Abstract. The roles of methionine metabolism in bovine preimplantation embryo development were investigated by using ethionine, an antimetabolite of methionine. In vitro produced bovine embryos that had developed to the 5-cell stage or more at 72 h after the commencement of in vitro fertilization (IVF) were then cultured until day 8 (IVF = day 0) in medium supplemented with 0 (control), 1, 5 and 10 mM ethionine. Compared with the blastocyst development in the control (40.0%), ethionine at 10 mM almost completely blocked blastocyst development (1.1%, P<0.001), and this concentration was used in the following experiments. Methionine added at the same concentration (10 mM, a concentration control of ethionine) did not cause such an intense developmental inhibition. Development to the compacted morula stage on day 6 was not affected by 10 mM ethionine treatment. S-adenosylmethionine (SAM) added to the ethionine treatment partly restored the blastocyst development. Semiquantitative reverse transcription-polymerase chain reaction analysis of cell lineage-related transcription factors in day 6 compacted morulae showed that the expressions of NANOG and TEAD4 were increased by ethionine treatment relative to the control (P<0.01). Furthermore, immunofluorescence analysis of 5-methylcytosine revealed that DNA was hypomethylated in the ethionine-treated day 6 morulae compared with the control (P<0.001). These results demonstrate that the disruption of methionine metabolism causes impairment of the morula-toblastocyst transition during bovine preimplantation development in part via SAM deficiency, indicating the indispensable roles of methionine during this period. The disruption of methionine metabolism may cause hypomethylation of DNA and consequently lead to the altered expression of developmentally important genes, which then results in the impairment of blastocyst development. Key words: Bovine, Methionine, One-carbon metabolism, Preimplantation embryo, S-adenosylmethionine (J. Reprod. Dev. 58: [91][92][93][94][95][96][97] 2012) M ethionine is a dietary essential amino acid that plays multiple biological roles, including those as the initiating amino acid in eukaryotic protein synthesis, a precursor of the essential methyl donor for methylation reactions (S-adenosylmethionine, SAM) and a source of redox regulators (cysteine and glutathione) [1,2]. Methionine metabolism can be seen as the trans-and remethylation cycle of methionine (methionine cycle) that intertwines with the cycle of folate metabolism (folate cycle) and the transsulfuration pathway from homocysteine, an intermediate in the methionine cycle [3], and these combined metabolic networks are called one-carbon metabolism [4,5]. Recently, mammalian oocytes and preimplantation embryos have been reported to express the key regulatory enzymes in one-carbon metabolism [6][7][8], suggesting that mammalian oocytes and preimplantation embryos can independently utilize and metabolize nutrients in one-carbon metabolism, such as methionine, choline, betaine, folates a...