126 EXPRESSION OF MYO-INOSITOL OXYGENASE IN BOVINE PRE-IMPLANTATION EMBRYOS AND ITS REGULATION BY ANTI-OXIDATIVE VITAMINS

Abstract

Myo-inositol (MI) added into in vitro culture media stimulates blastocyst development of mammals, including cattle (Holm et al. 1999 Theriogenology 52, 683–700), and these stimulatory effects are considered to be exerted via the incorporation of MI into phosphatidylinositol (Hynes et al. 2000 Mol. Reprod. Dev. 55, 265–269). Myo-inositol is catabolized by MI oxygenase (MIOX), which may affect bioavailability of MI for phosphatidylinositol pathway. In the present study, we investigated the expression pattern of MIOX transcripts in bovine pre-implantation embryos and the effects of anti-oxidative vitamins, which promote blastocyst hatching (Ikeda 2014 Reprod. Fertil. Dev. 26, 157), on the expression levels of MIOX in bovine blastocysts in vitro. Cumulus-enclosed oocytes obtained from slaughterhouse bovine ovaries were in vitro-matured for 22 h in modified SOF (mSOF) supplemented with 10% v/v FCS and 0.2 IU mL^–1 FSH. After in vitro maturation, the oocytes were subjected to IVF with Percoll gradient-selected sperm in an mSOF-based medium for 20 h. After IVF, presumptive zygotes were freed from 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 embryos at the 1-cell, 2-cell, 8-cell, morula, and blastocyst stages (n = 15 × duplicates for each stage) and reverse transcribed to cDNA using oligo (dT) primer in a 31.5 μL reaction volume. Transcripts for MIOX were examined by quantitative reverse transcription-PCR (qRT-PCR) using 1 μL of the cDNA solution and succinate dehydrogenase as a reference gene. Moreover, IVF-derived 8- to 16-cell embryos on Day 3 were cultured in mSOF supplemented with or without a vitamin mix (11 μM a-tocopherol and 9 nM β-carotene) up to Day 8, and blastocysts (n = 10 × 3 replicates) were collected from each treatment. The expression levels of MIOX transcripts in the blastocysts were compared between the treatments by using qRT-PCR. The data were statistically analysed by Mann–Whitney U test. The MIOX transcripts were undetectable at the 1-cell and 2-cell stages and then expressed from the 8-cell stage onward. The vitamin treatment significantly (P < 0.05) decreased the expression level of MIOX in blastocysts. These results suggest that MIOX is one of embryonic-activated genes in bovine pre-implantation embryos, and the level of its expression is regulated by anti-oxidative vitamins. The stimulatory effects of anti-oxidative vitamins on blastocyst development might be in part via the modulation of MI bioavailability.