|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1991 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1990 : ¥1,200,000 (Direct Cost : ¥1,200,000)
It has been shown that progesterone is the end product of steroidogenesis in quail granulosa cells and its production is stimulated by LH. During the development of a long-term cultures, we found that the granulosa cells rapidly lost the ability to produce progesterone. Moreover, progesterone levels in the media progressively decreased with time of culture. The present study was conducted to study the conversion of progesterone by the granulosa cells during long-term culture.
Granulosa cells, obtained from quail at 10 hrs before ovulation, were cultured in fibronectin-coated wells with serum-free medium supplemented with insulin, transferrin, and corticosterone at 39 C in 5% CO_2 and 95% air. To investigate progesterone metabolism, [ ^<14>C]progesterone was added to the medium and the culture was continued for 3 hrs. Then, radioactive steroids were extracted from the medium and analyzed by thin-layer chromatooraphy. The radioactive metabolites, recovered from thin-layer plates, were ide
ntified by comparisons of their chromatographic behavior with that of authentic preparations after oxidation or acetylation, and by the constant specific radioactivities of crystals during repeated crystallization with the authentic preparation.
Freshly isolated granulosa cells did not show any conversion of progesterone, but after 3 days in culture, the granulosa cells acquired the ability to convert radioactive progesterone into two major metabolites, identified as 5beta-pregnane-3, 20-dione and 3beta-hydroxy-5beta-pregnan20-one. Activity of 5beta-reductase, which was calculated from the amounts of these 5beta-reduced metabolites, increased during the culture. Inclusion of antioxidants to the medium, which was effective in preventing the spontaneous decrease in progesterone production during culture, prevented the increase of 5beta-reductase activity.
These results suggest that 5beta-reductase, which is newly induced or activated under the aerobic culture conditions, might contribute to the decrease in progesterone production in the granulosa cells during culture. Less