In Vitro Acute Exposure to DEHP Affects Oocyte Meiotic Maturation, Energy and Oxidative Stress Parameters in a Large Animal Model

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.

Oocyte mitochondrial bioenergy potential and oxidative stress: within-/between-subject, in vivo versus in vitro maturation, and age-related variations in a sheep model

“…To specifically investigate the distribution of active mitochondria, oocytes were washed in PBS with 3% bovine serum albumin (BSA) and incubated for 30 minutes in the same medium containing 280 nmol/L Mitotracker Orange CMTM Ros (Molecular Probes) at 38.5 C under 5% CO 2 (20,21). After incubation with the mt probe, oocytes were washed in PBS with 0.3% BSA and incubated for 15 minutes in the same medium containing 10 mmol/L 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (DCDHFDA) (22,23) to detect and localize intracellular sources of ROS.…”

“…Scanning was conducted with 25 optical series from the top to the bottom of the oocyte with a step size of 0.45 mm to allow three-dimensional distribution analysis. General criteria for oocyte mt patterns definition and intracellular ROS localization were adopted on the basis of earlier studies in other species (20)(21)(22)25).…”

The adjuvant effect induced by di-(2-ethylhexyl) phthalate (DEHP) is mediated through oxidative stress in a mouse model of asthma

“…Oxidative stress and inflammation have been found to be the root cause of some adverse health effects induced by phthalates in both humans (Ferguson et al, 2011;Ambruosi et al, 2011;Kim et al, 2014;North et al, 2014) and animals (Zhou et al, 2011;Kasahara et al, 2002;Zuo et al, 2014). For example, 1000 mg/kg DEHP exposure by gavage can cause lipid peroxidation in the liver of rats (Erkekoglu et al, 2014).…”

Effect of Antioxidants on Sperm Genetic Damage

“…ROSs are formed during the intermediate steps of oxygen reduction. However, modern life has obviously increased fertility problems through pesticide-induced ROS generation: xenoestrogens, endocrine-disrupting chemicals involved in plastic technology such as polychlorinated biphenyls, bisphenol A, phthalates and alkyl phenols, di-(2-ethylhexyl) phthalate (DEHP) (Ambruosi et al 2011 ). There are little data as to whether it is possible to increase DNA repair capacity in the oocyte; that is why in ART, the yield in terms of take-home baby rate per oocyte retrieved is no higher than 5 % (Patrizio and Sakkas 2009 ).…”