Abstract
Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos even though further studies are necessary to confirm this possibility.