NPFFR1基因对鹅卵泡颗粒细胞激素分泌和细胞凋亡的影响研究  

The Function of NPFFR1 on Hormone Secretion and Apoptosis of Follicle Granulosa Cell in Geese

作　　者：张克山[1,2] 高广亮 李琴[1,2] 赵献芝 李静[1,2] 王启贵 ZHANG Keshan;GAO Guangliang;LI Qin;ZHAO Xianzhi;LI Jing;WANG Qigui(Chongqing Academy of Animal Sciences, Chongqing 402460, China;Chongqing Engineering Research Center of Goose Genetic Improvement, Chongqing 402460, China)

机构地区：[1]重庆市畜牧科学院,重庆402460 [2]重庆市肉鹅遗传改良工程技术研究中心,重庆402460

出　　处：《畜牧兽医学报》2021年第10期2822-2831,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：重庆市自然科学基金面上项目(cstc2019jcyj-msxmX0065);重庆市科研院所绩效激励引导专项(19535);“重庆英才计划”鹅遗传育种科研创新团队(CQYC20200309103);财政部和农业农村部:国家现代农业产业技术体系资助(CARS-42-51)。

摘　　要：神经相关肽受体(RFamide-related peptide receptor,NPFFR1)是促性腺激素抑制激素的主要亲和受体,它在调控动物繁殖方面起着重要作用。为了解NPFFR1对鹅卵巢卵泡发育的作用,本研究以42周龄健康产蛋四川白鹅为试验材料(n=9),利用RT-qPCR法检测NPFFR1基因在等级前和等级卵泡颗粒细胞中的mRNA表达规律;在颗粒细胞中过表达NPFFR1基因,酶联免疫吸附法检测颗粒细胞上清液(n=9)中雌二醇(estradiol,E2)、孕酮(progesterone,P4)和抗缪勒管激素(anti-Mullerian hormone,AMH)的浓度变化,剩余贴壁细胞作一步法TUNEL检测细胞凋亡情况;转录组测序方法筛选大黄卵泡(8~10 mm)颗粒细胞过表达NPFFR1前后表达差异显著基因,并对差异表达基因进行功能聚类分析。结果显示,除F1等级外,其余等级卵泡颗粒细胞NPFFR1表达量均极显著高于等级前卵泡(P<0.01);过表达NPFFR1后,等级颗粒细胞上清液中的E2和等级前颗粒细胞上清液AMH的含量显著(P<0.05)降低,但孕酮P4含量变化不显著(P>0.05);转录组测序共筛选到267个差异表达基因(119个下调,148个上调),这些基因主要富集在生物节律过程、繁殖进程等生物学过程中;同时,与对照组相比,差异基因AMH显著下调表达(P<0.05),Clock(clock circadian regulator)、FOS(proto-oncogene,AP-1 trans-cription factor subunit)、Per(period circadian regulator)和ANTXR2(cell adhesion molecule 2)分别极显著(P<0.01)或显著(P<0.05)上调表达。上述试验结果提示,NPFFR1可从激素、细胞凋亡和生物节律等多个环节影响卵泡颗粒细胞,参与调控卵泡的时序等级发育。RFamide-related peptide receptor(NPFFR1)is a primary affinity receptor of the gonadotropin-inhibitory hormone(GnIH),which plays an important role in controlling animal reproduction.In order to explore the effect of the NPFFR1 on follicle development of geese,nine healthy laying Sichuan white geese of 42-week-old were used as experimental animals.The RT-qPCR technology was used to determine the expression pattern of NPFFR1 in the granulosa cells(GCs)of pre-hierarchical and hierarchical follicles.The NPFFR1 gene expression was upregulated using the overexpression plasmids technique in GCs,and the concentration of reproduction-related hormones(E2,P4 and AMH)was determined by ELISA in the cellular supernatant,the TUNEL assay was employed to detect apoptosis of the remaining anchorage-dependent cell.The RNA sequencing was performed to screen the differentially expressed genes(DEGs)before and after overexpressing NPFFR1 in GCs of 8-10 mm follicles,respectively.The functional cluster analysis of differentially expressed genes was carried out.The results showed that the gene expression of NPFFR1 was extremely significantly different between the pre-hierarchal and hierarchal periods follicular GCs in geese(P<0.01),except F1 follicles.After overexpressed the NPFFR1 gene(72 h),the concentration of E2 in the supernatant of GCs isolated from hierarchal follicles and AMH in the supernatant of GCs isolated from pre-hierarchal follicles were significantly decreased(P<0.05);There were 267 DEGs(119 down-regulated and 148 up-regulated)were selected.The DEGs were mainly enriched in the process of biological rhythm,follicles rhythm.The qPCR results showed that,compared to the control group,the AMH gene was significantly down-regulated(P<0.05),the Clock,FOS,Per and ANTXR2 were extremely significantly(P<0.01)or significantly(P<0.05)up-regulated,respectively.Therefore,it was specu-lated that the NPFFR1 gene might be involved in follicular development by regulating the expression levels of the rhythmic genes,affecting the steroid hormone

关 键 词：NPFFR1基因 鹅 卵泡等级发育 性激素 细胞凋亡 周期节律基因 

分 类 号：S835.2[农业科学—畜牧学]