Molecular biologic analysis of gonadotropin genes in patients with ovulation disorders caused by pituitary dysfunction

女性的生殖功能受下丘脑-垂体-卵巢系统的调节。下丘脑脉冲式释放的促性腺激素释放激素(GnRH)刺激垂体促性腺激素(FSH和LH)的分泌，不仅诱导卵巢卵泡生长和排卵，而且还产生卵巢类固醇激素(雌激素和孕酮)。这些激素的合成通过反馈机制相互调节。这个系统的异常会导致排卵障碍。在垂体功能障碍患者中，可能存在以下方面的异常：1)对GnRH(GnRH受体)的反应；2)GnRH与GnRH受体结合后的信号转导；3)促性腺激素基因表达的调节元件；因此，在本研究项目中，我们试图找出垂体功能障碍导致排卵障碍的原因。6例女性垂体功能障碍患者在名古屋大学医院就诊。采集患者外周血淋巴细胞，采用金刚烷法提取DNA。以GnRH受体、LHβ亚基和促性腺激素α亚基上游300个碱基的DNA为模板，采用聚合酶链式反应技术扩增出GnRH受体、LHβ亚基和GnRHα亚基上游300个碱基的基因。通过TA克隆将扩增出的DNA克隆到载体上，并进行测序。结果显示，所有患者均未发现上述基因异常。此外，还发现促性腺激素亚基结构与功能的关系以及促黄体生成素异常的新发现。此外，还研究了促性腺激素对卵母细胞和颗粒细胞某些基因表达的影响以及促性腺激素释放激素激动剂的临床意义。

Female reproductive function is regulated by hypothalamic-pituitary-ovarian system. Pulsatile released gonadotropin-releasing hormone (GnRH) from hypothalamus stimulates the secretion of pituitary gonadotropins (FSH and LH) that not only induce ovarian follicle growth and ovulation but also produce ovarian steroid hormones (estrogen and progesterone) . The syntheses of these hormones are regulated each other through the feed-back mechanism. Abnormalities in this system causes ovulation disorders. In the patients with pituitary dysfunction, abnormalities in the following parts are speculated : 1) Response to GnRH (GnRH receptor), 2) Signal transduction after binding of GnRH to GnRH receptor, 3) Regulatory elements for gonadotropin gene expression, and 4) Gonadotropin genes. Thus, we tried to detect the causes of ovulation disorders by pituitary dysfunction in the present research project.Six female patients with ovulation disorders by pituitary dysfunction were prepared at Nagoya University Hospital. Lymphocytes were collected from the patients, and DNAs were extracted by guanidium method. Genes of GnRH receptor, LHbeta-subunit, and 300-bp upstream of gonadotropin alpha-subunit were amplified by PCR technic using these DNA as templates. The amplified DNA was cloned to plasmid through TA cloning and sequenced. As results, no abnormalities were found in the above genes in all the patients.Additionally, new findings in anomalous LH and relationship between structure and function of gonadotropin subnits were gotten. Moreover, gonadotropin effects to some gene expression in oocyte and granulosa cells and clinical significance of GnRH agonist were also studied.