Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran 14174-66191, Iran.
Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol 46156-64616, Iran.
Int J Mol Sci. 2024 Oct 30;25(21):11653. doi: 10.3390/ijms252111653.
The in vitro generation of spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs) offers a viable approach for addressing male infertility. A multitude of molecules participate in this intricate process, which requires additional elucidation. Despite the decline in SSCs in aged testes, SSCs are deemed immortal since they can multiply for three years with repeated transplantation. Nonetheless, the examination of aging is challenging due to the limited quantity and absence of precise indicators. Using a microarray, we assessed genome-wide transcripts (about 55,000 transcripts) of fibroblasts and SSCs. The WGCNA approach was then used to look for SSC-specific transcription factors (TFs) and hub SSC-specific genes based on ATAC-seq, DNase-seq, RNA-seq, and microarray data from the GEO databases as well as gene expression data (RNA-seq and microarray data). The microarray analysis of three human cases with different SSCs revealed that 6 genes were upregulated, and the expression of 23 genes was downregulated compared to the normal case in relation to aging genes. To reach these results, online assessments of Enrich Shiny GO, STRING, and Cytoscape were used to forecast the molecular and functional connections of proteins before identifying the master routes. The biological process and molecular function keywords of cell-matrix adhesion, telomerase activity, and telomere cap complex were shown to be significantly altered in upregulated differentially expressed genes (DEGs) by the functional enrichment analysis. According to our preliminary research, cell-specific TFs and TF-mediated GRNs are involved in the creation of SSCs. In order to maximize the induction efficiency of ESC differentiation into SSCs in vitro, hub SSC-specific genes and important SSC-specific TFs were identified, and sophisticated network regulation was proposed. According to our research, these genes and the hub proteins that they interact with may be able to shine a light on the pathophysiologies of infertility and aberrant germ cells.
从胚胎干细胞 (ESCs) 体外生成精原干细胞 (SSCs) 为解决男性不育提供了一种可行的方法。许多分子参与了这个复杂的过程，需要进一步阐明。尽管衰老睾丸中的 SSCs 数量减少，但由于可以通过反复移植繁殖三年，因此它们被认为是不朽的。然而，由于数量有限且缺乏精确的指标，对衰老的研究具有挑战性。我们使用微阵列评估了成纤维细胞和 SSCs 的全基因组转录物（约 55,000 个转录物）。然后，我们使用 WGCNA 方法根据 ATAC-seq、DNase-seq、RNA-seq 和 GEO 数据库中的微阵列数据以及基因表达数据（RNA-seq 和微阵列数据）寻找 SSC 特异性转录因子 (TFs) 和枢纽 SSC 特异性基因。对来自三个不同 SSCs 的人类病例的微阵列分析表明，与正常病例相比，与衰老基因相关的 6 个基因上调，23 个基因下调。为了得出这些结果，我们使用在线评估 Enrich Shiny GO、STRING 和 Cytoscape 来预测蛋白质的分子和功能连接，然后确定主要途径。功能富集分析显示，上调差异表达基因 (DEGs) 的生物学过程和分子功能关键词发生了显著改变，包括细胞基质粘附、端粒酶活性和端粒帽复合物。根据我们的初步研究，细胞特异性 TF 和 TF 介导的 GRNs 参与了 SSCs 的产生。为了最大限度地提高 ESC 体外分化为 SSCs 的诱导效率，我们确定了枢纽 SSC 特异性基因和重要的 SSC 特异性 TF，并提出了复杂的网络调控。根据我们的研究，这些基因及其相互作用的枢纽蛋白可能能够揭示不孕和异常生殖细胞的病理生理学。
