Effects Of The Cytokines BFGF And LIF On Development Of Chicken Primordial Follicles

The development of early follicles in poultry ovaries is closely related to the material transmission and information communication between oocytes and granulosa cells,and cytokines play an important mediating role between them.Cytokines can promote cell proliferation and growth,and also regulate cell migration,differentiation and mitosis.There are many kinds of cytokines.Various cytokines have effects on follicle formation and development through paracrine or autocrine and material transport between oocytes and somatic cells:regulating the proliferation and differentiation of granulosa cells,hormone secretion and other cytokines generate etc.However,there are few studies on cytokines involved in early follicle growth and development in poultry.The reproductive performance of poultry is a major concern in animal husbandry,and early follicular development has a decisive influence on the final reproductive performance.Therefore,it is meaningful to conduct in-depth research on the regulatory role of cytokines in early follicular development.In this experiment,chicks were used as the research object to detect changes in gene expression during follicle development in chicks and to use established models of ovarian culture in vitro to study the regulation and mechanism of basic fibroblast growth factor(bFGF)and leukemia inhibitory factor(LIF)in the early follicle development of chicks,providing a theoretical basis for the subsequent research on egg production performance of poultry.1.Expression changes of miRNA in early follicle development of chicken ovaryThe formation and development of early follicles in chicks involves many changes in gene expression.We studied the changes of miRNA expression in the ovary of chicks of different days over time,and RNA-seq experimental technology was used to study the expression of miRNA in early chick ovary.In this experiment,three chicken ovaries of D0,D4 and D7 were taken and rinsed to remove excess accessory tissues and used for RNA-seq analysis.In the RNA-seq library construction results,performed quality inspection on miRNA,and screened out enough genes with significant differential expression to verify RT-qPCR.The verification results were consistent and the RNA-seq results were credible.The results showed that compared with D0 ovarian tissue,D4 ovarian tissue had a total of 10 miRNAs with significant difference,including 8 up-regulated,2 down-regulated genes;compared with D0 ovarian tissue,D7 ovarian tissue had 50 different expressions,including 35 up-regulated,15 down-regulated genes;compared with D4 ovarian tissue,D7 ovarian tissue had a total of 18 miRNAs with significant difference,including 12 up-regulated,6 down-regulated genes.When summarizing the target genes of differentially expressed miRNAs,we found that in D4 vs D0,the target genes were related to the anti-apoptosis and cytokine effects of cells;in D7 vs D0,the target genes were related to the cell anti-apoptosis,cytokine effects and hormone synthesis;in D7 vs D4,target genes were associated with gap junctions,cytokine effects,and MAPK signaling pathway.The target genes involved were GJA5,HLF,MAP3K14,etc.The above results indicate that miRNA might affect the development of chick follicles through gap junctions,cell anti-apoptosis,ovarian development signal related pathways and cytokine effects.2.Expression changes of lncRNA in early follicle development of chicken ovaryIn this study,the ovary of chicks of different days was used as experimental material,and RNA-seq was used to analyze the change of lncRNA levels in the ovary of chicks at different time.The genes with significant differential expression were screened and verified by RT-qPCR as in the previous chapter.The GO database was used to enrich the lncRNA target genes with differential expression.The KEGG database was used to annotate the differentially expressed lncRNA target genes.The results showed that compared with D0 ovarian tissue,there were 106 genes with significantly different expressions in D4 ovarian tissue,including 65 up-regulated,41 down-regulated genes;compared with D0 ovarian tissue,there were 305 expressions with significant differences in D7 ovarian tissue,including 214 up-regulated,91 down-regulated genes;compared with D4 ovarian tissue,D7 ovarian tissue had a total of 109 genes with significantly different expressions,including 52 up-regulated genes and 57 down-regulated genes.RT-qPCR verification experiments confirmed the reliability of RNA-seq results.The results of the GO database indicated that the differentially expressed lncRNA target genes were mostly enriched in antigen receptor-mediated signaling pathways,cytokine synthesis,and non-membrane transprotein tyrosine kinase activity and other physiological activities.Most of the target genes were related to cytokine-cytokine receptor interactions,cell adhesion molecules and signaling pathways related to follicle development.The results of D7 vs D4 showed that the expression of many lncRNAs whose target genes were cytokines or cytokine receptors increased significantly,including FGF2,IL6R and GDF9 