Cui Zhifu, Amevor Felix Kwame, Tang Bincheng, Qin Simeng, Lan Xi, Liu Lingbin, Liu Anfang
College of Animal Science and Technology, Southwest University, Beibei, 400715 Chongqing, PR China.
Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, PR China.
Int J Biol Macromol. 2025 Jan;286:138520. doi: 10.1016/j.ijbiomac.2024.138520. Epub 2024 Dec 6.
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 Ca 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 Ca 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.
提高家禽蛋壳质量是一个关键的育种目标，识别调控蛋壳钙化的遗传标记对于加速遗传进展至关重要。本研究聚焦于识别调控鸡子宫中蛋壳钙化的关键基因和分子机制。结果表明，蛋壳快速矿化在鸡蛋进入子宫约4小时后开始，这与子宫组织中观察到的形态学和组织学变化相对应。这与能量需求增加和离子转运蛋白的产生有关。转录组分析确定钙结合蛋白-1（CALB1）、质膜钙转运ATP酶2（ATP2B2）和gga-miR-34b-3p在蛋壳形成过程中差异表达。CALB1和ATP2B2是gga-miR-34b-3p的预测靶标，在维持细胞钙离子平衡中发挥作用。双荧光素酶报告基因检测证实gga-miR-34b-3p直接靶向抑制CALB1表达，尽管在ATP2B2野生型与gga-miR-34b-3p模拟物共转染时未观察到荧光素酶活性有显著变化。验证实验表明，在蛋壳钙化过程中，鸡子宫中CALB1和ATP2B2的mRNA和蛋白水平显著增加，CALB1主要表达于子宫管状腺细胞的细胞质中。此外，使用细胞角蛋白18免疫荧光鉴定的原代子宫管状腺细胞表明，沉默CALB1和ATP2B2会增加这些细胞内的钙离子浓度。综上所述，这些发现表明gga-miR-34b-3p/CALB1调控轴维持子宫管状腺细胞中的钙离子稳态，以促进蛋壳持续高效钙化，从而提高鸡的蛋壳质量。
