Overall aim:
Our hypothesis is that altered female reproductive function and health can be due to the reprotoxic effects of environmental factors acting mainly during foetal life. Since our preliminary data in the sewage sludge ovine model indicate that DEHP and PCBs preferentially accumulate in the foetus, we aim to focus on the impact of these chemicals, as well as a real-life cocktail of environmental chemicals (ECs) in sewage sludge, on the developing female gonad.
Specific objectives
Examine the effects of sewage sludge exposure during specific periods of foetal ovarian development in the sheep
By breaking gestation down into discrete biologically relevant periods (i. sex determination and early ovary development; ii. around primordial follicle formation; iii. secondary and then antral follicle formation) and comparing sewage sludge exposed to non-exposed animals, we will be able to investigate the impact of real-life, environmentally relevant EC cocktails on critical events during ovary genesis. Studying maternal and foetal genes, proteins, hormones and ovarian morphology we will unravel key effects of such exposure and identify candidate genes involved in the mechanism of action of EC cocktails.
Examine the effects of environmental concentrations of DEHP and PCBs on female reproductive development in the sheep and mouse
Our previous studies of sewage sludge exposures and the literature highlight PCB101 & 118 and DEHP as biologically active at environmental concentrations. In sewage sludge-exposed pregnant ewes these chemicals are present at higher concentrations in the foetus than in the mother. We will study the specific effects of these chemicals on ovary development in the sheep. These studies will be paralleled by matching studies in the mouse to allow more varied exposures, the use of large scale gene arrays and also imprinting/methylation analyses. The importance of the latter is the apparent transgenerational effects of developmental insults to the foetus. Because of the latter data we will also investigate the effects of PCBs and DEHP, using both in vivo and in vitro exposures, on methylation, metabolism and key developmental markers in stem cells, oocytes and pre-implantation embryos.
Investigate the transgenerational effects of DEHP and PCB exposure on F2 sheep and mouse ovaries
In order to specifically assess the effects of EC exposure across generations, unexposed foetuses from previously exposed sheep (sewage sludge) and mice (DEHP and PCBs) will be extensively investigated for key markers of ovarian development while novel target genes will be identified by proteomic and gene array studies. In addition, transgenerational effects of ECs on DNA methylation will be determined in the mouse model.
Examine the effects of DEHP and PCBs on cultured human and sheep foetal ovaries
In order to test the toxicity of selected ECs (DEHP and PCBs), an in vitro model allows more precise dosage and separates foetal from maternal effects. This is important in terms of informing on potential mechanisms leading to perturbed foetal ovarian development. Developmental similarities between sheep and human justify the use of both species in vitro. Once the culture system is established in the sheep, human and sheep exposure studies will be performed and changes in key and novel developmental genes and proteins will be established.
Integrate human and animal models
Our ultimate concern is to determine how the developing human female foetus might be affected by exposure to ECs during development. One of our key objectives therefore is to integrate the mouse and sheep exposure studies with the human. An essential process in this is to determine the expression and localisation of EC-sensitive genes in the human foetal ovary itself.