Effects of di-(2-ethylhexyl) phthalate and cadmium mixture during IVM on prepubertal sheep oocyte nuclear maturation and bioenergetic/oxidative parameters.

Di-(2-ethylhexyl) phthalate (DEHP) and Cadmium (Cd) are ubiquitous environmental contaminants [1]. In vitro studies, performed to date, separately analyzed their damaging effects on female germ cells [2-4]. The aim of the present study was to analyze the effects of a DEHP/Cd mixture on nuclear maturation and bioenergetic/oxidative status of prepubertal sheep oocytes compared with those of each single compound. Cumulus-oocyte complexes (COCs) recovered from slaughterhouses ovaries underwent in vitro maturation (IVM) [4] in presence of DEHP, Cd or DEHP/Cd mixture. Cd and DEHP concentrations were chosen based on a previous study on Cd (0.1µM) [4] and preliminary trials with DEHP (0.5 μM). COCs cultured in IVM medium with MeOH, as DEHP vehicle, were used as control (CTR). After IVM, nuclear maturation and bioenergetic/oxidative parameters were evaluated [4]. Data were analyzed by Chi-square test and one-way ANOVA (significance at p<0.05). To select an effective DEHP concentration (0.1 or 0.5μM), 301 oocytes were analyzed in 3 replicates. The maturation rate was not affected at both tested concentrations. However, 0.5μM DEHP reduced the percentage of oocytes showing healthy perinuclear/pericortical (pp) mitochondria (mt) distribution pattern (10/41, 24% vs 17/34, 50%, p<0.05), mt activity (275±94 vs 368±147, p<0.05), intracellular ROS levels (164±104 vs 323±169, p<0.001) and mt/ROS colocalization (0.4±0.1 vs 0.7±0.1, p<0.001) compared to CTR. For the mixture test, 265 oocytes were analyzed in 3 replicates. No effects were noticed on oocyte maturation rate at any tested conditions (DEHP/Cd mixture, Cd, DEHP, CTR). However, the mixture and both compounds reduced the rate of mature oocytes with pp mt pattern (2/33, 6%; 4/29, 14%; 8/34, 24% vs 15/27, 56% for mix, DEHP, Cd and CTR respectively, p<0.05), mt activity (316±235, 180±158, 298±244 vs 607±455 for mix, DEHP, Cd, mix and CTR respectively, p<0.01) and mt/ROS colocalization (0.4±0.3, 0.3±0.2, 0.4±0.2 vs 0.6±0.2 for mix, DEHP, Cd and CTR respectively, p<0.01) compared to CTR. As well, intracellular ROS levels were reduced at any tested condition, but they attained statistical significance only in MII oocytes exposed to DEHP (260±185, 140±133, 239±169 vs 476±325 for mix, DEHP, Cd, and CTR respectively, p<0.05, ns,ns). In conclusion, the DEHP/Cd mixture altered bioenergetic/oxidative status of prepubertal sheep oocytes, similarly to what observed for DEHP and Cd separetely.