Proteomic analysis of male and female chinese soft-shelled turtle (Pelodiscus sinensis) during key gonadal differentiation stages

The Chinese soft-shelled turtle is an important aquatic economic animal. The economic value of male individuals is significantly higher than that of female individuals due to their advantage in rapid growth. However, the molecular mechanism that influences the process of sex differentiation between male and female Chinese turtles remains unclear. Therefore, we conducted proteomic analysis on male and female Chinese soft-shelled turtles ( Pelodiscus sinensis ) during key gonadal differentiation stages (13, 15, 17, 19, and 21 days post-fertilization) to explore the molecular mechanisms underlying sex differentiation. A total of 6189 proteins in males and 6224 proteins in females were identified, including 745 male-specific and 780 female-specific proteins. Fuzzy C-Means clustering revealed dynamic expression patterns of proteins during gonadal differentiation stages: in males, Cluster 3 (557 proteins) showed a sustained downward trend, while Cluster 9 (493 proteins) exhibited a sustained upward trend; in females, Cluster 1 (540 proteins) and Cluster 9 (402 proteins) displayed similar opposing trends. The KEGG pathway enrichment analysis indicated that male-specific proteins were mainly involved in pathways such as Spliceosome and Apoptosis, while female-specific proteins were enriched in Ubiquitin-mediated proteolysis, TGF-beta signaling, and other pathways. The expression levels of key ubiquitination-related genes such as CUL3, CUL2, UBA6 and sex-related genes such as SOX9, WNT4 were significantly increased with development in females but decreased in males. Protein-protein interaction (PPI) network analysis further revealed complex interactions among these key proteins. These findings highlight that ubiquitination pathways and conserved signaling pathways such as TGF-beta play critical roles in regulating gonadal differentiation in P.sinensis , providing new insights into the molecular mechanisms of sex determination in genotypic sex determination (GSD) reptiles and a theoretical basis for all-male breeding in aquaculture. • 6189 and 6224 proteins identified in males and females, including 745 male-specific and 780 female-specific proteins. • Male-specific proteins enriched in Spliceosome and Apoptosis; female-specific proteins enriched in Ubiquitin-mediated proteolysis and TGF-beta signaling. • Key genes (e.g., CUL3 , CUL2 , UBA6 , SOX9 , WNT4 , etc.) increase during female development but decrease in males.