Yuan Junsong, Deng Yan, Zhang Yingying, Gan Xiang, Gao Shanyan, Hu Hang, Hu Shenqiang, Hu Jiwei, Liu Hehe, Li Liang, Wang Jiwen
Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, 611130, PR China.
Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, 611130, PR China.
Anim Reprod Sci. 2019 Jan;200:86-95. doi: 10.1016/j.anireprosci.2018.11.014. Epub 2018 Dec 1.
Bone morphogenetic protein 4 (BMP4) has an important role in regulating cellular proliferation, differentiation and apoptosis. It, however, is still unclear as to the mechanisms by which BMP4 regulates the apoptosis of granulosa cells (GCs) in geese. In the present study, there was cloning of the full-length coding sequence of goose BMP4 gene, which consisted of 1212 nucleotides encoding 403 amino acids. Its deduced amino acid sequence comprised one signal peptide, one TGFβ pro-peptide and one mature peptide domain. Results from conducting the quantitative real-time PCR (qPCR) indicated the relative abundances of BMP4 mRNA in geese GCs increased gradually from the relative abundances in pre-hierarchical follicles that were 4 to 6 mm in diameter to that in the fifth largest (F5) follicle and then relative abundances of BMP4 mRNA decreased with further development as the largest (F1) follicle. Results from use of the TUNEL assay indicated that overexpression of the goose BMP4 gene suppressed GC apoptosis and this was confirmed when relative abundances of the CAD, Caspase-9 and Caspase-3 proteins were determined using western blotting. In addition, overexpression of the BMP4 gene induced phosphorylation of AKT, which was inhibited with use of the PI3K inhibitor, LY294002. Co-transfection of BMP4 and LY294002 resulted in increased relative abundances of Caspase-9 and CAD proteins but had no effect on that of Caspase-3. Taken together, these results suggested that expression of the BMP4 gene resulted in a reduction in Caspase-9 protein leading to inhibition of GC apoptosis via the PI3K/AKT signaling pathway in geese.
骨形态发生蛋白4（BMP4）在调节细胞增殖、分化和凋亡方面具有重要作用。然而，BMP4调节鹅颗粒细胞（GCs）凋亡的机制仍不清楚。在本研究中，克隆了鹅BMP4基因的全长编码序列，该序列由1212个核苷酸组成，编码403个氨基酸。其推导的氨基酸序列包括一个信号肽、一个TGFβ前肽和一个成熟肽结构域。定量实时PCR（qPCR）结果表明，鹅GCs中BMP4 mRNA的相对丰度从直径4至6毫米的前等级卵泡中的相对丰度逐渐增加至第五大（F5）卵泡中的相对丰度，然后随着卵泡进一步发育至最大（F1）卵泡，BMP4 mRNA的相对丰度降低。TUNEL检测结果表明，鹅BMP4基因的过表达抑制了GC凋亡，这在使用蛋白质印迹法测定CAD、Caspase-9和Caspase-3蛋白的相对丰度时得到了证实。此外，BMP4基因的过表达诱导了AKT的磷酸化，而使用PI3K抑制剂LY294002可抑制这种磷酸化。BMP4和LY294002的共转染导致Caspase-9和CAD蛋白的相对丰度增加，但对Caspase-3的相对丰度没有影响。综上所述，这些结果表明，BMP4基因的表达导致Caspase-9蛋白减少，从而通过PI3K/AKT信号通路抑制鹅GC凋亡。
