80 Cumulus Cells Reflect the Success of IVF of Porcine Oocytes Treated with Dimethylthiourea

Abstract

The aim of this study was to evaluate the effect of the antioxidant dimethylthiourea (DMTU) as a supplement during the collection and washing of cumulus–oocyte complexes (COC) on the apoptosis in cumulus cells (CC) and the competition for oocyte development in vitro. The COC were obtained by follicular aspiration. The aspiration and washing medium was TCM-199 with 10% porcine follicular fluid, 0.3 mM sodium pyruvate, and penicillin/streptomycin. Treated groups were supplemented with 2 or 20 mM DMTU. After 44 h of in vitro maturation (IVM), the percentage of nuclear maturation was determined by staining of the denuded oocytes with Hoechst 33442 (n = 357). After 18 h of in vitro fertilization (IVF) with 15°C refrigerated spermatozoa from boars of proven fertility, the suspected zygotes were stained with Hoechst 33342 to evaluate the percentages of sperm penetration (SP), monospermic penetration (MP), male pronucleus formation (MPN), and IVF efficiency (monospermic oocytes/total oocytes) (n = 260). In post-IVM CC, flow cytometry was performed after staining with annexin V-propidium iodide to evaluate apoptosis and viability (n = 90,000). Data were analysed with Flowing V 2.5.1 (http://flowingsoftware.btk.fi/). A comparison of proportion among categories was used (Fisher’s exact test), and significant differences were determined at P < 0.05. No effects were observed in the percentages of nuclear maturation. Treatments with DMTU significantly increased the SP (Control: 35%; 2 mM: 72.9%; 20 mM: 67.8%) without modifying the MP. Although in both treatments, the amount of MPN was always greater than in the control, the percentages of MPN formation did not show significant differences. The 20 mM treatment significantly increased the efficiency of IVF (control: 22.2%; 2 mM: 31.9%; 20 mM: 45.7%). In the CC of the treated groups, a significant increase in viability (control: 7.4%; 2 mM: 12.2%; 20 mM: 14.7%) and a decrease in early apoptosis (control: 60.4%; 2 mM: 55.5%; 20 mM: 57.1%) were observed. Late apoptosis decreased in the 20 mM treatment (27.3%) compared with the other experimental groups (control: 31.3%; 2 mM: 31.2%). The addition of DMTU during COC collection and washing reduce the ROS-mediated apoptosis in CC and optimize the efficiency of IVF. Besides, CC quality reflects the effect of DMTU on oocytes, so viability and apoptosis analysis on CC constitutes an interesting noninvasive method to assess oocyte competence.