Schiffer Z, Keren-Tal I, Deutsch M, Dantes A, Aharoni D, Weinerb A, Tirosh R, Amsterdam A
Physics Department, Schottenstein Cellscan Center for Early Detection of Cancer, Bar-Ilan University, Ramat-Gan, Israel.
Mol Cell Endocrinol. 1996 Apr 19;118(1-2):145-53. doi: 10.1016/0303-7207(96)03774-4.
We have established granulosa cell lines which express constitutively the rat FSH receptors by cotransfection of primary granulosa cells obtained from preovulatory follicles with SV40 DNA, Ha-ras oncogene and a plasmid expressing FSH receptors. These cells respond specifically to ovine and human FSH by cell rounding, intracellular cAMP accumulation, and progesterone secretion in a dose-dependent manner. A new method for the demonstration and quantitation of changes in cell shape-Small Angle Laser Light Scattering (SALLS) analysis-has been utilized for measurement of cell rounding in response to FSH stimulation in these cells. When cells were incubated with increasing doses of either ovine or human FSH, partial rounding of cells was observed at FSH concentrations as low as 24 pM, while complete rounding of cells was observed at a range of 0.24-2.4 nM of FSH. Following aldehyde fixation, hormone-treated cells were examined using the method of SALLS analysis. Histograms obtained by applying SALLS analysis on FSH stimulated GFSHR-17 cells were a reflection of the structural changes induced by the hormone. FSH- and forskolin-incubated cells yielded structured distributions with defined mean size and standard deviations. Moreover, the increase in sharpness of dominant peak in the histogram was correlated with elevated concentration of FSH in a dose dependent manner. In conclusion, cellular response to FSH is correlated with a specific pattern of light scattered in immortalized granulosa cells expressing functional FSH receptors. Therefore, SALLS analysis may serve as a useful tool for in vitro bioassay of the gonadotropic hormone. Moreover, this method may lend itself to in vitro bioassay of any hormone that induces specific morphological changes in target cells.
我们通过将从排卵前卵泡获得的原代颗粒细胞与SV40 DNA、Ha-ras癌基因和表达促卵泡激素（FSH）受体的质粒共转染，建立了组成型表达大鼠FSH受体的颗粒细胞系。这些细胞通过细胞变圆、细胞内cAMP积累和孕酮分泌以剂量依赖性方式对绵羊和人FSH产生特异性反应。一种用于证明和定量细胞形状变化的新方法——小角激光光散射（SALLS）分析——已被用于测量这些细胞中FSH刺激引起的细胞变圆。当用递增剂量的绵羊或人FSH孵育细胞时，在低至24 pM的FSH浓度下观察到细胞部分变圆，而在0.24 - 2.4 nM的FSH范围内观察到细胞完全变圆。醛固定后，使用SALLS分析方法检查激素处理的细胞。通过对FSH刺激的GFSHR - 17细胞应用SALLS分析获得的直方图反映了激素诱导的结构变化。FSH和福斯可林孵育的细胞产生具有确定平均大小和标准差的结构化分布。此外，直方图中主峰锐度的增加与FSH浓度的升高呈剂量依赖性相关。总之，细胞对FSH的反应与表达功能性FSH受体的永生化颗粒细胞中散射光的特定模式相关。因此，SALLS分析可作为促性腺激素体外生物测定的有用工具。此外，该方法可能适用于对任何在靶细胞中诱导特定形态变化的激素进行体外生物测定。
