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.
Among Chinese soft-shelled turtles, Pelodiscus sinensis, males have a richer nutritional value and higher market price than females. All-male offspring were obtained by 17β-estradiol (E2). However, the molecular mechanisms of E2 inducing sexual reversal remain unclear. In this study, we cloned estrogen nuclear receptors (ERs) from P. sinensis and investigated their expression profiles. We examined the responses of ERα and ERβ after treatment with different concentrations of 1.0, 5.0, and 10 mg/mL E2. ERs showed abundant expressions in the adult gonad, ERα for ovary, and ERβ for testis. E2 can up-regulate the expression of ERα, which showed a remarkable increase while the promotion of ERβ was unobvious. They reached a high level at stage 17 after the treatment of E2, genes of the female-related genes Rspo1, Wnt4, β-catenin, Foxl2, Cyp19a1, and Sox3 exhibited a significant raise at stage 17 with the increase in the concentration of E2 while the male-related genes Sox9, Dmrt1, and Amh were significantly inhibited. Our study cloned the full length of ERs and analyzed their structures and expressions, laying a foundation for the further study of the effect of estrogen on sex determination.
The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important freshwater aquaculture animal in China. The Wnt gene family plays important regulatory roles in the development and growth of mammals. However, the precise function of these family genes has not been well understood in the sex differentiation of Chinese soft-shelled turtles. Here, we cloned a member of the Wnt family, Wnt2, which obtained a 1077 bp open reading frame that encoded a 358-aa protein. The putative amino acid sequences of proteins are exceeded 80% identical to other turtles. The expression level of Wnt2 peaked at the 14th stage both in female and male embryos during the early gonadal differentiation period of Chinese soft-shelled turtles, which occurred before gonadal differentiation. Wnt2 mRNA was expressed at higher levels in the brains and gonads of mature P. sinensis females compared with those in mature males. Wnt agonists significantly affected the expression level of Wnt2 during the gonadal differentiation period. After Wnt agonists (1.0 μg/μL, 2.5 μg/μL, 5.0 μg/μL) treatment, the expression level of the Wnt2 generally appeared to have an inverted-V trend over time in female embryonic gonads. The results suggested that Wnt2 may participate in the regulation of gonad development in P. sinensis during the early embryonic stages. These results could provide a theoretical basis for the reproduction process of the Chinese soft-shelled turtle.
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