| Co-Investigator(Kenkyū-buntansha) |
TANAKA Yudai Keio University, Department of Medicine, assistant, 医学部, 助手 (40317194)
YOSHIDA Hiroyuki Keio University, Department of Medicine, assistant, 医学部, 助手 (10327596)
YOSHIMURA Yasunori Keio University, Department of Medicine, professor, 医学部, 教授 (10129736)
KOMATSU Setsuko Department of Molecular Genetics National Institute of Agrobiological Sciences Tsukuba, Head of Laboratory, 遺伝子応答研究チーム長 (90355751)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
In this study, we tried to profile all the proteins expressed in the human sperm, analyzed the glycoproteins using lectin, and examined the effects of cryopreservation, association with infertility, and the relationship between spermatic functions and phosphorylation.
We observed 560 and 318 independent spots, respectively, for proteins extracted from the human and mouse spermatic membranes, and were able to identify the proteins represented by 118 and 94 spots, respectively. The proteins identified in both mouse and human sperm were as follows : calreticulin and HSP90, the existence of which has already been confirmed by previous reports, as well as the nicotinic acetylcholine receptor, G-protein coupled receptor, ZFP35, and a cAMP response element binding protein. These findings are expected to contribute usefully to elucidation of spermatic functions in the future.
In the mouse model, we examined the relationship between the signal transduction system via cAMP in the sperm and the acr
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osomal responses. We cultured sperm activated with a PKA stimulator, i.e., H8, in the presence of ^<32>P-ATP. This induced an acrosomal response, and a 45-kD protein with intensified phosphorylation was observed on autoradiography. We are currently analyzing this protein to determine its identity.Jn relation to the clinical aspect, we found that specimens from infertile males could be roughly divided into three groups according to the spot patterns appearing on the gel. At present, we are in the process of identifying the proteins present in markedly different quantities among the three groups, and analyzing the association of the expression pattern with the spermatic functions. Pattern comparison of the proteins extracted from the human spermatic membrane before and after freezing revealed four spots exhibiting obvious decrease in quantity after the cryopreservation. We did not succeed in our attempt to detect glycoproteins by lectin staining. In the future, we may need to adopt a different approach to stain the transcribed membrane with lectin, such as developing the extracted solution after treatment in the lectin column. Less
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