Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd., Pathum Wan, Bangkok, 10330, Thailand.
Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd., Pathum Wan, Bangkok, 10330, Thailand.
Theriogenology. 2022 Jun;185:34-42. doi: 10.1016/j.theriogenology.2022.03.021. Epub 2022 Mar 25.
Reproductive failure caused by porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by embryonic death and weak-born piglets and is associated with placental cell apoptosis and impairment of endometrial integrity. Here, we aimed to determine whether endometrial epithelial barrier function and viability were altered following PRRSV type 1 or type 2 infection. PRRSV inoculation was examined at the apical or basolateral side of porcine glandular endometrial epithelial cell cultures isolated from 4- to 6-month-old PRRSV-free herd gilts (n = 7 pigs). On the apical side, four days postinfection (4 dpi) with type 2 PRRSV, transepithelial electrical resistance decreased by 31% ± 5%, and paracellular permeability to fluorescein isothiocyanate-dextran (4 kDa) increased by 10-fold as compared with the mock and type 1 infection. Real-time polymerase chain reaction results revealed that both PRRSV types upregulated the mRNA expression of the barrier builder tight junction protein (TJ) Cldn5, but downregulated pore-forming TJ Cldn7. Additionally, the expression of other TJ genes, i.e., Cldn3 and Cldn8, was differentially increased by PRRSV type 1 and that of zonula occludens-1 was increased by PRRSV type 2. MTT assays indicated an increase in porcine glandular endometrial epithelial cell culture at 2-6 dpi following type 2 infection. Analysis of apoptosis using Annexin/propidium iodide staining combined with flow cytometry showed that the percentage of viable cells decreased, accompanied by a significantly higher dead cell population following PRRSV type 2 infection at 2-4 dpi. PRRSV type 1 infection also induced dead cells (>4%) at 2 dpi; however, the cell population recovered at 4 dpi. In conclusion, PRRSV type 2 infection caused more severe TJ barrier dysfunction and reduced cell viability compared with PRRSV type 1 infection in the porcine endometrium. Impairment in the membrane integrity of the maternal glandular endometrium may be the underlying mechanism of PRRSV-induced reproductive failure in pregnant sows.
猪繁殖与呼吸综合征病毒（PRRSV）引起的繁殖失败的特征是胚胎死亡和弱仔，与胎盘细胞凋亡和子宫内膜完整性受损有关。在这里，我们旨在确定 PRRSV 1 型或 2 型感染后是否改变了子宫内膜上皮屏障功能和活力。从 4 至 6 月龄 PRRSV 阴性猪群后备母猪中分离的猪腺状子宫内膜上皮细胞培养物的顶端或基底外侧侧接种 PRRSV（n = 7 头猪）。在感染 2 型 PRRSV 的第 4 天（4 dpi），顶端侧的跨上皮电阻降低了 31%±5%，而对荧光素异硫氰酸酯-葡聚糖（4 kDa）的旁通透性增加了 10 倍，与mock 和 1 型感染相比。实时聚合酶链反应结果表明，两种 PRRSV 类型均上调了屏障构建紧密连接蛋白（TJ）Cldn5 的 mRNA 表达，但下调了孔形成 TJ Cldn7。此外，PRRSV 1 型差异上调了其他 TJ 基因（即 Cldn3 和 Cldn8）的表达，而 PRRSV 2 型则上调了 zonula occludens-1 的表达。MTT 测定表明，感染 2 型后，猪腺状子宫内膜上皮细胞培养物在 2-6 dpi 时增加。使用 Annexin/碘化丙啶染色结合流式细胞术分析凋亡表明，感染 2 型后，活细胞百分比降低，同时在 2-4 dpi 时死细胞群显著增加。PRRSV 1 型感染也在 2 dpi 时诱导了死亡细胞（>4%）；然而，在 4 dpi 时细胞群恢复。总之，与 1 型 PRRSV 感染相比，2 型 PRRSV 感染在猪子宫内膜中引起更严重的 TJ 屏障功能障碍和降低的细胞活力。母体腺状子宫内膜膜完整性的损害可能是 PRRSV 引起妊娠母猪繁殖失败的潜在机制。
