| Summary: | Purpose: The purpose of this study was to screen the genes and pathways that are involved in spermatogonia stem cell (SSC) differentiation regulation during the transition from A<sub>undiff</sub> to A<sub>1.</sub> Methods: RNA sequencing was performed to screen differentially expressed genes at 1 d and 2 d after SSC differentiation culture. KEGG pathway enrichment and GO function analysis were performed to reveal the genes and pathways related to the initiation of early SSC differentiation. Results: The GO analysis showed that <i>Rpl21</i>, which regulates cell differentiation initiation, significantly increased after 1 day of SSC differentiation. The expressions of <i>Fn1</i>, <i>Cd9</i>, <i>Fgf2</i>, <i>Itgb1</i>, <i>Epha2</i>, <i>Ctgf</i>, <i>Cttn</i>, <i>Timp2</i> and <i>Fgfr1</i>, which are related to promoting differentiation, were up-regulated after 2 days of SSC differentiation. The analysis of the KEGG pathway revealed that RNA transport is the most enriched pathway 1 day after SSC differentiation. Hspa2, which promotes the differentiation of male reproductive cells, and Cdkn2a, which participates in the cell cycle, were significantly up-regulated. The p53 pathway and MAPK pathway were the most enriched pathways 2 days after SSC differentiation. <i>Cdkn1a</i>, <i>Hmga2</i>, <i>Thbs1</i> and <i>Cdkn2a</i>, microRNAs that promote cell differentiation, were also significantly up-regulated. Conclusions: RNA transport, the MAPK pathway and the p53 pathway may play vital roles in early SSC differentiation, and <i>Rpl21</i>, <i>Fn1</i>, <i>Cd9</i>, <i>Fgf2</i>, <i>Itgb1</i>, <i>Epha2</i>, <i>Ctgf</i>, <i>Cttn</i>, <i>Timp2</i>, <i>Fgfr1</i>, <i>Hspa2</i>, <i>Cdkn2a</i>, <i>Cdkn1a</i>, <i>Hmga2</i> and <i>Thbs1</i> are involved in the initiation of SSC differentiation. The findings of this study provide a reference for further revelations of the regulatory mechanism of SSC differentiation.
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