Data Sheet 1_In vitro culture of olfactory epithelial cells from Megalobrama amblycephala and their response to amino acid mixtures and prostaglandin F2α.pdf

Olfaction is essential for the survival and reproduction of fish, as it facilitates foraging, food localization, mate selection, and breeding. The in vitro cultured olfactory epithelial cells will provide an important resource for research on how fish use olfaction to detect odor molecules in their environment. In this study, olfactory epithelial cells from Megalobrama amblycephala were cultured in vitro to investigate their responses to various odors, amino acids, and prostaglandin F2α (PGF2α). Initially, the olfactory epithelial cells were cultured in vitro using the explant method and collagenase digestion technique. Based on observations of in vitro growth characteristics, collagenase digestion demonstrated superior growth stability and morphological features of ciliated neurons. The presence of olfactory neurospheres was identified through scanning electron microscopy (SEM). Immunofluorescence analysis revealed that most of the cells cultured were labeled with NEUN antibody. Additionally, the expression of olfactory receptors (ORs) was detected in the in vitro cultured olfactory epithelial cells using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). Stimulation with amino acids mixture and PGF2α significantly increased the number of olfactory epithelial cells labeled with pERK. RNA-seq analysis revealed that 1,276 differentially expressed genes (DEGs) were identified following PGF2α stimulation, with pathways related to olfaction and reproduction being significantly enriched. Collectively, this study successfully established an in vitro model of the olfactory epithelium cells in M. amblycephala and preliminarily investigated its response to odorant molecules, providing a valuable platform for research on fish olfactory function.

嗅觉对于鱼类的生存与繁衍至关重要，可协助其完成觅食、定位食物、择偶及繁殖等生命活动。体外培养的嗅觉上皮细胞（olfactory epithelial cells）将为解析鱼类通过嗅觉感知环境气味分子的机制提供重要的研究资源。本研究以团头鲂（Megalobrama amblycephala）的嗅觉上皮细胞为材料开展体外培养，以探究其对多种气味物质、氨基酸以及前列腺素F2α（PGF2α）的应答反应。研究首先采用组织块培养法与胶原酶消化技术进行细胞培养，通过观察体外生长特性发现，胶原酶消化法培养的细胞具备更优异的生长稳定性，且呈现出纤毛神经元的典型形态特征。借助扫描电子显微镜（scanning electron microscopy, SEM）可鉴定出嗅觉神经球的存在。免疫荧光分析结果显示，绝大多数培养细胞可被神经元特异性核蛋白抗体（NEUN）标记。此外，本研究通过荧光原位杂交（fluorescence in situ hybridization, FISH）与逆转录聚合酶链式反应（reverse transcription PCR, RT-PCR），在体外培养的嗅觉上皮细胞中检测到了嗅觉受体（olfactory receptors, ORs）的表达。经氨基酸混合物与PGF2α刺激后，被磷酸化细胞外调节蛋白激酶（pERK）标记的嗅觉上皮细胞数量显著升高。RNA测序（RNA-seq）分析结果显示，PGF2α刺激后共鉴定出1276个差异表达基因（differentially expressed genes, DEGs），且与嗅觉及繁殖相关的通路显著富集。综上，本研究成功构建了团头鲂嗅觉上皮细胞的体外模型，并初步探究了其对气味分子的应答反应，为鱼类嗅觉功能的相关研究提供了极具价值的实验平台。