Effect Of Ovarian Angiogenesis On Development Of Follicles In The Laying Chickens

Laying performance depends on the follicular development.During the follicular development,more than 95%ovarian follicles undergo atresia,only less than 5%follicles can develop into preovulatory stage and ovulation.In the process of research,we found that the number of pre-hierarchical follicles,especially the SYFs and LWFs in low yield laying hens is fewer than high yield laying hens.The pre-hierarchical follicles were suspended through the pedicle and radiates with the theca layer.Blood vessels can transport growth factor,hormones and nutrients from the ovary into follicles through the pedicle.The number of blood vessels plays an important role in the development of follicles.In this study,the key genes and signaling pathways during the follicular development were investigated by histomorphology observation,cell and molecular biological detection and transcriptome sequencing in the Hy-line white chickens.And we compared the follicular differences between low yield phase and high yield phase in chicken.Meanwhile,we explore the effect of the follicle-stimulating hormone and N-carbamylglutamate on follicles development via regulating ovarian angiogenesis.In this experiment,we coincidentally discovered the function of NCG on egg shell quility.Therefore,this study will provide the method to improve the rate of laying eggs and reduce the rate of breaking eggs.1.Transcriptome analysis of follicles in high-yield laying hensThe egg laying of poultry is determined by the follicular development.In the process of research,we found that the number of pre-hierarchical follicles in low yield laying hens is remarkably fewer than high yield laying hens.The levels of plasma E2 and P4 were all decreased in low yield laying hens than high yield laying hens.In order to explore the mechanism of pre-hierarchical follicles decrement,RNA sequencing technology were adopted to evaluate the predominant genes involved in the development of the small white follicle(SWF),small yellow follicle(SYF)and F6(hierarchical follicle).Analysis of the pathways enriched by differentially expressed genes in the SWFs,SYFs and F6 revealed that VEGF,steroid hormone biosynthesis,PPAR-γ signaling pathway,tight junction and gap junction signaling pathways were enriched.Results indicated that greater changes in the gene expression profiles when SWFs developed into SYFs compared with SYFs developed into F6.Therefore,the comparison between SWFs and SYFs was selected in the verified experiment.Based on Western blot analysis,the expression of angiogenesis-related proteins,including VEGF,VEGFR1,VEGFR2,ANGPT1 and ANGPT2 were all upregulated significantly in SYF when compared with the SWF while the expression of pigment epithelium derived factor declined significantly(P<0.05).Meanwhile,the levels of E2 and P4 in SYFs were increased 131.6%and 22.2%respectively.Furthermore,compared with the SWF,ApoD,APOA1,APOLD1,PPAR and VLDL were up-regulated,whereas occludin was down-regulated in the SYFs(P<0.05).The expression of VLDLR and PPAR-y were up-regulated while the expression of occludin was down-regulated(P<0.05).In conclusion,RNA sequencing technology and gene expression profile analysis indicated that angiogenesis,steroid hormone biosynthesis and yolk deposition were elevated when SWFs developed into SYFs.The promotion of E2 concentration in SYFs enhanced yolk synthesis and boost follicles development.Simultaneously,the increment of vasculature in SYFs provides the function for yolk rapidly deposition in the oocyte.2.Increased thecal angiogenesis was involved in FSH-enhanced follicular development in low-yield laying chickensIn our previous study,we found that the number of prehierarchical follicles,especially the number of SWFs and large white follicles(LWFs)in low yield laying hens is fewer than high yield laying hens.Meanwhile,the area of vasculature in atretic follicles is less than healthy follicles.And we found that the expression of follicle-stimulating hormone(FSH)and follicle-stimulating hormone receptor(FSHR)was higher in small yellow follicles than in small white follicles while it was lowest in atretic follicles(P<0.05).So,what is the relationship between FSH and follicular development,and how is it regulated?Here we used immunofluorescence,immunohistology,BrdU,TUNEL,Western bolt and RT-qPCR methods to investigate effects of FSH on the follicular development in vivo and vitro.Results indicated that FSH addition prominently promoted the number of prehierarchical and hierarchical follicles in vivo.The expression of steroid hormones(STAR,CYP19A1,CYP17A1 and CYP11A1)mRNA were enhanced remarkably(P<0.05)as well as the levels of plasma estradiol(E2),progesterone(P4)and luteinizing hormone(LH)were increased significantly(P<0.05).Meanwhile,results of immunofluorescence,RT-qPCR and Western bolt indicated that proliferation of follicular cells was accelerated by FSH via FSH-HIF-1 signaling and apoptosis was inhibited.Furthermore,FSH increased follicular angiogenesis through elevated the expression of vascular endothelial growth factor(VEGF),VEGFR and angiopoietins(ANGPT).In addition,FSH improved liver yolk synthesis and reduced the number of tight junction between granular cells.In summary,FSH promoted an increase in steroid hormones levels,which in turn promoted angiogenesis,yolk synthesis and reduced the expression of tight junction.The increased number of blood vessels promoted the yolk deposition.Moreover,the decreased tight junction between