Trpv4 links environmental temperature to testicular differentiation in hermaphroditic ricefield eel
- Institute of Hydrobiology, Chinese Academy of Sciences, China
- College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, China
- Hubei Hongshan Lab, China
- Huzhou Normal University, China
Figures
Figure 1 with 1 supplement
Warm temperature promotes gonadal transformation in ricefield eel.
(A) The distribution of four 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 the Wuhan area (Hubei Province). n=200 for each group. (C) Diagram showing the design of long-term temperature experiments. Two temperatures were used: 25℃ (cool temperature), 33℃ (warm temperature). n=400 for each group. (D) Representative hematoxylin and eosin (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. *p<0.05, ***p<0.001.
-
Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig1-data1-v1.zip
Figure 1—figure supplement 1
The phenotypic characteristics of ricefield eel.
(A) The graph showing the annual temperature dynamics by month for 30 years in the 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 is born as female. After 2 year growth, they reach sexual maturity. After spawning (ranging from May to August each year), females will enter 1–2 years of intersex stage before becoming functional males. The average lifespan of wild ricefield eel is around 10 years. (C) Hematoxylin and eosin (H&E) staining images showing the typical cell types in an ovary of a 2-year-old female, before spawning. Bar: 200 µm. (D) The average body length of fish that were raised for 90 and 180 days at the indicated temperatures. (E) The average body weight of fish that were raised for 90 and 180 days at the indicated temperatures. ns: not significant. The experiments were repeated two times.
-
Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig1-figsupp1-data1-v1.zip
Figure 2 with 1 supplement
trpv4 is highly responsive 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. n=3. (B) In situ hybridization (ISH) images showing trpv4 expression in ovaries, ovotestes, and testes. Black arrows pointing to trpv4-expressing cells. Bar: 200 µm. (C) The expression of the indicated trp and sex-biased genes in in vitro cultured ovaries at cool and warm temperatures. n=5 per group. (D) Quantitative real-time PCR (qPCR) results showing the expression patterns of trpv4 and male sex genes in repeated temperature shifting experiments in in vitro cultured ovaries. n=5 per group. (E) Confocal images showing the calcium signaling in cultured ovarian cells at the indicated conditions. After shifting to 34℃ for 0.5 hr, Cal-520 was added and calcium signal imaging was performed. The Trpv4 agonist GSK1016970A and antagonist RN1734 were administered in the culture medium, and 15 min later, calcium signal imaging was performed. Bar: 100 µm. (F) 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℃. n=5 per group. (G) ISH images showing the dynamic expression of trpv4 at the indicated time points before and after shifting to 34℃. At 25℃, trpv4 was moderately expressed in follicles of various stages of developing oocytes, and interstitial cell types. After shifting to 34℃, trpv4 signals became much stronger compared to 25℃. Bar: 200 µm. (H) Representative immunofluorescence (IF) images showing the co-localization of Trpv4- and Foxl2-expressing cells. Bar: 50 µm. n=10. *p<0.05, **p<0.01, ***p<0.001. ns: not significant. All experiments were repeated at least three times.
-
Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig2-data1-v1.zip
Figure 2—figure supplement 1
The effect of ovarian tissue heating of ricefield eel on the expression of sex genes.
(A) The quantitative real-time PCR (qPCR) results showing the expression patterns of the indicated female sex genes in repeated temperature shifting experiments of in vitro cultured ovaries. n=5 per group. (B) The qPCR results showing the expression of trpv4 at the indicated time points of in vitro cultured ovarian explants. n=5 per group. (C) The qPCR results showing the expression of the indicated temperature responding- and sex-biased genes in ovaries of females that were reared at 25℃ and after 1 day exposure to 34℃. n=5 per group. *p<0.05, **p<0.01, ***p<0.001. All experiments were repeated at least three times.
-
Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig2-figsupp1-data1-v1.zip
Figure 3 with 2 supplements
Warm temperature-induced male gene expression depends on Trpv4.
