Developmental stage specific effect of Mito-TEMPO on the expression outline of antioxidant genes, ROS balance and cryo-resilience of bovine IVP embryos

This is the authors' abstract. We don't add key points for this paper.

In vitro culture impairs mitochondrial metabolism of IVP derived bovine embryos resulting in accumulation of reactive oxygen species. Recently, the antioxidant Mito-TEMPO has attracted attention due to its capability to accumulate within mitochondria. In order to investigate the potential of Mito-TEMPO to improve quality of IVP derived embryos, this study analyzed the developmental stage specific effect of Mito-TEMPO on developmental capacity, ROS balance, expression outline of antioxidative genes and cryo-resilience of blastocysts. In three subsequent experiments Mito-TEMPO (1 μM) was added either to the maturation medium (MTM), the culture medium (MTC) or to both the maturation medium and the culture medium (MTMC). Concerning cleavage- and blastocyst rates, no effect of Mito-TEMPO supplementation could be detected, although MTM and/or MTC groups revealed significantly (p < 0.05) lower levels of ROS. Expression outline of antioxidative genes under study was not affected in MTM group, whereas down-regulation of the proapoptotic gene BAX was observed in MTM as well as MTMC groups. Moreover, Mito-TEMPO significantly affected expression outline of genes with antioxidative functions within mitochondria ( SOD2, GPX1, GSTA4 ) in MTC and/or MTMC groups and peroxisomes ( CAT ) in MTMC group. In contrast, expression of genes acting predominately outside mitochondria ( NFE2L2 and PRDX1 ) was not affected. Of high impact, the present study revealed for the first time greatly improved reexpansion and hatching rates of bovine vitrified-warmed embryos as a consequence of supplementation of Mito-TEMPO to culture media. Collectively, the present study successfully proved that Mito-TEMPO alleviates negative effects of the in vitro culture environment in bovine pre-implantation embryos. • Mitochondria-Targeted Antioxidant: By targeting mitochondrial ROS specifically, Mito-TEMPO offers a pathway-independent strategy to improve bovine embryo cryotolerance. • Pathway-Specific Action: Mito-TEMPO selectively affects specific antioxidant enzymes, downregulating GPX1, GSTA4, and SOD2 , while upregulating CAT , without influencing PRDX1 or NFE2L2 expression. • Enhanced Cryotolerance: The addition of Mito-TEMPO improves post-cryopreservation outcomes, with higher blastocyst expansion and hatching rates, suggesting enhanced embryo quality and developmental potential.