Effects Of Phytoestrogen Daidzein On Ovarian And Follicular Development In Chickens

The chicken embryonic ovarian germ-somatic cell coculture model was established and evaluated by germ cell proliferation stimulated by ginsenosides (GS), gonadotropin and steroid. The estrogenic and antioxidant effects of the phytoestrogen daidzein (DAI) on germ cell proliferation were evaluated by this coculture model. Meanwhile, the effects of DAI on laying performance and follicle development-related genes (gonadotrophin receptors: FSHR, LHR), P450 aromatase (P450arom) and prolactin receptor (PRLR)) mRNA expressions in Isa hens and local white silky fowls were investigated, and explored its possible molecular mechanisms on regulating follicle development. The above results will boost applications of flavonids such as DAI on local poultry reproduction.1. Establishment, confirmation and application of chicken ovarian germ-somatic cell coculture modelThe serum-free germ-somatic cell coculture model was established in vitro to evaluate the effects of the endogenous hormones and exogenous factors. Ovarian cells of 18-day-old chicken embryos were dispersed and cultured in the medium supplemented with insulin, transferrin and selenite (ITS medium). After culture for 24h, somatic cells attached at the bottom of the culture plate, grew as shapes of shuttle and almost spread around the whole bottom at 48h of culture. Germ cells as round or oval in shape were found in the surface of the somatic cells and their diameters were between 15-25μm. It was clear to observe the morphological changes of germ cells in ITS medium as the consequence of the monolayer formed by somatic cells. Germ cells were characterized by expression of a specific antibody for stem cell factor receptor c-kit. The effect of GS on germ cell proliferation was evaluated and the mechanism involving protein kinase C (PKC) pathway was investigated. Results showed that GS significantly increased germ cell proliferation and this stimulating effect was further increased by PKC activator (PMA), but inhibited by PKC inhibitor (H₇) in a dose-dependent manner. Moreover, GS elevated proliferating cell nuclear antigen (PCNA) expression and the PCNA -labeling index (LI) of germ cells displayed similar changes with the increased numbers of germ cells. These results indicated that GS stimulated proliferation of ovarian germ cells with involvement of the PKC-mediated system and the serum-free germ-somatic cell coculture model was an effective in vitro model for evaluating the effects of the endogenous hormones and exogenous bioactive materials.2. Follicle-stimulating hormone promoted proliferation of the cultured ovarian germ cells through protein kinases A and C-mediated systemsThe effect of follicle-stimulating hormone (FSH) on germ cell proliferation was evaluated by a chicken ovarian germ-somatic cell coculture model and possible involvement of protein kinase A (PKA) and PKC pathways was investigated. Cultured cells were dispersed from 18-day-old embryos and challenged with FSH alone or in combinations with forskolin (FRSK), PKA inhibitor (H₈₉) PMA or H₇ for 48h. The number of germ cells was counted and the proliferating cells were identified by Immunocytochemistry of PCNA. The PCNA-LI was determined for germ cells. Results showed that FSH (10-1000 ng/mL) significantly increased the number of germ cells (P<0.05) and this stimulating effect was increased by FRSK (10^-7-10^-5 M) and PMA (10^-8-10^-6 M), but was inhibited by H₈₉ (10^-7-10^-5 M) and H7 (10^-7-10^-5 M) in a dose-dependent manner. Furthermore, PCNA-LI of germ cells displayed similar changes with the number of germ cells. These results indicated that FSH stimulated proliferation of cultured ovarian germ cells with involvement of the protein kinases A and C-mediated systems.3. Stimulating effects of androgen on proliferation of cultured ovarian germ cells through androgenic and estrogenic actions in embryonic chickensThe effect of androgen on germ cell proliferation was evaluated by a chicken ovarian germ-somatic cell coculture model and the mechanisms were explored. Cultured cells were dispersed from 18-day-old embryos and challenged with testosterone (T) alone or in combinations with androgen receptor antagonist flutamide (FLU), estrogen receptor antagonist tamoxifen (TAM) or aromatase inhibitor letrozole (LET) for 48h. The number of germ cells was counted and the proliferating cells were identified by Immunocytochemistry of PCNA. Results showed that T (10^-7-10^-6M) significantly increased the number of germ cells (P<0.05) and this stimulating effect was inhibited by FLU (10-1000 ng/mL), TAM (10-1000 ng/mL) or LET (10^-9-10^-7 M) in a dose-dependent manner. Furthermore, PCNA-LI of germ cells displayed similar changes with the number of germ cells. These results indicated that T stimulated proliferation of cultured ovarian germ cells through both androgenic and estrogenic actions in embryonic chickens. 