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Promoting Effect of ß-Mercaptoethanol on In Vitro Development under Oxidative Stress and Cystine Uptake of Bovine Embryos¹

^a Department of Animal and Grassland Research, National Agricultural Center for Kyushu Okinawa Region, Kumamoto 861-1192, Japan ^b Developmental Biology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan ^c Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan ^d Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Tsukuba Norin-kenkyu-danchi, Ibaraki 305-0901, Japan

The effects of ß-mercaptoethanol (ß-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O₂ supplemented with ß-ME. Addition of ß-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O₂, to almost the same rates when they were cultured in 5% O₂. To investigate whether the growth-promoting effect of ß-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without ß-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without ß-ME. In contrast, addition of ß-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by ß-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by ß-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of ß-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that ß-ME has a protective effect in embryo development against oxidative stress and that the effect of ß-ME is associated with the promotion of cystine uptake of low availability in embryos.

First decision: 1 June 2001.

¹ This research was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan.

² Correspondence: Masashi Takahashi, 2421 Suya, Nishigoshi, Kikuchi, Kumamoto 861-1192, Japan. FAX: 81 96 249 1002; masashi{at}affrc.go.jp

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