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| 1 | Effects of MEK inhibitor U0126 on meiotic progression in mouse oocytes: microtuble organization, asymmetric division and metaphase Ⅱ arrest显示文摘In this study we used U0126, a potent and specific inhibitor of MEK, to study the roles of MEK/ERK/p90rsk signaling pathway in the meiotic cell cycle of mouse oocytes. The phosphorylation of MAP kinase and p90rsk in the oocytes treated with 1.5 μMU0126 was the same as that in oocytes cultured in drug-free medium. With 1.5 μM U0126 treatment, the spindles appeared normal as they formed in oocytes, but failed to maintain its structure.Instead, the spindle lost one pole or elongated extraordinarily. After further culture, some oocytes extruded gigantic polar bodies (>30 μm) that later divided into two small ones. Some oocytes underwent symmetric division and produced two equal-size daughter cells in which normal spindles formed. In oocytes with different division patterns,MAP kinase was normally phosphorylated. When the concentration of U0126 was increased to 15 mM, the phosphorylation of both MAPK and p90rsk were inhibited, while symmetric division was decreased. When incubating in medium containing 15 μM U0126 for 14 h, oocytes were activated, but part of them failed to emit polar bodies. MII oocytes were also activated by 15 μM U0126, at the same time the dephosphorylation of MAP kinase and p90rsk was observed. Our results indicate that 1) MEK plays important but not indispensable roles in microtubule organization;2) MEK keeps normal meiotic spindle morphology, targets peripheral spindle positioning and regulates asymmetric division by activating some unknown substrates other than MAP kinase/p90rsk; and 3) activation of MEK/ERK/p90rsk cascade maintains MII arrest in mouse oocytes. | CHAOTONG HENGYUFAN DAYUANCHEN XIANGFENSONG HEIDESCHATTEN QINGYUANSUN | 2003 | Cell Research2003，13，5： | 7 |
| 2 | 泛素羧基末端水解酶L1参与性成熟前小鼠卵母细胞的选择性剔除(英文)显示文摘异常卵母细胞的凋亡是性成熟前小鼠卵巢中具有缺陷的卵母细胞剔除的必要途径。泛素羧基末端水解酶L1(ubiquitin carboxyl-terminal hydrolase L1,UCH-L1)参与构成的泛素依赖性蛋白降解系统通过降解特定蛋白调节细胞凋亡,而UCH-L1依赖性的细胞凋亡对于睾丸的生精作用是必要的。本文利用免疫组化技术考察了性成熟前小鼠卵巢中不同发育阶段的卵泡数目的变化和UCH-L1在卵母细胞中的表达情况,结果显示卵巢中卵泡总数在出生后第21至28天显著减少;而UCH-L1蛋白在形态上出现异常的卵母细胞中表达显著增强,可能以某种程度与Jab1和p27Kip1蛋白关联。免疫荧光共定位显示UCH-L1在形态异常的卵母细胞中密度较高,而Jab1出现平行性的变化。免疫亲和分析显示卵巢中UCH-L1和Jab1发生作用。以上结果提示,UCH-L1在小鼠性成熟前的发育过程中异常卵母细胞的剔除方面具有重要作用,其作用可能以与Jab1和p27Kip1蛋白相关联的方式发生。 | 顾艳琼 陈秋菊 顾正 石燕 姚余卫 王健 孙兆贵 左嘉客 | 2009 | 生理学报2009，61，2： | 6 |
