Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40-50% of these cases. The cause of male infertility is still poorly diagnosed and treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the ESX1 gene. The aim of this study was to activate the ESX1 gene using CRISPRa technology in human germ cells (testicular seminoma cells-TCam-2). Successful activation of the ESX1 gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the ESX1 gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (p < 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the ESX1 gene was determined. Finally, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the ESX1 gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted.
Scientific reports. 2023 Oct 02*** epublish ***
Agnieszka Malcher, Zuzanna Graczyk, Hermann Bauer, Tomasz Stokowy, Andrea Berman, MikoĊaj Smolibowski, Dominika Blaszczyk, Piotr Jedrzejczak, Alexander N Yatsenko, Maciej Kurpisz
Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland. ., Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland., Department of Developmental Genetics, Max Planck Institute for Molecular Genetics, Berlin, Germany., IT Division, University of Bergen, Bergen, Norway., Department of Biological Sciences, University of Pittsburgh, Pittsburgh, USA., Department of Cell Biology, Center of Obstetrics, Gynecology and Infertility Treatment, University of Medical Sciences, Poznan, Poland., Department of OB/GYN and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA., Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland. .