1. Developmental Biology

Trpv4 links environmental temperature to testicular differentiation in hermaphroditic ricefield eel

  1. Zhi Yang
  2. Tingting Luo
  3. Yimin Zhang
  4. Yuhua Sun  Is a corresponding author
  1. Institute of Hydrobiology, Chinese Academy of Sciences, China
  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, China
  3. Hubei Hongshan Lab, China
  4. Huzhou Normal University, China
https://doi.org/10.7554/eLife.108272.4
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  1. Zhi Yang
  2. Tingting Luo
  3. Yimin Zhang
  4. Yuhua Sun
(2026)
Trpv4 links environmental temperature to testicular differentiation in hermaphroditic ricefield eel
eLife 14:RP108272.
https://doi.org/10.7554/eLife.108272.4
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eLife Assessment

This study presents useful findings on the molecular mechanisms driving female-to-male sex reversal in the ricefield eel (Monopterus albus) during aging, which would be of interest to biologists studying sex determination. The manuscript describes an interesting mechanism potentially underlying sex differentiation in M. albus. However, the current data are incomplete and would benefit from more rigorous experimental approaches for Western blotting.

https://doi.org/10.7554/eLife.108272.4.sa0

Abstract

The ricefield eel (Monopterus albus), an economically important aquaculture species in China, is a freshwater teleost fish that exhibits protogynous hermaphroditism. Although progress has been made in understanding the sex determination and differentiation of this species, the underlying mechanisms remain unclear. Here, we show that warm temperature promotes gonadal transformation by up-regulating testicular differentiation genes, such as dmrt1/sox9a in ovaries. Trpv4, a Ca2+-permeable cation channel expressed in gonadal somatic cells, is highly sensitive to ambient temperature and links environmental temperature to testicular differentiation in ricefield eel. In female fish reared at cool temperature, injection of Trpv4 agonist into the ovaries leads to a significant upregulation of male pathway genes and in female fish exposed to warm temperature, Trpv4 inhibition or trpv4 siRNA knockdown suppresses warm temperature-induced male gene expression. pStat3 signaling is downstream of Trpv4 and transduces Trpv4-controlled calcium signaling into the sex determination cascades. Inhibition of pStat3 activity prevents the up-regulation of testicular differentiation genes by warm temperature treatment and ovarian injection of Trpv4 agonist, whereas activation of pStat3 is sufficient to induce the expression of male genes in the presence of Trpv4 antagonist. pStat3 binds and activates jmjd3/kdm6b, an activator of the dmrt1 gene. Consistently, ovarian injection of Kdm6b inhibitor blocks the up-regulation of testicular differentiation genes by warm temperature exposure. We propose that environmental factors, such as temperature, promote gonadal transformation of ricefield eel by inducing the expression of male pathway genes in ovaries via the Trpv4-pStat3-Kdm6b-dmrt1 axis. Our results provide new insights into the molecular mechanism underlying natural sex change of ricefield eel, which will be useful for sex control in aquaculture.

Introduction

Sex determination in animals is intriguing and fascinating. In mammals, sex is determined genetically (genotypic sex determination, GSD). In lower vertebrates, such as fish and reptiles, however, sex regulators are diverse. Their sex can be influenced by various environmental factors, including temperature, pH, breeding density and social status (Gutzke and Crews, 1988; Honeycutt et al., 2019; Mei and Gui, 2015; Todd et al., 2019), the so-called environmental sex determination (ESD). The temperature-dependent sex determination (TSD) is one of the best studied forms of ESD. In red-eared slider turtle (Trachemys scripta), American alligator (Alligator mississippiensis), and Atlantic silverside (Menidia menidia), sex is determined solely by the temperature during the thermosensitive period of embryogenesis. In Australian central bearded dragon (Pogona vitticeps) and Nile tilapia (Oreochromis niloticus), which display GSD, temperature can override the genetic materials to control the gonadal sex differentiation (Holleley et al., 2015; Deveson et al., 2017; Xiong et al., 2020). Analysis of expression data during embryogenesis of normal ZW females and temperature sex reversed ZZ females has provided important insights into temperature-driven sex determination in the bearded dragon (Whiteley et al., 2021). Irrespective of TSD or GSD + TE (temperature effects), the downstream components are fairly conserved, including the epigenetic factors, such as jmjd3/kdm6b and sex determination genes, such as dmrt1 (Castelli et al., 2020; Ge et al., 2018; Weber et al., 2020; Whiteley et al., 2022; Martínez-Pacheco et al., 2024; Wu et al., 2024; Lu et al., 2025).

Most vertebrates, including the TSD reptiles, exhibit gonochorism. However, approximately 6% of fish species exhibit hermaphroditism, including protandrous, protogynous, and bidirectional hermaphroditism (Todd et al., 2019). The majority of them are marine fish, appearing in 27 families (Shao et al., 2014; Todd et al., 2019). Compared to marine fish, natural sex change in freshwater fish is very rare. The ricefield eel (Monopterus albus), also called Asian swamp eel, was first discovered as a protogynous hermaphroditic fish by Liu, 1944; Zhou and Gui, 2016; Luo et al., 2026. The species begins life as a female and then develops into a male through an intersex stage, thus displaying a female-to-male sex reversal during aging. Females are small in size (<25 cm), and during and after sex change, there is a gradual increase in body size (>55 cm for the majority of males). Among the described teleost fish species, ricefield eel has the fewest chromosome pairs (n=12) with the fewest number of chromosome arms (Cheng and Zhou, 2022), and is emerging as an important model animal for studying sex determination and differentiation as well as adaptive evolution (Ji et al., 2001). As early as the Ming Dynasty in ancient China in 1578, pharmacist Shi-Zhen Li has described the medicinal value of ricefield eel in the treatment of human diseases in his famous pharmacy monograph, the Bencao Gangmu, also called ‘Compendium of Materia Medica’ (Cheng and Zhou, 2022). Nowadays, ricefield eel has been developed as one of the most important economic fish in freshwater aquaculture in China, with annual production exceeding 350,000 tons (Song et al., 2022). Unfortunately, the wild population has declined rapidly in the wild due to degradation of the natural environment and human activity, such as overfishing. The reproductive mode of ricefield eel, which leads to many more females than males in spawning season, severely affects the sex ratio, and decreases the productivity of broodstock. Moreover, adult females lay limited eggs (~200) due to its small size, which is a limiting factor for massive production of seedlings for the aquaculture industry. Thus, the elucidation of the mechanisms underlying the sex determination/differentiation is important, which will aid in developing strategies/techniques for sex control that would break the bottleneck in the aquaculture industry (Wu et al., 2024).

