Effects Of Antioxidant Bioactive Substances On Prehierarchical Follicular Development In Laying Hens

The ovary of the domestic chicken constitutes an ideal model for studies of ovarian biology and follicle development. The follicular granulosa cells represent a crucial symbol for follicle development due to their unique morphology and pivotal roles in the process of follicular development. In this study, we established two culture models of chicken whole prehierarchical follicles and separated granulosa cells to investigate the effects and mechanisms of hormones, growth factors and antioxidant bioactive substances involved in follicular development. From these studies, we expected to provide theoretically the mechanisms of development and selection of preovulatory follicles and practically improve the reproductive performance in laying hens.1.Developmental morphological changes in the follicular cells and effects of FSH/prostaglandin on development of chicken prehierarchical follicular cellsTheca cell (TC) and granulosa cell (GC) of the domestic chickens constitute ideal model system for studies of follicular development. At the beginning, we evaluated the morphological changes of chicken TCs and GCs in preovulatory follicles in laying hens. Samples of prehierarchical small and large white follicles (SWF, LWF), small and large yellow follicle (SYF, LYF) were collected in laying hens and fixed in 4% prafoxmaldehyde for 24 h. The paraffin-embedded follicles were serially sectioned at 5μm thickness and placed on coated slides for haematoxylin/eosin staining or immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Morphological changes showed that there is only one stratum of GCs in SWF, double stratum of GCs in LWF, multiple stratum of GCs in SYF and LYF. Moreover, statistic analysis confirmed that the thicknesses of TCs layer and GCs layer were increased in a follicular size-dependent manner. The thickness was the largest in SYF and LYF (P<0.05). Furthermore, the number of GCs per mm2 manifested an increasing trend with the thickness in SWF, LWF, SYF, LYF (P<0.05). During the follicular development, an increase in the area of GCs layer was accompanied by an increase in the size of follicles (P<0.05). PCNA staining showed that PCNA label index (PCNA-LI) of GCs in SWF, LWF and SYF was significantly higher compared with LYF (P<0.05). Therefore, GCs and TCs manifested a stage-relevant pattern in morphology and proliferating activity during the course of follicular development. Meanwhile, effects of follicle-stimulating hormone (FSH) and prostaglandin E1 (PGE1) on theca and granulosa cell proliferation were investigated through whole follicle suspension culture in vitro. Results showed that FSH significantly stimulated the proliferation of GCs, but not the TCs in the prehirarchical follicles; PGE1 significantly stimulated the proliferation of both GCs and TCs, suggesting that FSH and PGE1 promoted hierarchical development mainly through stimulating proliferation of the GCs in these follicles.2. Effects of ginsenosides on follicular development and proliferation of GCs from chicken prehierarchical folliclesGinsenosides (GS) are the main molecular components responsible for the actions of ginseng and exert varying effects on a myriad of cells and tissues, including pharmacological responses to the central nervous systems, cardiovascular, endocrine and immune systems. In this experiment, the effects of GS on GCs proliferation was investigated by the suspension culture model of whole follicles. The follicles were cultured in Medium 199 supplemented with 0.5% fetal calf serum (FCS). After 16 h, the medium was replaced with serum-free medium and challenged with GS alone or in combination with PKC inhibitor H7 for 24 h. Statistical analysis showed that GS significantly increase the number of GCs in SWF, LWF, SYF and LYF, suggesting GS promoted the follicular development via stimulating GCs proliferation. Meanwhile, the separated GCs culture system was adopted to investigate the specific mechanism of the stimulatory effect of GS on chicken GCs. GCs were isolated from SYF and cultured in Medium 199 supplemented with 0.5% FCS.After 16 h, the medium was replaced with serum-free medium and challenged with GS alone or in combination with PKC inhibitor H7 or activator phorbol 12-myristate 13-acetate (PMA) for 24 h. The results showed that GS (0.1-10μg/ml) significantly increased the number of GCs of SYF in a dose-dependent manner, and this stimulatory effect was inhibited by H7, but enhanced by PMA.Meanwhile, the PCNA-LI of GCs displayed similar changes with the number of cells. Western blot analysis showed that 10μg/ml GS promoted the PKC translocation from the cytosolic compartment to the membrane compartment in GCs, which was blocked by combined H7treatment. Furthermore, GS up-regulated the mRNA expression of cyclin D1/CDK6 and cyclin E/CDK2 in GCs. However, inhibition of PKC with H7 attenuated this stimulatory effect of GS. These results indicated that GS could promote proliferation of chicken GCs through PKC-involved up-regulation of cyclin D1/CDK6 and cyclin E/CDK2.3. Effects of daidzein on follicular development and proliferation of GCs from chicken prehierarchical folliclesThe aim of this study was to evaluate the role of a phytoestrogen daidzein (DAI) on proliferation of GCs from prehierarchical follicles of laying hens. The effect of DAI on theca and granulosa cell proliferation was investigated via suspension culture model of whole follicles. The follicles were cultured in Medium 199 supplemented with 0.5% FCS. After 16 h, the medium was replaced with serum-free medium and the cells were challenged with 1μg/ml DAI for 24 h. Results showed that DAI significantly stimulated the proliferation of GCs and TCs in SWF,LWF,SYF and LYF, suggesting that DAI could promote prehierarchical follicles entering hierarchical development through stimulating the proliferation of follicular cells. Meanwhile, the pure GCs were separated by mechanic method and dispersed into single cells. After 16 h pre-incubation in 0.5% FCS-supplemented medium, the medium was replaced with serum-free medium, which was supplemented with 10μg/ml insulin,5μg/ml transferrin and 3×10-8 M selenite. GCs were challenged with 10-1000 ng/ml DAI for 24 h and assessed for proliferation. DAI(10-1000 ng/ml) significantly increased the number of GCs (P<0.05) and this stimulatory effect was inhibited by an estrogen receptor antagonist tamoxifen in a dose-dependent manner. Furthermore, BrdU-LI of GCs displayed similar changes with the number of GCs. These results indicated that DAI promoted proliferation of cultured chicken GCs via estrogenic action.In conclusion, through the methods of morphology and immunohistochemistry, we investigated the developmental changes in number and density of both GCs and TCs during the course of prehierarchical follicular development. GCs and TCs manifested a stage-relevant pattern in morphology and proliferating activity. By the whole follicle suspension culture, it was proved that both FSH and PGE1 promoted hierarchical development through stimulating the proliferation of GCs in these follicles. Moreover, plant-derived antioxidants GS and DAI could stimulate the proliferation of GCs in SWF,LWF, STF and LYF. By using the pure GCs culture model, we found that GS could promote proliferation of chicken GCs through PKC-involved up-regulation of cyclin D1/CDK6 and cyclin E/CDK2; DAI promoted proliferation of GCs from SYF involving estrogenic action. These results could be contributed to elucidate the mechanism of prehierarchical follicular development and selection, as well as to represent a reference for application of dietary-derived antioxidant bioactive substances to improvement of poultry reproductive performance.