Effect of embryo vitrification on the expression of brain tissue proteins in mouse offspring

Abstract

Vitrification is widely used in assisted reproductive technologies. However, the nervous system of vitrification offspring is of concern, and research on this is lacking. Vitrification-born mice (vitrification group), conventional in vitro fertilization-embryo transfer pregnancy-born mice (IVF group), and natural pregnancy-born mice (control group) were used to study the effects of vitrification of mouse embryos on protein levels in the brain of offspring. Proteins differentially expressed among the three groups were analyzed using proteomic methods, including two-dimensional electrophoresis, mass spectrometry, and bioinformatics analysis. Immunohistochemistry was used to verify the expression of differentially expressed proteins, such as Actb and Actg1, in each group. Twenty differentially expressed proteins in the brain tissue were identified using two-dimensional protein electrophoresis and mass spectrometry. Bioinformatics analysis revealed that these proteins were related to the development of anatomical structure, signal transduction, transport, cell differentiation, and stress response (biological processes) and the binding of molecules in vivo (molecular functions). The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the differentially expressed proteins were involved in 54 pathways, including phagosome, metabolic pathway, apoptosis, and cysteine and methionine metabolism. Thus, embryo vitrification may cause some changes in the mouse brain at the protein level, necessitating further safety assessment.

摘要

玻璃化冷冻技术广泛应用于辅助生殖技术。然而, 玻璃化冷冻术所出生后代的神经系统是令人关注的, 这方面的研究是缺乏的。采用玻璃化小鼠（玻璃化组）、常规体外受精-胚胎移植孕产小鼠（体外受精组）和自然孕产小鼠（对照组）研究玻璃化小鼠胚胎对子代脑组织蛋白水平的影响。应用蛋白质组学方法, 包括双向电泳、质谱和生物信息学分析, 分析三组间表达差异的蛋白。应用免疫组化方法验证各组差异表达蛋白Actb、Actg1的表达情况。应用二维蛋白电泳和质谱技术鉴定脑组织中20个差异表达蛋白。生物信息学分析显示, 这些蛋白与解剖结构的发展、信号转导、转运、细胞分化、应激反应（生物学过程）和体内分子的结合（分子功能）有关。《京都基因和基因组百科全书》通路分析显示, 差异表达蛋白参与了54条通路, 包括吞噬体、代谢通路、凋亡、半胱氨酸和蛋氨酸代谢。因此, 胚胎玻璃化冷冻可能在蛋白质水平上引起小鼠脑内的一些变化, 需要进一步的安全性评估。

Keywords:

• In vitro fertilization
• vitrification
• embryo
• ICR mouse
• brain

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

The data that support the findings of this study are available from the corresponding author, ZW, upon reasonable request.

Additional information

Funding

This work was supported by the Guangdong Provincial Key Laboratory of Reproductive Medicine under Grant no. [2012A061400003] and Science Fund of the National Natural Science Foundation of China under Grant no. [81200473].