The life history of ricefield eel implies that environmental factors initiate and promote the gonadal transformation via epigenetic mechanisms. Consistently, histone demethyltransferase/methyltransferase genes, such as kdm6b/kmt2 and DNA methylation enzyme genes, such as dnmt1/3, were dynamically expressed throughout the sex change process, and the expression levels of the master sex determination/differentiation genes are closely correlated to the levels of DNA and histone methylation, which can be impacted by environmental exposure (Wang et al., 2020; Fan et al., 2021; Jiang et al., 2022; Fan et al., 2022; Hu et al., 2022; Jiang et al., 2022). However, these epigenetic regulators are not inherently responsive to the environmental cues, implying that certain molecular sensors exist and serve as the link between environmental stimuli and the sex determination pathway. We have recently proposed that there is a temperature-induced sex reversal (TISR) mechanism in ricefield eel (Zhang et al., 2025), similar to that of embryonic bearded dragon (P. vitticeps) (Whiteley et al., 2018). Importantly, isolated ovarian explants are responsive to temperature stimuli, suggesting that the perception of temperature is executed by certain sensors expressed in ovarian cells. While preliminary data have suggested that the Ca2+-permeable, non-selective cation channel Trpv4 (Transient Receptor Potential Vanilloid 4 channel) might be a potential thermosensor, how Trpv4-regulated signals are transduced into the sex determination cascades remains unclear (Zhang et al., 2025).

In this work, we hypothesized that Trpv4 may bridge environmental temperature and the sex differentiation pathway in ricefield eel. By using small molecule agonist and antagonist of Trpv4 as well as siRNA-mediated knockdown of trpv4, we provided solid evidence that temperature-evoked Trpv4 activity promotes the sex change of ricefield eel via the downstream Ca2+-pStat3-Kdm6b-Dmrt1 axis.

Results

Warm temperature promotes gonadal transformation

Natural populations of ricefield eel are mainly distributed across East and Southeast Asia. Previous studies have reported that at the onset of sex change, wild fish from different geographic populations and habitats vary in age, body weight, and length. For instance, it is around 16 cm long (18-month-old) in Bandung area of Indonesia (Liem, 1963), and ~20 cm in Hainan and Guangzhou areas of southern China (Chan and Phillips, 1967; Wang and Zeng, 2006), ~30 cm (2-year-old) in Wuhan area of central China (Wang et al., 2008), and 35–40 cm (3-year-old) in Tianjin area of northern China (Liu and Wang, 1987; Fan et al., 2022; Fan et al., 2021). The average annual temperature in Hainan, Guangzhou, Wuhan, and Tianjin areas is approximately 25, 22, 17, and 13℃, respectively (Figure 1A). This observation implies that higher temperature facilitates the sex change of ricefield eel. To directly investigate this, during June-July, 2024, we have obtained ~200 2-year-old wild ricefield eels from the southernmost Hainan and central Wuhan, and examined their gonads. We found that less than 5% of the fish from Wuhan area were intersex animals, whereas approximately 24% of the fish from the Hainan area were in the intersex stage (Figure 1B).

Figure 1 with 1 supplement see all
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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

In the Wuhan area (Hubei province, China), the reproductive season of wild ricefield eel usually runs from May to July, when the average monthly temperature is 25–33℃ (Figure 1—figure supplement 1A). Immediately after spawning, the females (2-year-old) may undergo extensive ovarian tissue degeneration and physiological change (Liu and Gu, 1950), which leads to an irreversible commitment to becoming male via an intersex stage (Figure 1—figure supplement 1B–C). This observation again supported that the onset of sex change of ricefield eels is closely related to the external environment, in particular warm temperature.

The above observations prompted us to hypothesize that warm temperature plays an important role in initiating and driving the sex change of ricefield eel. To directly test this, long term temperature experiments were performed using females from the Wuhan Area (Figure 1C). One-and-a-half-year-old females (about 50 g) were randomly divided into two groups and reared at 25/26℃ (cool temperatures, CT) and 33/34℃ (warm temperatures, WT), for a period of 6 months. At days 30, 90, and 180, the gonadal sex of randomly selected fish from different groups was determined by H&E staining and expression analysis of sex-biased genes (Figure 1D). The average body length and weight of ricefield eels were comparable between the WT group and the CT group (Figure 1—figure supplement 1D–E). In CT group at day 90, ~90% of gonads were ovaries, and ~10% were ovotestes. In WT group, however, ~65% gonads were ovaries, and ~35% were ovotestes (Figure 1E). In CT group at day 180, ~80% of gonads were ovaries, and ~20% were ovotestes. In WT group, however, ~25% of gonads were ovaries, and ~75% were ovotestes (Figure 1E). We concluded that warm temperature promotes gonadal transformation of ricefield eel.

Trpv4 is highly responsive to environmental temperature

We went on to investigate how the gonadal tissues are responding to temperature cues. Previous work has suggested that Trpv4 associates with environmental temperature and sex determination in TSD alligator and ricefield eel (Yatsu et al., 2015; Huang et al., 2024; Zhang et al., 2025). We hypothesized that ricefield eel Trpv4 is expressed in ovary and functions as a thermosensor that perceives the environmental temperature cues. The results of qPCR experiments showed that trpv4 was more highly expressed in gonadal tissues than in non-gonadal tissues, exhibiting the highest expression in testis (Figure 2A). RNA in situ hybridization (ISH) experiments confirmed that trpv4 levels increased from ovary to testis (Figure 2B), implying that it was functionally associated with testicular development during aging.

