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.

中文翻译：

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钌红通过调节线粒体的Ca提高胚泡发育能力²⁺在受精猪卵母细胞和线粒体功能的体外

钌红 (RR) 抑制钙 (Ca2+) 从细胞质进入线粒体，并参与维持哺乳动物细胞中的 Ca2+ 稳态。Ca2+ 稳态对于受精卵母细胞的进一步胚胎发育非常重要。然而，RR 对体外受精 (IVF) 期间线粒体-Ca2+ (mito-Ca2+) 水平的影响对猪随后的囊胚发育能力的影响尚不清楚。本研究探讨了使用 RR 和/或组胺在受精卵母细胞中调节 mito-Ca2+ 水平及其对猪囊胚发育能力的影响。与成熟卵母细胞相比，IVF 后 6 小时受精卵中 mito-Ca2+ 检测染料 Rhod-2 的红色荧光强度显着增加（P < 0.05）。基于这些结果，我们使用 Rhod-2 染色和 mito-Ca2+ 摄取 1 (MICU1) 蛋白质水平作为使用蛋白质印迹分析的 mito-Ca2+ 摄取指标，研究了推定受精卵中 RR（10 和 20 μM）对 mito-Ca2+ 的变化。正如预期的那样，与 IVF 后 6 小时未处理的受精卵相比，RR 处理的受精卵显示出 MICU1 和 Rhod-2 红色荧光强度的蛋白质水平降低。由于线粒体功能的改善，20 μM RR 处理的受精卵的囊胚发育率在 IVF 后 6 小时显着增加（P < 0.05）。相反，组胺（mito-Ca2+ 抑制剂，100 nM）处理的受精卵的囊胚发育率显着降低（P < 0.05）。集体结果表明，RR 通过调节体外受精后的 mito-Ca2+ 和 MICU1 表达来改善猪胚胎的囊胚发育。