Gga-miR-34b-3p targets calbindin 1 to regulate cellular calcium ion homeostasis during eggshell calcification in...

Improving eggshell quality in poultry is a key breeding goal, and identifying genetic markers that regulate eggshell calcification is essential for accelerating genetic advancements. This study focused on identifying the keys genes and molecular mechanisms that regulate eggshell calcification in the chicken uterus. The results showed that rapid eggshell mineralization began approximately 4 h after the egg enters the uterus, corresponding with observed morphological and histological changes in the uterine tissue. This is associated with increased energy demands and the production of ion transport proteins. Transcriptome analysis identified calbindin-1 (CALB1), ATPase plasma membrane Ca²⁺ transporting 2 (ATP2B2), and gga-miR-34b-3p as differentially expressed during eggshell formation. CALB1 and ATP2B2 were predicted targets of gga-miR-34b-3p, with roles in maintaining cellular calcium ion balance. A dual-luciferase reporter assay confirmed that gga-miR-34b-3p directly targeted inhibited CALB1 expression, although no significant changes in the luciferase activity were observed with the co-transfection of ATP2B2 wild-type and gga-miR-34b-3p mimic. Validation experiments showed significant increases in CALB1 and ATP2B2 mRNA and protein levels of CALB1 and ATP2B2 in the chicken uterus during eggshell calcification, with CALB1 predominantly expressed in the cytoplasm of uterine tubular gland cells. Furthermore, primary uterine tubular gland cells, identified using immunofluorescence for Cytokertin 18, demonstrated that silencing CALB1 and ATP2B2 increased intracellular Ca²⁺ concentration in these cells. Taken together, these findings suggest that the gga-miR-34b-3p/CALB1 regulatory axis maintains calcium ion homeostasis in the uterine tubular gland cells, to facilitate continuous and efficient eggshell calcification and thereby enhancing eggshell quality in chickens.