1997 Fiscal Year Final Research Report Summary
Studies on molecularbiological approach of male infertility-Isolation of androgen responsive genes by genomic binding-site method.
| Project/Area Number |
07671745
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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 |
Urology
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| Research Institution | Yokohama City University |
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Principal Investigator |
TAKEDA Mitsumasa Yokohama City Univ., Urology, assistant professor, 医学部・附属病院, 講師 (70244499)
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| Co-Investigator(Kenkyū-buntansha) |
HOSAKA Masahiko Yokohama City Univ., Urology, professor, 医学部, 教授 (30106330)
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| Project Period (FY) |
1995 – 1997
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| Keywords | androgen / androgen receptor / androgen responsive gene / epididymis / rat |
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| Research Abstract |
To obtain a better understanding of molecular mechanism of androgen action on spermatogenesis and spermiogenesis, we performed genomic binding-site method to isolate androgen responsive genes. We have expressed histidine -tag fusion proteins encoding the DNA-binding domain of the rat androgen receptor (AR-DBD) in Escherichia col. using the pET1 5b vector. Large quantities of fusion proteins were produced and purified by nickelchelating resin. Gel shift assay revealed that AR-DBD protein could bind specifically with authentic androgen response elements(ARE). Rat genomic DNA digested by restriction enzymes was mixed with the AR-DBD protein and the fragments that bound to it were selected by nitrocellulose filter. These fragments were cloned into a plasmid vector and amplified. This sellection-amplification procedure were repeated five to six times and finally seven clones were isolated. Using these fragments as probes, northern analysis disclosed that one of the clones was strongly expressed in the rat epididymis and disappeared following withdrawal of testosterone by treatment of LH-RH analogue.
It was concluded that we isolated the androgen regulated gene that was highly expressed in the rat epididymis. Further studies should be necessary to evaluate the function of this gene on male reproduction.
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