Microanalysis of hormone responsive ovarian interstitial gland cells in miniature culture

Small cell aggregates of functional interstitial tissue were isolated from ovaries of 21- to 24-day-old rats, and their biochemical properties were studied in miniature cultures of 500-4000 cells. the isolated interstitial cells expressed high amounts of the mitochondrial cholesterol side-chain cleavage cytochrome P-450, visualized by immunofluorescent staining. Freshly isolated cells also expressed high activities of 17α-hydroxylase and 17:20-lyase, which were assayed by TLC analysis of [³H]progesterone metabolites. the TLC technique revealed the immediate conversion of progesterone to 5α-reduced progestins. Consequently, no aromatizable androgens were produced but, accumulation of androsterone resulted instead from the direct action of 17α-hydroxylase on 3α-hydroxy-5α-pregnane- 20-one (pregnanolone). Both the immunoreactive levels of cholesterol side-chain cleavage cytochrome P-450 and the rate of the 17:20-lyase activity declined rapidly during culture. However, addition of LH to the medium restored both enzymes, indicating the presence of functional LH receptors. the latter were also demonstrable by their ability to evoke cAMP formation in response to LH, but not to FSH. the activity of 5α-reductase in the interstitial cells was much higher (3-fold) than its activity in the granulosa cells. Unlike 17:20 lyase, the activity of 5areductase did not decay in culture, nor was it affected by LH. We thus established a novel and sensitive experimental method to isolate and study a minute population of ovarian cells which, unlike the follicular granulosa and theca cells, are enigmatically differentiated at the early stages of ovarian development.