Warm temperature promotes gonadal transformation in ricefield eel.

(A) The distribution of 4 geographic populations of ricefield eels in China, showing the average annual temperature of each. (B) Bar graph showing the percentage of intersex animals in 2-year-old wild-caught ricefield eels from Hainan (Hainan province) and Wuhan area (Hubei Province). (C) Diagram showing the design of long term temperature experiments. Two temperatures were used: 25 °C (cool temperature), 33 °C (warm temperature). (D) Representative H&E staining images showing the gonad types of animals that were reared at cool and warm temperatures at the indicated time points. Bar: 200 µm. (E) Bar graph showing the percentage of intersex animals after 180 days of cool and warm temperature treatment. The experiments were repeated at least two times.

trpv4 is highly responding to environmental temperatures.

(A) Relative expression levels of trpv4 in 10 different tissues in adult ricefield eels. B: brain, H: heart, E: eye, K: kidney, S: spleen, L: liver, M:muscle, O: ovary, OT: ovotestis, T: testis. (B) ISH images showing trpv4 expression in ovaries, ovotestes, and testes. Black arrows pointing to trpv4 expressing cells. Bar: 200 µm. (C) Relative expression levels of the indicated trp and sex-biased genes of in vitro cultured ovaries at cool and warm temperatures. (D) qPCR results showing the expression patterns of trpv4 and male sex genes in repeated temperature shifting experiments of in vitro cultured ovaries. (E) qPCR results showing the expression of trpv4 at the indicated time points of in vitro cultured ovarian explants. (F) Confocal images showing the calcium signaling in cultured ovarian cells at the indicated conditions. Bar: 100 µm. (G) qPCR results showing the dynamic expression of the indicated genes in gonads of female ricefield eel at 25 □, and at the indicated time points after shifting to 34 □. day 1: day 1 after shifting to 34 □. (H) ISH images showing the dynamic expression of trpv4 at the indicated time points after shifting to 34 □. At 25 □, trpv4 was moderately expressed in primary growth oocytes. After shifting to 34 □, trpv4 signals became stronger in follicles of various stages of developing oocytes, and interstitial cell types. Bar: 200 µm. *: P< 0.05, **: P< 0.01, ***: P< 0.001, and ****: P< 0.0001. ns: not significant. All experiments were repeated at least three times.

Animal experiments show that warm temperature induced male gene expression depends on Trpv4.

(A) Cartoon showing the design of experiments. Female eels kept at cool (25 □) and warm (34 □) temperatures were injected with the Trpv4 agonist GSK1016790A and antagonist RN1734 into the ovaries, respectively. After 2-3 days of injection, the ovaries were isolated and processed for the subsequent experiments. (B) qPCR results showing the relative expression levels of the sex-biased genes at the indicated conditions, based on the animal experiments. (C) Representative WB images showing the expression of the indicated markers at the indicated conditions. GSK: GSK1016790A. (D) Representative IF images showing the expression of male/testicular differentiation genes at the indicated conditions. GSK: GSK1016790A. Bar: 200 µm. (E) quantification of panel D. *: P< 0.05, **: P< 0.01, ***: P< 0.001, and ****: P< 0.0001. ns: not significant. All experiments were repeated at least three times.

siRNA-mediated trpv4 knockdown abolishes the abnormal up-regulation of male genes by warm temperature treatment.

(A) qPCR results showing the relative expression of the indicated genes at the indicated conditions in animal experiments. (B) Representative WB images showing the expression of sex biased proteins at the indicated conditions in animal experiments. *: P< 0.05, **: P< 0.01, ***: P< 0.001, and ****: P< 0.0001. ns: not significant. All experiments were repeated at least three times.

The JAK/Stat3 signaling is downstream of Trpv4.

(A) Heat map showing the expression of the indicated genes of different groups. (B) IF images showing the expression of pStat3 at the indicated conditions in animal experiments. The white arrows indicated the location of pStat3 expressing cells. The experiments were repeated at least two times. Bar: 200 µm. (C) Quantification of calcium signals at the indicated conditions. Ovarian explants were cultured at the indicated conditions, and calcium signals were determined by calcium indicator dye Cal-520 acetoxymethyl ester. (D) Representative WB images showing the expression of the indicated makers in ovaries, early ovotestes, and middle ovotestes. (E) pStat3 levels after the addition of A23187 and BAPTA-AM in cultured ovarian cells at 25 □ and 33/34 □ conditions. All experiments were repeated two times.

