| Project/Area Number |
62570746
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | FUKUI MEDICAL SCHOOL |
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Principal Investigator |
ASO Takeshi Fukui Medical School, 医学部, 助教授 (60093176)
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| Co-Investigator(Kenkyū-buntansha) |
NEGAMI Akira Fukui Medical School, 医学部, 助手 (50201695)
KAMITANI Naoyuki Fukui Medical School, 医学部, 講師 (30161219)
TOMINAGA Toshiro Fukui Medical School, 医学部, 教授 (10026908)
竹内 譲 福井医科大学, 医学部, 助手 (90197278)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1987: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Ovary / Granulosa cell / Steroidogenesis / Aromatase / Cell culture / FSH / Caイオン / ステロイド生能 / エストラゴオール / テストステロン / 細胞増殖 |
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| Research Abstract |
The influence of varlous factors on steroldogenlc functlons of granulosa cells (G-cells) were investigated using in vitro cell culture methods. Human and porcine G-cells were obtained by aspiration from the follicles of middle size. After separation of G-cells by centrifugation, the cells were incubated in the medium containing Waymouth : Hanks : FCS/6:3:1, at 37 C in humidified 95% air and 5% CO_2. The cells exhibited 100% of attachment after 120 minutes of incubation and reached a confluence stage after 7 days of culture. Apparent enhancement of cell attachment and growth were observed after incubation with FSH. The stimulative effect of FSH on estradiol production from testosterone was already detected after 6 hours incubation and the extent of enhancement exhibited a time dependent manner up to 24 hours of incubation. Additional increase of estradiol production was obtained when the cells were stimulated by LH. The influence of pulsatile FSH stimulations (1 hour with and 3 hours wi
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thout FSH) did not show any significant concomitant increase of estradiol production during the period of of incubation with FSH. On the contray,intermittent addition of testosterone to the medium with contiuous FSH stimulation induced pormpt estradiol production. These results suggest that testosterone is readily converted to estradiol in the cells whose aromatase activity has been fully activated by FSH stimulation. The changes of intra-cellular calcium concentration Induced by FSH were analysed by measuring the fluorescent intensity using the specific chemical reaction between Fura 2 and Fura 2 - AM. Intra-cellular caleium concentration elevated promptly and significantly after the exposure of G-cells to FSH in dose dependent manner. This calcium increase provoked by FSH appears to play a key role for aromatase Induction and estradiol production. For further aproach to study the functions of G-cell, the possibilities of co-culture and cell perfusion systems were investigated. The results of basic experiments indicated that co-cuture of granulosa and theca cells with collagen membrane, cell perfusion system using cytodex beads were promissing methods to clarify the factors influencing the G-cells functions. Less
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