4. Estrogenic and antioxidant effects of a phytoestrogen daidzein on ovarian germ cells in embryonic chickensThe estrogenic and antioxidant effects of the phytoestrogen DAI on germ cell proliferation were evaluated. Cultured cells were dispersed from 18-day-old embryos and challenged with DAI alone or in combinations with TAM for 48h. Results showed that DAI significantly increased the number of germ cells (P<0.05) and this stimulating effect was inhibited by TAM in a dose-dependent manner. Furthermore, PCNA-LI of germ cells displayed similar changes with the number of germ cells. To estimate the antioxidant action of DAI, ovarian cells were exposed to the reactive oxygen species (ROS)-producing system hypoxanthine/xanthine oxidase (HX/XO). The changes of superoxide dismutase (SOD) activity and glutathione (GSH) level were measured for estimation of the antioxidant status. Ovarian cells were severely damaged by free radicals and this deteriorating effect could be prevented by DAI. Moreover, HX/XO-induced decrease in SOD activity and GSH level was restored by DAI (P<0.05). These results indicated that DAI promoted proliferation of cultured ovarian germ cells by estrogenic action and attenuated ROS-induced toxicity by antioxidant action in embryonic chickens.5. Effects of daidzein on laying performance and follicle development in Isa hens and white silky fowlsThe effects of DAI on laying performance and follicle development-related gene (gonadotrophins receptors FSHR and LHR), P450 aromatase (P450arom) and prolactin receptor (PRLR)) mRNA expressions in Isa hens and local white silky fowls were evaluated. The egg laying rate, mean egg weight, yolk and albumen weight were determined for estimation of the laying performance. The mRNA expressions of related genes were measured by the semi-quantitative RT-PCR. Results showed that DAI significantly increased the performance of egg laying during the late stage of the egg laying cycle in Isa hens and white silky fowls. White silky fowls were more sensitive to DAI than Isa hens. The relative mRNA abundance of the related gene (FSHR, LHR, P450arom and PRLR) was imcreased after DAI treatment. These results indicated that DAI improved the egg laying performance of Isa hens and white silky fowls after the laying peak and up-regulated the expressions of FSHR, LHR, P450arom and PRLR mRNAs in both species.In conclusion, a serum-free germ-somatic cell coculture model was established to evaluate the effects of the endogenous hormones and exogenous bioactive materials. The results showed ovarian germ cells were c-kit positive and thus c-kit represented a molecular marker for germ cells. GS stimulated proliferation of ovarian germ cells with involvement of the PKC-mediated system; T stimulated proliferation of cultured ovarian germ cells through both androgenic and estrogenic actions in embryonic chickens; FSH stimulated proliferation of cultured ovarian germ cells with involvement of the protein kinases A and C-mediated systems. These experiments further verified that the germ-somatic cell coculture model was an effective in vitro model to evaluate the effects of the endogenous hormones and exogenous materials. Phytoestrogen DAI promoted proliferation of cultured ovarian germ cells by estrogenic action and attenuated ROS-induced toxicity by antioxidant action in embryonic chickens. Furthermore, DAI improved the egg laying performance of Isa hens and white silky fowls after the laying peak and up-regulated the expressions of FSHR, LHR, P450arom and PRLR mRNAs in Isa hens and white silky fowls. The above results will surely boost applications of DAI on poultry (particularly local poultry) reproduction.