FOLLICULOGENESIS AND EMBRYO DEVELOPMENT EXPOSURE TO A MIXTURE OF TOXICANTS: EVALUATION IN THE RABBIT MODEL. – FEDEXPO

Epidemiological, clinical and animal data argue for noxious effects of environmental toxicants on human reproduction notably in men. Concerns are also rising regarding folliculogenesis, quality of the oocyte and of the embryo derived from it, since all of them are targets for large epigenetic reprogramming.
Therefore, our project aims to evaluate the effect of a mixture of 7 environmental toxicants (selected pertinently from the human pregnancy exposome) on folliculogenesis and/or preimplantation embryo development. Moreover, we will assess modifications of the placenta and long term health consequences on progeny, notably its gametes, providing clues for a potential transgenerational inheritance.

A preliminary pharmacokinetic study will decide of doses and frequencies of exposure for each toxicant to obtain internal exposures similar to those observed in pregnant women. Then, 4 groups of 12 female rabbits will be constituted:
- Group F (folliculogenesis): exposed to the 7 toxicants from post-natal week 2 (start of folliculogenesis) to 19 (full sexual maturity) then artificially inseminated with the sperm from a non-exposed male
- Group FED (folliculogenesis + embryo development): as Group F but prolonging the exposure for 80 hours after insemination (blastocyst stage) to decipher additional effects of an embryonic exposure
- Group ED (embryo development): exposed from sperm insemination to 80 hours post insemination (p.i.), in order to describe any specific effect on the preimplantation window of exposure, independently from the follicular/oocyte exposure notably for chemicals with long half-life
- Group NE: exposed to vehicle only until 80h post insemination (p.i).

Then our project will be divided into 3 work-packages.
Work-package 1 aims to described folliculogenesis in 2 groups (F and NE) through ovarian hormones measurements in blood, ovarian histological evaluation of follicular stages and density, ovarian immuno-histological analysis of 4 proteic markers of cell proliferation (PCNA), activation of early stage follicles (GDF-9) and steroïdogenic enzymes (CYP17 and CYP19). Follicular apoptosis will be compared between groups using a TUNEL fluorescent method.

Work-package 2 aims to assess the effects of the exposure on the F1 preimplantation embryo. Embryos will be collected 80h p.i in the 4 groups to assess embryo stages, cell lineage allocation within the blastocyst, gene expression and whole genome DNA methylation in blastocysts using a custom rabbit microarray and Reduced Representation Bisulfite Sequencing (RRBS).

Work-package 3 is dedicated to the evaluation of feto-placental and post-natal long term effects on F1 progeny. Embryos from groups FED, ED and NE will be transferred into non-exposed foster mothers to exclude any effects on the uterus. Gestation will be followed ultrasonographically. Placenta from 4 F0 females per group will be collected for histological, transcriptome (rabbit array) and methylome analysis (RBSS). Six males and 6 females F1 offspring per group will be followed and evaluated until 6 months for growth, arterial tension and blood metabolic parameters. At 6 months, F1 semen will be analyzed and sperm DNA extracted for methylome analysis (RBSS). Gonads from 3 F1 females and 3 males will be collected for histological analysis. F1 cumulus-oocyte complexes punctured from ovaries will be dissociated for transcriptome and methylome analysis, while oocytes, due to the scarcity of material, will be tested only for the methylation status of several genes detected in WP1 as affected in the FED blastocysts by bisulfite conversion and pyrosequencing. Non-sacrificed F1 adults will be bred for an eventual multigenerational study (out of the scope of this project).

This project should allow to discriminate the oocyte-driven (via the folliculogenesis) from the embryo-driven impacts on long term health after a human-based exposure to a complex mixture of toxicants.