Research on role in uterus and ovary of receptors LGR homologous to gonadotropin receptors

促性腺激素（LH和FSH）受体和TSH受体是7个跨膜受体的成员，它们具有大的n端胞外重复序列（LGR: leucine-rich repeats containing G-protein偶联受体）。本研究采用同源受体LGR4、LGR5、LGR6、LGR7和LGR8。LGR4、LGR5和LGR6是孤儿受体。由于无法证实受体通过配体结合细胞膜表达，因此将M1表位添加到受体蛋白的n端区域，从而可以通过DNA转染在293T细胞表面的M1抗体进行证实。当受体被引入几种引起LH受体中cAMP合成激活的氨基酸突变时，尽管M1表位检测到受体细胞表面表达，但cAMP的基础水平并未增加，表明cAMP不可能是第二信使的候选者。利用子宫内膜进行RT-PCR检测，证实LGR4和LGR5的表达量较高。将cAMP响应性启动子驱动基因表达的荧光素酶蛋白表达载体与LGR7表达载体共转染293T细胞，建立用受体系统代替免疫反应性松弛素测量生物活性松弛素的检测系统。荧光素酶反应性太低，无法测量临床样本。采用RT-PCR方法检测卵巢、颗粒细胞、子宫内膜、肌瘤、输卵管和绒毛组织中松弛素、INSL3、LGR7、LGR8的表达。松弛素和INSL3在所有细胞中均有表达。另一方面，虽然LGR7受体仅在子宫内膜中表达，但LGR8并没有在每个组织中表达。这些基因的表达同样用睾丸组织检测。利用TESE收集的睾丸组织进行RT-PCR。不论精子是否存在，松弛素和INSL3均被检测到。虽然LGR7表达相似，但当标本中没有精子时，LGR4无法检测到。在LRG8出现的情况下，精子存在的可能性是可预测的。少

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 … More 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