Effect Of Prostaglandin And Quercetin On Development Of Chicken Prehierarchical Follicles

Egg production is one of the most efficiency industries in husbandry. The reproductive performance of poultry was mainly regulated by the level of ovarian follicle development in addition to species, environment, management and other factors. During the adult life of the poultry, only about 5% follicles could develop to the small yellow follicles. Subsequently the follicle was selected into hierarchy development and ovulation in the end. In addition, compared to the mammals, the follicles of the poultry were arranged from small to big and easy to observe and manipulate. Therefore, the ovary of the poultry constitutes an ideal model system for studies of ovarian follicular development and atresia. Furthermore, study the regulation of poultry follicular development has an important significance for improving the performance of egg production and quality. In this study granulosa cells (GCs) and theca externa cells (TECs) were isolated from prehierarchical small yellow follicles (SYF) of laying hens. In addition, the effect of prostaglandin (PG), insulin-like growth factor I (IGF-Ⅰ) and arachidonic acid (AA) on proliferation of cultured TECs were evaluated, together with the underlining mechanisms. Meanwhile, the attenuating effect of quercetin on cadmium-induced oxidative damage and apoptosis was investigated in cultured GCs from chicken ovarian follicles. From these studies, we expected to provide theoretic guidance for selection of preovulatory follicle and improved the reproductive performance of poultry.1. Establishment and application of theca externa cell culture modelThe theca externa layers from prehierarchical SYF were separated by mechanic method and dispersed into single cells by collagenase, and then cells were cultured in 0.5% stripped-serum media. After 12 h incubation, the cells attached to the plate bottom and spindle growing, subsequently characterized by 3β-hydroxysteroid dehydrogenase (3β-HSD) reaction. Meanwhile, BrdU incorporation evaluates the role of luteinizing hormone (LH) on proliferation of TECs. The results indicated that TECs grow well in stripped-serum media and this model could be used for studies the regulation of TECs proliferation and differentiation.2. Effect of prostaglandin on luteinizing hormone-stimulated proliferation of theca externa cells from chicken prehierarchical folliclesThe effect of PG on proliferation of chicken TECs from prehierarchical SYF was evaluated and involvements of PKA and PKC signaling pathway as well as mRNA expression of cyclins, cyclin-dependent kinases (CDK) and CREB1 were explored. TECs were separated and dispersed into single cells. After 12 h pre-incubation in medium with 0.5% stripped fetal calf serum (FCS), the medium was replaced with serum-free medium and cells were challenged with PGE1 and LH for 24h and assessed for proliferation. Results showed that PGE1 (0.1-10 ng/ml) displayed a similar proliferating effect as LH on TECs, and this stimulating effect was restrained by PGE receptor 2 antagonist SC19220 at 10-7-10-5 M. Prostaglandin synthase antagonist indomethacin (10-7-10-5 M) suppressed LH-induced increase in cell number with a dose-dependent manner. PGE1-stimulated proliferation of TECs was hindered by Hg9 (PKA inhibitor) but not by H7 (PKC inhibitor). In addition, proliferation of TECs was increased by dbcAMP treatment, but not by a PKC activator PMA. Meanwhile, BrdU incorporation displayed similar changes with the cell number. Immunocytochemical staining showed that PGE1 elevated expression of transcription factor CREB1 (cAMP response element binding protein). Furthermore, we found that H89, SC19220 and indomethacin abolished the PGE1-stimulated increase in the expression of cyclin CCND1/CDK6 and CCNE1/CDK2, CREB1. In conclusion, PGE1 promoted the proliferation of TECs from chicken SYF and was also involved in mediating LH-stimulated intracellular PKA signal transduction, subsequently activated CREB1 signaling pathway and up-regulation of cyclin D1/CDK6 and cyclin E1/CDK2 mRNAs, suggesting that PG promotes the proliferation of TECs of chicken prehierarchical follicles that may be related to dominant follicle development.3. Interactive actions of prostaglandin and insulin-like growth factor I on proliferation of theca externa cellsThe interactive actions of PG and Insulin-like growth factor I (IGF-Ⅰ) on proliferation of TECs was investigated in prehierarchical SYF of laying hens. Results showed that IGF-I (1-100 ng/ml) manifested a similar proliferating effect as PGE2 (0.1-10 ng/ml) on TECs, and this stimulating effect was restrained by AG1024 (IGF-I receptor inhibitor), KP372-1 (PKB&AKT inhibitor), NS398 (COX-2 inhibitor). Moreover, AG1024, KP372-1 and NS398 abolished the IGF-I-stimulated the increase of COX-2 mRNA and PGE2 production. Meanwhile, the rescue experiment indicated that combined treatment of PGE2 showed better rescuing effect on NS398 inhibition, which implies COX-2 plays the important role in mediating IGF-I action. In addition, PGE2-stimulated cell proliferation was also predominantly hindered by KP372-1 instead of SC19220. Furthermore, KP372-1 