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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

In cultured primary ovarian explants, trpv4 was one of the most up-regulated trp genes induced by warm temperature exposure (Figure 2C). Temperature-shifting experiments showed that trpv4 was highly sensitive to temperature cues (Figure 2D), displaying a pattern similar to male sex genes, such as dmrt1/sox9a, but inverse to that of female sex genes, such as cyp19a1a/foxl2 (Figure 2—figure supplement 1A). trpv4 was elevated as early as 4 hr post warm temperature treatment (Figure 2—figure supplement 1B), suggesting that it was an early temperature-response gene. The trpv4 gene encodes a constitutively active Ca2+-permeable cation channel that can be activated by warm temperature (Güler et al., 2002). Consistently, when Ca2+ indicator Cal-520 was added to the cells, a marked increase in the calcium signal was observed at 34℃ in comparison to 26℃ (Figure 2E), which suggested that Ca2+ signaling mediates the temperature-controlled Trpv4 activity, similar to the embryonic dragon (Whiteley et al., 2021). This was further supported by our RNA-seq data that trpv4 and many genes involved in Ca2+ transport and sequestration were upregulated in ovotestes compared to ovaries (Zhang et al., 2025).

While trpv4 is highly responding to temperature changes in cultured ovarian cells, it is not known whether this was the case in vivo. To explore this, female fish were transiently reared at cool temperature (25℃) for 1 day, and then shifted to warm temperature (34℃) for 10 days. trpv4 mRNA in ovaries was already elevated on day 1 after shifting to 34℃ (Figure 2—figure supplement 1C), and its levels progressively increased over time, exhibiting a pattern similar to that of testicular differentiation genes (Figure 2F). To understand in more detail the role of trpv4, we studied its expression pattern in ovaries by performing an ISH experiment. The ISH results showed that trpv4 was moderately expressed in ovarian somatic and interstitial cells around the oocytes at cool temperature (Figure 2G). Warm temperature exposure led to an increase of trpv4 gene expression in ovarian follicles of different developmental stages, and its levels were positively correlated with the duration of temperature exposure.

To explore the identity of the Trpv4-expressing follicle cells, double immunofluorescence (IF) experiments were performed using antibodies against Trpv4 and Foxl2, a granulosa cell marker. The IF results showed that Trpv4 protein was expressed in a portion of Foxl2-positive granulosa cells (Figure 2H). The observation suggested that these trpv4-expressing follicle cells in the ovary may play an important role in gonadal transformation in response to warm temperature.

Temperature-induced male gene expression depends on Trpv4

The co-upregulation of trpv4 and male pathway genes prompted us to ask whether the thermosensor Trpv4 in ovaries functions to transduce the temperature cues into the sex determination cascades. To investigate this, animal experiments were performed by injecting into ovaries with small molecules RN1734 and GSK1016790A, known Trpv4 specific antagonist and agonist, respectively (Liu et al., 2021). The temperature shifting experiments were performed, and females were divided into four groups: 25℃+DMSO; 25℃+GSK1016790 A; 34℃+DMSO; 34℃+RN1734 (Figure 3A). Three to ten days post-injection, the gonads were isolated and used for the subsequent analyses.

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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

Compared to the cool temperature group, warm temperature exposure increased the expression of testicular differentiation genes, such as dmrt1 and gsdf, accompanied by moderately decreased expression of ovarian differentiation genes, such as cyp19a1a and foxl2 (Figure 3B). Injection of RN1734 into ovaries blocked the up-regulation of testicular differentiation genes by warm temperature treatment. Injection of GSK1016790A in fish reared at 25℃ was sufficient to induce the expression of testicular differentiation genes to an extent similar to warm temperature treatment, accompanied by decreased expression of ovarian differentiation genes. The effects of small molecules on gene expression were dose-dependent (Figure 3—figure supplement 1A). The effects of RN1734 or GSK1016790A treatment on above sex determination factors were confirmed at the protein levels, as revealed by Western blot (WB) and IF experiments (Figure 3C–F). The experiments were also repeated using cultured ovarian explants and/or cells, and similar results were observed (Figure 3—figure supplement 1B–F). The specificity of the key antibodies was effectively verified through siRNA-mediated knockdown (Figure 3—figure supplement 2). Activation and inhibition of Trpv4 ion channel function by the small molecules was correlated with increased and decreased calcium signaling, respectively (Figure 2E), suggesting that calcium signaling mediated Trpv4 in the control of sex-biased gene expression.

The above data suggested that Trpv4 is sufficient and necessary to induce the expression of testicular differentiation genes in ovaries, via its ion channel function. To further demonstrate this, trpv4 siRNA was injected into the ovaries. trpv4 expression was markedly down-regulated by 0.1 µmol siRNA injection (Figure 4A). Importantly, siRNA injection led to a marked decrease in expression of male pathway genes that were up-regulated by warm temperature exposure, and a slight increase in expression of ovarian differentiation genes that were repressed by warm temperature treatment. Similar results were observed at the protein levels as revealed by WB experiments (Figure 4B–C). We concluded that warm temperature-induced male gene expression is dependent on Trpv4 activity.

