Sommersberg B, Bulling A, Salzer U, Fröhlich U, Garfield R E, Amsterdam A, Mayerhofer A
Anatomisches Institut, Technische Universität München, D-80802 München, Germany.
Biol Reprod. 2000 Dec;63(6):1661-8. doi: 10.1095/biolreprod63.6.1661.
Follicle-stimulating hormone is the major regulator of growth and development of antral follicles in the ovary. Granulosa cells (GCs) in these follicles are coupled via gap junctions (GJs) consisting of connexin 43 (Cx 43). Because we and others have found that Cx 43 and GJs, respectively, are more abundant in large antral follicles compared with small antral and preantral follicles, we hypothesized that FSH may control Cx 43 gene expression, GJ formation, and intercellular communication. To directly address these points, we chose a rat GC line (GFSHR-17) expressing the FSH receptor and the Cx 43 gene. The functionality of FSH receptors was shown by the effects of porcine FSH, namely cell rounding, reduced cellular proliferation, and stimulation of progesterone production of GFSHR-17 cells, which are effects that were detectable within hours. Treatment with FSH also statistically significantly increased Cx 43 mRNA levels, as shown after 6 to 9 h in Northern blots. These effects were antedated by altered GJ communication, which was observed within seconds. Using a single-cell/whole-cell patch clamp technique, we showed that FSH rapidly and reversibly enhanced electrical cell coupling of GFSHR-17 cells. Increased GJ communication was associated with statistically significantly decreased phosphorylation of Cx 43, which was observed within 10 min after FSH addition, during immunoprecipitation experiments. Our results demonstrate, to our knowledge for the first time, that the gonadotropin FSH acutely and directly stimulates intercellular communication of GFSHR-17 cells through existing GJs. Moreover, FSH also increases levels of Cx 43 mRNA. These changes are associated with reduced proliferation and enhanced differentiation of GFSHR-17 cells. In vivo factors in addition to FSH may be involved in the regulation of GJ/GJ communication between GCs in the follicle, but our results suggest that improved cell-to-cell coupling, enhanced Cx 43 gene expression, and possibly, formation of new GJs are direct consequences of FSH receptor activation and may antedate and/or initiate the pivotal effects of FSH on GCs.
促卵泡激素是卵巢中窦状卵泡生长和发育的主要调节因子。这些卵泡中的颗粒细胞（GCs）通过由连接蛋白43（Cx 43）组成的间隙连接（GJs）相互连接。因为我们和其他人发现，与小窦状卵泡和窦前卵泡相比，Cx 43和GJs在大窦状卵泡中分别更为丰富，所以我们推测促卵泡激素可能控制Cx 43基因表达、GJ形成和细胞间通讯。为了直接解决这些问题，我们选择了一种表达促卵泡激素受体和Cx 43基因的大鼠GC系（GFSHR-17）。猪促卵泡激素的作用证明了促卵泡激素受体的功能，即细胞变圆、细胞增殖减少以及GFSHR-17细胞孕酮分泌增加，这些作用在数小时内即可检测到。促卵泡激素处理在统计学上也显著增加了Cx 43 mRNA水平，如Northern印迹显示在6至9小时后。这些作用之前先出现了GJ通讯的改变，这在数秒内即可观察到。使用单细胞/全细胞膜片钳技术，我们表明促卵泡激素迅速且可逆地增强了GFSHR-17细胞的电细胞偶联。GJ通讯增加与Cx 43磷酸化在统计学上显著降低相关，这在免疫沉淀实验中促卵泡激素添加后10分钟内即可观察到。据我们所知，我们的结果首次证明，促性腺激素促卵泡激素通过现有的GJs急性且直接地刺激GFSHR-17细胞的细胞间通讯。此外，促卵泡激素还增加了Cx 43 mRNA水平。这些变化与GFSHR-17细胞增殖减少和分化增强相关。除促卵泡激素外，体内因素可能参与卵泡中GCs之间GJ/GJ通讯的调节，但我们的结果表明，改善的细胞间偶联、增强的Cx 43基因表达以及可能新GJs的形成是促卵泡激素受体激活的直接后果，并且可能先于和/或启动促卵泡激素对GCs的关键作用。
