Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
J Neuroendocrinol. 2020 Jun;32(6):e12857. doi: 10.1111/jne.12857. Epub 2020 May 20.
Kisspeptin plays a critical role in governing gonadotrophin-releasing hormone (GnRH)/gonadotrophin secretion and subsequent reproductive function in mammals. The hypothalamic arcuate nucleus (ARC) kisspeptin neurones, which co-express neurokinin B (NKB) and dynorphin A (Dyn) and are referred to as KNDy neurones, are considered to be involved in GnRH generation. The present study aimed to establish cell lines derived from goat KNDy and GnRH neurones. Primary-cultured cells of female Shiba goat foetal hypothalamic ARC and preoptic area (POA) tissues were immortalised with the infection of lentivirus containing the simian virus 40 large T-antigen gene. Clones of the immortalised cells were selected by the gene expression of a neuronal marker, and then the neurone-derived cell clones were further selected by the gene expression of KNDy or GnRH neurone markers. As a result, we obtained a KNDy neurone cell line (GA28) from the ARC, as well as two GnRH neurone cell lines (GP11 and GP31) from the POA. Immunocytochemistry revealed the expression of kisspeptin, NKB and Dyn in GA28 cells, as well as GnRH in GP11 and GP31 cells. GnRH secretion from GP11 and GP31 cells into the media was confirmed by an enzyme immunoassay. Moreover, kisspeptin challenge increased intracellular Ca2+ levels in subsets of both GP11 and GP31 cells. Kisspeptin mRNA expression in GA28 cells, which expressed the oestrogen receptor alpha gene, was significantly reduced by 17β-oestradiol treatment. Furthermore, the transcriptional core promoter and repressive regions of the goat NKB gene were detected using GA28 cells. In conclusion, we have established goat KNDy and GnRH neurone cell lines that could be used to analyse molecular and cellular mechanisms regulating KNDy and GnRH neurones in vitro, facilitating the clarification of reproductive neuroendocrine mechanisms in ruminants.
Kisspeptin 在调节促性腺激素释放激素（GnRH）/促性腺激素分泌和随后的哺乳动物生殖功能方面起着关键作用。下丘脑弓状核（ARC） kisspeptin 神经元，共同表达神经激肽 B（NKB）和强啡肽 A（Dyn），并被称为 KNDy 神经元，被认为参与 GnRH 的产生。本研究旨在建立源自山羊 KNDy 和 GnRH 神经元的细胞系。通过感染含有猿猴病毒 40 大 T 抗原基因的慢病毒，使雌性柴山羊胎仔下丘脑 ARC 和视前区（POA）组织的原代培养细胞永生化。通过神经元标志物的基因表达选择永生化细胞的克隆，然后通过 KNDy 或 GnRH 神经元标志物的基因表达选择神经元衍生的细胞克隆。结果，我们从 ARC 获得了一个 KNDy 神经元细胞系（GA28），从 POA 获得了两个 GnRH 神经元细胞系（GP11 和 GP31）。免疫细胞化学显示 GA28 细胞中 kisspeptin、NKB 和 Dyn 的表达，以及 GP11 和 GP31 细胞中 GnRH 的表达。通过酶免疫测定证实了 GP11 和 GP31 细胞向培养基中分泌 GnRH。此外，Kisspeptin 刺激增加了 GP11 和 GP31 细胞中某些亚群的细胞内 Ca2+水平。表达雌激素受体α基因的 GA28 细胞中 kisspeptin mRNA 的表达在 17β-雌二醇处理后显著降低。此外，使用 GA28 细胞检测了山羊 NKB 基因的转录核心启动子和抑制区。综上所述，我们已经建立了山羊 KNDy 和 GnRH 神经元细胞系，可用于体外分析调节 KNDy 和 GnRH 神经元的分子和细胞机制，有助于阐明反刍动物的生殖神经内分泌机制。
