BLOC1S1 promotes proliferation of goat spermatogonial stem cells / 生物工程学报

ABSTRACT

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.