The Research Of Calcium Transporting In Chicken Endometrial Cells And The Mechanisms Of Calcium Deposition In Initial Stage Of Eggshell Calcification

Object: We investigate the effect of progesterone on the expressions of proteins and genes in the chicken uterus, and research the mechanism of calcium deposition in the early stage of calcification.Methods: In the present study, chicken endometrial cells were cultured and treated with different concentrations of sex hormones in vitro. The cells growth rules and the effect of sex hormones on the proliferation of cells were detected. Make use of RT-PCR and RTQ-PCR to determine the effects of sex hormones on the m RNA expression changes of Cabp-d28 k, Ca2+-ATPase, Carbonic anhydrase(CA), TRPV6, Osteopontin(OPN), Ovotransferrin(OVO), Ovalbumin(OVA), Ovocleidin 17(OC17) and Clusterin(Clu) in chicken endometrial epithelium cells. And then we treat the hens with progesterone or progesterone receptor antagonist before eggshell calcification, the eggshell and uterus were gathered in the early stage of calcification(4.5-5.5 h after ovulation). The ultrastructures of eggshell in different groups were observed by SEM, and the expression rules of different genes in different groups were detected by RTQ-PCR. The correlation analyses between gene expressions and eggshell ultrastructures were conducted. At last we employ immunohistochemistry and Western Blotting to verify the quantities and distributions of protein Cabp-d28 k and Ovalbumin in the uterus of different groups.Results: We cultured the chicken endometrial cells in vitro successfully. And we found that the growth curves of chicken endometrial epithelial and stromal cells are similar. The chicken endometrial cells treated with 100 n M progesterone proliferated significantly(P<0.05). The results of different genes’ expression in uterus showed that, the progesterone have negative regulatory impacts on the Cabp-d28 k,CA and OPN m RNA expression in the chicken uterus significantly(P<0.05) or extremely significantly(P<0.01), but the expression of OVA, Clu and OC17 m RNA in the uterus lower than the control group significantly(P<0.05) or extremely significantly(P<0.01) under the influence of progesterone. The progesterone receptor antagonist promoted the OVO, Clu and OC17 m RNA expression, but decreased the expression of CA, TRPV6 and OPN m RNA in the uterus, the differences were significant(P<0.05) or extremely significant(P<0.01). In addition, the thickness of eggshell membrane and mastoid layer, the fiber and nodule diameter were increased, the mastoid and fiber clearance became smaller than the control group under the action of progesterone, the differences were significant(P<0.05) or extremely significant(P<0.01). And there are correlations between the Cabp-d28k、OVA、OVO and OPN m RNA expressions in uterus and the eggshell ultrastructures. The results of immunohistochemistry and western blotting showed that, Cabp-d28 k and Ovalbumin distributed in uterus of all groups, but expression quantities of Cabp-d28 k in progesterone treated groups higher than other groups, whereas a contrary trend was observed for Ovalbumin.Conclusion: The progesterone inhibited the expressions of Cabp-d28 k and OPN in chicken uterus significantly(P<0.05), diminished the mastoid areas, mastoid diameters and fiber clearances of eggshell,promoted the expressions of OVA and OVO in chicken uterus, multiplied the Fiber and nodules diameter, and thicken the membrane and mastoid layer significantly(P<0.05). From the results, we speculated that the progesterone can regulate the expressions of the genes and proteins witch associated with eggshell calcification in chicken uterus, then tighten up the structure of eggshell fiber membrane and mastoid layer, and increase the intensity of of eggshell. This study provided a theory and experiment foundation for the further research of calcium deposition mechanism of eggshell calcification.