2000 Fiscal Year Final Research Report Summary
Molecular basis of apoptosis in human granulosa cells
| Project/Area Number |
11671620
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Tottori University |
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Principal Investigator |
LZAWA Masao Tottori University Faculty of Medicine, Associate Professor, 医学部, 助教授 (50032222)
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| Co-Investigator(Kenkyū-buntansha) |
HARADA Tasuku Tottori University Faculty of Medicine, Assistant Professor, 医学部, 講師 (40218649)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Human granulosa cells / Apoptosis / Apoptosis-related gene / Caspase-9 variant / DFF / Androgen / Apaf-1 / Immortalized cell line |
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| Research Abstract |
Apoptosis of granulosa cells during follicular atresia and corpus luteum regression is a crucial aspect of ovarian physiology, and the abnormal regulation may lead to ovarian dysfuncti on. As an initial step to clarify the molecular basis of apoptosis , we previously demonstrated the expression of a set of apo- ptotic machinery in granulosa cells from patients undergoing IVF.However, the primary culture system had disadvantages for further analysis of the apoptotic machinery. We established immortalized cell line s from granulosa cells of normal ovaries by transfection with SV-40 large T antigen. Using one of these cell lines as a model system, we demonstrated the expression of apoptotic machinery, which had been demonstrated in granulosa cell s from IVF pa-tients. The cell line constitutively expresses transcripts for Apaf-1, Bcl-X _L, Bax, Caspase-3, Caspase-9 and its splicing variants as well as DFF-45, DFF-40, and AIF.In addition, expression of phosphatidylserine (PS) receptor, which plays a role for phagocytosis of apoptotic cells, was also demonstrated. Treatment with staurosporine of these cells induced apoptosis in a caspase-dependent and -independent manner. We examined several endogenous factors as potential apoptosis inducers. Among those factors examined, testosterone (Test) efficiently induced apoptosis in a dose-dependent manner. Within 6 hours after treatment with 3 μM Test, PS was expressed on the cell surface accompanying with nuclear and DNA fragmentation . The cell death process was partially suppressed by a caspase inhibitor Z-VAD- fmk. Concomitant treatment with 17β- estradiol exhibited no effect. In conclusion, we have demonstrated for the first time the androgen-dependent induction of apoptosis in human granulosa cells.
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