granulosa cells made yolk precursors(VLDL)can readily accessed into the oocyte,facilitating the rapid yolk incorporation.In general,FSH promoted follicular development through the regulation of angiogenesis,steroid hormones production tight junction and yolk deposition.3.Effect of dietary N-carbamylglutamate on development of ovarian follicles via enhanced angiogenesis in the chickenN-carbamylglutamate(NCG),an analogue of N-acetyl-L-glutamate(NAG),can increase arginine synthesis in mammals and improve the reproductive performance.However,the effect of NCG on poultry laying performance is still unclear.The purpose of this study was to investigate the effect of NCG on chicken follicular development with the technology of histomorphological observation,the cellular and molecular level test combined with transcriptome sequencing(RNA-seq).The dosage and timing for NCG administration were evaluated for its effect on follicular development.Results showed that supplementation with 1%NCG in the diet for 14 d led to accelerated development of growing follicles(over 60μm in oocyte diameter)and significantly increased feed intake and feed efficiency(P<0.05).Plasma amino acids(AA)analysis showed that feeding with 1%NCG significantly increased of plasma AA levels(P<0.05).RNA-seq analysis revealed that NCG supplementation upregulated expression of genes related to angiogenesis(VEGFA,VEGFC)and cell proliferation(CCND1、CDK2、CDK3),but downregulated expression of apoptosis-related genes(BAX、caspase-3).Moreover,NCG enhanced plasma NO level;upregulated expression of PKG-I,Raf1,and p-p38;and increased angiogenesis of the ovaries.In conclusion,dietary NCG(1%for 14 d)can promote development of ovarian follicles by increasing angiogenesis in ovaries of the chicken.4.Improvement of eggshell quality by dietary N-carbamylglutamate supplementation in laying chickensLater in its development,oocytes enter the reproductive tract and form eggs.Egg quality defects seriously reduce the quality grade and increase egg breakage in egg marketing activities.According to statistics,in the production of poultry eggs,the loss caused by eggshell breakage accounted for 6%-8%of the total egg production.Therefore,reducing the rate of eggshell breakage is also an important measure to increase the production of eggs.In this study the effect of N-carbamylglutamate(NCG)on eggshell quality was investigated by evaluating calcium absorption and calcification in laying hens.A total of 30 newly hatched female Hy-line chicks were randomly assigned to control group(basal diet)and treatment group(basal diet supplemented with 1%NCG).At 25 weeks,eggs from each group were obtained to assess egg quality parameters.Blood samples were collected for analysis of mineral,hormone and amino acids levels at 16 h after laying egg.Uterine tissues were removed and fixed in 4%neutral paraformaldehyde solution or kept in liquid nitrogen for mineral determination,quantitative PCR and Western blot.Results showed that the egg quality(eggshell thickness,strength and percentage,egg specific gravity and eggshell effective thickness)was significantly increased while effective thickness of mastoid layer,width of mastoid gap and mammillary knobs were significantly decreased by dietary NCG supplementation(P<0.05).The levels of minerals(Ca,P,Fe,Mg,Na,K)in eggshell,plasma and uterus were remarkably elevated(P<0.05).Meanwhile,the concentrations of calcium metabolism-related hormones(17β-estradiol,parathyroid hormone and calcitonin)were increased in NCG group(P<0.05).Moreover,expression of calbindin 1,carbonic anhydrase 2,ovalbumin,ovotransferrin,ovocleidin-17,ovocleidin-116 and clusterin mRNAs as well as calbindin 1 and ATP2A1 proteins in uterus,duodenum and kidney was all upregulated in hens fed with NCG(P<0.05).In addition,the number of blood vessels in uterus,height of uterine mucosal folds,villus length in endometrial and areas of uterine mucosal folds were significantly increased in NCG group(P<0.05).In conclusion,dietary 1%NCG supplementation during 0-25 weeks can improve eggshell quality through changing morphology of endometrial,promoting angiogenesis in uterus,regulating the expression of calcium metabolism-related genes and secretion of related hormones to promote eggshell formation in the laying hens.In summary,several signaling pathways and key genes were identified in the development of prehierarchical by RNA-seq.Angiogenesis,steroid hormone biosynthesis and yolk deposition were important signaling pathways that regulate S WFs developed into SYFs.Meanwhile,the down-regulation of tight junction provided a rapid transport channel for yolk deposition during the follicular development.FSH promoted the synthesis of steroid hormones as well as the angiogenesis by increasing the expression of HIF-1 which facilitated the transport of the yolk precursors synthesized in the liver to the follicles.The decreased of the tight junction between the granulosa cells promoted the deposition of the yolk precursor transported into the oocyte and accelerated the development of the follicle.Meanwhile,NCG increased the ovarian angiogenesis and promoted the proliferation of ovarian cells while inhibited cell apoptosis and finally facilitated the development of follicles via enhancing the plasma NO level.Furthermore,NCG improved eggshell quality through changing the morphology of endometrial,promoting the angiogenesis in uterus,regulating the expression of calcium metabolism-related genes and secretion of related hormones to promote eggshell formation in the laying hens.