| 3 | 蟾蜍泛素羧基末端水解酶(tUCH)以不依赖其UCH活性的方式参与卵母细胞成熟调控显示文摘本实验室已报道中华大蟾蜍卵母细胞的p28蛋白具泛素羧基末端水解酶活性,称作tUCH,它和哺乳类中发现的UCH L1的氨基酸序列具高度同源性,二级结构同源性比较发现,二者可能具类似的功能。本文实验表明:未成熟卵母细胞和成熟卵母细胞的可溶性蛋白中均含有tUCH,约占提取物中蛋白质总量的2%。根据测定所得到的GST-tUCH和GST-UCH L1对底物Ub-AMC的酶动力学参数,说明卵母细胞中tUCH可能与小鼠UCH L1有类似的生物学功能;anti-tUCH单抗可以与原核细胞表达的tUCH和显性失活突变类型tUCH C(90)S特异结合,但不识别小鼠的UCH L1。Anti-tUCH单抗能够和tUCH结合但不能封闭它的UCH活性。当anti-tUCH单抗注入卵母细胞内,则孕酮诱导的生发泡破裂(germinal vesicle breakdown,GVBD)过程受到抑制,足见tUCH参与GVHD调节并不依赖其UCH活性。 | 孙兆贵 孔维华 颜山 顾正 左嘉客 | 2003 | 实验生物学报2003，36，2： | 4 |
| 4 | MiR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine granulosa cells显示文摘Background: Granulosa cells(GCs) proliferation and estradiol synthesis significantly affect follicular development.The miR-214-3p expression in the ovarian tissues of high-yielding sows is higher than that in low-yielding sows,indicating that miR-214-3p may be involved in sow fertility. However, the functions and mechanisms of miR-214-3p on GCs are unclear. This study focuses on miR-214-3p in terms of the effects on GCs proliferation and estradiol synthesis.Results: Our findings revealed that miR-214-3p promotes proliferation and inhibits estradiol synthesis in porcine GCs. MiR-214-3p can increase the percentage of S-phase cells, the number of EdU labeled positive cells, and cell viability. However, E2 concentration was reduced after miR-214-3p agomir treatment. We also found that miR-214-3p up-regulates the expression of cell cycle genes including cell cycle protein B(Cyclin B), cell cycle protein D(Cyclin D), cell cycle protein E(Cyclin E), and cyclin-dependent kinase 4(CDK4) at the transcription and translation levels, but down-regulates the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1(CYP11A1), cytochrome P450 family 19 subfamily A member 1(CYP19A1), and steroidogenic acute regulatory protein(StAR)(i.e., the key enzymes in estradiol synthesis). On-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target genes of miR-214-3p in proliferation and estradiol synthesis are Mfn2 and NR5A1, respectively.Conclusions: Our findings suggest that miR-214-3 p plays an important role in the functional regulation of porcine GCs and therefore may be a target gene for regulating follicular development. | Shengjie Shi Xiaoge Zhou Jingjing Li Lutong Zhang Yamei Hu Yankun Li Gongshe Yang Guiyan Chu | 2021 | Journal of Animal Science and Biotechnology2021，12，1： | 3 |
