| Project/Area Number |
16580229
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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 |
Applied animal science
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| Research Institution | Kobe University |
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Principal Investigator |
HARAYAMA Hiroshi Kobe University, Graduate School of Science and Technology, Associate Professor, 自然科学研究科, 助教授 (30281140)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAKE Masashi Kobe University, Graduate School of Science and Technology, Professor, 自然科学研究科, 教授 (60093316)
MURASE Tetsuma Gifu University, Faculty of Applied Biological Sciences, Associate Professor, 応用生物科学部, 助教授 (30303514)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | fertilization / sperm / intracellular signaling / protein kinase / phospholipase / cAMP / hyperactivation / pig / イノシトール3リン酸 / カルシウムストアー / タンパク質キナーゼC / cAMP / チロシンキナーゼ / SYK / リン酸化 |
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| Research Abstract |
1.Identification of cAMP-dependent protein tyrosine kinase : A spleen tyrosine (Y) kinase (SYK) was activated via the cAMP-PKA signaling in the sperm connecting and principal pieces. The mechanism for SYK activation is quite different between spermatozoa and lymphocytes, suggesting that the activation of sperm SYK is controlled by a novel signaling system.
2.Observation of relationship between SYK activation and expression of sperm fertilizing ability : The cAMP analog-treated spermatozoa exhibited flagellar hyperactivation coincidently with the SYK activation. This indicates a role of SYK as a regulator for sperm fertilizing ability.
3.Analyses of cDNA sequence of testicular SYK : The sequence of PCR product (590 bps) amplified from testicular cDNA was same as that of pig spleen syk gene (the C-terminal catalytic domain of SYK), confirming production of SYK in the testis.
4.Identification and functional analyses of sperm SYK substrate : The SYK substrate was a phospholipase Cγ1 (PLCγ1). This enzyme, which was cAMP-dependently phosphorylated at the Y352 residue, bound to and activated PLCγ1 by the phosphorylation at the Y783 residue. Additionally, chemical inhibition of PLCγ1 with U-73122 was valid to the suppression of over-expression of flagellar hyperactivation.
5.Trial to introduce PLCγ1 peptide fragments into spermatozoa : Although two kinds of commercial kits were used in this experiment, we faced problems of methodology that unknown factors included in the reagents and introduction protocol severely damaged sperm motility. A possible method for peptide introduction remains to be improved.
6.Identification and functional analyses of other regulatory factors of sperm fertilizing ability : Sperm PKC was controlled via the cAMP-PKA signaling. The chemical inhibition of sperm PKC with Ro-32-0432 was valid to the suppression of over-expression of flagellar hyperactivation.
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