Expression Regulation And Function Of C1QTNF6 In Chicken Ovarian Granulosa Cells

Ovarian development is a dynamic process regulated by many factors,and the state of ovarian development directly affects the performance of egg laying,and granulosa cells are the earliest follicular cells,which are closely related to follicular development and selection.C1q/Tumor necrosis factor associated protein 6(C1QTNF6)is a newly discovered paradiponectin homolog,belonging to the CTRPs family,which is an adipokine with regulatory effects on metabolism,inflammation and cardiovascular system.Its molecular structure mainly includes four domains: N-terminal signal peptide,short-variation domain,gel-as-is domain and C-terminal globule domain.Early studies have found that C1QTNF6 is widely expressed in mammalian ovaries,suggesting that C1QTNF6 plays an important role in regulating follicular development,but what function it plays in chicken ovaries is still unknown.Therefore,in this study,Hyline brown laying hens were selected as the research object,and C1QTNF6 gene was screened out by RNA-seq data of ovarian tissue at 15 W,20W,30 W and 68 W,combined with the results of adiponectin in chicken ovarian granulosa cells in our laboratory at the previous stage,and it was speculated that it might be related to ovarian and granulosa cell development.Therefore,this study took Hyline brown laying hens as the research object to explore the distribution and localization of C1QTNF6 in follicles and its regulation and function in follicular granulosa cells,and the following results were obtained:Experiment 1.Tissue expression analysis of C1QTNF6 gene,CDS cloning and bioinformatics analysis of its sequence were performedIn this study,q RT-PCR was used to detect the expression of C1QTNF6 in different tissues of chicken,and it was found that C1QTNF6 was highly expressed in ovary of chicken.C1QTNF6 gene coding protein region(CDS)was cloned 777 bp,encoding 259 amino acids.Phylogenetic tree showed that C1QTNF6 was in line with the law of species evolution,and had the closest relationship with Turkey and quail,and the furthest relationship with mammals,indicating that C1QTNF6 was highly conserved in the interspecies evolution.In addition,fluorescence probe of C1QTNF6 was designed and in situ hybridization method was used to find that C1QTNF6 was mainly expressed in the granular layer of pregrade and grade follicles.Therefore,C1QTNF6 may be involved in follicular development in poultry.Experiment 2.Regulation and function of C1QTNF6 on chicken ovarian granulosa cellsIn order to study the functional regulation of C1QTNF6 gene in ovarian granulosa cells,the overexpression vector C1QTNF6-PCDNA3.1-EGFP containing the target gene C1QTNF6 was successfully constructed in this study,and the si RNA interference fragment was designed according to the CDS sequence of C1QTNF6 gene.Overexpression of C1QTNF6 gene was found in Prehierarchical Granulosa cells(Ph GCs)(P<0.05)decreased the expression of steroid synthesis-related genes(STAR,CYP11A1,3B-HSD);CCK8 showed that C1QTNF6 overexpressed in Ph GCs at 12 h,36 h,48 h,60 h and 72 h could promote Ph GCs activity.The results of flow cycle and apoptosis showed that the proliferation of Ph GCs(P<0.05)and inhibited Ph GCs apoptosis(P<0.05),and the interference results were opposite.Preovulatory granulosa cells(Po GCs)were found to be significant(P<0.05)decreased the expression of genes related to steroid synthesis;CCK8 showed that overexpression of C1QTNF6 in Po GCs at 12 h,36 h,48 h,60 h and 72 h could inhibit the activity of Po GCs.Flow cycle and apoptosis results showed that inhibition of cell proliferation(P<0.05)and promoted apoptosis(P<0.05),and the interference results were opposite.After statistical analysis of the middle egg data of local laying hens(Gushi laying hens)in Henan Province at 28 W and 36 W laying peak in the early stage of the project team,these two week-old laying hens were divided into high yield group and low yield group,and the expression of C1QTNF6 m RNA in 28 W and 36 W ovaries was detected by q RT-PCR.The results showed that,the expression of C1QTNF6 gene in Gushi laying hens at 28 and 36 weeks of age was significantly higher than that in low yield group(P< 0.05)higher than that in high-yield group,suggesting that C1QTNF6 gene may play a negative role in follicular development of laying hens.Experiment 3.Effects of overexpression/interference of C1QTNF6 and Adipo Ron cotreatment on steroid hormone expression in chicken ovarian granulosa cellsEffects of overexpression or interference of C1QTNF6 and Adipo Ron co-treatment on the expression of reproductive hormones and receptor related genes in chicken ovarian granulosa cells for 24 h.The results showed that Adipo Ron alone increased the expression of Adipo Q and Adipo R1 in Ph GCs and Po GCs,and promoted the expression of E2,P and anabolic steroid hormone-related marker genes.In Ph GCs,overexpression of C1QTNF6 decreased the expressions of St AR,CYP11A1,3β-HSD,E2 and P,while co-treatment with overexpression of C1QTNF6 and Adipo Ron significantly promoted the expressions of Adipo Q and Adipo R1,and significantly reduced the expression of P.In Po GCs,Overexpression of C1QTNF6 significantly reduced the expressions of Adipo R1,3β-HSD,St AR,CYP11A1 genes as well as E2 and P,while co-treatment with overexpression of C1QTNF6 and Adipo Ron promoted the expressions of Adipo Q,Adipo R1,3β-HSD and CYP11A1.In conclusion,C1QTNF6 can inhibit the expression of Adipo R1,and there is no synergistic effect between C1QTNF6 and Adipo Ron in steroid hormone production.In conclusion,C1QTNF6 gene is highly expressed in ovarian tissue.The full length of CDS sequence is 777 bp,encoding 259 amino acids,which is mainly distributed in ovarian granulosa cells.It can affect follicular development by regulating the proliferation and differentiation of chicken ovarian granulosa cells and the synthesis of steroid hormones.In addition,C1QTNF6 is also involved in the regulation of follicular development by pituitary hormones(FSH and LH),and as a collateral homolog of adiponectin,there is no synergistic effect between C1QTNF6 and Adipo Ron in steroid hormone production.These results provide theoretical and experimental basis for clarifying the molecular regulation mechanism of C1QTNF6 gene during follicular development and maturation of chicken follicles.