Purification of ovarian theca-interstitial cells by density gradient centrifugation

The study of the biochemical mechanisms regulating the differentiation of the ovarian androgen-producing cells has been difficult because of the lack of a method for isolating the theca-interstitial cells (TIC) from the granulosa and other cell types. We report here a simple, rapid and reproducible method for obtaining large numbers of highly enriched TIC using density gradient centrifugation. When dispersed cells from ovaries of hypophysectomized immature rats were centrifuged in a Percoll gradient (20-70%), the cells were separated into five incompletely resolved bands. Of these, band V contained the androgen-producing cells. The band V cells produced 3.2- and 3.9-fold more cAMP and androgen, respectively, than the whole ovarian cells and contained 3.8- and 3.5-fold more 125I-hCG binding and 3 beta-HSDH labeled cells, respectively. These data indicated that the TIC were 65% pure. In order to improve the degree of purification, a discontinuous density procedure was used. When the ovarian cells were centrifuged in d = 1.055 g/ml Percoll, the purified TIC produced 4.7- and 5.9-fold more cAMP and androgen than the whole ovarian cells and they contained 5.3-fold more 125I-hCG binding. The TIC were cleanly separated from the granulosa cells and histochemical staining for 3 beta-HSDH activity revealed that the TIC were 93.0 +/- 1.3% pure. This method provides, for the first time, the opportunity to study TIC differentiation under defined conditions without granulosa cell contamination.