Regulation Of Local Growth-Promoting Factors On Follicular Development In The Laying Chickens

At present, people's consumption and market force the poultry industries to choice high-reproductive performance individuals in the breeding. While the reproductive performance depends on the follicular development of poultry. Thus, study of follicular development is the theoretical basis for understanding the regulation of reproductive function and improving the laying performance in poultry. Moreover, the poultry ovary constitutes an ideal model for studies of ovarian biology and follicular development. The growth and maturation of poultry follicles depend on a complex process controlled by a series of hormones and growth factors. In this study, the morphology of developmental follicles in laying hens was detected by histological method. The granulosa and theca cells were isolated from prehierarchical small yellow follicles (SYF) of laying hens, and germ cells were isolated from ovary of 18-day-old chick embryos. In addition, we evaluated the effects of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), stem cell factor (SCF) and ghrelin on the development of follicles and their related mechanism. From these studies, we expected to provide theoretic guidance for improving the reproductive performance of poultry.1. Morphology of developmental follicles in the laying chickensDevelopment of poultry follicles is a complex process with histological changes. The physiological functions depended on the tissue structure. It was benefited to study the histology of follicles in the development for understand the mechanism of follicular development. We performed histological methods to learn the development of chicken follicles in all stages. The results showed that primordial germ cells had migrated to the gonadal ridge to form the undifferentiated gonads at 6th hatching day. At stage of 9th hatching day, the gonads became differentiated and the feature of ovary had appeared and oocytes consisted branches. Primordial follicles become development after born. The ovarian follicles of the laying chicken maintain hierarchy at different stages of development. Furthermore, histology and proliferation of granulosa and theca cells were at specific stages. In conclusion, development of follicles was a process of histological changes with maturation of oocytes and development of follicular cells.2. Effects of EGF on follicular development from chicken prehierarchical follicles The selection of follicles in the poultry was affected by the endo/auto/paracrine factors and integrated regulated by the complex circulating hormones and local factors. Follicle stimulating hormone (FSH) and EGF played decisive role in the process of poultry follicular development. The mechanisms of them and interaction were worthy of further study. In this study the effects of EGF and FSH on the follicular development were evaluated in the prehierarchical follicles of laying chickens. RT-PCR results showed that EGF and its receptor (EGFR) mRNAs displayed high expression in granulosa cells from the prehierarchical follicles including the large white follicle (LWF) and SYF, and thereafter decreased markedly till the largest preovulatory follicle. After treatment with EGF and FSH for 24 h, the thicknesses of granulosa and theca layers and density of granulosa cells were significantly increased in both LWF and SYF in suspension follicle culture system (P<0.05). An additive effect of EGF with FSH was demonstrated. And the result of giemsa staining showed that both EGF and FSH increased the mitotic index of granulose cells in the prehierarchical follicles. Subsequently the proliferation of granulosa cells from SYF was remarkably increased by treatment of either EGF or FSH (0.1-100 ng/ml) in dose-dependent manner for 24 h. The results were confirmed by elevated proliferating cell nuclear antigen (PCNA) expression and decreased apoptosis. Furthermore, EGF-induced cell proliferation was accompanied with increased mRNA expression of EGFR, FSH receptor and the cell cycle-regulating genes (cyclins D1 and El, cyclin-dependent kinases 2 and 6), as well as decreased expression of luteinizing hormone receptor mRNA (P<0.05). However, the EGF or FSH-elicited increase in PCNA immunocytochemistry and decrease in apoptosis was reversed by simultaneous treatment of EGFR antagonist AG1478, leading to inhibition of EGF and FSH-induced cell proliferation. In conclusion, EGF and EGFR mRNA expression manifested stage-specific changes in follicle development. EGF promoted cell mitosis, retarded cell apoptosis and differentiation, regulated cell cycle and interacted with FSH to enhance the proliferation of granulose cells in the prehierarchical follicles, thus regulated follicle growth before selection in the laying chicken.3. Effects of bFGF on chicken embryo ovary development and follicular development from chicken prehierarchical folliclesBasic FGF is one of the potent mitogens tissue development and cellular function. In this study, the expression of FGF receptor 1 (FGFR1) from 18-day-old embryos and small yellow follicles in the chickens was determined, and the effects of bFGF on the ovary germ cells and prehierarchical follicular development were evaluated in the chickens. Results showed that FGFR1 was abroad expressed in the cells of ovary cortex and medullary, and strongly expressed in the granulosa layer. In vitro, the proliferation of ovary germ cells and granulosa cells was remarkably