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 17 alpha-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 5 alpha-reduced progestins. Consequently, no aromatizable androgens were produced but, accumulation of androsterone resulted instead from the direct action of 17 alpha-hydroxylase on 3 alpha-hydroxy-5 alpha-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 5 alpha-reductase in the interstitial cells was much higher (3-fold) than its activity in the granulosa cells. Unlike 17:20 lyase, the activity of 5 alpha-reductase 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.
Bitzur
S
S
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Orly
J
J
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000378:metabolism; D000728:Androgens / Q000096:biosynthesis; D000818:Animals; D002449:Cell Aggregation; D002469:Cell Separation; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D007986:Luteinizing Hormone / Q000494:pharmacology; D010053:Ovary / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013254:Steroid 17-alpha-Hydroxylase
D000728:Androgens; D011374:Progesterone; D007986:Luteinizing Hormone; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D013254:Steroid 17-alpha-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase; D000446:Aldehyde-Lyases