Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China.
Gene. 2022 Apr 30;820:146227. doi: 10.1016/j.gene.2022.146227. Epub 2022 Feb 3.
Spermatogonial stem cells (SSCs) are the basis of spermatogenesis. Systematically exploring the critical factors associated with the formation of SSCs will provide new insight to improve the formation efficiency, and their practical application. Here we explore the regulatory mechanism of the ECM-receptor interaction signaling pathway and related genes during differentiation of SSCs in chicken. Firstly, the positive cell rate of SSCs protein marker was detected by immunofluorescence and flow cytometry and qRT-PCR was used to identify, the expression of related marker genes after 10 days of RA-induction. Secondly, the ESCs on 0d/ 4d /10d after RA- induction/self-differentiation were collected, and the total RNA was then extracted from cells. Finally, high-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. After PCA analysis of the RNA-seq data, Venny analysis, GO and KEGG enrichment were further used to find the key signaling pathways and genes in the RA-induction process. The results showed that on day 10 of RA-induction, grape cluster growth cells expressed integrinβ1, the specific marker protein of SSCs cells, and the integrinβ1 positive rate was 35.1%. Also, SSCs marker genes CVH, Integrinβ1, Integrinα6 were significantly up-regulated during RA-induction. Moreover, the significantly enriched pathway, ECM-receptor interaction signaling, in current study may play a crucial role in RA-induction. Then, JASPAR was used to predict the differential gene transcription factors in the signaling pathway, finding that RA receptor was a transcription factor of COL5A1, COL5A2 and COL3A1. The qRT-PCR results showed that the expression levels of RA receptors (RXRA, RARA and RXRG) and the predicted genes (COL5A1, COL5A2 and COL3A1) were both significantly increased during RA-induction. Also, dual-luciferase reporter assay showed that RA could affect the luciferin activities of COL5A1, COL5A2 and COL3A1. These results suggest that RA plays a crucial role in the formation of chicken spermatogonial stem cells via the transcription levels of COL5A1, COL5A2 and COL3A1 to regulate the ECM-receptor interaction signaling pathway. Additionally, knockdown of COL5A1/COL5A2/COL3A1 could effectively reduce the formation efficiency of SSCs. This indicated that the interference of RA receptor binding genes in the ECM-receptor interaction signaling pathway could decrease the efficiency of RA induced SSCs formation. Therefore, this study concludes that RA promotes formation of chicken spermatogonial stem cells by regulating the ECM-receptor interaction signaling pathway.
精原干细胞（SSCs）是精子发生的基础。系统地探讨与 SSCs 形成相关的关键因素，将为提高形成效率及其实际应用提供新的见解。在这里，我们研究了 ECM-受体相互作用信号通路和鸡 SSCs 分化过程中相关基因的调控机制。首先，通过免疫荧光和流式细胞术检测 SSCs 蛋白标志物的阳性细胞率，并通过 qRT-PCR 鉴定 RA 诱导后 10 天相关标记基因的表达。其次，收集 RA 诱导/自分化后 0d/4d/10d 的 ESCs，然后从细胞中提取总 RNA。最后，使用高通量分析方法（RNA-seq）对这些细胞的转录组进行测序。对 RNA-seq 数据进行 PCA 分析后，进一步进行 Venny 分析、GO 和 KEGG 富集，以寻找 RA 诱导过程中的关键信号通路和基因。RNA-seq 数据的 PCA 分析结果显示，在 RA 诱导的第 10 天，葡萄簇生长细胞表达了 SSCs 细胞的特异性标志物整合素β1，其整合素β1 阳性率为 35.1%。此外，在 RA 诱导过程中，SSCs 标记基因 CVH、Integrinβ1、Integrinα6 显著上调。此外，目前研究中显著富集的通路——ECM-受体相互作用信号通路，可能在 RA 诱导中发挥关键作用。然后，使用 JASPAR 预测信号通路中差异基因的转录因子，发现 RA 受体是 COL5A1、COL5A2 和 COL3A1 的转录因子。qRT-PCR 结果表明，RA 受体（RXRA、RARA 和 RXRG）和预测基因（COL5A1、COL5A2 和 COL3A1）的表达水平在 RA 诱导过程中均显著增加。此外，双荧光素酶报告基因检测表明，RA 可影响 COL5A1、COL5A2 和 COL3A1 的荧光素酶活性。这些结果表明，RA 通过调节 COL5A1、COL5A2 和 COL3A1 的转录水平，在鸡精原干细胞的形成中发挥关键作用。此外，COL5A1/COL5A2/COL3A1 的敲低可有效降低 SSCs 的形成效率。这表明干扰 ECM-受体相互作用信号通路中的 RA 受体结合基因会降低 RA 诱导 SSCs 形成的效率。因此，本研究得出结论，RA 通过调节 ECM-受体相互作用信号通路促进鸡精原干细胞的形成。
