The Role Of Chicken ATP2A3 Gene In Regulating Calcium Transport In Uterine Epithelial Cells During Eggshell Mineralization

Eggs’quality is closely related to eggshell,the low quality of eggshell problem which including soft-shelled eggs,broken shell eggs and thin-shelled eggs,will cause economic losses that can not be underestimated for enterprises.How to solve the problem of eggshell quality has become a hot topic for scholars.Calcium carbonate is the main component of eggshell which content up to 98%,and Ca²⁺transport in the uterus during eggshell formation influences calcium carbonate formation and thus eggshell quality.Therefore,current studies focus on the regulation of calcium transport in eggshell mineralization,and trying to improve the quality of eggshell by regulating the mineralization process.In this study,candidate genes related to the regulation of calcium transport during eggshell formation are analyzed and their functions are verified based on the m RNA transcriptome databases of chicken uterus.The results are as follows:1.The candidate gene ATP2A3 was found to be involved in the calcium regulation process of uterine cells during eggshell formation through the data analysis.R was used to analyze the m RNA transcriptome sequencing of chicken uterus,the results showed 270 differentially expressed genes were found before and after eggshell formation.GO and KEGG analysis of DEGs revealed that 65 genes were involved in calcium ion transport,among them,the expression of ATP2A3 was up-regulated,especially during the growth stage of eggshell formation,the expression increased significantly（P<0.01）.The functional bioinformatics analysis of chicken ATP2A3 using various online tools showed that chick ATP2A3 is an acidic,hydrophilic and stable transmembrane protein with eight transmembrane helices.It catalyzes ATP hydrolysis to transport Ca²⁺from the cytosol into the endoplasmic reticulum which dependent on Mg²⁺.These results suggest that ATP2A3 is involved in the regulation of calcium transport process in uterine cells during eggshell formation,especially in the growth stage of eggshell formation.2.Serum calcium was found to be decreased significantly during the growth stage of eggshell formation.Blood samples were collected at different stages of eggshell formation,and the changes of blood calcium ions were detected by serum biochemical analysis.The results showed that the blood calcium concentration showed a downward trend during eggshell formation,especially during the growth stage of eggshell formation（P<0.05）.3.The expression of ATP2A3 was different at different stages of eggshell formation.Collecting chicken uterus during different stage of eggshell formation for fluorescence quantitative PCR analysis,the results showed that ERS was occursed during eggshell mineralization,and the expression of ATP2A3 in uterine epithelial cells was significantly increased during eggshell formation（P<0.05）,especially the growth stage of eggshell mineralization,whose ATP2A3 expression reached the highest level（P<0.01）.4.The role of ATP2A3 in regulating calcium transport during eggshell formation was validated by treating chicken uterine cells with ATP2A3inhibitors and agonists.The intracellular and extracellular Ca²⁺concentration were detected as well as ATP2A3 expression of uterine epithelial cells treated by inhibitors and agonists.It shows that 10n M thapargargin effectively inhibited Ca²⁺transport by ATP2A3,and 100n M resveratrol effectively increased ATP2A3 activity.ATP2A3 was confirmed to be involved in the regulation of calcium transport in chicken uterine cells.5.E₂ and different Ca²⁺concentrations had no significant effect on calcium transport in uterine cells.Using E₂ and different Ca²⁺concentrations treated the uterine epithelial cells,and no significant changes in intracellular and extracellular Ca²⁺concentrations were found.Therefore,estrogen is not directly involved in Ca²⁺regulation at the eggshell gland,and calcium concentration has no significant effect on Ca²⁺transport at the eggshell gland.In conclusion,ATP2A3 is involved in the regulation of calcium transport in chicken uterus cells during eggshell mineralization.This study provides ideas and theoretical basis for the futural study on genes related to eggshell mineralization.