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.

Figure 1. Effects of DEHP on cumulus cell chromatin fragmentation and condensation.

At all tested concentration, DEHP increased cumulus cells chromatin fragmentation and condensation in equine COCs (A). In separate evaluation of morphological features of apoptosis, such as marginated chromatin (Type A), single small densely stained nucleus (Type B), multiple densely nuclear fragments (Type C), apoptotic bodies (Type D), a significant increase of apoptotic bodies could be observed after IVM in presence of DEHP (B). Cumulus cell chromatin morphology was affected irrespectively to oocyte maturation stage (C). Numbers of analyzed cumuli oophori per group, from which cells analyzed where obtained, are indicated on the top of each histogram. One-way ANOVA: a, b P<0.001; c,d P<0.01.

Figure 2. Mitochondrial distribution pattern and ROS localization in equine matured oocytes exposed to DEHP.

For each oocyte, corresponding bright-field (A,B,C), UV light (A1,B1,C1) and confocal laser scanning images showing mt distribution pattern (A2,B2,C2), intracellular ROS localization (A3,B3,C3) and mt/ROS merge (A4,B4,C4) are shown. Oocytes are representative of heterogeneous (pericortical/perinuclear; A), homogeneous (B) and abnormal (C) mitochondrial distribution pattern, respectively. Scale bar represents 60 µm.

Figure 3. Effects of DEHP on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in matured oocytes.

Dose-response curve of the in vitro effects of DEHP on mitochondrial activity and intracellular ROS levels in single equine metaphase II stage oocytes expressed as Mitotracker Orange CMTM Ros (A) and DCF (B) fluorescence intensities. Oocytes treated with DEHP showed significantly higher ROS levels compared to controls. Values are expressed as arbitrary densitometric units (ADU). Pearson's correlation coefficents of Mitotracker Orange CMTM Ros and DCF fluorescent labelling in oocytes cultured in presence of DEHP (C). Numbers of analyzed oocytes per group are indicated on the top of each histogram. Student's t-Test: a, b P<0.05.

Figure 4. Effects of DEHP on total ATP content and total SOD activity in single equine matured oocytes.

ATP content and total SOD activity in single equine MII oocytes cultured in presence of 12 µM DEHP. Control oocytes were cultured in absence of DEHP. Numbers of analyzed oocytes per group are indicated on the top of each histogram. Oocytes treated with 12 µM DEHP showed significantly higher ATP content compared to controls. Values are expressed as pmol/oocyte (A). Total SOD activity did not vary upon 12 µM DEHP exposure in matured equine oocytes compared with controls. Values are expressed as IU/mg protein (mean±sd of 9 evaluations per oocyte; B). Student's t-Test: a, b P<0.05.

Figure 5. Effects of DEHP and DEHP/NAC co-treatment on cumulus cell apoptosis and intracellular ROS levels.

At the concentration of 12 and 1200 µM, DEHP increased cumulus cell apoptosis in equine COCs. NAC reversed DEHP-induced apoptosis observed at 12 µM (A). At the concentrations of 12 and 1200 µM, DEHP increased CC intracellular ROS levels and co-treatment with NAC reduced the DEHP-induced ROS increase observed at 1200 µM (B). Numbers of analyzed cumuli oophori per group (from MII+NM oocytes), from which analyzed cells where obtained, are indicated on the top of each histogram. Representative images of equine CCs after IVM in presence of DEHP subjected to TUNEL analysis (C) to determine apoptosis and DCF staining (D) to evaluate intracellular ROS levels. In C, corresponding bright-field and UV light images are also provided. One-way ANOVA: comparisons between used DEHP doses: a,b,c P<0.0001; comparisons between DEHP and DEHP/NAC: _*,_** P<0.0001.