Animal experiments showing that warm temperature induced male gene expression depends on pStat3.

(A) Cartoon showing the design of the experiments. Female eels kept at cool (25 □) and warm (34 □) temperatures were injected with the pStat3 agonist Colivelin and antagonist HO3867 into the ovaries, respectively. After 2-3 days of injection, the ovaries were isolated and processed for the subsequent experiments. (B) qPCR results showing the relative expression of the indicated genes at the indicated conditions. (C) IF images showing the expression of male biased genes at the indicated conditions. Bar: 200 µm. (D) Quantification of panel C. (E) Relative expression of the indicated genes at the indicated conditions. *: P< 0.05, **: P< 0.01, ***: P< 0.001, and ****: P< 0.0001. ns: not significant. All experiments were repeated at least three times.

pStat3 binds and activates the kdm6b gene.

(A) Cartoon showing the conserved pStat3 binding motifs upstream the TSS of the kdm6b gene. (B) ChIP experiments showing the enrichment of pStat3 at the kdm6b locus in ovarian tissues of fish reared at cool temperature (CT) and warm temperature (WT) in the absence and presence of HO-3867. (C) ISH images showing the expression of kdm6b in ovary and testis. Bar: 200 µm. (D) Cartoon showing the mechanism of Trpv4-mediated temperature induced sex change in ricefield eel.

(A) The graph showing the annual temperature dynamic by month for 30 years in Wuhan area, Hubei province, China. The highest and lowest temperatures per month were shown. (B) Schematic of the whole process of sex change, summarizing morphology, length and gonadal histology across time (10 years). The ricefield eel are born as females. After 2 year growth, they reach sexual maturity. After spawning (ranging from May to August each year), fish will enter 1-2 years of intersex stage before becoming functional males. The average lifespan of wild ricefield eel is around 10 years. (C) H&E staining images showing the typical cell types in an ovary of a 2-year-old female, before spawning. Bar: 200 µm.

(A) The qPCR results showing the expression patterns of the indicated female sex genes in repeated temperature shifting experiments of in vitro cultured ovaries. (B) Relative expression levels of the indicated temperature responding- and sex-biased genes in ovaries of females that reared at 25 □ and after 1 day exposure to 34 □. (C) ISH over-staining images showing the expression of trpv4 in ovaries of females that reared at 25 □ and 34 □ for 3 days. Black arrows indicating the trpv4 positive cells. *: P< 0.05, **: P< 0.01, ***: P< 0.001, and ****: P< 0.0001. ns: not significant. All experiments were repeated at least three times.

(A) Relative expression of the indicated male- and female-biased genes in ovarian explants cultured at 25 □ and 34 □ with increasing doses of small molecule RN1734. (B) qPCR results showing the relative expression levels of the sex-biased genes at the indicated conditions, based on in vitro cultured ovarian explants. (C) WB images showing the relative expression levels of the indicated proteins at the indicated conditions, based on in vitro cultured ovarian explants. (D) IF images showing the relative expression levels of Dmrt1 at the indicated conditions, based on in vitro cultured ovarian explants. *: P< 0.05, **: P< 0.01, ***: P< 0.001, and ****: P< 0.0001. ns: not significant. All experiments were repeated at least three times.

(A-B) IF images showing increased expression levels of pERK and Amh from ovaries to ovotestes. The experiments were repeated at least two times.

(A) qPCR results showing the relative expression of the indicated genes at the indicated conditions, based on in vitro cultured ovarian explants. (B) IF images showing the expression levels of pStat3 at the indicated conditions, based on in vitro cultured ovarian explants. (C) qPCR results showing the relative expression of the indicated genes at the indicated conditions, based on in vitro cultured ovarian explants. *: P< 0.05, **: P< 0.01, ***: P< 0.001, and ****: P< 0.0001. ns: not significant. All experiments were repeated at least three times.