Ruthenium red improves blastocyst developmental competence by regulating mitochondrial Ca²⁺ and mitochondrial...

Ruthenium red (RR) inhibits calcium (Ca²⁺) entry from the cytoplasm to the mitochondria, and is involved in maintenance of Ca²⁺ homeostasis in mammalian cells. Ca²⁺ homeostasis is very important for further embryonic development of fertilized oocytes. However, the effect of RR on mitochondria-Ca²⁺ (mito-Ca²⁺) levels during in vitro fertilization (IVF) on subsequent blastocyst developmental capacity in porcine is unclear. The present study explored the regulation of mito-Ca²⁺ levels using RR and/or histamine in fertilized oocytes and their influence on blastocyst developmental capacity in pigs. Red fluorescence intensity by the mito-Ca²⁺ detection dye Rhod-2 was significantly increased (P < 0.05) in zygotes 6 h after IVF compared to mature oocytes. Based on these results, we investigated the changes in mito-Ca²⁺ by RR (10 and 20 μM) in presumptive zygotes using Rhod-2 staining and mito-Ca²⁺ uptake 1 (MICU1) protein levels as an indicator of mito-Ca²⁺ uptake using western blot analysis. As expected, RR-treated zygotes displayed decreased protein levels of MICU1 and Rhod-2 red fluorescence intensity compared to non-treated zygotes 6 h after IVF. Blastocyst development rate of 20 μM RR-treated zygotes was significantly increased 6 h after IVF (P < 0.05) due to improved mitochondrial functions. Conversely, the blastocyst development rate was significantly decreased in histamine (mito-Ca²⁺ inhibitor, 100 nM) treated zygotes (P < 0.05). The collective results demonstrate that RR improves blastocyst development in porcine embryos by regulating mito-Ca²⁺ and MICU1 expression following IVF.