Figure 4
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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

Next, we evaluated the effect of Trpv4 inhibition in testes of male adults. To this end, RN1734 was injected into gonads of male fish, and qPCR was performed to assess the expression of female and male pathway genes. The results showed that Trpv4 inhibition by RN1734 treatment led to an increase in the expression of female pathway genes in testes, at the expense of male pathway genes (Figure 4D). The results suggested that Trpv4 activity is persistently required for the expression of male pathway genes in testis.

pStat3 signaling is downstream of Trpv4

We next asked how Trpv4-controlled Ca2+ flux was interpreted and transduced into the sex determination cascades. In TSD turtles and TISR central bearded dragon, Stat3/4 are activated through phosphorylation by temperature-evoked calcium signaling, and is directly involved in sex determination (Weber et al., 2020; Whiteley et al., 2021). Based on our RNA-seq data, we found that stat3, and the Jak/Stat3 pathway target genes, such as socs3 and egr1/2, were significantly upregulated in early ovotestes compared to ovaries (Figure 5A). We, therefore, hypothesized that pStat3 signaling is downstream of Trpv4-controlled calcium influx to promote the expression of male pathway genes in ovaries. In fact, several lines of evidence were in favor of this hypothesis. First, activation of Trpv4 ion channel by GSK1016790A at cool temperature led to elevated pStat3 levels and calcium signals in ovarian explants, similar to that by higher temperature treatment, and inhibition of Trpv4 by RN1734 at warm temperature decreased pStat3 levels and calcium signals (Figure 5B–D). Second, pStat3 signals were detected in the gonadal somatic cells around the oocytes and interstitial cells, analogous to that of trpv4 (Figure 5B; Figure 2G). Third, the levels of pStat3 were gradually increased from ovaries to testes in wild-caught ricefield eels, along with the male sex promoting factors, such as Amh (Figure 5E–F; Figure 5—figure supplement 1). Fourth, WB blot analysis of A23187- or BAPTA-AM-treated ovarian cells showed that exposure to the ionophore A23187 increased phosphorylation levels of Stat3 at 25℃, whereas chelating of calcium with BAPTA-AM led to diminished activation of Stat3 (Figure 5G–H). Collectively, these observations strongly suggested that Trpv4 cell autonomously controls phosphorylation of Stat3 via the regulation of calcium signal in gonadal somatic cells.

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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

To explore the function of the pStat3 signaling, animal experiments were performed by injecting into ovaries with small molecules HO-3867 or Colivelin. HO-3867, a curcumin analogue, is a selective pStat3 inhibitor, which blocks pStat3 activity by directly binding to Stat3 DNA-binding domain, and Colivelin is a potent synthetic peptide activator of pStat3, which increases pStat3 levels by acting through the GP130/IL6ST complex (Wu et al., 2024). The injected females were divided into four groups based on the temperatures and the small molecules injected: 25℃+DMSO; 25℃+Colivelin; 34℃+DMSO; 34℃+HO-3867 (Figure 6A). Three to ten days post injection (dpj), the ovaries were isolated and subjected to the subsequent analyses. Injection of HO-3867 blocked the up-regulation of male pathway genes by warm temperature exposure, whereas injection of Colivelin at 25℃ was sufficient to induce the expression of these genes to an extent similar to warm temperature treatment (Figure 6B). Similar results were observed at the protein levels as revealed by IF experiments (Figure 6C–D). We also repeated the experiments using ovarian explants and/or cells, which produced similar results (Figure 6—figure supplement 1A–B).

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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

To functionally demonstrate that pStat3 signaling is downstream of Trpv4, rescue experiments were performed by injecting into ovaries with individual and combined small molecules as described below. The females were divided into six groups based on the temperatures and the small molecules injected: 25℃+DMSO; 25℃+GSK1016790 A; 25℃+GSK1016790A+HO-3867; 34℃+DMSO; 34℃+RN1734; 34℃+RN1734+Colivelin. Three to five days post-injection, the ovaries were isolated and subjected to the qPCR analysis. The results showed that mRNA up-regulation of testicular differentiation genes by the administration of Trpv4 agonist at 25℃ was abolished by the treatment with pStat3 inhibitor, and that expression of testicular differentiation genes inhibited by Trpv4 antagonist treatment at 34℃ can be partially restored by the injection with pStat3 agonist (Figure 6E). Similar results were obtained using in vitro cultured ovarian explants (Figure 6—figure supplement 1C).

To further confirm that Trpv4 functions upstream of pStat3, Trpv4 agonist GSK1016790A and stat3 siRNA were simultaneously injected into the ovaries of fish reared at 25℃, and the expression of sex-biased genes was examined by qPCR analysis. The results showed that the testicular differentiating genes failed to be activated by GSK1016790A in the presence of stat3 siRNAs (Figure 6F). Taken together, we concluded that Trpv4 promotes male sex gene expression in ovaries via the activation of the downstream pStat3 signaling.

pStat3 binds and activates kdm6b

Kdm6b is known as a conserved activator at the top of the male sex determination pathway, via binding and activating dmrt1 through removing the repressive histone mark H3K27me3 (Whiteley et al., 2018; Yao et al., 2023; Chen et al., 2024; Dupont et al., 2025; Lu et al., 2025), and pStat3 can transcriptionally activate the kdm6b gene by directly binding to its upstream DNA motifs in reptiles (Weber et al., 2020; Wu et al., 2024). We reasoned that pStat3 had a similar function in ricefield eel. When analyzing –5 kb promoter sequences upstream of the TSS of the kdm6b gene, we found that there were three conserved pStat3 binding sites (TTCnnnGAA) (Figure 7A). Chromatin immunoprecipitation (ChIP) experiments using pStat3 antibodies showed that pStat3 levels were significantly higher at kdm6b promoter in ovaries of females reared at warm temperature than at cool temperature, which was abolished in the presence of pStat3 inhibitor HO-3867 (Figure 7B). To explore whether pStat3 directly activates kdm6b, luciferase assay was performed. pGL4-kdm6b-luc construct was constructed by cloning ~3.5 kb kdm6b promoter sequences into the pGL4-luc plasmid. A mutant construct (pGL4-kdm6bM-luc) was generated by replacement of ‘TTCAGAGAA’ with ‘TTAAAAGAA.’ pGL4-kdm6b-luc and pGL4-kdm6bM-luc were transfected into HEK293T cells in the presence and absence of pStat3 agonist Colivelin, and the luciferase activities were measured. The results showed that the activities of pGL4-kdm6b-luc were significantly higher than that of pGL4-kdm6bM-luc in the presence of Colivelin (Figure 7C).

