Universitat Autònoma de Barcelona, Departament de Ciència Animal i Dels Aliments, 08193, Bellaterra, Barcelona, Spain.
Università Degli Studi di Sassari, Dipartimento di Medicina Veterinaria. V.Vienna 2, 07100, Sassari, Italy.
Cryobiology. 2020 Apr;93:56-61. doi: 10.1016/j.cryobiol.2020.02.011. Epub 2020 Feb 25.
This work studies the effect of vitrification of in vitro matured (IVM) prepubertal goat oocytes on: 1) oocyte damage assessed by reactive oxygen species (ROS) level and apoptosis and 2) embryo development after Intracytoplasmic sperm injection (ICSI) and Parthenogenic Activation (PA). Oocytes were IVM in supplemented TCM-199 for 22-24 h. Control group oocytes matured during 24 h were directly used for the analysis after IVM. Vitrified/warmed IVM-oocytes were vitrified after 22 h of IVM in 15% ethylene glycol (EG), 15% dimethyl sulfoxide (MeSO) and 0.5 M sucrose and after subjected to warming procedure. Oocyte ROS level was measured by staining denuded IVM-oocytes with 10 μM 2'7' dichlorodihydrofluorescein diacetate. Apoptosis was analyzed by Annexin V (AV) Apoptosis Detection kit and Propidium iodide (PI) signal and oocytes were classified as: Live (AV PI), early apoptotic (AV PI), dead non-apoptotic (AV PI) and necrotic (AV PI). Developmental competence of vitrified/warmed oocytes was assessed by PA (5 min in 5 μM Ionomycin plus 4 h in 2 mM 6-Dimethylaminopurine), and by ICSI fertilization. Presumptive zygotes were in vitro cultured for 8 days in commercial media BO-IVC. Vitrified/warmed oocytes showed higher ROS levels (P < 0.0001), lower live oocytes (44 vs. 66%; P: 0.0025) and higher dead non-apoptotic oocytes (33 vs. 13% P: 0.023) compared to control. No differences were found on normal zygote formation (2 PN) (32 vs. 25%) or blastocyst development (0 vs. 4%) after ICSI fertilization. However, after PA, significant differences were found in cleavage rate (59 vs.78%; P < 0.0343) and blastocyst formation (1 vs. 25%; P < 0.0001). In conclusion, vitrification reduced oocyte competence by increasing dead oocytes and ROS levels.
这项工作研究了玻璃化对体外成熟（IVM）未成年山羊卵母细胞的影响：1）通过活性氧（ROS）水平和凋亡评估卵母细胞损伤，2）胞质内精子注射（ICSI）和孤雌激活（PA）后的胚胎发育。卵母细胞在补充有 TCM-199 的培养基中 IVM 22-24 小时。对照组的卵母细胞在 IVM 24 小时后直接用于 IVM 分析。在 IVM 22 小时后，将 IVM 后的卵母细胞用 15%乙二醇（EG）、15%二甲基亚砜（MeSO）和 0.5 M 蔗糖进行玻璃化/解冻，并进行解冻程序。通过用 10 μM 2'7'二氯二氢荧光素二乙酸酯染色去透明化的 IVM 卵母细胞来测量卵母细胞的 ROS 水平。通过 Annexin V（AV）凋亡检测试剂盒和碘化丙啶（PI）信号分析凋亡，将卵母细胞分为：活（AV PI）、早期凋亡（AV PI）、死亡非凋亡（AV PI）和坏死（AV PI）。通过 PA（5 分钟在 5 μM 离子霉素中，4 小时在 2 mM 6-二甲基氨基嘌呤中）和 ICSI 受精评估玻璃化/解冻卵母细胞的发育能力。假设合子在商业培养基 BO-IVC 中体外培养 8 天。与对照组相比，玻璃化/解冻后的卵母细胞 ROS 水平更高（P<0.0001），活卵母细胞更少（44% vs.66%；P：0.0025），死亡非凋亡卵母细胞更多（33% vs.13%；P：0.023）。ICSI 受精后，正常合子形成（2PN）（32% vs.25%）或囊胚发育（0% vs.4%）没有差异。然而，PA 后，卵裂率（59% vs.78%；P<0.0343）和囊胚形成率（1% vs.25%；P<0.0001）有显著差异。总之，玻璃化通过增加死亡卵母细胞和 ROS 水平降低了卵母细胞的活力。
