Mechanism Of VIP Gene Regulating Granulosa Cell Apoptosis In Laying Hens

Ovary is an important reproductive organ of poultry,which produces a large number of follicles.Follicles may be blocked at various stages of growth and development due to the regulation of different factors,which ultimately leads to reproductive performance decline.Granulosa cells play an important role in regulating the growth and development of follicles,and their apoptosis can induce follicular atresia.There are many factors that induce apoptosis of granulosa cells,including hormones,cytokines,oxidative stress,mitochondria and other in vitro factors.When granulosa cells undergo apoptosis,follicles in poultry begin to lose biological function and the regulation between follicular cells is unbalanced,which promotes the apoptosis of oocytes and membrane cells in follicles,and ultimately leads to follicular atresia.The growth factors,gonadal steroids and cytokines secreted by granulosa cells in the survival state can reduce the oxidative damage of oocytes,prevent mitochondrial DNA damage caused by excessive intracellular reactive oxygen species(ROS),and avoid granulosa cell apoptosis caused by mitochondrial dysfunction.The purpose of this study was to elucidate the molecular mechanism of VIP gene regulating the proliferation and apoptosis of follicular granulosa cells in laying hens.Our previous study found that the content of VIP in hens in the shading group was significantly higher than that in the normal light group.Therefore,this study took VIP gene as the main research object to explore the effect of VIP on the reproductive performance of laying hens and its molecular mechanism.By constructing VIP gene overexpression vector,the expression changes of key genes of oxidative stress and apoptosis were detected by Q-PCR,and the survival status of granulosa cells was detected by flow cytometry and CCK-8 method.The effects of VIP gene on proliferation,apoptosis and oxidative stress of follicular granulosa cells in laying hens were clarified.By constructing the VIP promoter deletion fragment,the core promoter region and the transcription factors binding to the promoter were screened,and the wild-type vector and mutant vector of the transcription factor were constructed.The wild-type vector and the mutant vector were co-transfected with the VIP core promoter fragment into chicken granulosa cells,and the dual luciferase was used to detect the change of its promoter activity,and the interaction effect between VIP and transcription factors was studied.The JAK/STAT signaling pathway is closely related to apoptosis.After transfection of VIP overexpression gene,Q-PCR was used to detect the expression of key genes in JAK/STAT signaling pathway,so as to study the molecular mechanism of VIP gene regulating follicular granulosa cell apoptosis in laying hens.The main results are as follows :(1)In order to further verify the regulatory effect of VIP gene on follicular granulosa cells of laying hens,the overexpression vector pcDNA3.1-VIP of VIP gene was successfully constructed,and pcDNA3.1-VIP was transferred into follicular granulosa cells of laying hens.The results of Q-PCR showed that pcDNA3.1-VIP significantly promoted the expression of VIP compared with empty vector pcDNA3.1(P<0.01).The above results showed that pcDNA3.1-VIP gene overexpression vector was successfully constructed.(2)The constructed pcDNA3.1-VIP was transfected into chicken granulosa cells.The results of Q-PCR showed that the expression of pro-apoptotic gene tumor necrosis factor(FAS)in granulosa cells was significantly increased(P<0.01),the expression of transcriptional regulator Myc(C-myc)was significantly decreased(P<0.01),and the expression of anti-apoptotic gene BCL2 was significantly decreased(P<0.05).The granulosa cells transfected with pcDNA3.1-VIP were detected by flow cytometry at 24 hours after transfection.The results showed that there was no significant difference in the number of normal cells and dead cells between the overexpression group and the empty vector control group.The number of late dead cells was different but not significant(P>0.05),and the number of early dead cells was significantly different(P<0.05).The cell activity was significantly lower than that of the control group at 24 h and 36 h after overexpression of VIP gene(P<0.05).At the same time,the effect of VIP gene overexpression on the cell cycle of granulosa cells was detected by flow cytometry.The results showed that compared with the control group,the number of G1 phase cells in the overexpression group increased significantly(P<0.01),the number of S phase cells decreased significantly(P<0.01),and the number of G2 phase cells increased significantly(P<0.01).Based on the above results,the overexpression of VIP gene can inhibit the proliferation of granulosa cells and promote the occurrence of apoptosis.(3)After transfection of VIP overexpression vector,the expression of JAK2,STAT3,Pim1 and BCL2 in JAK/STAT signaling pathway decreased significantly(P<0.01),STAT3 and Pim1 decreased significantly(P < 0.05),and the expression of anti-apoptotic gene BCL2 decreased significantly(P< 0.01).It is speculated that VIP gene may regulate the apoptosis level of granulosa cells by regulating JAK/STAT signaling pathway.(4)In order to study the regulation of the upstream transcription factor of VIP gene on VIP gene,six promoter deletion fragments primers were designed according to the2000 bp sequence upstream of the CDS region of chicken VIP gene.Using cDNA as template and dual luciferase pGL3.0 plasmid as vector,the VIP truncated fragment expression vector from p-VIPF1 to p-VIPF6 was successfully constructed by double enzyme digestion.The 6 truncated fragments were transfected into follicular granulosa cells,and the dual luciferase activity of different groups was detected after 24 hours.The results showed that there was a significant difference in activity between p-VIPF5 and pVIPF6 fragments(P<0.01).Therefore,it was speculated that the core promoter existed in p-VIPF5,which was-359 bp to-737 bp.Bioinformatics methods were used to predict the transcription factors binding to this region,and it was found that there was a binding site of SOX11 in this region.It was preliminarily speculated that there was a binding relationship between SOX11 transcription factor and VIP gene core promoter.(5)In order to verify whether there is a binding relationship between the SOX11 transcription factor and the VIP gene core promoter,p-SOX11 wild-type vector and pSOX11 mut mutant vector were constructed in this study.Both of them were cotransfected with p-VIPF5 into the follicular granulosa cells of laying hens,and the control group p-VIPF5 and the blank group pGL3.0 were set up.The results of dual luciferase assay showed that the activity of p-VIPF5 + p-SOX11 was significantly higher than that of the control group and the blank group.The fluorescence activity of p-VIPF5 + pSOX11 mut was not significantly different from that of the control group.Based on the above results,it is proved that there is a binding relationship between SOX11 transcription factor and VIP gene.In summary,VIP gene has a regulatory effect on the apoptosis of granulosa cells in laying hens and promotes the apoptosis of granulosa cells.VIP gene regulates the apoptosis of granulosa cells in laying hens through JAK/STAT signaling pathway.In addition,the transcription factor SOX11 has a binding relationship with VIP gene.This study provides a theoretical basis for exploring the mechanism of the effect of VIP gene on follicular development after granulosa cell apoptosis,and lays a foundation for further exploring the possibility of reducing follicular atresia by reducing the level of granulosa cell apoptosis,thereby improving the reproductive performance of poultry.