The aim of the present study was to clarify whether or not our vitrification procedure at the germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was upregulated in 4–8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL gene in embryos.

中文翻译：

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生囊期猪卵丘-卵母细胞复合物的玻璃化不会触发卵母细胞和早期胚胎的细胞凋亡，但会激活4 细胞期后的抗凋亡Bcl-XL基因表达

本研究的目的是澄清我们在生发囊泡 (GV) 阶段的玻璃化过程是否会触发卵母细胞和后续胚胎的凋亡级联反应。未成熟的猪卵丘-卵母细胞复合物被玻璃化和加热（玻璃化组）或经受冷冻保护剂（CPA 组）或未经任何处理培养（对照）。对所有治疗组的卵母细胞进行体外成熟（IVM）、受精和胚胎培养。在 IVM 培养结束时的活卵母细胞和体外受精 (IVF) 后的卵裂期胚胎中检测细胞凋亡。我们在 IVM 结束时和所有组的早期胚胎中检测到卵母细胞中促凋亡 Bax 和 CASP3 基因的 DNA 断裂频率、半胱天冬酶活性水平、磷脂酰丝氨酸外化和 mRNA 水平相似。然而，在玻璃化组中，抗凋亡 Bcl-XL 基因在 4-8 个细胞胚胎中上调，与对照组和 CPA 组相比，Bcl-XL/Bax mRNA 比值显着增加了 8 倍（P < 0.05)。总之，通过我们的方法在 GV 阶段对猪卵母细胞进行玻璃化并不会触发卵母细胞和后续胚胎中的凋亡级联反应，而是触发了胚胎中抗凋亡 Bcl-XL 基因的上调。