and so on.The above results indicated that lncRNA might promote the formation and development of follicles in early chicks through cytokines,cell junction,and signaling pathways related to follicle growth.The relevant cytokines might be FGF2,LIF and GDF9,etc.3.Regulation of bFGF on the development of primordial follicles in chicksMany cytokines play an important role in promoting the activation of female primordial follicles.The RNA-seq results showed that the target gene of lncRNA with significantly increased expression during the development of D4 chicken ovary to D7 was FGF2.At the same time,there have been previous reports that FGF2 can promote the activation of primordial follicles in rats.There are few studies on the role of FGF2 in the regulation of primordial follicle activation in avian.In this experiment,we investigated the reciprocal stimulating effect of the cytokine bFGF and FSH on primordial follicle development and the possible signaling mechanism involving protein kinase B(AKT)and extracellular regulated protein kinases(ERK)pathway.4-day-old chicken ovaries were treated with bFGF and FSH for 3 days in culture to investigate the effect of bFGF and FSH on primordial follicle development.HE staining,Immunohistochemistry,quantitate real-time PCR(qRT-PCR),Western blot,immunofluorescence were used in this study.A correlated change of the bFGF receptor(FGFR1)mRNA expression and time course of primordial follicle activation was revealed in the early chick ovaries.Reciprocal stimulating effect of bFGF and FSH was demonstrated on primordial follicle activation,along with accelerated granulosa cells proliferation and decreased cell apoptosis.The promoting effect of bFGF was attenuated by an FGFR1 inhibitor SU5402 in that the percentage of growing follicles number was decreased.AKT and ERK signaling pathway mediated the action of bFGF and FSH on promoting primordial follicle activation.The above results indicate that the cytokine bFGF and FSH imposed reciprocal stimulating effects on granulosa cells proliferation and anti-apoptosis to promote primordial follicle activation via PI3K-AKT and ERK signaling pathways in the early chick ovaries.4.Regulation of LIF on the development of primordial follicles in chicksThe RNA-seq results showed that the target gene of lncRNA that increased significantly during the development of D4 chicken ovary to D7 was IL6R,which was related to the receptor composition of LIF.There is little research on the effect of LIF on primordial follicle activation of chicks.In this experiment,we examined the role and correlation of cytokines LIF and bFGF in the development of primordial follicles,as well as the signaling pathways involved the process,including AKT,ERK and Wnt/β-catenin signaling pathway.Ovarian tissue was collected from chicks 4 days after hatching,and treated with LIF and bFGF alone or in combination for 3 days to observe the changes in follicular development.HE staining,immunochemistry,Western blot,transmission electron microscopy and immunofluorescence were used in this study.The results showed that there was a time correlation between the changes in protein expression of LIF with its receptor(LIFR)and the development of early follicles.LIF and bFGF have a synergistic effect on the activation of primordial follicles,whose specific performance is to promote the proliferation of follicular cells and reduce the apoptosis of follicular cells.However,SC 144,the antagonist of LIFR,can inhibit this effect of LIF to promote the activation of primordial follicles.Simultaneously,AKT and ERK signaling pathways were also involved in the processes of both LIF and bFGF promoting proliferation and suppressing apoptosis,and Wnt/β-catenin signaling pathway may also had some mediating effect.The above results indicate that LIF and bFGF may promoted the activation of primordial follicles by promoting the proliferation and inhibiting the apoptosis of chicken follicular cells.This process involved AKT,ERK and Wnt/β-catenin signaling pathways.In conclusion,RNA-seq was used to detect changes in gene expression during the development of ovarian follicles in chicks.LncRNA and miRNA with significant expression differences were summarized,compared and supplemented.It was found that in the primordial follicle activation stage of chicks,expression of lncRNA whose target genes associated with FGF2 and LIF increased significantly;The established ovarian tissue culture model in vitro was used to study the regulatory role of bFGF and LIF in primordial follicle activation in chick ovary.It was found that FSH and bFGF play a synergistic role,possibly through the PI3K-AKT and ERK signaling pathways to stimulate the proliferation of granulosa cells and anti-apoptosis in early ovaries of chickens to promote follicle activation;while LIF and bFGF may promote follicular cell proliferation through a synergistic effect,and inhibit its apoptosis,and then promote the activation of follicles through AKT,ERK and Wnt/β-catenin signaling pathway.These results will be a preliminary step for further research on the regulatory mechanism of follicle development in poultry and improving egg production performance of poultry.