Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan.
Am J Physiol Endocrinol Metab. 2013 Mar 15;304(6):E566-75. doi: 10.1152/ajpendo.00432.2012. Epub 2013 Jan 8.
The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by follicle-stimulating hormone (FSH). Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 (Per2)-dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erbα (Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 days with FSH expressed higher mRNA levels of Per2, Rev-erbα, Bmal1 (Arnt1), Lhcgr, and connexin (Cx) 43 (Gja1) compared with the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by Western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erbα transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43.
本研究旨在评估缝隙连接与促卵泡激素（FSH）刺激下大鼠颗粒细胞时钟系统成熟之间的关系。通过穿刺已用己烯雌酚和马绒毛膜促性腺激素（eCG）处理过的 Period2（Per2）-dLuc 报告基因转基因鼠的卵巢，分别制备未成熟和成熟的颗粒细胞。暴露于地塞米松（DXM）同步化的成熟颗粒细胞显示出几个 Per2-dLuc 振荡和时钟基因的节律性表达。有趣的是，我们观察到明确的证据表明，FSH 刺激显著增加了颗粒细胞中 Per2 振荡的幅度，这一点通过 Per2 和 Rev-erbα（Nr1d1）mRNA 水平的升高得到了证实。FSH 还诱导了 Per2 振荡的主要相位提前移位。用 FSH 培养 2 天的成熟颗粒细胞表达的 Per2、Rev-erbα、Bmal1（Arnt1）、Lhcgr 和连接蛋白（Cx）43（Gja1）mRNA 水平高于未成熟颗粒细胞。同样，我们的免疫荧光结果显示，在 eCG 刺激的窦卵泡中存在丰富的 Cx43 蛋白，而在初级和窦前卵泡中则存在较弱或没有 Cx43 的荧光信号。Western 印迹分析也证实了类似的结果。两种缝隙连接阻滞剂林丹和 carbenoxolone（CBX）显著降低了 Per2 振荡的幅度，进一步导致 Per2 和 Rev-erbα 转录水平的显著降低。此外，林丹和 CBX 都诱导了 Per2 振荡的明显相位延迟移位。这些发现表明，FSH 通过增加 Cx43 的表达诱导时钟系统的发育。