(A) Cartoon showing the design of animal 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. n=40. (B) Quantitative real-time PCR (qPCR) results showing the relative expression levels of the sex-biased genes at the indicated conditions, based on the animal experiments. n=5 per group. (C) Representative Western blot (WB) images showing the expression of the indicated markers at the indicated conditions. (D) Relative quantification of the indicated proteins in Panel C. WB was repeated three times. (E) Representative immunofluorescence (IF) images showing the expression of the indicated markers at the indicated conditions. Vimentin was used to show all cell types in ovaries. GSK: GSK1016790A. Bar: 200 µm. n=10, and 9/10 showed induced expression of Dmrt1/Sox9a. (F) Quantification of panel (E). *p<0.05, **p<0.01, ***p<0.001. ns: not significant. All experiments were repeated at least three times.
-
Figure 3—source data 1
PDF file containing original western blots for Figure 3C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-data1-v1.zip
-
Figure 3—source data 2
Original files for western blot analysis in Figure 3C.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-data2-v1.zip
-
Figure 3—source data 3
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-data3-v1.zip
Figure 3—figure supplement 1
In vitro cultured ovarian explant experiments show that warm temperature-induced male gene expression depends on Trpv4.
(A) Quantitative real-time PCR (qPCR) results showing the expression of the indicated male- and female-biased genes in ovarian explants cultured at 25℃ and 34℃ with increasing doses of small molecule RN1734. n=5 per group. (B) qPCR results showing the expression of the sex-biased genes at the indicated conditions, based on in vitro cultured ovarian explants. n=5 per group. (C) Western blot (WB) images showing the expression levels of the indicated proteins at the indicated conditions, based on in vitro cultured ovarian explants. (D) Quantification of panel C. (E) Immunofluorescence (IF) images showing the expression levels of Dmrt1 at the indicated conditions, based on in vitro cultured ovarian explants. n=5 per group. (F) Quantification of panel C. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. All experiments were repeated at least three times.
-
Figure 3—figure supplement 1—source data 1
PDF file containing original western blots for Figure 3—figure supplement 1C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-figsupp1-data1-v1.zip
-
Figure 3—figure supplement 1—source data 2
Original files for western blot analysis in Figure 3—figure supplement 1C.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-figsupp1-data2-v1.zip
-
Figure 3—figure supplement 1—source data 3
Source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-figsupp1-data3-v1.zip
Figure 3—figure supplement 2
Antibody specificity was detected by siRNA knockdown and Western blot (WB).
(A) Antibody specificity was detected by siRNA knockdown. (B) Quantitative real-time PCR (qPCR) assay for the knockdown effect of siRNA. n=3. (C) Quantification of the panel A. n=3. ***p<0.001.
-
Figure 3—figure supplement 2—source data 1
PDF file containing original western blots for Figure 3—figure supplement 2A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-figsupp2-data1-v1.zip
-
Figure 3—figure supplement 2—source data 2
Original files for western blot analysis in Figure 3—figure supplement 2A.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-figsupp2-data2-v1.zip
-
Figure 3—figure supplement 2—source data 3
Source data for Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig3-figsupp2-data3-v1.zip
Figure 4
siRNA-mediated trpv4 knockdown abolishes the abnormal up-regulation of male genes by warm temperature treatment.
(A) Quantitative real-time PCR (qPCR) results showing the relative expression of the indicated genes at the indicated conditions in animal experiments. (B) Representative Western blot (WB) images showing the expression of sex biased proteins at the indicated conditions in animal experiments. (C) Quantification of panel B. (D) qPCR results showing the relative expression of the indicated genes at the indicated conditions. RN1734 and DMSO were injected into gonads of male fish reared at 25/26℃. Female fish injected with DMSO were used as control. *p<0.05, **p<0.01, ***p<0.001. ns: not significant. All experiments were repeated at least three times.
-
Figure 4—source data 1
PDF file containing original western blots for Figure 4B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig4-data1-v1.zip
-
Figure 4—source data 2
Original files for western blot analysis in Figure 4B.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig4-data2-v1.zip
-
Figure 4—source data 3
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig4-data3-v1.zip
Figure 5 with 1 supplement
The JAK/Stat3 signaling is downstream of Trpv4.
