Degradation And Functions Of The Regressed Ovarian Follicles In The Laving Chickens

Laying performance depends largely on the growth and development of ovarian germ cells and follicles in laying hens,while the regulation of follicle development is the core issue of reproductive performance.No more than 5%chicken ovarian follicles reach preovulatory stage and ovulation occurs eventually.After ovulation,the remaining tissues collectively referred to as postovulatory follicle(POF)and suffer structural and functional regression.However,the other 95%follicles which fail to reach preovulatory stage will undergo atresia.These follicles termed atretic follicles.POF and atretic follicles are two main parts of regressed follicles in the chicken ovary.In this study,we investigated the regulation of follicle growth by POFs and screened the candidate genes controlling atretic follicles in the Hy-line white chickens.Elucidation of the promoting action of POFs on progressive development of the prehirarchical follicles and clarification of measurements for reducing follicle atrasia will be beneficial for full-utiliztion of the ovarian follicle pool.Therefore,this study will provide theoretical basis of the POF functions and practical guidance for increasing laying performance.1.Coherent apoptotic and autophagic activities involved in regression of postovulatory folliclesAfter ovulation in mammals,rupture of mature follicles is reorganized into the corpus luteum that secrets progesterone(P4)to stimulate endometrial development.The situation in birds differs considerably.Beyond ovulation the ruptured avian follicle forms a postovulatory follicle(POF)that is not considered analogous to mammalian corpus luteum.The function and regression mechanisms of avian POFs remain poorly understood.Here we investigated the changes in apoptotic and autophagic activities that were involved during POF degradation.Using HE,oil red staining,TEM and P4 level detecting,results showed that the structure and secretory function of POF3 manifested the most apparent deterioration during whole processes of regression.A TUENL assay revealed that the granulosa layer maintained longer viability than the theca layer.Importantly,mitochondrial apoptosis and endoplasmic reticulum(ER)stress-associated genes and proteins reached their highest levels in the granulosa cells of POF3.Beclinl was distributed mainly in theca cells and coupled with LC3?-? accumulation,Sequestosome-1(p62)degradation and Beclinl elevation confirmed that autophagic activity had increased dramatically in the theca layer of POFs.These results indicate that the apoptosis of the granulosa cells from POFs occurs by mitochondrial apoptosis and ER stress and that a coherence of Beclinl-induced autophagy and caspase-induced apoptosis results in regression of theca layers of avian POFs.2.Promotion of the prehierarchical follicle growth by postovulatory follicles involving PGE2-EP2 signalingThe postovulatory follicle(POF)in birds is an enigmatic structure,the function of which remains largely unknown.Previous studies on chickens have shown that the removal of POFs leads to the postponement of oviposition and the disturbance of broody behavior.One suggestion is that POFs may secrete some crucial hormones or cytokines to act on reproductive organs.However,such secretions and their specific target organs remain to be identified.Here,we investigate the putative functions of POFs in promoting the development of prehierarchical follicles in chickens and explore the possible signaling mechanisms controlling these processes.By establishing POF,granulosa cells from POF and SWFs culture,and POF co-cultured SWFs and granulosa cells from POF co-cultured SWFs,we measure the protein and mRNA level of SWFs.Results show that POFs express steroidogenic acute regulatory protein(STAR),cholesterol side-chain cleavage enzyme(CYP11A1),cyclooxygenase 1(COX1),and COX2 in granulosa cells(GCs),and,most notably,that POF1 produces more prostaglandin E2(PGE2)or prostaglandin F2? than do the Fl follicle or the other POFs.Using coculture systems,we also found that POF1 or GCs from POF1(POF1-GCs)significantly promote the proliferation of theca externa cells of small white follicles(SWFs,one phase of the prehierarchical follicle).Treatment with PGE2 significantly facilitates theca externa cell proliferation in SWFs.This POF-stimulating effect on SWF growth was prevented by treatment with indomethacin(COX inhibitor)or TG6-10-1(PGE2 type 2 receptor[EP2]antagonist).Therefore,POF1 may secrete PGE2 to stimulate the progression of SWF by