Delayed effects of early-life exposure to ethinylestradiol on the DNA methylation landscape, proteome and physiology of the self-fertilizing mangrove rivulus Kryptolebias marmoratus

Abstract

Early-life represents a critical plastic window during which environmental factors can have long-term repercussions on the phenotype later in life. Although this concept is now widely accepted, the molecular mechanisms by which early exposures influence the propensity of disease and phenotype later in life remain elusive. Accumulating evidence points towards the role of epigenetic processes in mediating stable changes in gene expression resulting from developmental exposures. Endocrine disrupting chemicals (EDCs) are of particular interest as they can interfere with hormone processes that are crucial for the normal development and homeostasis in organisms. Hence, the general objective of this thesis was to investigate the molecular mechanisms mediating the long-term effects of developmental exposure to a model xenoestrogen, ethinylestradiol (EE2), in the mangrove rivulus, Kryptolebias marmoratus. This self-fertilizing hermaphrodite fish model allows to work on a single homozygous and isogenic lineage, thus eliminating the effects of genetic variability on the observed phenotypic plasticity. To this end, we first investigated the effects of a 28-days exposure of hatchlings to a low (environmental - 4 ng/L) and a high concentration (120 ng/L) of EE2 on the phenotype throughout development until adulthood, by monitoring growth, reproduction and steroid hormone levels. While fish growth was impaired during exposure, compensatory growth, reduced fecundity and modification of the endocrine status were observed months after the exposure ceased. To further our understanding of the mechanisms underlying the delayed effects of EE2, we used a label-free quantitative proteomic approach on the brain, liver and gonads of adult fish. Overall, we have observed that EE2 impacts at the proteome level were tissue- and concentration-specific, with most effects triggered in the liver by the lower concentration of 4 ng/L. We show for the first time that estrogen-responsive pathways, such as lipid metabolism, inflammation, and the innate immune system, that are typically affected after acute exposure to EE2 in several species, were persistently affected months after the exposure ceased. As the liver presented the most interesting protein expression responses, we investigated the genome-wide DNA methylation patterns of 168 dph adult livers to highlight persistent epigenetic marks that may drive the stable changes in gene expression, underlying the observed effects on the proteome and organismal phenotype. Similar to the proteome, most effects on specific DNA methylation were triggered at the environmentally relevant concentration (4 ng/L), emphasizing the non-monotonic behavior of endocrine disruptors. Although no common genes were identified in the two omic approaches, networks related to lipid metabolism and inflammation were also identified in the methylome study. This study is the first to investigate the long-term effects of an early-life exposure by combining proteomic and epigenetic approaches and provides a robust basis for further studies aimed at deciphering the molecular mechanisms underlying the delayed effects of endocrine disruptor exposure.

Date of Award	2 May 2018
Original language	English
Awarding Institution	University of Namur
Sponsors	Fonds de la Recherche Scientifique F.R.S.-FNRS
Supervisor	Frederic Silvestre (Supervisor), Karine Van Doninck (Co-Supervisor), Caroline Nieberding (Jury), Ryan L. Earley (Jury), Maria Victoria Suarez Ulloa (Jury) & Marie-Agnès Coutellec (Jury)