Satou Kazuhiro, Mochimaru Yuta, Nakakura Takashi, Kusada Tomoyuki, Negishi Jun, Musha Shiori, Yoshimura Nanaka, Kato Yukio, Tomura Hideaki
Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan.
Department of Anatomy, Graduate School of Medicine, Teikyo University, Tokyo 173-8605, Japan.
J Reprod Dev. 2017 Apr 21;63(2):199-204. doi: 10.1262/jrd.2016-174. Epub 2017 Jan 15.
Reproduction is regulated by gonadotropins secreted from gonadotrophs. The production and secretion of gonadotropins are mainly regulated by gonadotropin-releasing hormone (GnRH). Agonists or antagonists that influence GnRH action on gonadotrophs are important to regulate reproduction; however, these factors have not been fully characterized due to the lack of simple and easy-to-use techniques to detect gonadotropin secretion from gonadotropin-producing cells. In the present study, we found that Gaussia luciferase (Gluc), which was expressed in LβT2 cells, can be secreted like a luteinizing-hormone (LH) upon stimulation with GnRH. The Gluc secreted into the medium was easily monitored as luminescence signals. The detection range of the GnRH-induced Gluc activity was comparable to that of the enzyme-linked immunosorbent assay for LH. In addition, when the Gluc was expressed in AtT20 cells, which produce adrenocorticotropic hormone (ACTH), the Gluc activity in the medium increased in parallel with the ACTH secretion upon stimulation with corticotropin-releasing hormone. Thus, the Gluc assay in the present study can be easily used for high-throughput screening of factors that influence LH or ACTH secretion from LβT2 or AtT20 cells, respectively.
生殖受促性腺激素细胞分泌的促性腺激素调节。促性腺激素的产生和分泌主要受促性腺激素释放激素(GnRH)调节。影响GnRH对促性腺激素细胞作用的激动剂或拮抗剂对调节生殖很重要;然而,由于缺乏简单易用的技术来检测促性腺激素产生细胞的促性腺激素分泌,这些因素尚未得到充分表征。在本研究中,我们发现LβT2细胞中表达的高斯荧光素酶(Gluc)在GnRH刺激下可像促黄体生成素(LH)一样分泌。分泌到培养基中的Gluc可作为发光信号轻松监测。GnRH诱导的Gluc活性的检测范围与LH的酶联免疫吸附测定相当。此外,当Gluc在产生促肾上腺皮质激素(ACTH)的AtT20细胞中表达时,促肾上腺皮质激素释放激素刺激后,培养基中的Gluc活性与ACTH分泌平行增加。因此,本研究中的Gluc测定可轻松用于高通量筛选分别影响LβT2或AtT20细胞LH或ACTH分泌的因子。