| The follicle is the basic unit of mammalian ovary structure and function,and granulosa cells(GCs),as the largest cell group in the follicle,provide nutrients and a microenvironment for the growth and maturation of oocytes.In mammalian ovaries,only 1% of follicles develop and ovulate,and the remaining 99% of follicles undergo atresia during development.Current studies have shown that granulosa cell apoptosis is the main cause of follicle atresia,and the normal development of follicles is also inseparable from the proliferation of GCs and the synthesis of estradiol.Fibroblast growth factor 21(FGF21),a metabolic factor mainly secreted by the liver,relies on the activation of a complex composed of FGFR1 and β-klotho(KLB)for signal transduction.Studies have shown that FGF21 affects female fertility through the hypothalamic-pituitary-ovarian axis.However,in addition to FGF21 acting through endocrine,it has also been found in recent years that FGF21 plays an important role in regulating the browning of white adipocytes in an autocrine manner.In this study,FGF21,its receptor FGFR1,and co-receptor KLB exist in porcine ovarian GCs,so it is speculated that FGF21,in addition to regulating female fertility in an endocrine manner,can also act as an autocrine factor in ovarian GCs to directly regulate GCs function affect follicle development.In this study,porcine ovarian GCs were used as the research object,and the effect and mechanism of FGF21 on the proliferation,apoptosis and estradiol synthesis of GCs were explored from the cellular and molecular levels by interfering and overexpressing FGF21.And the effect and mechanism of FGF21 on follicular development were verified in FGF21 knockout mice.The main results of this study are as follows:1.FGF21 is expressed in ovarian GCs and promotes estradiol synthesis.By Western blot and q RT-PCR detection,it was found that the expression of FGF21 in the ovaries of high yielding sows was significantly higher than that of low yielding sows(P < 0.05).Histological immunofluorescence staining showed that FGF21 and its receptors were mainly expressed in porcine ovarian GCs.The expression of FGF21 in GCs increased with the increase of follicle diameter.FGF21-si RNA and r FGF21 in porcine ovarian GCs cultured in vitro found that FGF21 significantly increased the expression of cholesterol side chain lyase(CYP11A1)and aromatase(CYP19A1)(P < 0.05),the key genes for steroid hormone synthesis,and increased the level of estradiol synthesis in GCs.2.FGF21 promotes the proliferation of GCs and inhibits apoptosis.In this study,GCs were treated with si FGF21 and r FGF21.Using flow cytometry,Ed U staining,CCK-8,real-time quantification and Western blot detection,it was found that FGF21 promoted the expression of cell cycle-related factors Cyclin B,Cyclin D and Cyclin E(P < 0.05),and the expression of p21(P < 0.05),a negative regulator of cell proliferation,is reduced,and the number of cells in the proliferation phase is increased at the same time.In addition,the overexpression of FGF21 in GCs showed that the level of anti-apoptotic factor BCL2 was significantly increased(P < 0.05),while the level of pro-apoptotic factor BAX was significantly decreased(P < 0.05).Flow cytometry showed that FGF21 reduced the proportion of early apoptotic cells.3.FGF21 activates PI3K/AKT/m TOR signaling pathway to promote GCs proliferation and estradiol synthesis.Through the analysis of transcriptome sequencing data of GCs with si FGF21 and r FGF21,compared with si FGF21 and the control group,r FGF21(0ng/m L)added group and r FGF21(1 ng/m L)added group,it was found that 246 and 712 genes were significantly up-regulated and 246 and 849 genes were significantly down-regulated,respectively.KEGG enrichment analysis of differential genes(DEGs)and found that DEGs were highly enriched in the PI3K-AKT signaling pathway.GCs were further treated with the PI3 K inhibitor LY294002,and it was verified that FGF21,its receptor FGFR1 and the coreceptor KLB formed a complex to activate the PI3K/AKT/m TOR signaling pathway,and regulates the expression of key steroidogenesis genes CYP11A1 and CYP19A1 and cell cyclerelated factors Cyclin B,Cyclin D,Cyclin E and p21,and promotes the proliferation of GCs and the synthesis of estradiol.4.FGF21 enhances mitochondrial biosynthesis in GCs and maintains the balance of cellular oxidative-antioxidant system to inhibit the cell apoptosis.After r FGF21 treatment,there was no difference in the expression of death receptor genes in GCs,whereas mitochondrial-related genes changed significantly.In this study,by treating GCs with si FGF21 and r FGF21 and it was found that r FGF21 significantly increased the number of mitochondria and DNA copy number of GCs(P < 0.05),cellular ATP concentration and total antioxidant activity(T-AOC).At the same time,the expression of key genes ND3,COX2 and COX3 in the process of mitochondrial biosynthesis was significantly increased(P < 0.05),indicating that FGF21 promotes mitochondrial biosynthesis in GCs and maintains the balance of intracellular oxidation-antioxidant system to inhibit the occurrence of apoptosis in GCs.5.FGF21 knockout mice have blocked follicle development.In order to further verify the effect of FGF21 on follicular development,HE staining of ovaries and all ovarian sections of 100-day-old wild-type mice and FGF21 knockout mice were performed to count the number of follicles at all levels.It was found that FGF21 KO mice had fewer large follicles in the ovaries,more the follicles are in the preantral stage.Ovarian PCNA staining and Tunel staining showed that the proliferation activity of ovarian GCs in FGF21 KO mice was significantly decreased(P < 0.0001)and the degree of apoptosis was significantly increased(P < 0.05).Statistics found that the estrous cycle of FGF21 KO mice was disordered,and the litter size was significantly reduced(the average litter size of FGF21 KO mice: 3;the average litter size of wide-type mice: 8)(P < 0.001).It shows that the degree of apoptosis of GCs in FGF21 knockout mice was increased and the follicle development was blocked.In conclusion,FGF21 and its receptor FGFR1 and coreceptor KLB were mainly expressed in porcine ovarian GCs,and the expression of FGF21 increased with the increase of follicle diameter.At the cellular and molecular levels,we found that FGF21 promoted the synthesis of estradiol in GCs,increased the proliferation activity,and inhibited the occurrence of apoptosis.Mechanistically,the complex formed by FGF21,FGFR1 and KLB activated the PI3K/AKT signaling pathway to increase the expression of key estradiol synthesis genes and cell cycle factors,and promoted the synthesis and proliferation of estradiol in GCs.And after the activation of the PI3K/AKT signaling pathway,the mitochondrial biosynthesis of GCs is enhanced,and the balance of the cellular oxidation-antioxidant system is maintained to resist the occurrence of apoptosis.These results were initially validated in the ovaries of FGF21 KO mice,explaining why follicle development is blocked by loss of FGF21.It provides theoretical basis for further elucidating the potential regulatory mechanism of porcine follicle development,and provides molecular markers for improving ovulation number and litter size of pigs from molecular breeding in the future. |
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