Ruthenium red improves blastocyst developmental competence by regulating mitochondrial Ca and mitochondrial functions in fertilized porcine oocytes in vitro.

Ruthenium red (RR) inhibits calcium (Ca) entry from the cytoplasm to the mitochondria, and is involved in maintenance of Ca homeostasis in mammalian cells. Ca homeostasis is very important for further embryonic development of fertilized oocytes. However, the effect of RR on mitochondria-Ca (mito-Ca) levels during in vitro fertilization (IVF) on subsequent blastocyst developmental capacity in porcine is unclear. The present study explored the regulation of mito-Ca levels using RR and/or histamine in fertilized oocytes and their influence on blastocyst developmental capacity in pigs. Red fluorescence intensity by the mito-Ca 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-Ca by RR (10 and 20 μM) in presumptive zygotes using Rhod-2 staining and mito-Ca uptake 1 (MICU1) protein levels as an indicator of mito-Ca 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-Ca inhibitor, 100 nM) treated zygotes (P < 0.05). The collective results demonstrate that RR improves blastocyst development in porcine embryos by regulating mito-Ca and MICU1 expression following IVF.