Li Yating, Jing Jiongjie, Dang Wenqing, Han Qi, Guo Xiangyu, Jia Kaiqi, Cheng Ying, Wang Kai, Kebreab Ermias, Lyu Lihua
College of Animal Science, Shanxi Agricultural University, Taigu, 030801, Shanxi, China.
College of Agricultural and Environmental Sciences, University of California, Davis, 95616, CA, USA.
Theriogenology. 2021 Sep 1;171:55-63. doi: 10.1016/j.theriogenology.2021.05.009. Epub 2021 May 13.
Notch signaling pathway plays an important regulatory role in the development of mammalian follicles. This study aimed to explore the effect of Notch2 on the function of bovine follicles luteinized granulosa cells (LGCs). We detected that the coding sequence (CDS) of bovine Notch2 gene is 7416 bp, encoding 2471 amino acids (AA). The homology of Notch2 AA sequence between bovine and other species is 86.04%-98.75%, indicating high conservatism. Immunohistochemistry found that Notch2 receptor and its ligand Jagged2 localize in granulosa cells (GCs) and theca cells in bovine antral follicles. And immunofluorescence found that positive signals of Notch2 and Jagged2 overlap in bovine LGCs, speculating that Notch2 receptor may react with Jagged2 ligand to activate Notch signaling pathway and play an important role in bovine LGCs. To further investigate the function of Notch2, Notch2 gene was silenced by short hairpin RNA (shRNA) and CCK-8 analysis showed that the proliferation rate of LGCs was downregulated significantly (P < 0.01). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the mRNA expression of apoptosis related gene Bcl-2/Bax decreased (P < 0.01) and Caspase3 increased (P < 0.05), cell cycle related gene CyclinD2/CDK4 complex decreased (P < 0.01) and P21 increased (P < 0.05), steroidogenesis gene STAR and 3β-HSD decreased (P < 0.01) while CYP19A1 and CYP11A1 had no significant difference (P > 0.05). In addition, Enzyme-linked immunosorbent assay (ELISA) showed that there was no difference in estradiol (E) secretion (P > 0.05) while the progesterone (P) secretion decreased (P < 0.01). In conclusion, Notch2 plays an important role in regulating bovine LGCs development.
Notch信号通路在哺乳动物卵泡发育中起重要调节作用。本研究旨在探讨Notch2对牛卵泡颗粒黄体细胞（LGCs）功能的影响。我们检测到牛Notch2基因的编码序列（CDS）为7416 bp，编码2471个氨基酸（AA）。牛与其他物种之间Notch2氨基酸序列的同源性为86.04%-98.75%，表明高度保守。免疫组织化学发现Notch2受体及其配体Jagged2定位于牛窦状卵泡的颗粒细胞（GCs）和卵泡膜细胞中。免疫荧光发现Notch2和Jagged2在牛LGCs中的阳性信号重叠，推测Notch2受体可能与Jagged2配体反应以激活Notch信号通路，并在牛LGCs中起重要作用。为进一步研究Notch2的功能，通过短发夹RNA（shRNA）沉默Notch2基因，CCK-8分析显示LGCs的增殖率显著下调（P<0.01）。定量逆转录聚合酶链反应（qRT-PCR）显示凋亡相关基因Bcl-2/Bax的mRNA表达降低（P<0.01），Caspase3增加（P<0.05），细胞周期相关基因CyclinD2/CDK4复合物降低（P<0.01），P21增加（P<0.05），类固醇生成基因STAR和3β-HSD降低（P<0.01），而CYP19A1和CYP11A1无显著差异（P>0.05）。此外，酶联免疫吸附测定（ELISA）显示雌二醇（E）分泌无差异（P>0.05），而孕酮（P）分泌降低（P<0.01）。总之，Notch2在调节牛LGCs发育中起重要作用。
