Abstract
This study employed primary uterine epithelial cells from Sansui ducks (Anas platyrhynchos) at the peak of lay as an in vitro model. Using shRNA-mediated interference, we generated single knockdowns of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), inositol 1,4,5-trisphosphate receptor type 2 (IP3R2), and inositol 1,4,5-trisphosphate receptor type 3 (IP3R3), as well as dual knockdowns of IP3R1+IP3R2, IP3R1+IP3R3, and IP3R2+IP3R3, and a triple knockdown targeting all three IP3R isoforms. These treatments were designed to elucidate the regulatory effects of IP3R expression on intracellular calcium ion (Ca²⁺) concentrations and the expression of genes related to ion transport. After 48 h of transfection, fluorescence labeling with the Fluo-3/AM probe showed that intracellular Ca²⁺ concentrations were significantly higher in all treatment groups compared with the control group (P < 0.01). The real-time qPCR (RT-qPCR) analysis of ten ion transport-related genes revealed that the expression of transient receptor potential cation channel subfamily V member 6 (TRPV6), ORAI calcium release-activated calcium modulator 1 (ORAI1), epithelial sodium channel gamma subunit (SCNN1G), solute carrier family 8 member A1 (SLC8A1), ATPase plasma membrane Ca²⁺-transporting 1 (ATP2B1), and ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺-transporting 2 (ATP2A2) was significantly downregulated in multiple IP3R knockdown groups compared with the NC group (P < 0.05). The expression levels of these genes also showed significant correlations with intracellular Ca²⁺ levels (P < 0.05), including both positive and negative correlations. In the IP3R1 knockdown group, TRPV6, ORAI1, and SCNN1G were negatively correlated with Ca²⁺ concentration (P < 0.05). In contrast, ATP2B1 exhibited a positive correlation with Ca²⁺ in the IP3R2 knockdown group (P < 0.01), whereas SLC8A1 expression was positively correlated with Ca²⁺ in the IP3R1 + IP3R3 dual knockdowns group (P < 0.05). Collectively, the knockdown of IP3Rs not only reduced intracellular calcium ion (Ca²⁺) release but also altered the expression of ion channel-related genes, thereby affecting Ca²⁺ homeostasis. These findings provide new molecular evidence for the regulatory role of IP₃Rs in ion transport networks and Ca²⁺ balance in uterine epithelial cells of laying ducks.