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°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 3β-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 3β-HSD did not vary by day 15 but decreased from 141.6±16.5×10³ cells/CL on day 15 to 113.8±13.2×10³ cells/CL on day 20 of pregnancy. However, 3β-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 3β-HSD-positive cells migrated faster than the smaller 3β-H SD-positive cells on each day of pregnancy. The diameters of 3β-HSDpositive 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 3β-HSD-positive cells from Fr. 2 (mean diameter; 24.96 μm) than from Fr.3, Fr.4 and Fr.5 (mean diameters; 27.20, 30.79 and 31.28 pan, 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-incubations of cells from Fr. 2 with cells from Fr. 3 or Fr. 4 on days 15 and 20 of pregnancy.