Direct Full-Length RNA Sequencing Reveals an Important Role of Epigenetics During Sexual Reversal in Chinese Soft-Shelled Turtle

Abstract

Sex dimorphism is a key feature of Chinese soft-shelled turtle (Pelodiscus sinensis). The males (M) have higher econosmic value than females (F) due to wider calipash and faster growth. Exogenous hormones like estradiol and methyltestosterone can induce sexual reversal to form new phenotypes (pseudo-female, PF; pseudo-male, PM) without changing the genotype. The possibility of inducing sexual reversal is particularly important in aquaculture breeding, but the underlying biological mechanisms remain unclear. Here we applied a direct RNA sequencing method with ultralong reads using Oxford Nanopore Technologies to study the transcriptome complexity in P. sinensis. Nanopore sequencing of the four gender types (M, F, PF, and PM) showed that the distribution of read length and gene expression was more similar between same-sex phenotypes than same-sex genotypes. Compared to turtles with an M phenotype, alternative splicing was more pronounced in F turtles, especially at alternative 3′ splice sites, alternative 5′ splice sites, and alternative first exons. Furthermore, the two RNA methylation modifications m5C and m6A were differentially distributed across gender phenotypes, with the M type having more modification sites in coding sequence regions, but fewer modification sites in 3′UTR regions. Quantitative analysis of enriched m6A RNAs revealed that the N6-methylated levels of Odf2, Pacs2, and Ak1 were significantly higher in M phenotype individuals, while the N6-methylated levels of Ube2o were reduced after sexual reversal from both M and F phenotypes. Taken together, these findings reveal an important role of epigenetics during sexual reversal in Chinese soft-shelled turtles.

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

“…Breeding of all male turtles has always been a hot issue in Chinese soft-shelled turtles. The gonad tissues of four type of Chinese soft-shelled turtles (i.e., M, F, PM, and PF) can develop normally and produce gametes for reproduction, as previously studied [ 28 ]. Therefore, we analyzed the changes in protein levels among four types of Chinese soft-shelled turtles (ie., M, F, PM, and PF) using TMT-based quantitative proteomics to explore the molecular mechanism of sex reversal procedure in P. sinensis .…”

“…Mature female and male individuals were cultured in a sunny pond with a water depth of about 1 m. E2 and MT can induce sex reversal in female or male P. sinensis at the embryo stage before sex differentiation [ 28 ]. E2 and MT were diluted with ethanol to 10 mg/mL.…”

Role of Sox3 in Estradiol-Induced Sex Reversal in Pelodiscus sinensis

“…E2 powder was dissolved in anhydrous ethanol to achieve a final concentration of 10 mg/mL, and 5 µL was injected after corroding the egg shell with a small amount of hydrochloric acid. Previous studies showed that 5 µL of 10 mg/mL E2 was the most effective dose to induce sexual reversal in Chinese soft-shelled turtle [ 9 ]. The control group was injected with an equal volume of anhydrous ethanol.…”

“…Liang et al developed sex-specific markers to accurately identify the sex of P. sinensis using RAD-Seq. A previous study reported that when P. sinensis eggs were incubated at 30 °C, sexual differentiation began on day 16 (stage 13) and ended on day 22 (stage 20) [ 9 ]. Treatment with exogenous hormones such as estradiol (E2) and methyltestosterone (MT) during sexual differentiation induce sex reversal in P. sinensis [ 3 ].…”

Effect of Exogenous Hormone on R-Spondin 2 (Rspo2) and R-Spondin 3 (Rspo3) Gene Expression and Embryo Development in Chinese Soft-Shelled Turtle (Pelodiscus sinensis)

“…During the critical period of sex determination, estrogen can inhibit the expression of male sex-related genes amh and dmrt1, thus effectively blocking the development of the male phenotype and facilitating that of the female phenotype [11]. During gonadal development in animals, estradiol (E2) can induce sex reversal through the Wnt/β-catenin signaling pathway in female sex determination [12]. Exogenous hormones can also affect sex determination in P. sinensis [13].…”