enhanced in a dose-dependent manner after treatment with bFGF (0.1-100 ng/ml), But the bFGF-induced cell proliferations were decreased by the FGFR1 antagonist SU5402. Subsequently, the activators or inhibitors of protein kinases A and C were added into the culture cells alone or in combine with 10 ng/ml bFGF to evaluate the signaling pathway. Results showed that cell proliferations were remarkablely promoted by PMA and inhibited by H7 (P<0.05). Whereas, FRSK and H89 had no significantly effects. Further, the result of injection bFGF into embryos confirmed the pro-proliferation of bFGF on the ovary germ cells of chicken embryos. The bFGF-elicited proliferation of the granulosa cells was accompanied with increased mRNA expression of the cell cycle-regulating genes, including CCND1ICDK6 and CCNE1ICDK2, whereas H7 inhibited the up-regulation of the genes. In conclusion, the above results indicated that bFGF promoted cell proliferation through binding with FGFR1 and involving PKC, up-regulated cell cycle, thus to regulate the development of ovary germ cells from chicken embryos and prehierarchical follicle in the laying chickens.4. Effects of SCF on proliferation of theca cells from chicken prehierarchical folliclesSCF is an earliest discovered paracrine factor, which had promoting follicle recruitment function. In the ovary, SCF was secreted by granulose cells. SCF was called "theca cell organizer", involving recruitment and differentiation of theca cells. In this study, the effects of SCF on the proliferation of theca cells and involving signaling pathway were evaluated in the prehierarchical follicles of laying chickens. In vitro, after 16 h pre-culture, the proliferation of theca cells from small yellow follicles was remarkably enhanced in a dose-dependent manner after treatment with SCF (0.1-100 ng/ml). With the proliferation of theca cells, the rate of cell apoptosis decreased after treatment with SCF. Immunohistochemistry results showed that the expression of c-Kit increased after treatment with SCF. But the SCF-induced proliferation of theca cells was decreased by c-kit antibody. In order to discuss the signaling pathway, the activators or inhibitors of protein kinases A and C were added into the culture theca cells. Results showed that the proliferation of granulosa cells was remarkablely inhibited by H7, and promoted by PMA (P<0.05). Whereas, FRSK and H89 had no significantly effects. In conclusion, the above results indicated that SCF promoted proliferation of theca cells through binding with c-Kit and involving PKC signalling pathway, hence to regulate the development of prehierarchical follicle of chickens.5. Effects of Ghrelin on follicular development from chicken prehierarchical folliclesGhrelin is a polypeptide mainly secreted by the stomach. It was reported that ghrelin had many important physiological roles in the release of growth hormone, food intake, and energy metabolism. Recent studies showed that ghrelin and its receptor (GHSR) were expressed in the ovary and had different regulating actions on follicular development. The purpose of this study was to study the effects of ghrelin on the follicular development from chicken prehierarchical follicles. The expression of GHSR mRNA was determined by RT-PCR, and the effects of ghrelin on the morphology of follicles and the proliferation of granulose cells were estimated by histological and immunohistochemical methods in follicles or cells cultured system. The results showed that GHSR mRNA was expressed in both granulosa and theca layers, and the expression increased with follicular development till large white follicle and small yellow follicle, and then decreased thereafter. After treatment with 100 ng/ml ghrelin for 24 h, the thicknesses of granulosa and theca layers and density of granulosa cells were significantly increased in both suspension cultured LWF and SYF (P<0.05). In vitro, after pre-culture for 12 h, the proliferation of granulose cells from small yellow follicles was remarkably enhanced in a dose-dependent manner after treatment with ghrelin (1-1000 ng/ml). Immunohistochemistry results showed that the expression of both protein kinases A and C increased after treatment with 100 ng/ml ghrelin. The proliferation of granulosa cells was remarkablely inhibited with H89 and H7, whereas, FRSK and PMA had the inverse effects. In addition, the labelling indexes of granulosa cells laminin and connexion 43 were remarkably elevated on the cultured follicles and cells (P<0.05). In conclusion, the above results indicated that ghrelin involved PKA and PKC signalling pathway and promoted the expression of laminin and connexion 43 in granulosa cells to enhance cell to cell interactions, subsequently stimulates the proliferation of granulosa cells, thus regulated chicken prehierarchical follicle development.In conclusion, we investigated the developmental changes of chicken follicles by the methods of morphology and immunohistochemistry and the effects of EGF, bFGF, SCF and ghrelin on the follicular development by suspension follicle culture and cell culture. The results showed that the loacal growth-promoting factors promoted proliferation of cells via their respective receptors and protein kinase signalling pathways, hence to regulate the development of chicken follicles.These results could be contributed to elucidate the mechanism of follicular development and selection, as well as to represent a reference for improvement of poultry reproductive performance.