Zhai Bo, Liu Huiyu, Li Xu, Dai Lisheng, Gao Yan, Li Changhong, Zhang Lianjiang, Ding Yu, Yu Xianfeng, Zhang Jiabao
College of Animal Science, Jilin University, Changchun 130062, China.
Cell Physiol Biochem. 2013;32(2):264-78. doi: 10.1159/000354435. Epub 2013 Jul 31.
Bone morphogenetic protein-15 (BMP15) is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now.
In this study, flow cytometry (FCM) was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1)/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi) method was used to inhibit the expression of (MCP-1)/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR) and western blotting.
The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1)/CCL2 and fibrillin1 (FBN1) were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced.
The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries.
骨形态发生蛋白15（BMP15）是哺乳动物繁殖所必需的母体基因。BMP15在卵母细胞中的表达随着卵泡的生长和发育而增加。目前，BMP15在猪卵丘细胞凋亡过程中的功能及调控机制尚不清楚。
本研究采用流式细胞术（FCM）分析不同浓度的BMP15对卵丘细胞凋亡的影响。利用高通量测序技术筛选与BMP15密切相关的调控基因。为了证实单核细胞趋化蛋白-1（MCP-1）/CCL2和原纤蛋白1（FBN1）在卵丘细胞凋亡中的作用，采用RNA干扰（RNAi）方法抑制MCP-1/CCL2和FBN1的表达。通过FCM、3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐、定量实时聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测经小干扰RNA（siRNA）转染技术处理的卵丘细胞的凋亡和增殖情况。
结果显示BMP15处理的卵丘细胞凋亡水平呈剂量依赖性显著降低。MCP-1/CCL2和FBN1的表达均受BMP15调控。转染后BMP15处理使FBN1沉默时猪卵丘细胞的增殖显著增加，卵丘细胞凋亡受到抑制。CCL2沉默时卵丘细胞的增殖显著降低，凋亡率显著增加。
本研究结果首次表明CCL2和FBN1是BMP15在防止猪卵巢卵丘细胞凋亡中的重要调控因子。
