Importance of Methionine Metabolism in Morula-to-blastocyst Transition in Bovine Preimplantation Embryos

Abstract

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...

Both the folate cycle and betaine‐homocysteine methyltransferase contribute methyl groups for DNA methylation in mouse blastocysts

“…These effects appear to be specifically due to inhibition of SAM production, rather than nonspecific or toxic effects, because addition of either the direct SAM precursor methionine or SAM to the medium rescued global 5-MeC levels in the ICM. This interpretation is consistent with the effects in bovine embryos of directly blocking methionine conversion to SAM with ethionine, where 5-MeC levels were also reduced (22). Therefore, we propose that, unlike virtually all nonhepatic cells, the mouse ICM has 2 pathways for SAM production that are at least partly redundant.…”

“…A methyl pool is almost certainly produced by pre‐implantation embryos. Exogenously supplied methionine is converted to SAM by both mouse and bovine preimplantation embryos (21, 22). Preimplantation embryos also likely have a fully functional folate cycle (15, 23) because all the relevant enzymes of 1‐carbon metabolism are expressed in the embryos of several species (24, 25), and inhibiting the folate cycle using the antifolate, methotrexate (MTX), causes preimplantation developmental arrest in vitro due to depletion of thymidylate and purines (25, 26).…”

Astaxanthin ameliorates heat stress-induced impairment of blastocyst development In Vitro: –astaxanthin colocalization with and action on mitochondria–

“…Culture media for in vitro production of bovine embryos Culture media for in vitro maturation (IVM) of immature oocytes recovered from ovaries, in vitro fertilization (IVF) of matured oocytes, and in vitro culture (IVC) of zygotes up to the blastocyst stage were prepared following a previously published protocol based on synthetic oviduct fluid (SOF) containing amino acids [46] with some modifications: the concentration of sodium pyruvate was increased to 0.5 mM, and the media were modified for each of the applications according to a previous report [18]. In brief, the medium for IVM (IVMM) was supplemented with 5.6 mM glucose, 10 % (v/v) fetal calf serum, and 0.2 IU/ml follicular-stimulating hormone (Kyoritsu Seiyaku, Kawasaki, Japan); the medium for IVF (IVFM) was not supplemented with glucose, and the medium for IVC (IVCM) was supplemented with 1.5 mM glucose.…”

“…Groups of 10 CEOs were matured in vitro in 50-μl drops of IVMM covered with mineral oil (Nacalai Tesque, Kyoto, Japan). Matured oocytes were fertilized in vitro with frozen-thawed bull sperm as described [39], except with regard to the medium used [18]. In brief, frozen-thawed bull semen was layered onto a discontinuous Percoll gradient solution (45 % and 90 % [v/v]) and centrifuged at 700×g for 30 min.…”

The Effects of Calcitonin on the Development of and Ca²⁺ Levels in Heat-shocked Bovine Preimplantation Embryos <i>In Vitro</i>

“…Groups of 10 CEOs were matured in vitro for 22 h at 38.5 C in 50 µl drops of IVMM. Matured oocytes were fertilized in vitro with frozen-thawed sperm from a single Japanese Black bull as described previously [28], except with regard to the medium used [29]. In brief, frozen-thawed bull semen was layered onto a discontinuous Percoll (Sigma, St. Louis, MO, USA) gradient solution (45% and 90% [v/v]) and centrifuged at 700 × g for 30 min.…”