Figure 7
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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

kdm6b mRNA was expressed in immature oocytes in ovaries, was induced by warm temperature exposure, and was expressed at higher levels in testes than in ovaries in ricefield eel (Figure 3—figure supplement 1B; Figure 6—figure supplement 1A; Figure 6B; Figure 7D), analogous to that of trpv4. The expression of kdm6b was elevated by the injection (animal experiments) or addition (cell culture experiments) of GSK1016790A and Colivelin at 25℃, and was decreased by the injection or addition of RN1734 or HO-3867 at 34℃ (Figure 3B; Figure 6B; Figure 3—figure supplement 1B; Figure 6—figure supplement 1A). Thus, the kdm6b gene displayed an expression pattern similar to that of the testicular differentiation genes, which strongly supported that kdm6b is a male pathway gene downstream of Trpv4-Ca2+-pStat3 axis.

If kdm6b is downstream of Trpv4-pStat3 to regulate the expression of dmrt1, inhibition Kdm6b demethyltransferase activity should prevent up-regulation of testicular differentiation genes by warm temperature treatment, similar to HO-3867 and RN1734 treatment. GSK-J4, a Kdm6b specific-inhibitor, was injected into the ovaries of ricefield eel, and the expression of sex-biased factors were examined by the qPCR and IF analyses. The results showed that GSK-J4 injection significantly down-regulated the expression of testicular differentiation factors at the expense of ovarian differentiation factors (Figure 6B–D). Similar results were obtained in cultured ovarian cells (Figure 6—figure supplement 1A). We propose that there exists a Trpv4-pStat3-Kdm6b axis that links the environmental temperature and the male sex determination pathway (Figure 7E).

Discussion

Since the first discovery that ricefield eel is a teleost fish of hermaphroditism (Liu, 1944), the underlying mechanism has been under intensive investigation. However, it is still mysterious, partially because it is challenging to perform genetic studies due to the unique reproductive strategy and life history of the species (Song et al., 2022). In this work, we provided solid evidence that warm temperature promotes the sex change of adult ricefield eel, and that the ion channel protein Trpv4 functions as an important molecular linker connecting the environmental temperature and the sex determination pathway. Our results support a model in which temperature-driven sex change is achieved via the Trpv4-pStat3-Kdm6b-Dmrt1 axis. In this model, warm temperature exposure leads to increased calcium influx via Trpv4 in ovarian granulosa cells, which promotes phosphorylation of pStat3 and expression of the male pathway genes, eventually and gradually resulting in transformation of ovary to testis via an ovotestis. Our work for the first time provides the mechanistic explanation of how natural sex change occurs in aging ricefield eel, which may serve as a paradigm to study natural sex change of other adult animals, including the hermaphroditic marine fish.

The initial perception and translation of environmental cues into the sex determination cascades remain unclear in any species. Previous studies have proposed endoplasmic reticulum chaperones, heat shock proteins, and transmembrane ion channels as potential temperature sensors (He et al., 2010; Shi et al., 2024). In contrast to Cdc2-like kinases (Clks) that respond to subtle temperature change in homeothermic organisms, the transient receptor potential (TRP) cation channels may play important roles in poikilothermic animals that are exposed to strong ambient temperature change (Haltenhof et al., 2020; Huang et al., 2024). The TRP channel family can be divided into at least seven subfamilies, including TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, and TRPV. The thermo-TRPs contain nine members, such as TRPV (TRPV1-4). TRPV1-3 are mainly activated by temperatures above 35 °C, respectively (Mo et al., 2022). Of note, TRPV4 is activated by warm temperatures (27–35°C) (Güler et al., 2002; Goikoetxea et al., 2021), which are physiologically relevant to the spawning and/or the onset of sex change of ricefield eel. Trpv4 has been shown to be abundantly expressed during testicular/sperm development (Kumar et al., 2015; Mundt et al., 2018). Activation of Trpv4 by various endogenous/exogenous stimuli increases Ca2+ influx, participating in multiple downstream biological events, including gonadal homeostasis (Güler et al., 2002; Vrenken et al., 2016; Liu et al., 2021; Luo et al., 2023; Yamamoto et al., 2024). Importantly, Trpv4 has been shown to associate with temperature and sex determination in TSD alligator, and pharmaceutical activation and inhibition of Trpv4 alters testis differentiation (Yatsu et al., 2015). In ricefield eel, trpv4 was one of the most up-regulated trp genes by warm temperature treatment, and its expression was associated with testicular development. Thus, we focused on the role of Trpv4 in this work. Based on in vitro and in vivo experiments, we demonstrated that a portion of Trpv4-expressing gonadal somatic cells are highly sensitive to temperature cues and are functionally important in linking environmental temperature to sex determination pathways in the ricefield eel.

Previous work in the red-eared slider turtle (T. scripta) and the bluehead wrasse (Thalassoma bifasciatum) has indicated that Stat3 phosphorylation and epigenetic regulation are involved in TSD or sex change (Todd et al., 2019). In turtles, pStat3 is likely to be involved in sex determination by transcriptionally activating the female pathway genes, such as foxl2 and/or repressing the epigenetic factors, such as kdm6b (Holleley et al., 2015; Deveson et al., 2017; Whiteley et al., 2018; Weber et al., 2020; Chen et al., 2024; Wu et al., 2024). The function of jmjd3/kdm6b in sex determination in embryonic TISR fish, such as tilapia has been well established (Yao et al., 2023). Overexpression and knockdown of kdm6b cause female-to-male or male-to-female sex reversal, respectively. Our ChIP and luciferase reporter experiments suggested that pStat3 directly binds and activates the kdm6b gene in ricefield eel. In turtles, pStat3 functions as a repressor of kdm6b (Weber et al., 2020), whereas in ricefield eel and the bluehead wrasse, pStat3 exerts an opposite effect (Todd et al., 2019). We reasoned that a yet-unidentified co-factor may determine whether Stat3 is a transcriptional repressor or activator. A comparison of promoter sequences of kdm6b between turtle and ricefield eel supported this (data not shown).

