270 MITOCHONDRIA AND REACTIVE OXYGEN SPECIES COLOCALIZATION IN IN VIVO AND IN VITRO MATURED OOCYTES FROM SUPEROVULATED ADULT EWES

Abstract

Analyses of energy and redox status parameters are emerging technologies to improve oocyte quality assessment. Mitochondria (mt) play a vital role in the oocyte to support maturation, fertilization, and pre-implantation development. They are the major source of reactive oxygen species (ROS) produced during oxidative phosphorylation, which are not only by-products of cell metabolism but also important molecules for regulation of intracellular cell signaling. The aim of the present study was to test for mt/ROS colocalization in oocytes recovered from superovulated adult ewes and examined after in vivo or in vitro maturation (IVM). Cumulus–oocyte complexes of 8 superovulated (fluorogestone acetate + D-cloprostenol for oestrus synchronization, pFSH/pLH and eCG for superovulation) adult (2 to 8 years of age) ewes were recovered (ovariohysterectomy by midventral laparotomy performed 54 h after vaginal sponge removal) either from flushing oviducts (oviducal oocytes) or from ovarian growing follicles (1–5 mm in diameter; follicular oocytes). Follicular oocytes were analysed after IVM (Ambruosi et al. 2009 Theriogenology 71, 1093–1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mt, and ROS evaluation. Hoechst 33258 and Mitotracker Orange CMTM Ros were used to label nuclear chromatin and mt (Ambruosi et al. 2009) and 2′,7′-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353–360). Oocytes at the metaphase II (MII) stage showing regular ooplasmic size (>130 μm in diameter) and morphology were selected for confocal analysis of mt/ROS fluorescence distribution, intensity, and colocalization. Forty oviducal MII oocytes recovered from 8 ewes were analysed. Thirty-two oocytes recovered from the ovaries of 4 ewes underwent IVM, and 23 out of 32 (72%) reached nuclear maturation and were analysed. The rate of oocytes showing perinuclear mt distribution pattern did not differ between oviducal and IVM oocytes (33%, 13/40 v. 43%, 10/23; not significant). In these oocytes, fluorescent intensity of mt labelling and intracellular ROS levels did not differ between oviducal and IVM ooocytes (996.27 ± 363.57 v. 798.13 ± 275.91; not significant; and 1808.11 ± 442.78 v. 1473.29 ± 662.49, for mt and ROS, respectively; not significant), whereas mt/ROS colocalization was significantly higher in ovulated oocytes than in IVM oocytes (Pearson coefficient 0.67 ± 0.11 v. 0.39 ± 0.19, respectively; P < 0.001). In conclusion, in oocytes of adult ewes, mt aggregation, apparent energy status, and intracellular ROS levels do not differ between ovulated and IVM oocytes, but mt/ROS colocalization differs between the 2 groups. As it was reported for other cell systems that such a difference can be indicative of healthy status of ovulated oocytes, we suggest that mt/ROS colocalization could be considered as a suitable marker of oocyte quality.