돼지 미성숙 난포란의 체외성숙 시 β-mercaptoethanol의 첨가가 체외성숙, 체외수정 및 Glutathione 수준에 미치는 영향
Effect of β-Mercaptoethanol Supplement during In Vitro Maturation on IVM, IVF and Glutathione Level in Porcine Oocytes
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본 연구는 돼지 미성숙 난포란의 체외성숙 시 β-mercaptoethanol(ME)의 첨가가 체외성숙, 체외수정 후 웅성전핵의 형성 및 세포질 내의 glutathione의 수준에 미치는 영향을 알아보고자 실시하였다. 체외성숙 시 25μM의 β-ME를 첨가한 경우 다른 처리구에 비하여 성숙율이 약간 증가하는 것으로 나타났으며 36시간 체외성숙(81.0%) 보다 46시간 체외성숙(82.5%)에서 높은 성숙율을 나타냈다. 체외수정후 웅성전핵 형성에 있어서는 50μM의 β-ME 첨가구에서 가장 높게 나타났으며(P<0.05), 36시간 체외성숙(84.1%) 보다 46시간 체외성숙(84.2%)에서는 유의적 차이가 나지 않았다. Glutathione의 수준은 체외성숙 후 50μM의 β-ME 첨가구가 다른 처리구에 비교하여 높게 나타났으며(P<0.05), 36시간(25.4pmol)보다 46시간(28.1pmol)에서 높은 결과를 보였다. 체외수정 후 웅성전핵이 형성된 다음 세포질 내 glutathione의 수준 역시 50μM의 β-ME 첨가구에서 가장 높은 결과를 나타냈으며, 36시간 체외성숙(19.7mol)보다 46시간 체외성숙(20.1pmol)에서 유의적으로 높은 결과를 나타냈다(P<0.05).
Abstract
Experiments were conducted to determine the effects of beta-mercaptoethanol (β-ME) supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and intracellular glutathione (GSH) concentration. Porcine cumulus-intact oocytes were matured in TCM-199 medium containing porcine follicular fluid, sodium pyruvate, D-glucose, FBS, hormonal supplements, and β-ME (0, 25, 50 and 100μM) for 36 to 46h. After culture, cumulus-free matured oocytes were co-incubated with epididymal spermatozoa for 18h. There were no significant differences in the maturation rate among treatment groups. However, increases (P<0.05) in intracellular GSH concentration before and after fertilization were observed in 50μM β-ME supplements to the IVM medium. Also, increases (P<0.05) in male pronuclear formation after IVF were observed in same treatment group. In conclusion, supplementing β-ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.
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