Hormonal And Nutritional Regulation Of Prehierarchical Follicular Development In Laying Hens

The ovary of the domestic chicken constitutes an ideal model system for studies of ovarian biology and follicle development. Chicken granulosa cells are easily isolated and play an important role in follicle development, so it was looked as a crucial symbol in follicle development. In this study, we established the culture model of granulosa cell and discussed the effect and mechanism of hormones, growth factors and nutriment which has relationship with follicle development. From these studies, we expected to provide theoretic guidance for selection of preovulatory follicle and improve the performance of chicken.1.Establishment and application of granulosa cell culture modelThe granulosa layers from prehierarchical small yellow follicles (SYF) were separated by mechanic method and dispersed into single cells by collagenase, and then cells were cultured in different serum media. Cell viability was quantified by measurement of the mitochondrial reduction of MTT to produce a dark-blue formazan product. The results indicated that cultured cell showed the proper proliferation and growth, afterl6h pre-incubation in 0.5% FCS medium. Furthermore, the feasibility of this model was validated by follicle-stimulating hormone (FSH). FSH stimulated proliferation of granulosa cells in a dose-dependent manner from 0.1 to 10 ng/ml. Action of protein kinases A and C system in FSH-stimulated proliferation of granulosa cells was revealed that FSH promoted the proliferation of granulosa cells from SYF was primary mediated by the intracellular PKA signal transduction. The PCNA-LI of granulosa cells displayed similar changes with the number of cells.The above results indicated that this model could be used for studies about regulation of granulosa cell proliferation and differentiation.2. Study in the proliferation effect of prostaglandin on granulosa cells from chicken prehierarchical folliclesThe aim of the present study was to evaluate the role of prostaglandin (PG) on proliferation of granulosa cells from prehierarchical small yellow follicles (SYF) of buff laying hens. The granulosa layers were separated by mechanic method and dispersed into single cells. After 16h pre-incubation in 0.5% FCS medium, the medium was replaced with serum-free medium, which was supplemented with 10μg/ml insulin,5μg/ml transferrin and 3×10μM selenite. Cells were challenged with PGE1 and FSH for 24h and then assessed for proliferation.The results showed that PGE1 (0.1-10 ng/ml) had a similar proliferating effect as FSH on granulosa cells, and these stimulating effects were restrained by the PGE receptor antagonist SC-19220 at 10-7-10-5 M. Prostaglandin synthase antagonist indomethacin (10-7-10-5M) suppressed FSH-induced increase in the number of granulosa cells in a dose-dependent manner. Downstream activation of protein kinase A by forskolin-activated adenylate cyclase resulted in elevated proliferation of granulosa cells, an effect unobserved by phorbol-12-myristrate-13-acetate-activated protein kinase C.In addition, PGE1-stimulated proliferation of granulosa cells was hindered by H89 (PKA inhibitor) but not by H7 (PKC inhibitor). Furthermore, the proliferating cell nuclear antigen labeling index (PCNA-LI) of granulosa cells displayed similar changes with the number of cells. Immunocytochemical staining showed that PGE1 induced the expression of transcription factor CREB1 (cAMP response element binding protein) in granulosa cells, and CREB1 expression stimulated by forskolin was higher than that of PGE1.These results indicated that PGE1 promoted the proliferation of granulosa cells from SYF and was also involved in mediating FSH-stimulated intracellular PKA signal transduction.3.Interactive actions of prostaglandin (PG) and epidermal growth factor (EGF) on proliferation of granulosa cellsThe interactive actions of prostaglandin (PG) and epidermal growth factor (EGF) on proliferation of granulosa cells was investigated in prehierarchical small yellow follicles (SYF) of laying hens. The granulosa layers were dispersed into single cells by 12.5μg/ml collagenase.After 16 h pre-incubation in 0.5% fetal calf serum-supplemented medium, the medium was replaced with serum-free medium.Immunocytochemical staining showed that granulosa cells expressed EGF and its receptor, and their expression was increased by PGE1 (1-100 ng/ml) or forskolin(10-7 to 10-5 M) treatments.EGF receptor was also induced by its ligand EGF. The specific prostaglandin synthase inhibitors SC560 (for COX-1) and NS398 (for COX-2) suppressed EGF-stimulated increase of the granulosa cell number. Furthermore, the effect of EGF was confirmed by the immunocytochemical staining of the proliferating cell nuclear antigen in granulosa cells. Though EGF promoted the expression of both COX-1 and COX-2, the rescue experiment indicated that combined treatment of PGE1 showed better rescuing effect on NS398 inhibition than SC560 at 10-6 M, which implies COX-2 plays the predominant role in mediating EGF action. The above results indicate that reciprocal stimulation of intracellular PG and EGF production may enhance proliferation of granulosa cells, hence to facilitate development of chicken prehierarchical follicles.4. Effect of Arachidonic acid (AA) on proliferation, cell viability and cell function of granulosa cells from prehierarchical small yellow folliclesArachidonic acid is a cis-polyunsaturated fatty acid that is a normal constituent of membrane lipids in animal cells. In addition to its role as a precursor of prostaglandins, AA itself may play an important role in the regulation of cell function. The AA on development and function of granulosa cells was investigated in prehierarchical small yellow follicles of laying hens. The result indicated that there were no significant difference on proliferation of granulosa cell between Arachidonic acid, Linoleic acid and Linolenic acid. Cell viability was measured by MTT showed that the cell viability with increasing of AA concentration(10-6-10-5M), but the cell viability was higher at high concentration of AA (10-4 M). Immunocytochemical staining showed that AA increased the expression of PKA, LN and Cx-43.Therefore, mediated by PKC signal pathway, AA promoted the cell viability and regulated the cell function by increase the production of extracellular matrix (ECM) and connexin (Cx). The above results indicated this cell culture model that cells pre-incubated in 0.5% fetal calf serum-supplemented medium for 16h then the medium was replaced with serum-free medium 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 expression of EGF, EGFR and CREB1 of granulosa cells from SYF and was also involved in mediating FSH-stimulated intracellular PKA signal transduction. Otherwise, EGF stimulated the production of PG predominant mediatied by COX-2. It forms a positive intracellular feedback cycle and regulated by "cross talk" between different signal pathways. Sequentially, the proliferation of granulosa cell was accelerated and follicle development was facilitated.