| Project/Area Number |
03455026
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
広領域
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| Research Institution | Kanazawa University |
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Principal Investigator |
OSANAI Minoru Kanazawa University, Dept.of Biology, Professor, 理学部, 教授 (50113486)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Kohichi Iwate University, Dept.of Applied Entomology, Associated professor, 農学部, 助教授 (20003791)
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| Project Period (FY) |
1991 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1992: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1991: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | apyrene spermatozoa / motility acquisition of spermatozoa / initiatorin / endopeptidase / glandula prostatica / spermatophore / Bombyx mori / Antheraea yamamai / イムノブロッティング / エンドペプチダーゼ / イニシヤトリン / 精包 / BAEEase / イムノブロッテング |
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| Research Abstract |
The silkmoth, Bombyx mori(Bombycidae) produces two types of spermatozoa, apyrene spermatozoa and eupyrene spermatozoa that fertilize eggs. In the vesicula seminalis of male moth, these spermatozoa are immotile. We found that the apyrene sperm-activating factor (AAF) is secreted from the glandula prostatica of the male reproductive tract, and that AAF is initiatorin, an endopeptidase of serine-protease type that splits proteins at the arginine residues on the C-side. Molecular weight of this enzyme was determined as 30KDa with SDS-PAGE.Antiserum was prepared from a band of this PAGE corresponding to initiatorin. Immunological analysis demonstrated that the rise in this enzyme activity at the emergence results in an increase in the enzyme molecules produced by translation of mRNA.Initiatorin was purified using HPLC from the spermatophore formed in the female bursa copulatrix. The enzyme activity was tested with BAEE as a preferred substrate of initiatorin. This protease is very heat-labile. After purification four isoforms, two 29KDa and two 26KDa were obtained. Although they have high specific activities of BAEEase (200-fold purified), their abilities inducing sperm motility is relatively not strong. They may partially degraded products of the true 30KDa-initiatorin found in the glandula prostatica. Twenty amino acids of a purified 29KDa preparation, initiatorin I was sequenced from the N-terminal. Examination with GENETYX showed that this had high homologies with eight endopeptidases of various mammalian tissues. The 30-KDa initiatorin preparation purified from the glandula prostatica showed a high motility-inducing activity. In a wild silkmoth, Antheraea yamamai(Saturniidae), initiatorin-like endopeptidase was also found. This enzyme activated Antheraea-sperm as well as Bombyx-sperm. The identical mechanism of acquisition of sperm motility was also found in the orthopteran insects.
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