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| 泛素羧基末端水解酶L1 C90S和I93M点突变不影响小鼠卵母细胞成熟 |
| 许亚军1,张瑜2,石燕2,印惠荣1,汪伟伟1,胡婷婷1,松迪1*,孙兆贵2* |
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(1. 海军军医大学(第二军医大学)长海医院生殖医学中心, 上海 200433;
2. 上海市生物医药技术研究院, 国家卫生健康委员会计划生育药具重点实验室, 上海 200032
*通信作者) |
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| 摘要: |
| 目的 通过观察泛素羧基末端水解酶L1（UCHL1）点突变蛋白与野生型蛋白过量对小鼠卵母细胞成熟的影响，并分析其在小鼠卵母细胞离体成熟过程中的作用及作用方式。方法 通过原核重组蛋白技术制备UCHL1点突变（C90S和I93M）和野生型蛋白与谷胱甘肽S-转移酶（GST）的融合蛋白，通过显微注射技术将UCHL1野生型蛋白与GST的融合蛋白、UCHL1点突变蛋白与GST的融合蛋白、GST、PBS分别注射到小鼠未成熟卵母细胞，或在体外培养基中添加UCHL1野生型蛋白与GST的融合蛋白，分析野生型UCHL1过量、UCHL1（I93M）点突变蛋白添加及泛素水解酶活性缺失的UCHL1（C90S）点突变蛋白添加对卵母细胞生发泡破裂（GVBD）率的影响。分离UCHL1（I93M）点突变小鼠与野生型小鼠的生发泡期卵母细胞，体外培养3 h后比较其GVBD率。结果 显微注射UCHL1野生型蛋白及其点突变体与GST的融合蛋白的各组之间、注射GST组、注射PBS组卵母细胞GVBD率差异均无统计学意义，在培养基中添加UCHL1蛋白与GST的融合蛋白组卵母细胞GVBD率与无注射无添加对照组相比差异也无统计学意义（P均>0.05）。注射UCHL1（C90S）与GST的融合蛋白组有少量生发泡期卵母细胞体外发育为MⅡ期卵母细胞后呈现极体较对照组偏大的现象。UCHL1（I93M）点突变小鼠生发泡期卵母细胞体外GVBD率与野生型小鼠比较差异无统计学意义（P>0.05）。结论 在小鼠卵母细胞中添加外源性UCHL1、有致病作用的UCHL1（I93M）突变体或泛素水解酶活性丧失的UCHL1（C90S）突变体均不影响卵母细胞成熟的GVBD进程，UCHL1（I93M）点突变小鼠卵母细胞GVBD率亦无异常，但UCHL1（C90S）点突变可能影响极体的形成。 |
| 关键词: 泛素羧基末端水解酶L1 生发泡破裂 卵母细胞 蛋白显微注射 重组蛋白质类 |
| DOI：10.16781/j.0258-879x.2021.11.1217 |
| 投稿时间：2021-08-01修订日期：2021-10-19 |
| 基金项目:上海市计划生育科学研究所青年科技创新基金（Q2016-5）. |
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| C90S and I93M mutations of ubiquitin C-terminal hydrolase L1 do not affect mouse oocyte maturation |
| XU Ya-jun1,ZHANG Yu2,SHI Yan2,YIN Hui-rong1,WANG Wei-wei1,HU Ting-ting1,SONG Di1*,SUN Zhao-gui2* |
(1. Department of Reproductive Medicine, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
2. NHC Key Lab of Reproduction Regulation (Shanghai Institute for Biomedical and Pharmaceutical Technologies), Fudan University, Shanghai 200032, China
*Corresponding authors) |
| Abstract: |
| Objective To observe the effects of ubiquitin C-terminal hydrolase L1 (UCHL1) mutant protein and wild-type protein overdose on mouse oocyte maturation, and analyze its role and interaction mode in mouse oocyte maturation in vitro. Methods The fusion proteins of UCHL1 mutations (C90S and I93M) and wild-type UCHL1 proteins with glutathione S-transferase (GST) were prepared by recombinant protein expression technology. The fusion proteins, GST and phosphate buffered solution (PBS) were injected into immature oocytes by microinjection technology, or wild-type proteins were added to in vitro culture medium to analyze the effects of wild-type UCHL1 overdose, UCHL1 (I93M) mutant protein addition or addition of UCHL1 (C90S) mutant protein with loss of ubiquitin hydrolase activity on oocyte germinal vesicle breakdown (GVBD). The germinal vesicle oocytes of UCHL1 (I93M) mutant mice and wild-type mice were isolated and cultured in vitro for 3 h, and then the GVBD rates were compared. Results There was no significant difference in the GVBD rates of oocytes between microinjection groups with UCHL1- or its mutants-GST fusion protein, GST injection group or PBS injection group (all P<0.05). There was also no significant difference in the GVBD rates of oocytes in the group added with UCHL1-GST fusion protein in the culture medium compared with the control group without injection or addition (P>0.05). A small number of germinal vesicle oocytes in the UCHL1 (C90S)-GST fusion protein injected group progressed to MⅡ stage oocytes in vitro, and the polar body was larger than that of the control group. There was no significant difference in GVBD rates of germinal vesicle oocytes in vitro between UCHL1 (I93M) mutant mice and wild-type mice (P>0.05). Conclusion The addition of exogenous UCHL1, UCHL1 (I93M) mutant with toxic effects, or UCHL1 (C90S) mutant with loss of hydrolase activity in mouse oocytes do not affect the GVBD process of oocyte maturation. The GVBD rate of mouse oocytes with UCHL1 (I93M) mutation is normal. However, the mutation of UCHL1 (C90S) may affect the formation of polar bodies. |
| Key words: ubiquitin C-terminal hydrolase L1 germinal vesicle breakdown oocytes protein microinjection recombinant proteins |
