1995 Fiscal Year Final Research Report Summary
Biological nature and cDNA cloning of sperm ligand binding to pZP1
| Project/Area Number |
06454481
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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 |
Obstetrics and gynecology
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| Research Institution | Hyogo College of Medicine |
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Principal Investigator |
KOYAMA Koji HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,PROFESSOR, 医学部, 教授 (00068496)
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| Co-Investigator(Kenkyū-buntansha) |
KOMORI Shinji HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,ASSISTANT, 医学部, 助手 (60195865)
YAMASAKI Noriyuki HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,ASSISTANT, 医学部, 助手 (50174644)
HASEGAWA Akiko HYOGO COLLEGE OF MEDICINE,DEPT.OF OB/GYN,ASSISTANT, 医学部, 助手 (50212402)
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| Project Period (FY) |
1994 – 1995
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| Keywords | Fertilization / Acrosome Reaction / Zona Pellucida / Sperm Ligand / Secondary Sperm Receptor / Proacrosin / pZP1 |
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| Research Abstract |
The zona pellucida is an extracellular matrix surrounding mammalian oocytes and possesses various important functions for fertilization. A component of mouse zona pellucida (mZP2) has been shown to function a secondary sperm receptor. Recently, we succeeded in cloning a cDNA coding for a porcine zona pellucida glycoprotein (pZP1). The cDNA sequence was 54% homologous to mZP2. The aim of this study is to examine the function of pZP1 using recombinant pZP1 (r-pZP1). The recombinant protein used in this study was the NH_2-terminal region (amino acid positions 1-198) of pZP1. Human sperm was prepared by washing and centrifugation in BWW medium containing BSA (3mg/ml). After preincubation in 5% CO_2 in air for 3 hours, the sperm was incubated for 15min in the medium containing ionophore A23187 (10 mu M) to induce the acrosome reaction (AR). r-pZp1 (1-198) was added to the sperm suspension at a final concentration of 10 mu g/ml. The methanol-fixed sperm was treated with a monoclonal antibody (MAb-5H4), which has been proven to recognize an epitope on pZP1 (50-59), and followed by thetreatment with FITC conjugated anti-mouse IgG.AR was assessed by PSA lectin staining. When r-pZP1 (1-198) was added to the AR induced human sperm suspension, the number of sperm bound the protein gradually increased during the incubation. While, the treatment of sperm with r-pZP1 (1-198) did not increase AR.When the r-pZP1 (1-198) was added to in vitro fertilization medium, neither sperm binding to human zona pelludica nor sperm penetration into zona-free hamster eggs was interfered. r-pZP1 (1-198) was shown to bind to the acrosome reacted sperm but not to acrosme intact sperm. This suggests that the NH2-terminal portion (1-198) of pZP1 might be functional for fertilization without carbohydrate residues. It is thus concluded that the domain of pZP1 (1-198) serves as a secondary sperm receptor in the same manner to mZP2.
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