PGE2-EP2 signaling.These results indicate that POF1 may serve as a transient supplementary endocrine gland in the chicken ovary that stimulates the development of the prehierarchical follicles through PGE2-EP2 signaling.3.Screening genes related with development and atresia of prehierarchical folliclesIn chicken production,the number of atretic follicles exerts a direct influence on laying rates.The detailed mechanisms of follicle atresia remain largely unknown Here we investigate the possible reason of follicle atresia.Small while follicles,small yellow follicles and atretic small yellow follicles were selected,we explored that the changes in morphology via HE staining and the difference in gene expression.Results showed that compared with the normal follicles,hyperplasia and loosen occure in the granulosa layer of atretic follicles,coupled with the decline in the expression of E-Cadherin and Bcl2 and the increase in the expression of caspase-3 and caspase-9.By detecting genes related hormone synthases,we found compared to the small yellow follicle,the expression of Cypllal,Hsd3b2,Cypl7al and Cypl9al decreased but COX1 and COX2 increased in the atretic small yellow follicle.There is an increase in the expression of FSHR,LHR,ER?,EP2,EP4 and FP in the small yellow follicle,but a decline in EP3.In addition,the increase in TGF?R1 expression and the the decease in FGFR1 and VEGFR1 were witnessed in the atretic small yellow follicle.However,compared to the small white follicle,there is an increase in BMPR1?,BMPR2,TGFPRl?FGFR1,VEGFR1 and VEFGR2 expressions,only the VLDLR expression were seen a decrease.These results indicates that the structure of atretic follicles were demaged,and the process of atresia is mediated by casepase.Although the capacities of the steroid hormone synthesis are gradual losing in atretic follicles,the higher expressions of the receptors of gonadotropins and steroid hormones help postpone follicular atresia.The increase in prostaglandins synthetase and their receptors are associated with the regression and reabsorption of atretic follicles.The abilities of prostaglandins synthesis in atretic follicles can medicate regression and reabsorption through their EP2,EP4 and FP receptors via autocrine signaling.4.bFGF medicates growth and yolk deposition in chicken prehierarchical folliclesAs we found above,results from previous experiments showed the expression of FGFR1 increased but VLDLR decreased with the follicle development.The major components of the yolk mass consist of very low density lipoprotein(VLDL)and vitellogenin(VTG),both of which are synthesized in the liver,transported to the ovary and taken up by maturing oocytes via very low density lipoprotein receptor(VLDLR)-mediated endocytosis.In this study,we used immunofluorescence,Western bolt and qRT-PCR essays to investigate whether bFGF exert impacts on growth,development and yolk deposition of chicken prehierarchical follicles.Results demonstrated that VLDLR was mainly expressed in the granulosa layer of the prehierarchical and preovulatory follicle,and its expression declined with follicles growth.Supplementation with SU5402(FGFR1 antagonist),we found that bFGF facilitate proliferation of follicular theca externa cells via FGFR1-AKT signaling,and promote angiogenesis in the theca layer.In addition,bFGF could inhibit the expression of VLDLR and p-ERK in the granulosa cells.Combined inhibition of FGFR1 and PPARy,we found that PPARy can also suppress the VLDLR expression in the granulosa cells.These results indicate that bFGF facilities growth and yolk deposition in chicken follicles,not only because it can promote the proliferation and angiogenesis in the theca layer,but because it is able to inhibit the VLDLR expression in the granulosa cells.In summary,these studies indicate the apoptosis of the granulosa cells from POFs occurs by mitochondrial apoptosis and ER stress and that a coherence of Beclinl-induced autophagy and caspase-induced apoptosis results in regression of theca layers of avian POFs;POF may secrete PGE2 to stimulate the progression of SWF by PGE2-EP2 signaling.Furthermore,we screened genes related development and atresia of chicken prehierarchical follicles in chickens.Using bFGF treatment,we found bFGF has a beneficial effects on growth,development and yolk deposition of chicken prehierarchical follicles.These results not only provide theoretical basis for the studies of degradation of regressed follicles and regulating the following follicle progress in chickens,but also offer references of improving poultry laying performance.