Trpv4, pStat3 and Kdm6b were expressed in somatic cells surrounding ovarian oocytes and interstitial cells. After warm temperature exposure, their levels were elevated over time, similar to that of the male pathway genes. We propose that the magnitude and duration of temperature exposure promote sex change of ricefield eel by driving the accumulation of testicular differentiation genes in sufficient quantities (Weber et al., 2020). In sex reversal animals, a long existing question is what cells in the gonad respond to environmental temperatures to initiate sex change. In groupers, such as Epinephelus akaara, certain pre-existing somatic cells, called testicular-inducing steroidogenic cells, trigger the sex change (Murata et al., 2021). Recently, it has been proposed that a group of Nr5a1+Trp- thermosensitive steroidogenic cells use temperature-dependent Ca2+ signals to transduce into the male sex determination pathway in turtles (Li et al., 2025; Ye et al., 2025). Here, using ricefield eel as a model, we showed that a portion of Trpv4-expressing granulosa cells in the ovary respond to temperature cues, and initiate the sex reversal by increasing the expression of male pathway genes. This is supported by the fact that Dmrt1 expression showed up within follicles in a typical granulosa cell location (Figure 3E; Figure 6C). We propose that warm temperature exposure may cell-autonomously reprogram a portion of Trpv4+ granulosa cells into Dmrt1/Sox9a-positive Sertoli precursor cells, thereby promoting the sex change of ricefield eel.

To summarize, this study used ricefield eel hermaphrodites to elucidate the molecular basis underlying the transduction of environmental temperature into an intracellular signal for sex determination. We have made a few important findings. First, the ovarian somatic cells in ricefield eel are highly responsive to the warm temperature. Warm temperature is sufficient to induce the expression of male pathway genes, without input of other factors, such as hormones. Second, we identified a group of Trpv4-expressing granulosa cells that can directly perceive and respond to ambient temperature changes. Third, the pStat3-Kdm6b-Dmrt1 axis is downstream and mediates temperature-evoked Trpv4 activation. Our work revealed a comprehensive TISR mechanism, which involves signals that initiate sex reversal (temperature) and the capture (Trpv4), sensing (Trpv4-controlled calcium influx), transduction and interpretation (the Jak/Stat3 pathway) of environmental signals into the sex determination pathway (kdm6b/dmrt1). Of course, this work has limitations. Due to the unique life history of ricefield eel, genetic evidences are not sufficiently provided to substantiate the conclusion. Direct evidence for Trpv4 control of Ca2+ signaling is lacking.

Materials and methods

Sampling and maintenance of ricefield eel

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Ricefield eels were purchased from the Baishazhou market, Wuhan, China. They were temporarily maintained in the lab at 25±1℃ under a 14 hr light/10 hr dark cycle, and fed daily with commercial diet. Female fish were usually less than 40 cm in length, males were more than 50 cm in length, and intersex animals were of medium length.

Animal experiments and treatments were performed according to the Guide for Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences (IHB, CAS, Protocol No. 2016–018).

RNA preparation and RNA-sequencing

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We have performed RNA-sequencing (RNA-seq) experiments using gonadal tissues from female, middle-late intersex, and male animals (Zhang et al., 2025). To explore the earliest events that trigger the onset of sex change of ricefield eel, gonads from female, early intersex, and middle intersex animals were isolated. Each gonad was examined by color and morphology, and in some cases, histological experiments were performed to confirm the gonadal identities. Two to three gonads from each group were pooled, and total RNAs were isolated using a TRIzol reagent (Thermo Scientific, USA). RNA sample quality was checked by the OD 260/280 ratio using a Nanodrop 2000. The total mRNAs were sent to the BGI (Beijing Genomics Institute) company (Shenzhen, China), where RNA-sequencing libraries were constructed and sequenced by a BGI-500 system. RNA-seq experiments were performed at least two times, with two technical repeats.

Quantitative real-time PCR (qPCR) experiments

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Total RNA was isolated using the Isolation Kit mRNA. dmrt1/sox9a were used as male-specific genes, and cyp19a1a/foxl2 were used as female-specific genes. The primers and the related information used in this work were listed in Supplementary file 1. qPCR analysis was used to determine gene expression levels. A total of 1 µg RNA was reverse transcribed into cDNAs using the TransScript All-in-One First-Strand cDNA synthesis Supermix (Transgen Biotech, China, AT341). qPCR amplification was carried out on a Bio-Rad CFX96 Touch Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in triplicate. The reaction mixture consisted of 5 µl PerfectStart Green qPCR SuperMix (Transgen Biotech, China), 3.6 µl ddH2O, 0.2 µl forward and reverse primers, and 1 µl cDNA. The cycling parameters used were 94℃ for 30 s, 94℃ for 5 s, 60℃ for 15 s, and 72℃ for 10 s for 40 cycles. Quantification cycle or cycle threshold values were determined using CFX Manager 3.1 (Bio-Rad, USA). Primers were 18–21 nucleotides long, with a melting temperature between 58 and 60℃ and a guanine-cytosine content between 50% and 60% generating an amplicon of 80–250 bp. Beta-actin (β-actin) was used as a reference gene (Jiang et al., 2022). All qPCR experiments were repeated three times, and the relative gene expression levels were calculated based on the 2−ΔΔCt method.

siRNA knockdown experiments

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To study in vivo function of Trpv4, we used siRNA to deplete the expression of the trpv4 gene. The sequences of the ricefield eel trpv4 gene (Accession Number: NW_018127903.1) were obtained from NCBI GenBank. The siRNA sequences are listed in Supplementary file 2. These siRNAs were purchased from Sangon Biotech (Shanghai, China). Eighteen female fish were equally divided into three groups: 25℃+MOCK; 34℃+MOCK; 34℃+trpv4-siRNA. For the 34℃-group setting, after 1–2 day of acclimation at 25℃, the temperature of water was gradually increased by 3℃ per day until reaching 34℃. For RNAi experiment, before increasing the temperatures, the siRNA was injected into ovaries through genital papilla. siRNAs were injected twice, for an interval of 2 days. Three trpv4-siRNAs were mixed in equal amounts; each fish was injected with 100 nmol/kg. The MOCK groups were injected with equal amounts of control siRNAs. Two days after the second siRNA injection, ovarian samples were processed for qPCR analysis, and/or cryopreserved for ISH.

