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比较猪模型中原核期卵母细胞玻璃化冷冻前后对细胞骨架完整性、受精和发育能力的影响。

Comparison of cytoskeletal integrity, fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model.

机构信息

Research Institute for Animal Breeding and Nutrition, Herceghalom H-2053, Hungary.

出版信息

Cryobiology. 2013 Dec;67(3):287-92. doi: 10.1016/j.cryobiol.2013.08.009. Epub 2013 Aug 28.

Abstract

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.

摘要

本研究旨在通过猪模型研究卵母细胞在体外成熟(IVM)之前或之后进行玻璃化冷冻对卵母细胞活力、细胞骨架完整性和体外受精(IVF)后体外发育能力的影响。来自屠宰场的猪卵巢卵母细胞在GV 期或 MII 期通过改良的固相玻璃化(SSV)进行玻璃化冷冻。通过立体显微镜观察评估卵母细胞活力,通过免疫荧光染色后共聚焦显微镜观察核期和微管及 F-肌动蛋白的形态。通过 Orcein 染色评估受精情况。MII 期卵母细胞的玻璃化冷冻存活率高于 GV 期卵母细胞。然而,GV 期玻璃化冷冻后存活卵母细胞达到 MII 期的能力与对照卵母细胞相似。此外,在 IVM 后,GV 期玻璃化冷冻的卵母细胞纺锤体和 F-肌动蛋白完整性优于 MII 期玻璃化冷冻的卵母细胞(MII 期玻璃化冷冻的卵母细胞)。与这一结果一致的是,与 MII 期玻璃化冷冻的卵母细胞相比,GV 期玻璃化冷冻的卵母细胞在体外受精(IVF)后有更好的第二极体排出和支持雄性原核形成的能力。受精率在各组之间没有差异。最后,GV 期玻璃化冷冻卵母细胞的发育能力优于 MII 期玻璃化冷冻卵母细胞。然而,与对照组相比,两个玻璃化冷冻组的囊胚发育能力均有所下降。总之,与 MII 期玻璃化相比,GV 期玻璃化冷冻更有利于猪卵母细胞获得更好的细胞骨架组织和发育为囊胚的能力。

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