Promoting effect of beta-mercaptoethanol on in vitro development under oxidative stress and cystine uptake of bovine embryos.
The effects of beta-mercaptoethanol (beta-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% O(2) supplemented with beta-ME. Addition of beta-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O(2), to almost the same rates when they were cultured in 5% O(2). To investigate whether the growth-promoting effect of beta-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 beta-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without beta-ME. In contrast, addition of beta-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by beta-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 beta-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of beta-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that beta-ME has a protective effect in embryo development against oxidative stress and that the effect of beta-ME is associated with the promotion of cystine uptake of low availability in embryos.
Takahashi
M
M
Department of Animal and Grassland Research, National Agricultural Center for Kyushu Okinawa Region, Kumamoto 861-1192, Japan. masashi@affrc.go.jp
Nagai
T
T
Okamura
N
N
Takahashi
H
H
Okano
A
A
D000818:Animals; D001826:Body Fluids; D002417:Cattle / Q000196:embryology*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D046508:Culture Techniques; D003553:Cystine / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000378:metabolism*; D005314:Embryonic and Fetal Development / Q000187:drug effects*; D005187:Fallopian Tubes / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D008623:Mercaptoethanol / Q000494:pharmacology*; D018384:Oxidative Stress; D010100:Oxygen / Q000008:administration & dosage; D013462:Sulfur Radioisotopes
D003470:Culture Media; D013462:Sulfur Radioisotopes; D003553:Cystine; D008623:Mercaptoethanol; D010100:Oxygen