and NS398 abolished the PGE2-stimulated the increase of COX-2 mRNA, AG1024 also inhibited the PGE2and IGF-I stimulated the expression of IGF-1R mRNA. Morever, in suspension culture model of whole follicles, treatment of IGF-I (100 ng/ml) and PGE2 (10 ng/ml) alone or in combination significantly increased cell density, thickness of theca externa layer from SYF. Furthermore, IGF-1 and PGE2 display an additive effect. In conclusion, IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis via PKB signal cascades, and then PGE2 could combined with corresponding receptor, stimulated IGF-IR mRNA expression and promoted TECs proliferation. These observations suggest that reciprocal stimulation of intracellular PG and IGF-I may enhance proliferation of TECs, hence to facilitate development of chicken prehierarchical follicles.4. Effect of arachidonic acid on proliferation, cell viability and cell function of theca externa cellsAA is a cis-polyunsaturated fatty acid that is a normal constituent of membrane lipids in animal cells. Moreover, AA play an important role in the regulation of reproductive system as a precursor of prostaglandins. The AA on development and function of TECs was investigated in prehierarchical small yellow follicles of laying hens. Results showed that AA (10-7-10-4M) have no significant promoting effect on TEC. However, the cell viability was activated with increasing of A A concentration (10-7-10-6 M), and this stimulating effect was restrained by H7 at 10-8-10-6 M. Furthermore, AA (10-6 M) elicited PKC translocation from cytosol to membrane, provoked expression of E-cadherin,β-catenin and connexin-43, dramatically enhaced cell adhesion to typeⅣcollagen (collagen IV) and inhibited cell apoptosis. In addition, we found that H7 abolished the AA-stimulated increase in the expression of E-cadherin,β-catenin, connexin-43 and cell adhesion to collagenⅣ. In conclusion, mediated by PKC signal pathway, AA promoted cell viability, inhibited cell apoptosis and regulated the cell function by enhanced expression of E-cadherin, P-catenin and Connexin-43 and cell adhesion to collagen IV. Therefore, AA may be facilitate dominant follicle selection and development of chicken prehierarchical follicles.5. Quercetin attenuates cadmium-induced oxidative damage and apoptosis in granulosa cells from chicken ovarian folliclesThe attenuating effect of quercetin on cadmium-induced oxidative damage and apoptosis was investigated in cultured granulosa cells from chicken ovarian follicles. Results showed that exposure to 5μM CdCl2 induced a decrease in granulosa cell number and viability, caused chromatin condensation and DNA fragmentation. Moreover, cadmium treatment markedly increased malondialdehyde level and decreased glutathione peroxidase and superoxide dismutase activities. Furthermore, cadmium provoked higher BAX expression, inhibited expression of BCL2 and X-linked inhibitor of apoptosis protein (XIAP) and activated caspase-3. However, simultaneous supplementation with 1μg/ml quercetin protected granulosa cells against cadmium-induced cytotoxicity through attenuating lipid peroxidation, renewing antioxidant enzymes activities and alleviating apoptosis by modulating XIAP, BAX and BCL2 expression, and inhibiting caspase-3 activity. Therefore, these results suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in granulosa cells through attenuating lipid peroxidation, elevating intracellular antioxidant status and inhibiting apoptosis to ensure reproductive health.The above results indicated this cell culture model that cells incubated in 0.5% strip fetal calf serum media was feasible. This model system enables studies of differentiation and morphology as well as of signal transduction events specific for this highly specialized follicular cell type. Finally, this system will be instrumental in answering key questions concerning follicular architecture, function, growth, and maturation. PGE1 promoted the proliferation of TECs from chicken SYF and was also involved in mediating LH-stimulated intracellular PKA signal transduction, subsequently activated CREB1 signaling pathway and cell cycle. In addition, IGF-Ⅰstimulated the expression of COX-2 and production of PGE2 via PKB signal pathway. It forms a positive intracellular feedback cycle and regulated by "cross talk" between different signal pathways. Sequentially, the proliferation of TEC was accelerated and follicle development was facilitated. Furthermore, mediated by PKC signal pathway, AA promoted cell viability, inhibited cell apoptosis and regulated the cell function by enhanced cell adhesion and intercellular information exchange. Therefore, AA and its metabolites-PG may be facilitate development of chicken prehierarchical follicles through regulate the proliferation and function of TECs. In addition, we demonstrated quercetin as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in GCs through attenuating lipid peroxidation, elevating intracellular antioxidant status and inhibiting apoptosis to ensure reproductive health. These findings provide theoretic guidance and experimental platform for selection of dominant follicle and improving poultry reproductive performance.