Rutin enhances mitochondrial function and improves the developmental potential of vitrified ovine GV-stage oocyte

Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca²⁺, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca²⁺, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca²⁺ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca²⁺ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.