| Project/Area Number |
09460154
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | University of Tsukuba |
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Principal Investigator |
BABA Tadashi University of Tsukuba, Institute of Applied Biochemistry, Professor, 応用生物化学系, 教授 (40165056)
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| Co-Investigator(Kenkyū-buntansha) |
OKABE Masaru Genome Information Research Center, Osaka University, Professor, 遺伝情報実験施設, 教授 (30089875)
TOSHIMORI Kiyotaka Department of Anatomy, Medical College of Miyazaki, Professor, 医学部, 教授 (20094097)
TOYODA Yutaka Obihiro University, Professor Emeritus, 名誉教授 (90050418)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥13,000,000 (Direct Cost: ¥13,000,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1998: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1997: ¥7,100,000 (Direct Cost: ¥7,100,000)
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| Keywords | fertilization / sperm / egg / zona pellucida / mouse / acrosome / acrosin / protease / 卵子 |
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| Research Abstract |
To elucidate the sperm function (sperm/egg interaction) in mammalian fertilization, we have carried out biochemical and biological experiments using genetically modified mutant mice, and have obtained the following results :
1) To elucidate the role of acrosin in fertilization, we have examined the involvement of acrosin in the acrosome reaction of sperm, using the acrosin-deficient mutant mice. When the ability of sperm to adhere (attach) and bind to the zona pellicida of cumulus-free eggs was assessed in vitro, no significant difference was observed between the wild-type and acrosin-deficient mice. Immunocytochemical analysis demonstrated that the release of several acrosomal proteins from the acrosome of AcrィイD2-/-ィエD2 mouse sperm was significantly delayed during the calcium ionophore- and solubilized zona pellucida-induced acrosome reaction, in spite of normal membrane vesiculation. These data indicate that the delayed sperm penetration of the zona pellucida in the acrosin-deficient
… More
mouse results from the altered rate of protein dispersal from the acrosome, and provide the first evidence that the major role of acrosin is to accelerate the dispersal of acrosomal components during acrosome reaction.
2) To identify a novel candidate(s) for acrosomal proteins that act on the sperm/egg interaction, a DNA fragment was PCR-amplified from a cDNA library of acrosin-deficient mouse testis, and then used as a probe to screen a mouse testis cDNA library. Complementary DNA clones encoding each of four similar but different serine proteases, TESP1, TESP2, TESP3, and TESP4, have been identified.
3) We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome. The time required for the dispersal of acrosomal components was demonstrated to be approximately 3 seconds after the onset of acrosome reaction.
4) A porcine homologue of the major secretory protein of human epididymis, HE1 was purified from cauda epididymal fluids. The HE1 homologue was found to be a major cholesterol-binding protein, suggesting that it is involved in the regulation of the lipid composition of the sperm membranes during sperm maturation in epididymis. Less
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