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

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。

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

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 secretion and apoptosis of follicular granulosa cells of goose.