| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
The gonadotropin (LH and FSH) receptor and TSH receptor are members of seven transmembrane receptors that have large N-terminal extracellular domain containing leucine-rich repeats (LGR : leucine-rich repeats containing G-protein coupled receptor). Homologous receptors, LGR4, LGR5, LGR6, LGR7, and LGR8 were used in the present study. LGR4, LGR5, and LGR6 are the orphan receptors. The M1 epitope was added to the N-terminal region of the receptor protein because the cell membrane expression by the ligand binding of the receptor was not able to be confirmed, allowing to be confirmed by the M1 antibody on cell surface of 293T cells by DNA transfection. When the receptors were introduced several kinds of the amino acid mutation that causes constitutive activation of cAMP production in the LH receptor, basal level of cAMP production was not increased in spite of receptor cell surface expression detected by M1 epitope, suggesting cAMP unlikely the candidate of second messenger. The expression
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of LGR4 and LGR5 was able to be confirmed by RT-PCR by using the endometrium.
The luciferase protein expression vector in which cAMP responsive promoter drives gene expression was co-transfected in 293T cell with expression vector of LGR7 for the establishment of the assay system to measure bioactive relaxin by receptor sysytem instead of measuring immunoreactive relaxin. Luciferase resposiveness was too low to measure clinical samples.
The expression of relaxin, INSL3, LGR7, and LGR8 was examined with RT-PCR by using the ovary, the granulosa cell, the endometrium, the myoma, the oviduct, and the villus. Relaxin and INSL3 were expressed in all cells. On the other hand, LGR8 was not ditected in each organization though receptors LGR7 were expressed only in the endometrium.
The expression these gene was similarly examined by using the testis tissue. RT-PCR was done by using the testis tissue that had been gathered by TESE. Relaxin and INSL3 were detected regardless of the presence of the sperm. LGR4 could not be detected when sperm was not found in the specimen though LGR7 expression was similar. The possibility that the presence of the sperm is predictable in the presence of the appearance of LRG8 was suggested. Less
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