Kisspeptin neurons located in the hypothalamic preoptic area (POA) are suggested to be responsible for the induction of the gonadotropin-releasing hormone (GnRH) surge and the following luteinizing hormone (LH) surge to regulate female mammals' ovulation. Accumulating evidence demonstrates that the preovulatory level of estrogen activates the POA kisspeptin neurons (estrogen positive feedback), which in turn induces a GnRH/LH surge. This study aimed to derive a cell line from goat POA kisspeptin neurons as an in vitro model to analyze the estrogen positive feedback mechanism in ruminants. Neuron-derived cell clones obtained by the immortalization of POA tissue from a female Shiba goat fetus were analyzed for the expression of kisspeptin (KISS1) and estrogen receptor α (ESR1) genes using quantitative real-time reverse transcription-polymerase chain reaction and three cell clones were selected as POA kisspeptin neuron cell line candidates. One cell line (GP64) out of the three clones showed significant increase in the KISS1 level by incubation with estradiol for 24 h, indicating that the GP64 cells mimic endogenous goat POA kisspeptin neurons. The GP64 cells showed immunoreactivities for kisspeptin and estrogen receptor α and retained a stable growth rate throughout three passages. Further, intracellular calcium levels in the GP64 cells were increased by the KCl challenge, indicating their neurosecretory ability. In conclusion, we generated a new KISS1-expressing cell line derived from goat POA. The current GP64 cell line could be a useful model to elucidate the estrogen positive feedback mechanism responsible for the GnRH/LH surge generation in ruminants.

中文翻译：

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雌激素增加来自山羊视前区的新产生的永生化 KISS1 表达细胞系中 KISS1 的表达

位于下丘脑视前区 (POA) 的 Kisspeptin 神经元被认为负责诱导促性腺激素释放激素 (GnRH) 激增和随后的促黄体生成素 (LH) 激增，以调节雌性哺乳动物的排卵。越来越多的证据表明，排卵前的雌激素水平会激活 POA Kisspeptin 神经元（雌激素正反馈），进而诱导 GnRH/LH 激增。本研究旨在从山羊 POA Kisspeptin 神经元中提取细胞系作为体外模型来分析反刍动物的雌激素正反馈机制。使用定量实时逆转录聚合酶链反应和三个细胞分析了通过永生化来自雌性 Shiba 山羊胎儿的 POA 组织获得的神经元衍生细胞克隆的 Kisspeptin (KISS1) 和雌激素受体α (ESR1) 基因的表达。克隆被选为 POA Kisspeptin 神经元细胞系候选者。通过与雌二醇孵育 24 小时，三个克隆中的一个细胞系 (GP64) 显示 KISS1 水平显着增加，表明 GP64 细胞模拟内源性山羊 POA 亲吻肽神经元。GP64 细胞对kisspeptin 和雌激素受体α 显示出免疫反应性，并在三个传代中保持稳定的生长速度。此外，GP64 细胞中的细胞内钙水平因 KCl 挑战而增加，表明它们的神经分泌能力。综上所述，我们从山羊 POA 中生成了一个新的 KISS1 表达细胞系。当前的 GP64 细胞系可能是阐明导致反刍动物 GnRH/LH 激增的雌激素正反馈机制的有用模型。