88 EXPRESSION OF ENZYMES RELATED TO HOMOCYSTEINE METABOLISM AND HOMOCYSTEINE SENSITIVITY IN BOVINE PREIMPLANTATION EMBRYOS

Abstract

Homocysteine is a nonessential amino acid produced through methionine metabolism. Elevation of homocysteine levels (hyperhomocysteinemia) increases intracellular S-adenosylhomocysteine (SAH). S-adenosylhomocysteine binds to methyltransferases (MT) with greater affinity than does S-adenosylmethionine, which is the universal methyl donor used by various cellular MT, including DNA and histone MT. Thus, SAH acts as a potent competitive inhibitor of methylation reactions. Therefore, disorder of homocysteine metabolism may affect cellular homeostasis in part through the process involving methylation reactions (Williams KT and Schalinske KL 2007 J. Nutr. 137, 311–314). Despite its predicted importance, the involvement of homocysteine metabolism in pre-implantation embryonic development remains unaddressed. In the present study, the expression of enzymes related to homocysteine metabolism in bovine pre-implantation embryos and the effects of homocysteine on the post-fertilization development of these embryos in vitro were investigated. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were in vitro-matured (IVM) for 22 h in modified synthetic oviduct fluid (mSOF) supplemented with 10% v/v FCS and 0.2 IU mL^–1 follicle-stimulating hormone. After IVM, the oocytes were subjected to IVF with Percoll gradient-selected sperm from one bull in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from the cumulus cells and cultured in mSOF up to Day 8 (IVF = Day 0). All cultures were performed at 38.5°C under 5% CO₂, 5% O₂, and 90% N₂. Total RNA was extracted from individual blastocysts on Day 7 to 8 and reverse transcribed to cDNA using oligo(dT) primer. Transcripts for methionine adenosyltransferase 2A (MAT2A), MAT2B, adenosylhomocysteinase, methionine synthase, betaine-homocysteine MT, serine hydroxymethyltransferase 1, and 5,10-methylenetetrahydrofolate reductase were examined by qualitative PCR using bovine-specific primers for each gene and the cDNA as templates. β-Actin transcripts were used as an internal control. Moreover, presumptive zygotes after IVF were cultured in mSOF supplemented with 0 (control), 10, and 100 μm homocysteine, and development to cleavage stage and blastocyst was assessed on Day 3 and Day 7 and 8, respectively. The cultures were replicated 4 times using 561 embryos. The development data were statistically analyzed by using the general linear model. Transcripts for all genes examined were detected. Homocysteine added to the culture medium of bovine IVF embryos did not affect the cleavage rate (86.8, 83.3, and 84.3% for control, 10 μm, and 100 μm, respectively); however, blastocyst rate significantly decreased (P = 0.02) on Day 7 (12.8, 9.3, and 7.5%, respectively). The blastocyst rate on Day 8 showed no difference (P = 0.33) among the groups (24.4, 20.4, and 20.1%, respectively). These results indicate that a system for homocysteine metabolism is present in bovine pre-implantation embryos and that high homocysteine levels affect the developmental kinetics of these embryos.

Supported by KAKENHI.