母猪健康和闭锁卵巢有腔卵泡的转录组测序比较分析

Transcriptome sequencing analysis of porcine granulosa cells in healthy and atretic follicles

[目的]本文旨在通过对猪卵泡颗粒细胞进行转录组测序了解卵泡闭锁相关基因的表达谱并确定闭锁卵泡的潜在分子标记物。[方法]通过HE染色对卵泡及颗粒细胞进行形态学观察;抽取健康卵泡和晚期闭锁卵泡的颗粒细胞,进行转录组测序分析,最后采用实时荧光定量PCR检测差异表达基因的相对表达水平。[结果]转录组数据表明,与闭锁卵泡颗粒细胞相比,健康卵泡颗粒细胞中有771个差异基因,其中204个为上调基因,567个为下调基因;GO功能富集分析显示,差异基因显著富集到320个生物进程中,主要与细胞增殖和血管生成有关;KEGG通路分析表明,差异基因富集到48条信号通路中,主要参与氧化应激、激素合成和能量代谢等方面的信号通路;实时荧光定量PCR结果表明,CYP1B1、PDE10A、NUPR1、NOX4、LDLR、LHCGR和CYP19A1表达趋势与测序结果一致。[结论]氧化应激、血管生成、能量代谢以及激素合成等多方面因素综合调控猪卵巢中颗粒细胞的凋亡,继而影响卵泡闭锁现象的产生。

[Objectives]Transcriptome sequencing analysis was performed on porcine granulosa cells in healthy and atretic follicles to understand the gene expression profiles associated with follicle atresia,and to identify potential molecular biomarkers for atretic follicles.[Methods]In this experiment,HE staining was performed on the porcine follicles and granulosa cells to observe the morphology.The granulosa cells of healthy follicles and advanced atretic follicles were extracted and analyzed using transcriptome sequencing.Quantitative real-time PCR was carried out to verify the relative expression levels of differentially expressed genes.[Results]The analysis of transcriptome data showed that 771 differentially expressed genes(204 up-regulated and 567 down-regulated)were identified in the healthy follicular granulosa cells compared with the atretic granulosa cells.GO analysis showed that a total of 320 biological process were significantly enriched,and these were mainly associated with cell proliferation and angiogenesis.KEGG analysis showed that differentially expressed genes were significantly enriched into 48 signaling pathways,and these pathways were mainly associated with oxidative stress,hormone synthesis and energy metabolism.The data of quantitative real-time PCR showed that the expression tendency of CYP1B1,PDE10A,NUPR1,NOX4,LDLR,LHCGR and CYP19A1 was similar with the data of transcriptome sequencing.[Conclusions]Oxidative stress,angiogenesis,energy metabolism,hormone synthesis and other factors comprehensively regulate the apoptosis of granulosa cells in porcine ovary,which affects the occurrence of follicular atresia.Our data provided a comprehensive theoretical basis for understanding the mechanism of follicular atresia.