【 TITLE 】Ruthenium red improves blastocyst developmental competence by regulating mitochondrial Ca²⁺ and mitochondrial functions in fertilized porcine oocytes in vitro

【 JOURNAL 】Journal of Reproduction and Development​(JRD)

【 NAME 】Ho-Geun JEGAL, Hyo-Jin PARK, Jin-Woo KIM, Seul-Gi Yang, Min-Ji KIM, Deog-Bon KOO

【 PUBLISHED 】22 April 2020

【 ISSN 】0916-8818

【 DOI 】https://doi.org/10.1262/jrd.2020-013

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【 ABSTRACT 】

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.​

【 KEYWORDS 】In vitro fertilization, Mito-Ca2+ uptake 1 (MICU1), Mitochondria-Ca2+, Porcine embryos, Ruthenium red​