| 5 | Uchl1及其关联蛋白参与小鼠实验性隐睾中精母细胞凋亡(英文)显示文摘以往研究显示Uchl1参与调节生理状况下小鼠精母细胞的凋亡。本文以小鼠实验性单侧隐睾为研究模型,以切除单侧睾丸和假手术作为对照,用苏木精-伊红(HE)染色和DNA末端标记(TUNEL)观察生精细胞的形态和凋亡情况;用免疫组化分析Uchl1及其相关蛋白Jab1和p27kip1在隐睾症的热应激反应导致精母细胞损失过程中的变化情况,并用亲和分析(pull-down)和免疫荧光共定位检测三种蛋白在精母细胞的关联性。结果显示,Jab1和p27kip1,与Uch1平行,在具凋亡形态的精母细胞中响应热应激而含量增加,而在多核巨细胞中无类似变化。Jab1可以与睾丸蛋白提取物中Uchl1结合,并与Uchl1和p27kip1特异性地在具凋亡形态的精母细胞中共定位。以上结果提示,Uchl1蛋白的积累参与隐睾中热应激诱导的生精细胞凋亡过程,但不影响接下来的多核巨细胞的形成,且Uchl1蛋白的作用机制涉及Jab1和p27kip1参与的一种新途径。 | 杜平 姚余卫 石燕 顾正 王健 孙兆贵 左嘉客 | 2014 | 生理学报2014，66，5： | 3 |
| 6 | 泛素羧基末端水解酶1基因缺失对雌性小鼠生殖系统发育的影响显示文摘目的研究泛素羧基末端水解酶L1 (ubiquitin C-terminal hydrolase L1,UCH-L1)基因缺失对雌性小鼠生殖系统的影响。方法利用阴道涂片、免疫组织化学、蛋白质印迹和RNA干扰等方法,分析UCH-L1基因缺失对小鼠卵巢功能及卵泡发育的影响。结果 UCH-L1基因缺失纯合子成年雌性小鼠的体质量降低,运动困难,且子宫偏细。在UCH-L1基因缺失纯合子小鼠的卵巢和子宫中检测不到UCH-L1蛋白表达,卵巢体积明显小于杂合子小鼠,其卵巢皮质层存在一些原始卵泡或闭锁卵泡,没有成熟卵泡和黄体。雌激素受体(estrogenreceptor,ER)、卵泡刺激素受体(follicle-stimulating hormone receptor,FSHR)、黄体生成素受体(luteinizing hormone receptor,LHR)及孕酮受体(progesterone receptor,PR)等生殖关键受体在UCH-L1缺失纯合子小鼠和杂合子小鼠卵巢中都有表达,其中纯合子小鼠卵巢中ER的表达水平明显低于杂合子小鼠。通过RNA干扰的方法下调生发泡(germinal vesicle,GV)期卵母细胞中UCH-L1的表达,能明显降低卵母细胞的成熟率。结论 UCH-L1基因缺失雌性小鼠不具备形成成熟卵泡及排卵的能力,卵巢中ER表达量显著降低。推测UCH-L1有可能通过下调卵巢中ER的表达,影响卵母细胞成熟及排卵过程。 | 吴比 梁珊珊 阮井玲 黄鑫 滕晓明 洪岭 | 2020 | 实验动物与比较医学2020，40，4： | 1 |
| 7 | 己烯雌酚促验进实动物卵泡发育同步化及其机制的初步研究显示文摘目的观察人工合成的非甾体化合物己烯雌酚(DEs)对性成熟前小鼠和大鼠卵巢中卵泡发育的影响,以及出现有卵泡同步化机理。方法取性成熟前雌性小鼠和大鼠,腹腔注射一定剂量的DES,然后取卵巢经连续切片和HE染色后,统计整个卵巢内三级卵泡数量。同时,取卵巢利用免疫组织化学和免疫印迹方法,分别检测泛素末端水解酶LI(UCH-L1),c-Jun结合蛋白l(Jabl)和细胞周期素依赖激酶抑制因子p27KipI在小鼠卵巢中的定位和含量。结果UCH-LI定位在卵巢中各级卵泡的卵母细胞中,Jab1分布于次级和三级卵泡的卵母细胞核中,而p27KipI广泛分布于卵巢各类细胞中。免疫组化结果显示,仅就卵母细胞而言,由于DES的处理,UCH—L1和Jab1的表达信号增强,而p27KipZ的信号变弱,与肿瘤细胞中的情况类似。然而,免疫印迹分析结果表明,以上3种蛋白在卵巢中相对b-actin的含量因DES处理而均呈现一定程度的上升变化趋势。青春前期的小鼠,在1-4周龄期间,卵巢中UCH.L1和p27KiPI的蛋白量随着周龄增加的趋势,而Jab1的变化趋势不明显。结论腹腔注射DES或导致卵巢内卵泡发育同步化,其中涉及的机理可能是,UCH-L1及其关联蛋白Jab1导致p27KiPI在卵母细胞核中的含量下降,进而导致了多个卵泡的卵母细胞同时发育启动。 | 顾艳琼 石燕 王健 孙兆贵 | 2013 | 实验动物与比较医学2013，33，2： | 0 |
| 8 | 泛素羧基末端水解酶L1C90S和I93M点突变不影响小鼠卵母细胞成熟显示文摘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。 | 许亚军 张瑜 石燕 印惠荣 汪伟伟 胡婷婷 松迪 孙兆贵 | 2021 | 第二军医大学学报2021，42，11： | 0 |
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