Reply: Exposure of human fallopian tube epithelium to elevated testosterone results in alteration of cilia gene expression and beating.
Reply: Exposure of human fallopian tube epithelium to elevated testosterone results in alteration of cilia gene expression and beating.
复制标题
答复：人类输卵管上皮暴露于升高的睾酮会导致纤毛基因表达和跳动的改变。
DOI：
10.1093/humrep/deab022
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Russo,Angela
中科院分区：
文献类型：
--
作者：
Jackson-Bey,Tia;Colina,José;Isenberg,BrettC;Coppeta,Jonathan;Urbanek,Margrit;Kim,JJulie;Woodruff,TeresaK;Burdette,JoannaE;Russo,Angela
关于我们需要薄层组织的论点，这对于获得高分辨率显微镜确实至关重要，因为纤毛需要靠近物镜以获得高分辨率视频。我们同意上皮并非完全没有基质。通过显微镜，你可以专注于细胞轴承纤毛，并揭示柱状结构，同时记录纤毛移动。基质可能代表定量PCR（qPCR）中的一个问题，但由于我们只考虑纤毛基因，在这篇特定的论文中，基质细胞mRNA的任何污染都不会改变这些转录本。对于未来的qPCR实验，我们确实包括基质对照。我们还包括免疫组织化学（IHC）图像，以确认纤毛细胞中的关键变化，如FOXJ 1。"浅表分泌物“不能通过单一的代表性IHC图片可靠地报告。重要的是要记住，这是从一个病人，有显着的变异性。在每个检测中，我们包括多个患者样本以解释变异性，但由于出版物的限制，我们显示一个图像。组织固定在4%，IHC方法会话中确实存在排印错误。对于图6，我们计算了折叠，但是我们有四个不同的患者，对于每个患者，我们计算折叠，然后将其平均。因此，对于每个患者为1的对照将给出平均值= 1和SEM= 0;对于高睾酮条件，也计算每个患者的倍数，平均值为0.75，因此< 1，此外，在这种情况下，四次测量不给出相同的值，因此SEM不等于零，这在论文中报道。我们将保留所有这些批评意见，以供将来研究之用。注：论文中的所有示意图均使用BioRender创建。com.
Regarding the argument that we required a thin layer of tissue, this is indeed critical to obtain high-resolution microscopy since the cilia need to be close to the objective to obtain a high-resolution video. We agree that the epithelium is not completely free of stroma. By microscopy, you can focus on cells bearing cilia, and reveal the columnar structure while recording cilia moving. Stroma could represent an issue in the quantitative PCR (qPCR), but since we are only considering cilia genes, in this specific paper, any contamination of stromal cell mRNA should not alter those transcripts. For future qPCR experiments, we are indeed including a stroma control. We also included immunohistochemistry (IHC) images to confirm key changes in the ciliated cells, such as FOXJ1. The ‘superficial secretion’is not reliably reported by a single, representative IHC picture. It is important to keep in mind that this is from one patient and there is significant variability. In each assay, we include multiple patient samples to account for the variability, but due to publication limitations, we show one image. The tissues were fixed in 4%, there is indeed a typo in the IHC method session. For Fig. 6, we calculated folds, but we had four different patients and for each patient we calculate fold and then averaged them. Therefore, the control, being 1 for each patient will give a mean= 1 and SEM= 0; for the high testosterone condition folds are also calculated for each patient and the mean is 0.75, so< 1, also, in this case, the four measurements do not give the same value and therefore SEM is not equal to zero, which is reported in the paper. We will keep all these critiques in consideration for future studies. Note: All schematics found in the paper were created using BioRender. com.