We have previously reported the effect of Anthocyanin and cAMP modulators on maturation and apoptosis of sheep oocytes. In this study, we investigated the reduction capacity and apoptotic gene expression results at MII sheep oocytes.
Materials and methods
Good ovine Cumulus-oocyte complexes (COCs) were transferred to Pre-IVM medium, then to two type IVM mediums: 1) 0.1μg/ml Delphinidin Chloride supplemented SPOM medium and 2) Simple SPOM medium for 24 hours. After maturation period, MII oocytes with first polar body were selected. Immunoﬂuorescence microscopy staining was used to evaluate intracellular GSH and ROS content of MII oocytes. In addition, the expression rate of some apoptotic genes (Bcl-2, Bax, Caspase 3, P53, C-myc) was investigated by Real-time PCR in MII oocytes. Statistical analyses were carried out by T test.
Anthocyanin treatment during IVM increased intracellular glutathione (GSH) levels and significantly reduced reactive oxygen species (ROS) (19.97 ± 1.29 and 26.77 ± 2.6 respectively, P<0.05). Real-time PCR results showed that the expression rates of P53 and Caspase 3 genes in group 1 significantly were lower than group 2 (0.43 ± 0.08 and 0.64 ± 0.02 respectively, P<0.05). However Bax (1.23 ± 0.02) and C-myc (1.43 ± 0.11, P<0.05) genes were lower in group 1. The highest expression rate of anti- apoptotic gene (Bcl-2) was seen in the group 2 (0.65 ± 0.16, P<0.05).
Anthocyanin treatment during IVM improved maturation and viability of sheep oocytes, most likely by increasing intracellular GSH synthesis and reducing ROS level. About apoptotic genes, the five studied genes did not appear to be involved in the control of oocyte death.