Miyauchi F, Midgley A R
Department of Obstetrics and Gynecology, Ehime Rosai Hospital, Niihama.
Endocrinol Jpn. 1990 Oct;37(5):649-63. doi: 10.1507/endocrj1954.37.649.
On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CL) were dissected and dissociated into single cell suspensions by enzyme treatments. The suspended luteal cells were allowed to sediment in a BSA gradient at 4 degrees C for 3.5 hours. Five fractions were collected from the top (Fraction (Fr.) 1) to the bottom (Fr. 5) of the gradient. Cells were incubated in serum-free DME-F12 for 20 hours with or without hCG (100 ng/ml) to test them functionally, and the accumulation of progesterone and testosterone was determined by radioimmunoassay. To assess 3,3-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used. Cells were examined by light microscopy, and the percentage of cells containing dark blue formazan deposits and their diameters were determined in at least 40 microscopic fields. The number of cells staining for 3 beta-HSD did not vary by day 15 but decreased from 141.6 +/- 16.5 X 10(3) cells/CL on day 15 to 113.8 +/- 13.2 X 10(3) cells/CL on day 20 of pregnancy. However, 3 beta-HSD-positive cells maintained the same levels of progesterone secretion until the advent of luteolysis, then they increased in size progressively throughout pregnancy. In BSA gradients, the relatively larger 3 beta-HSD-positive cells migrated faster than the smaller 3 beta-HSD-positive cells on each day of pregnancy. The diameters of 3 beta-HSD-positive cells differed significantly in Frs. 2, 3, 4 and 5 on days 15 and 20 of pregnancy. On day 15 of pregnancy, less progesterone accumulated in wells containing 3 beta-HSD-positive cells from Fr. 2 (mean diameter; 24.96 microns) than from Fr. 3, Fr. 4 and Fr. 5 (mean diameters; 27.20, 30.79 and 31.28 microns, respectively) but the Fr. 2 cells responded more to hCG stimulation. Fr. 2 also showed a higher ratio of testosterone accumulation to progesterone accumulation than the other fractions. The response to hCG stimulation of cells in Fr. 2 tended to be higher than that in Fr. 3 on day 20 of pregnancy. These data suggest that the steroidogenic rat luteal cells are comprised of morphologically and functionally different cell types after day 15 of pregnancy. No stimulating nor inhibiting effects were observed in co-incubation of cells from Fr. 2 with cells from Fr. 3 or Fr. 4 on days 15 and 20 of pregnancy.
在妊娠第5、10、15和20天，解剖大鼠黄体（CL），并通过酶处理将其解离成单细胞悬液。将悬浮的黄体细胞在4℃下于牛血清白蛋白（BSA）梯度中沉降3.5小时。从梯度的顶部（第1组分）到底部（第5组分）收集5个组分。将细胞在无血清的DME-F12中孵育20小时，添加或不添加人绒毛膜促性腺激素（hCG，100 ng/ml）以进行功能测试，并通过放射免疫测定法测定孕酮和睾酮的积累量。为了评估3β-羟基类固醇脱氢酶（HSD）活性，采用了组织化学悬浮染色法。通过光学显微镜检查细胞，并在至少40个显微镜视野中确定含有深蓝色甲臜沉淀的细胞百分比及其直径。妊娠第15天时，3β-HSD染色的细胞数量没有变化，但从妊娠第15天的141.6±16.5×10³个细胞/黄体减少到妊娠第20天的113.8±13.2×10³个细胞/黄体。然而，3β-HSD阳性细胞在黄体溶解出现之前保持相同的孕酮分泌水平，然后在整个妊娠过程中逐渐增大。在BSA梯度中，妊娠各天中相对较大的3β-HSD阳性细胞比较小的3β-HSD阳性细胞迁移得更快。妊娠第15天和20天时，第2、3、4和5组分中3β-HSD阳性细胞的直径存在显著差异。在妊娠第15天，来自第2组分（平均直径；24.96微米）的3β-HSD阳性细胞所在孔中积累的孕酮比来自第3、4和5组分（平均直径分别为27.20、30.79和31.28微米）的少，但第2组分的细胞对hCG刺激的反应更强。第2组分中睾酮积累与孕酮积累的比值也高于其他组分。妊娠第20天时，第2组分细胞对hCG刺激的反应往往高于第3组分。这些数据表明，妊娠第15天后，大鼠黄体中产生类固醇的细胞由形态和功能不同的细胞类型组成。在妊娠第15天和20天，将第2组分的细胞与第3或第4组分的细胞共同孵育，未观察到刺激或抑制作用。
