| Project/Area Number |
62440067
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Gunma University School of Medicine |
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Principal Investigator |
IBUKI Yoshito Gunma Univ. Sch. of Med., Associate Prof., 医学部, 助教授 (40008256)
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| Co-Investigator(Kenkyū-buntansha) |
HASEGAWA Yoshihisa Gunma Univ. Sch. of Med., Research Associate, 医学部, 助手 (40092001)
宮本 薫 群馬大学, 医学部, 助手 (30125877)
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| Project Period (FY) |
1987 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥27,200,000 (Direct Cost: ¥27,200,000)
Fiscal Year 1990: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1988: ¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1987: ¥11,000,000 (Direct Cost: ¥11,000,000)
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| Keywords | Inhibin / Activin / Gonadotropin / Receptor / Pituitary / Ovary / Hypothalamus / ヒト / ラット / 卵胞発育 / 妊娠 / FSH / 下性体ー性腺 / cDNA / RIA / 性同期 |
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| Research Abstract |
We established immunoaffinity chromatography for the purification of human inhibin in follicular fluid. Polyclonal antibodies directed against bovine inhibin were produced in three castrated male goats which were immunized intradermally with 500 ug purified 32 KDa bovine inhibin at monthly intervals for one year. Fifty ml antiserum from one of the goats was coupled with 50 ml Affigel 10. Human follicular fluid was obtained at oocyte collection in the IVF programme. Inhibin activity was measured by using rat pituitary cell culture system and bovine inhibin RIA. The inhibin-containing fraction eluted Matrex gel Red A column was applied to immunoaffinity column. A purification factor over several hundred folds could be achieved by the single procedure. Following Sephacryl S-200 gel filtration, the bioactive and immunoreactive inhibin were separated by the reverse phase HPLC. Both bioactive and immunoreactive fractions were separately purified to homogeneity by successive chromatographies.
… More
The purified bioactive inhibin preparation contained two isoforms of inhibin 34 KDa and 29 KDa which were stained with specific monoclonal antibody against A subunit (44H) and polyclonal antibody to a subunit (GBH1031) by using immunobltting method. On the other hand, the immunoreactive inhibin fraction containen two isoforms of 28 KDa and 25 KDa were stained with GBH1031, not with 44H. The purified bioactive human inhibin has a potent FSH release inhibiting activity similar to 32 KDa porcine or bovine inhibins.
To investigate the regulation of the LH/hCG receptor gene by gonadotropins, we examined the effect of PMSG and hCG on the expression of LH/hCG receptor in immature rat ovary. Northern blot analysis of ovarian RNA revealed a major mRNA of 5400 nucleotides and minor species of 7500, 3600, 2300 and 1200 nucleotides, and PMSG treatment slightly increased the intensity of all LH/hCG receptor messengers. Subsequently, hCG treatment decreased the number of LH/hCG receptor by day 2 and mRNA levels by 12h after injection. The level of mRNA recovered and increased 5-fold of control by day 6, then returned to control levela by day 10, followed by slower decline in LH/hCG receptor in plasma membrane. These studies demonstrate that the effects of PMSG and hCG on the number of LH/hCG receptor are closely related to the actions of these hormones on LH/hCG receptor messenger levels. Less
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