fetal sex determine the effects of low-level chemical mixture exposure on the fetal neuroendocrine system in sheep

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 hypothalamo-pituitary gonadal (HPG) function in sheep. This 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 fetal neuroendocrine system in a sex-specific manner. Maternal exposure prior to conception or during pregnancy only, altered the expression of key fetal neuroendocrine regulatory systems such as GnRH 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 fetuses 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 fetal 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

Structural and functional analyses of the DMC1-M200V polymorphism found in the human population

The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility

TOPAZ1, a Novel Germ Cell-Specific Expressed Gene Conserved during Evolution across Vertebrates

BACKGROUND: We had previously reported that the Suppression Subtractive Hybridization (SSH) approach was relevant for the isolation of new mammalian genes involved in oogenesis and early follicle development. Some of these transcripts might be potential new oocyte and granulosa cell markers. We have now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific PAZ domain gene. PRINCIPAL FINDINGS: Sheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with a peak during prophase I of meiosis, and in males during adulthood. In the mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved in vertebrates and specifically expressed in mouse and sheep gonads. It is localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse adult testis. CONCLUSIONS: We have identified a novel PAZ-domain protein that is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ lin

Effects of environmental pollutants on the reproduction and welfare of ruminants

Anthropogenic pollutants comprise a wide range of synthetic organic compounds and heavy metals, which are dispersed throughout the environment, usually at low concentrations. Exposure of ruminants, as for all other animals, is unavoidable and while the levels of exposure to most chemicals are usually too low to induce any physiological effects, combinations of pollutants can act additively or synergistically to perturb multiple physiological systems at all ages but particularly in the developing foetus. In sheep, organs affected by pollutant exposure include the ovary, testis, hypothalamus and pituitary gland and bone. Reported effects of exposure include changes in organ weight and gross structure, histology and gene and protein expression but these changes are not reflected in changes in reproductive performance under the conditions tested. These results illustrate the complexity of the effects of endocrine disrupting compounds on the reproductive axis, which make it difficult to extrapolate between, or even within, species. Effects of pollutant exposure on the thyroid gland, immune, cardiovascular and obesogenic systems have not been shown explicitly, in ruminants, but work on other species suggests that these systems can also be perturbed. It is concluded that exposure to a mixture of anthropogenic pollutants has significant effects on a wide variety of physiological systems, including the reproductive system. Although this physiological insult has not yet been shown to lead to a reduction in ruminant gross performance, there are already reports indicating that anthropogenic pollutant exposure can compromise several physiological systems and may pose a significant threat to both reproductive performance and welfare in the longer term. At present, many potential mechanisms of action for individual chemicals have been identified but knowledge of factors affecting the rate of tissue exposure and of the effects of combinations of chemicals on physiological systems is poor. Nevertheless, both are vital for the identification of risks to animal productivity and welfare

Contemporary genetic technologies and female reproduction

BACKGROUNDThe Fifth Evian Annual Reproduction (EVAR) Workshop Meeting discussed knowledge regarding contemporary genetics in female reproduction.METHODSSpecialist reproductive medicine clinicians and geneticists delivered presentations based on published literature and current research. The content of this report is based on the expert presentations and subsequent group discussions that took place during this Workshop.RESULTSNumerous ovarian genes with a role in infertility have been identified. Future challenges for genetic screening of patients, such as those with polycystic ovary syndrome, primary ovarian insufficiency or endometriosis, include the identification of high-throughput strategies and how to apply these findings to infertile patients. The identification of high-quality embryos in IVF using objective technologies remains a high priority in order to facilitate single-embryo transfer. Gene expression profiling of cumulus cells surrounding the oocyte, and proteomic and metabolomic approaches in embryo culture media may significantly improve non-invasive embryo quality assessment.CONCLUSIONSThe way forward in advancing the knowledge of genes involved in reproduction was considered to be through genome-wide association studies involving large numbers of patients. Establishing international collaboration is required to enable the application of such technologies in sufficient numbers of patients

Transcriptome profiling of sheep granulosa cells and oocytes during early follicular development obtained by Laser Capture Microdissection

<p>Abstract</p> <p>Background</p> <p>Successful achievement of early folliculogenesis is crucial for female reproductive function. The process is finely regulated by cell-cell interactions and by the coordinated expression of genes in both the oocyte and in granulosa cells. Despite many studies, little is known about the cell-specific gene expression driving early folliculogenesis. The very small size of these follicles and the mixture of types of follicles within the developing ovary make the experimental study of isolated follicular components very difficult.</p> <p>The recently developed laser capture microdissection (LCM) technique coupled with microarray experiments is a promising way to address the molecular profile of pure cell populations. However, one main challenge was to preserve the RNA quality during the isolation of single cells or groups of cells and also to obtain sufficient amounts of RNA.</p> <p>Using a new LCM method, we describe here the separate expression profiles of oocytes and follicular cells during the first stages of sheep folliculogenesis.</p> <p>Results</p> <p>We developed a new tissue fixation protocol ensuring efficient single cell capture and RNA integrity during the microdissection procedure. Enrichment in specific cell types was controlled by qRT-PCR analysis of known genes: six oocyte-specific genes (<it>SOHLH2</it>, <it>MAEL</it>, <it>MATER</it>, <it>VASA</it>, <it>GDF9</it>, <it>BMP15</it>) and three granulosa cell-specific genes (<it>KL</it>, <it>GATA4</it>, <it>AMH</it>).</p> <p>A global gene expression profile for each follicular compartment during early developmental stages was identified here for the first time, using a bovine Affymetrix chip. Most notably, the granulosa cell dataset is unique to date. The comparison of oocyte vs. follicular cell transcriptomes revealed 1050 transcripts specific to the granulosa cell and 759 specific to the oocyte.</p> <p>Functional analyses allowed the characterization of the three main cellular events involved in early folliculogenesis and confirmed the relevance and potential of LCM-derived RNA.</p> <p>Conclusions</p> <p>The ovary is a complex mixture of different cell types. Distinct cell populations need therefore to be analyzed for a better understanding of their potential interactions. LCM and microarray analysis allowed us to identify novel gene expression patterns in follicular cells at different stages and in oocyte populations.</p

Maternal and fetal tissue accumulation of selected endocrine disrupting compounds (EDCs) following exposure to sewage sludge-treated pastures before or after conception

This paper reports patterns of accumulation of selected EDCs in adult and fetal livers of sheep exposed to sewage sludge-treated pastures, at different times and at two stages of development