(A) Heat map showing the expression of the indicated genes in different groups. (B) Immunofluorescence (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. n=12, and 10/12 showed increased expression of pStat3. (C) Quantification of panel B. (D) Bar graph showing the relative 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. (E) Representative Western blot (WB) images showing the expression of the indicated markers in ovaries, early ovotestes, and middle ovotestes. n=5 per group. (F) Quantification of panel E for relative expression of Amh and pStat3. (G) pStat3 levels after the addition of A23187 and BAPTA-AM in cultured ovarian cells at 25℃ and 34℃ conditions. (H) Quantification of panel G. All experiments were repeated at least two times. **p<0.01, ***p<0.001.
-
Figure 5—source data 1
PDF file containing original western blots for Figure 5E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig5-data1-v1.zip
-
Figure 5—source data 2
Original files for western blot analysis in Figure 5E.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig5-data2-v1.zip
-
Figure 5—source data 3
PDF file containing original western blots for Figure 5G, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig5-data3-v1.zip
-
Figure 5—source data 4
Original files for western blot analysis in Figure 5G.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig5-data4-v1.zip
-
Figure 5—source data 5
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig5-data5-v1.zip
Figure 5—figure supplement 1
Expression levels of pERK and Amh are increased from ovaries to ovotestes.
(A–B) The representative immunofluorescence (IF) images showing the expression of pERK (A) and Amh (B) from ovaries, ovotestes, and testes. n=6 per group. Bar: 200 μm. The experiments were repeated at least two times.
Figure 6 with 1 supplement
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 HO-3867 into the ovaries, respectively. After 2–3 days of injection, the ovaries were isolated and processed for the subsequent experiments. n=50. (B) Quantitative real-time PCR (qPCR) results showing the relative expression of the indicated genes at the indicated conditions. n=5 per group. (C) Immunofluorescence (IF) images showing the expression of male-biased genes at the indicated conditions. Bar: 200 µm. n=10, and 8/10 showed increased expression of pStat3/Dmrt1. (D) Quantification of panel C. n=5 per group. (E) qPCR results showing the relative expression of the indicated genes at the indicated conditions. n=5 per group. (F) qPCR results showing the relative expression of the indicated genes at the indicated conditions. n=5 per group. *p<0.05, **p<0.01, ***p<0.001. The qPCR experiments were repeated at least three times.
-
Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig6-data1-v1.zip
Figure 6—figure supplement 1
In vitro cultured ovarian explant experiments showing that warm temperature-induced male gene expression depends on pStat3.
(A) Quantitative real-time PCR (qPCR) results showing the expression of the indicated genes at the indicated conditions, based on in vitro cultured ovarian explants. (B) Representative immunofluorescence (IF) images showing the expression of pStat3 at the indicated conditions, based on in vitro cultured ovarian explants. n=6 per group. (C) qPCR results showing the 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. All experiments were repeated at least three times.
-
Figure 6—figure supplement 1—source data 1
Source data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig6-figsupp1-data1-v1.zip
Figure 7
pStat3 binds and activates the kdm6b gene.
(A) Cartoon showing the conserved pStat3 binding motifs upstream the TSS of the kdm6b gene. (B) Chromatin immunoprecipitation (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) conditions, in the absence and presence of HO-3867. (C) The dual fluorescence reporter gene assay revealed that pStat3 dose-dependently activated the transcription of kdm6b. Different letters indicate significant differences. n=3. (D) Luciferase assay for kdm6b-luc and kdm6bM-luc activities in 293T cells, in the absence and presence of pStat3 agonist Colivelin. (E) The representative in situ hybridization (ISH) images showing the expression of kdm6b in ovary and testis. Bar: 200 µm. n=3 per group. (F) Cartoon showing how Trpv4 may link environmental temperature to the sex determination cascades via the downstream signaling pathways in ricefield eel. ***p<0.001. ChIP and Luciferase experiments were repeated two times.
-
Figure 7—source data 1
Source data for Figure 7.
- https://cdn.elifesciences.org/articles/108272/elife-108272-fig7-data1-v1.zip
Author response image 1
Author response image 2
Author response image 3
Additional files
-
Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/108272/elife-108272-supp1-v1.xlsx
-
Supplementary file 2
siRNA sequences used in vivo experiments.
- https://cdn.elifesciences.org/articles/108272/elife-108272-supp2-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108272/elife-108272-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Trpv4 links environmental temperature to testicular differentiation in hermaphroditic ricefield eel
eLife 14:RP108272.
https://doi.org/10.7554/eLife.108272.4
