Tandem Mass Tag-Based Quantitative Proteomics Analysis of Gonads Reveals New Insight into Sexual Reversal Mechanism in Chinese Soft-Shelled Turtles

SIMPLE SUMMARY: The Chinese soft-shelled turtle has obvious sex dimorphism, which is an important aquatic economic species in China. Exogenous hormones can cause the sexual reversal of P. sinensis, but the molecular mechanism remains unclear. Here, TMT-based quantitative proteomics analysis of four types of P. sinensis (i.e., female (F), male (M), pseudo-female (PF), pseudo-male (PM)) gonads were performed. We found that there were common pathways, such as focal adhesion, endocytosis, apoptosis, ribosome, and spliceosome, which both played crucial roles in two comparison groups, including F vs. PM and M vs. PF. Furthermore, these differentially expressed proteins were associated with various biological processes, such as embryonic development and catabolic process, which were closely related to the sexual reversal of P. sinensis. ABSTRACT: Chinese soft-shelled turtles display obvious sex dimorphism. The exogenous application of hormones (estradiol and methyltestosterone) can change the direction of gonadal differentiation of P. sinensis to produce sex reversed individuals. However, the molecular mechanism remains unclear. In this study, TMT-based quantitative proteomics analysis of four types of P. sinensis (female, male, pseudo-female, and pseudo-male) gonads were compared. Quantitative analysis of 6107 labeled proteins in the four types of P. sinensis gonads was performed. We identified 440 downregulated and 423 upregulated proteins between pseudo-females and males, as well as 394 downregulated and 959 upregulated proteins between pseudo-males and females. In the two comparisons, the differentially expressed proteins, including K7FKG1, K7GIQ2, COL4A6, K7F2U2, and K7FF80, were enriched in some important pathways, such as focal adhesion, endocytosis, apoptosis, extracellular matrix-receptor interaction, and the regulation of actin cytoskeleton, which were upregulated in pseudo-female vs. male and downregulated in pseudo-male vs. female. In pathways such as ribosome and spliceosome, the levels of RPL28, SRSF3, SNRNP40, and HNRNPK were increased from male to pseudo-female, while they decreased from female to pseudo-male. All differentially expressed proteins after sexual reversal were divided into six clusters, according to their altered levels in the four types of P. sinensis, and associated with cellular processes, such as embryonic development and catabolic process, that were closely related to sexual reversal. These data will provide clues for the sexual reversal mechanism in P. sinensis.