title: Toxicity evaluation of ethanol treatment during in vitro maturation of porcine oocytes and subsequent embryonic development following parthenogenetic activation and in vitro fertilization.
abstract: Ethanol is frequently used as a solvent in several techniques for in vitro production (IVP). It is also used for the parthenogenetic activation (PA) of oocytes. Although a number of studies have suggested that ethanol has detrimental effects on fibroblasts and neuronal cells, little attention has been paid to the effects of ethanol on porcine oocytes. Thus, the aim of this study was to evaluate the effects of the addition of ethanol to in vitro maturation (IVM) medium. We investigated the effects of ethanol (0, 1 and 3%) on the following parameters: nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic development following PA and in vitro fertilization (IVF). After 44 h of IVM, the 3% group showed a significant (P<0.05) decrease in nuclear maturation (34.0%) compared with the control group (70.3%). The 1 and 3% groups exhibited a significant (P<0.05) decrease in GSH levels and an increase in ROS levels compared with the control group. Compared with the control group, the 3% group had significantly (P<0.05) lower cleavage rates following PA (51.6 vs. 86.9%) and IVF (53.2 vs. 70.6%), as well as lower blastocyst formation rates and decreased total cell numbers following PA (11.3% and 31.8 vs. 53.6% and 65.4, respectively) and IVF (4.1% and 22.0 vs. 36.1% and 70.3, respectively). We evaluated the mRNA expression levels of DNA repair‑related and apoptosis‑related genes in the cumulus oocyte complexes (COCs). The 1% ethanol group showed significantly (P<0.05) higher mRNA expression levels of poly(ADP‑ribose) polymerase‑1 (PARP‑1), Bax, Bak and caspase‑3, and the 3% ethanol group had significantly (P<0.05) increased PARP‑1, Bax and caspase‑3 mRNA expression levels compared with the control group. Our results suggest that treatment with >1% ethanol during IVM exerts a toxic effect on the developmental potential of PA and IVF porcine embryos by decreasing the intracellular GSH level, thereby increasing the intracellular ROS level and upregulating the expression of apoptosis‑related genes.
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Sanghoon
authors.initials: S
authors.lastname: Lee
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Eunhye
authors.initials: E
authors.lastname: Kim
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361‑763, Republic of Korea.
authors.forename: Sang-Hwan
authors.initials: SH
authors.lastname: Hyun
mesh_terms: D000818:Animals; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D047108:Embryonic Development / Q000187:drug effects; D000431:Ethanol / Q000633:toxicity*; D005307:Fertilization in Vitro / Q000379:methods*; D005978:Glutathione / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D010312:Parthenogenesis / Q000187:drug effects*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D013552:Swine; D015854:Up-Regulation; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D000431:Ethanol; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3; D005978:Glutathione