小鼠生殖细胞特异性转录因子 NOBOX 调控初级卵泡发育机制的研究
作者:
作者单位:

1.中国医科大学实验动物部,沈阳 110000; 2.辽宁省转基因动物研究重点实验室,沈阳 110000

中图分类号:

R-33


Study of the mechanism by which the mouse germ cell-specific transcription factor NOBOX regulates primary follicle development
Author:
Affiliation:

1.Department of Laboratory Animal Science, China Medical University, Shenyang 110000, China. 2.Key Laboratory of Transgenetic Animal Research, Liaoning Province, Shenyang 110000

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    摘要:

    目的 NOBOX 是在原始卵泡激活过程中起关键作用的转录因子,可直接调控 Gdf9 转录,并参与 GDF9 / SMAD 通路影响颗粒细胞增殖分化,但其在初级卵泡及之后的具体调控机制并不清楚。 Kit 作为影响卵泡发育的重要基因,其启动子存在多个 NOBOX 结合位点。 本研究拟探索在初级卵泡发育阶段,转录因子 NOBOX 对 Kit 的转录调控作用,以及通过 GDF9 / SMAD 通路对卵泡发育的影响。 方法 体外培养初级卵泡统计发育时间;qRT- PCR 检测显微注射 Nobox-siRNA 的初级卵泡中 NoboxKitKitl、Gdf9 的 mRNA 表达变化;Western blot 检测显微注射 Nobox-siRNA 的初级卵泡中 NOBOX、Kit、P-SMAD的蛋白表达变化;ChIP 实验验证转录因子 NOBOX 在 Kit 基因启动子上的结合位点。 结果 初级卵泡在体外培养第 5 天可发育为次级卵泡,注射 Nobox-siRNA 的初级卵泡延缓 2 d 发育至次级;初级卵泡中注射 Nobox-siRNA 后,卵泡中 NoboxKitKitl、Gdf9 mRNA 表达显著下调,NOBOX、Kit、P- SMAD2 / 3 蛋白表达量降低;转录因子 NOBOX 可以结合于 Kit 基因启动子。 结论 NOBOX 是小鼠初级卵泡发育至次级卵泡的关键基因,NOBOX 可直接结合 Kit 基因启动子,且影响初级卵泡中 KitL/ Kit 和 GDF9 / SMAD 通路。

    Abstract:

    Objective NOBOX is a transcription factor that plays a key role in the activation of primary follicles. It directly regulates the transcription of Gdf9 and participates in the GDF9 / SMAD pathway to affect the proliferation and differentiation of granulosa cells. Kit, an important gene affecting follicular development, has multiple NOBOX binding sites in its promoter. In this study, we aimed to investigate the transcriptional regulation of Kit by the transcription factor NOBOX in the primary follicular development stage as well as the influence of the GDF9 / SMAD pathway on follicular development. Methods The development time of primary follicles in vitro was measured. qRT-PCR was used to detect the mRNA expression changes of Nobox, Kit, Kitl and Gdf9 in primary follicles injected with Nobox-siRNA. Western blot was used to measure protein expression changes for NOBOX, Kit and P-SMAD in primary follicles injected with Nobox-siRNA. ChIP assay was used to identify the binding site of NOBOX on the Kit gene promoter. Results Primary follicles developed into secondary follicles on the 5th day in vitro. Injection of Nobox-siRNA into primary follicles delayed their development into secondary follicles by 2 days. After Nobox-siRNA injection into primary follicles, the expression of Nobox, Kit, Kitl and Gdf9 mRNAs in follicles was significantly downregulated, and the expression levels of NOBOX, Kit and P-SMAD2 / 3 proteins were decreased. NOBOX showed binding to the Kit gene promoter. Conclusions NOBOX is a key gene in the development of primary follicles to secondary follicles in mice. NOBOX directly binds to the Kit gene promoter and affects the KitL/ Kit and GDF9 / SMAD pathways in primary follicles.

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张 雪,金美玉,于 洋,郑志红.小鼠生殖细胞特异性转录因子 NOBOX 调控初级卵泡发育机制的研究[J].中国比较医学杂志,2021,31(4):107~114.

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  • 收稿日期:2020-07-07
  • 在线发布日期: 2021-05-28
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