Aquaporins (AQPs), a family of small membrane‐spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17β‐Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT‐PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor‐mediated mechanism in a dose‐dependent manner. A 732‐bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8‐shRNA2 had more effective inhibition on target gene than AQP8‐shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8‐shRNA2 lentivirus infected GCs (p < .05), while Bcl‐2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen‐mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.

中文翻译：

---

AQP8通过调节颗粒细胞凋亡参与雌激素介导的水牛卵泡发育

水通道蛋白 (AQPs) 是一个小的跨膜蛋白家族，参与流体运输、细胞信号传导和繁殖。调节AQP8表达会影响小鼠颗粒细胞 (GCs) 的凋亡、卵巢卵泡发生、卵子发生和早期胚胎发育，但其作用从未在其他物种中得到研究。本研究的目的是表征水牛卵泡发育中的AQP8功能。免疫组化法分析AQP8在水牛卵泡中的表达模式。17β-雌二醇（E2）或雌激素受体拮抗剂ICI182780治疗体外培养的GCs，AQP8的表达使用 qRT-PCR 检测。通过shRNA技术研究了其在水牛GCs凋亡中的作用。发现 AQP8 在次级卵泡中表达较高 ( p  < .05)，并且其在 GC 中的 mRNA 水平通过受体介导的机制以剂量依赖性方式被 E2 上调。获得了一个 732 bp 的水牛AQP8编码区，在不同物种间的氨基酸水平上高度保守。AQP8 ‐shRNA2 对靶基因的抑制比AQP8 ‐shRNA1更有效（66.49% vs. 58.31%）（p  < .05）。敲除AQP8诱导 GC 在 G2/M 阶段停滞并发生细胞凋亡。与对照组相比，较高的Caspase9在AQP8 ‐shRNA2 慢病毒感染的 GC中观察到表达( p  < .05)，而Bcl-2和Bax表达水平无明显变化 ( p  > .05)。总之，上述结果表明AQP8通过调节细胞周期进程和 GC 的凋亡参与雌激素介导的水牛卵泡发育调节。