WB analysis

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WB was performed as previously described (Sun et al., 2020; Sun et al., 2023). The antibodies used in this work were: Amh (Huabio, #HA500137, China), Dmrt1 (home-made), Sox9a (home-made), Foxl2 (Thermofisher, #PA1-802, USA), Stat3 (Cell signaling, #9139, USA), pStat3 (Cell Signaling, #9145, USA). To validate the specificity of the antibodies, siRNA-mediated knockdown with immunoblot quantification with at least two replicates were performed.

IF experiments

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Anesthetized fish were fixed with 4% PFA, and gonads were isolated. The gonads were washed three times with PBS and dehydrated in sucrose solution (15% sucrose/PBS, 30% sucrose/PBS) for 2 hr at 4℃. Gonads were mounted in Tissue-Tek OCT compound (#4583, Sakura) and sectioned to 30 µm thickness on a cryostat. Slides containing sections were dried at room temperature for 30 min and washed three times for 5 min at room temperature with PBS. Sections on slides were blocked using 5% normal bovine serum (#A2153, Sigma Life Science) in PBS+0.1% Triton-X100 (#V900502, VETEC) for 2 hr. After wash, Primary antibodies, including Amh (Huabio, China), Foxl2 (ab5096, abcam), Trpv4 (Huabio, ER65407, China), Dmrt1 (home-made), Sox9a (home-made), Vimentin (OMA1-06001, Thermofisher, USA), were added at dilution of 1: 1000. Slides were washed three times with PBS for 5 min, followed by incubation in Alexa 488 or Alexa 555 (#A11008/A21428, Thermo Fisher, USA) secondary antibodies (1: 500) for 2 hr. The samples were counter-stained with DAPI (#D9542, Sigma, 1:1000) in 1×PBS at room temperature for 1 hr. After three times washing with PBS, slides were mounted using an anti-fade mounting medium (#HY-K1042, MedChemExpress, China). Mounted slides were imaged with a Leica Confocal Microscope (TCS SP8 STED, Germany).

In situ hybridization (ISH)

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To detect the expression of trpv4/kdm6b in the gonads, ISH experiments were performed. The cDNAs of ricefield eel trpv4 and kdm6b were amplified by gene-specific primers (Supplementary file 1). Digoxin-labeled RNA probes of Sp6-sense and T7-antisense were synthesized using the DIG RNA Labeling Kit Sp6/T7 (Roche, Basel, Switzerland). ISH was conducted following the methodology outlined below. Briefly, the fixed gonads were processed by dehydration, paraffin embedding, and serial sectioning (5 µm). Then the gonad slices were digested at 37℃ with 200 ng/ml proteinase K for 5 min. Hybridization was carried out for 16 hr at 60 ℃ using a probe concentration of 1 ng/µl in the hybridization buffer. The samples were incubated with the Anti-Digoxigenin-AP conjugate (Roche, Basel, Switzerland) at a 1:2500 dilution for 16 hr at 4℃, and stained in NBT/BCIP staining solution (Roche, Basel, Switzerland) in the dark for 0.5–1.5 hr at room temperature. The results were observed and photographed using an optical microscope (Zeiss, Oberkochen, Germany). Drawings and final panels were designed using Adobe Photoshop CS6 (San Jose, CA, USA).

ChIP experiments

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ChIP experiments were performed according to the Agilent Mammalian ChIP-on-chip manual. Briefly, gonadal tissues were processed into single cells and were fixed with 1% formaldehyde for 10 min at room temperature. The reactions were stopped by 0.125 M Glycine for 5 min with rotation. The fixed chromatin was sonicated to an average of (500–1000) bp (for ChIP qPCR) using the S2 Covaris Sonication System (USA) according to the manual. Then Triton X-100 was added to the sonicated chromatin solutions to a final concentration of 0.1%. After centrifugation, 50 µl of supernatants were saved as input. The remainder of the chromatin solution was incubated with Dynabeads previously coupled with 5 µg ChIP-grade pStat3 antibodies overnight at 4℃ with rotation. The next day, after 7 times washing with the wash buffer, the complexes were reverse cross-linked overnight at 65℃. DNAs were extracted by hydroxybenzene-chloroform-isoamyl alcohol and purified by a Phase Lock Gel (Tiangen, China). The ChIPed DNAs were dissolved in 100 µl distilled water. qPCR was performed using a Bio-Rad instrument. The enrichment was calculated relative to the amount of input as described. All experiments were repeated at least two times. The relative gene expression levels were calculated based on the 2−ΔΔCt method.

Luciferase assay

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HEK293T cells were seeded in 24-well plates in DMEM medium containing 10% FBS for 24 hr. The cells were then transiently transfected with the pGL4-kdm6b-luc or pGL4-kdm6bM-luc reporters using Lipofectamine 2000 (Invitrogen). pTKRenilla was used as an internal control. The luciferase activity was measured with the Dual-luciferase Reporter Assay system (Promega).

Hematoxylin and eosin (H&E) experiments

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H&E experiments were used for the identification of gonadal types in ricefield eels. The gonads were fixed in Bouin’s solution for at least 24 hr, and the H&E experiments were performed by Wuhan Icongene Biotechnology Company. Briefly, dehydration and paraffin embedding were then performed on the ASP6025S Automatic Vacuum Tissue Processor (Leica, Wetzlar, Germany). The samples were sectioned using the Leica microtome (Leica) at a thickness of 5 µm. After de-paraffinization, hydration and staining, the sections were examined on the Nikon ECLIPSE Ni-U microscope and micrographs were taken with the Digit Sight DS-Fi2 digital camera (Nikon).

Gonadal sex identification

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The gonadal types were initially identified according to morphological features, including the size, shape, and color. The gonadal sex of each fish was confirmed by histological sectioning and microscopic observation. In some cases, gene expression analysis was also used to confirm the gonadal sex types. Male genes, such as dmrt1, were not expressed in ovaries, slightly up-regulated in early ovotestes, and abundantly expressed in middle- and late-ovotestes.

