CHARACTERIZATION OF GRANULOSA-CELL SUBPOPULATIONS FROM AVIAN PREOVULATORY FOLLICLES BY MULTIPARAMETER FLOW-CYTOMETRY
CHARACTERIZATION OF GRANULOSA-CELL SUBPOPULATIONS FROM AVIAN PREOVULATORY FOLLICLES BY MULTIPARAMETER FLOW-CYTOMETRY
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DOI：
10.1210/endo-122-2-651
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发表时间：
1988-02-01
期刊：
影响因子：
4.8
通讯作者：
CRISSMAN, HA
中科院分区：
文献类型：
--
作者：
MARRONE, BL;CRISSMAN, HA
本研究的目的是用多参数流式细胞术来表征单个母鸡（Gallus alticus）排卵前卵泡在卵泡成熟的特定阶段的颗粒细胞群。将颗粒细胞固定并用选择性结合DNA（Hoechst 33342，蓝色）、RNA（派洛宁Y，红色）或蛋白质（异硫氰酸荧光素，绿色）的三种荧光染料染色。使用配备有三激光激发系统的流式细胞仪来分析来自染色细胞的三种颜色的荧光。对每个细胞进行前向角光散射和轴向光损失测量以确定相对细胞尺寸。此外，测量RNA与蛋白质和DNA与蛋白质的比率。结果表明：（1）卵泡成熟过程中，增殖细胞比例下降，细胞体积增大，颗粒细胞可分为高蛋白和低蛋白两个亚群;在卵泡成熟过程中，高蛋白亚群的细胞比例增加，低蛋白亚群的细胞比例减少：3）高蛋白亚群和低蛋白亚群在细胞周期分布、RNA含量和细胞大小上也存在差异;（4）细胞在两个亚群中的分布和增殖程度受排卵周期阶段的影响，主要是在最成熟的卵泡中。结果表明，颗粒细胞群体的动态异质性，从个别卵泡，并显示卵泡成熟和排卵周期的阶段对细胞生长和代谢的影响。
The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using muliparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.