Long-term temperature experiments

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The aim of this experiment was to assess the gonadal phenotypes of female fish that were reared at 25℃ (cool temperature, CT) vs 33/34℃ (warm temperatures, WT) over 6 months. The experiments were performed from September 3, 2024. 1.5-year-old wild female fish were transiently maintained for 3–5 days at the laboratory, and unhealthy animals were discarded. A total of approximately 400 female fish were randomly divided into the CT and WT groups, stocked in 10–12 tanks, at a density of 15–20 fish per tank. Fish were fed with commercial diet.

1, 3, and 6 months later, one tank in each group was randomly selected, and fish were anaesthetized and measured for body length and weight. The gonads were isolated and subjected to the histological analysis to determine the gonadal sex types. In some cases, gene expression analysis was used to determine the gonadal sex.

Short-term temperature experiments

Animal experiments

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The experiments were used to explore how warm temperature treatment affects the expression of sex differentiation genes in ovaries in a short period of time (3–10 days). The ricefield eels were reared at 25℃ (cool temperature) and at 34℃ (warm temperature). The temperature-increase protocol started at 25℃, with a progressive increase of 3℃ per day until reaching 34℃. Temperature was monitored twice a day throughout the experiment. Fish were fed with Artemia daily.

The ricefield eels from the Baishazhou market were transiently raised for 1–2 days at cool temperature (25℃). The fish were then divided into four groups based on the temperature and the injected small molecules: 25℃+DMSO; 25℃+GSK1016790 A; 34℃+DMSO; 34℃+RN1734. For the 34℃ group setting, the temperature of water was gradually increased by 3℃ every day until reaching 34℃. Before increasing the temperatures, the small molecules of appropriate doses were injected into ovaries. The final concentrations used were 0.02 mg/kg body weight for RN1734, 0.01 mg/kg body weight for HO-3867 or GSK1016790A or Colivelin, and a similar volume of 1% DMSO were injected and served as control.

To determine the upstream and downstream relationships between Trpv4 and pStat3, rescue experiments were performed by injecting the small molecules into the ovaries. Six groups were set up based on the temperature and the injected small molecules: 25℃+DMSO; 25℃+GSK1016790 A; 25℃+GSK1016790A+HO-3867; 34℃+DMSO; 34℃+RN1734; 34℃+RN1734+Colivelin.

To investigate the role of Kdm6b, 0.5 µM GSK-J4, a Kdm6b-specific inhibitor, was injected into the ovaries or added to the cultured ovarian explants and/or cells.

Ovarian explant or cell culture

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The ovaries were isolated from female ricefields and washed with cold 2% pen/strep PBS three times. The ovaries were cut into 2 mm3 pieces, and/or were digested with 0.25% TrypLE for 30 min into single cells. For single-cell culture, after filtration, ovarian cells were plated and cultured in 12-well plates.

For pharmaceutical experiments for Trpv4, the cells were divided into four groups: 26℃+DMSO; 26℃+GSK1016790 A; 33℃+DMSO; 33℃+RN1734. For the 33℃ group setting, after 1–2 days of acclimation at 25℃, the temperature of water was gradually increased by 3℃ every day until reaching 33℃. The doses of small molecules were optimized, and the final concentrations used were at 10 μM for RN1734, 100 nM for GSK1016790A, and a similar volume of 1% DMSO were added and served as control. For pharmaceutical experiments for pStat3 function, 2 μM HO-3867 and 20 μM Colivelin were added.

To determine the upstream and downstream relationships between Trpv4 and pStat3, rescue experiments were performed. Six groups were set up based on the temperature and the small molecules: 25℃+DMSO; 25℃+GSK1016790 A; 25℃+GSK1016790A+HO-3867; 33℃+DMSO; 33℃+RN1734; 33℃+RN1734+Colivelin.

Statistical analysis

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For gene expression analyses, differences in mean values between two groups were assessed using Student’s t-test. A one-way ANOVA was used to compare the expression levels of each target and the differences were determined using Tukey’s post hoc test. Significance was defined as *p<0.05, **p<0.01, ***p<0.001. Data are presented as mean± SEM (standard error of the mean). Statistical analyses were conducted and graphs.

Data availability

All the electronic data can be found in the article or the supplementary files. All antibodies and plasmids generated in this study are available. Further information and requests for materials should be directed to and will be fulfilled by the lead contact, Yuhua Sun (sunyh@ihb.ac.cn).

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Article and author information

Author details

  1. Zhi Yang

    1. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
    2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
    Present address
    Huzhou Normal University, Huzhou, China
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  2. Tingting Luo

    1. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
    2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  3. Yimin Zhang

    1. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
    2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared

  4. Yuhua Sun

    1. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China
    2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
    3. Hubei Hongshan Lab, Wuhan, China
    4. Huzhou Normal University, Huzhou, China
    Contribution
    Conceptualization, Resources, Supervision
    For correspondence
    sunyh@ihb.ac.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7902-9022

Funding

National Key Research and Development Program of China (2022YFD2400101)

  • Yuhua Sun

National Natural Science Foundation of China (32674024)

  • Yuhua Sun

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2022YFD2400101) to YH Sun. We thank Tanhong Eel Industry Aquaculture Co., Ltd (Chibi city, Hubei Province) for collecting the fish materials and fish farming. We thank Yue Ou from Sun’s lab for drawing the schematic role of Trpv4. We are grateful to Prof. Jianzhen Li from Northwest Normal University for critical comments on this work.

Ethics

Animal experiments and treatments were performed according to the Guide for Animal Care and Use Committee of the Institute of Hydrobiology, Chinese Academy of Sciences (IHB, CAS, Protocol No. 2016-018).

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  1. Zhi Yang
  2. Tingting Luo
  3. Yimin Zhang
  4. Yuhua Sun
(2026)
Trpv4 links environmental temperature to testicular differentiation in hermaphroditic ricefield eel
eLife 14:RP108272.
https://doi.org/10.7554/eLife.108272.4

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