Trpv4 mediates temperature induced sex change in ricefield eel
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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.
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Abstract
The ricefield eel ( Monopterus albus ), an economically important aquaculture species in China, is a freshwater teleost fish that exhibits protogynous hermaphroditism. While progress has been made toward understanding the sex determination and differentiation of this species, the underlying mechanisms remain elusive. Here we show that warm temperature promotes gonadal transformation by up-regulating testicular differentiation genes such as dmrt1/sox9a in ovaries. Trpv4, a Ca 2+ -permeable cation channel expressed in gonads, is highly sensitive to ambient temperature and mediates warm temperature-driven sex change of ricefield eel. In female fish reared at cool temperature, injection of Trpv4 agonist into the ovaries leads to significant up-regulation of testicular differentiation 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 male gene dmrt1 . Consistently, ovarian injection of Kdm6b inhibitor blocks the up-regulation of testicular differentiation genes by warm temperature treatment. 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.
Highlights
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Warm temperature promotes gonadal transformation of ricefield eel
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Trpv4 links environmental temperature and the sex determination pathway
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pStat3 is downstream of Trpv4-controlled calcium signaling
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pStat3 binds and activates kdm6b/jmjd3
Article activity feed
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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.
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Reviewer #1 (Public review):
Summary:
This study investigates the molecular mechanism by which warm temperature induces female-to-male sex reversal in the ricefield eel (Monopterus albus), a protogynous hermaphroditic fish of significant aquacultural value in China. The study identifies Trpv4 - a temperature-sensitive Ca²⁺ channel - as a putative thermosensor linking environmental temperature to sex determination. The authors propose that Trpv4 causes Ca²⁺ influx, leading to activation of Stat3 (pStat3). pStat3 then transcriptionally upregulates the histone demethylase Kdm6b (aka Jmjd3), leading to increased dmrt1 gene expression and ovo-testes development. This work aims to bridge ecological cues with molecular and epigenetic regulators of sex change and has potential implications for sex control in aquaculture.
Strengths:
(1) This …
Reviewer #1 (Public review):
Summary:
This study investigates the molecular mechanism by which warm temperature induces female-to-male sex reversal in the ricefield eel (Monopterus albus), a protogynous hermaphroditic fish of significant aquacultural value in China. The study identifies Trpv4 - a temperature-sensitive Ca²⁺ channel - as a putative thermosensor linking environmental temperature to sex determination. The authors propose that Trpv4 causes Ca²⁺ influx, leading to activation of Stat3 (pStat3). pStat3 then transcriptionally upregulates the histone demethylase Kdm6b (aka Jmjd3), leading to increased dmrt1 gene expression and ovo-testes development. This work aims to bridge ecological cues with molecular and epigenetic regulators of sex change and has potential implications for sex control in aquaculture.
Strengths:
(1) This study proposes the first mechanistic pathway linking thermal cues to natural sex reversal in adult ricefield eel, extending the temperature-dependent sex determination paradigm beyond embryonic reptiles and saltwater fish.
(2) The findings could have applications for aquaculture, where skewed sex ratios apparently limit breeding efficiency.
Weaknesses:
(A) Scientific Concerns:
(1) There is insufficient replication and data transparency. First, the qPCR data are presented as bar graphs without individual data points, making it impossible to assess variability or replication. Please show all individual data points and clarify n (sample size) per group. Second, the Western blotting is only shown as single replicates. If repeated 2-3 times as stated, quantification and normalization (e.g., pStat3/Stat3, GAPDH loading control) are essential. The full, uncropped blots should be included in the supplementary data.
(2) The biological significance of the results is not clear. Many reported fold changes (e.g., kdm6b modulation by Stat3 inhibition, sox9a in S3A) are modest (<2-fold), raising concerns about biological relevance. Can the authors define thresholds of functional relevance or confirm phenotypic outcomes in these animals?
(3) The specificity of key antibodies is not validated. Key antibodies (Stat3, pStat3, Foxl2, Amh) were raised against mammalian proteins. Their specificity for ricefield eel proteins is unverified. Validation should include siRNA-mediated knockdown with immunoblot quantification with 3 replicates. Homemade antibodies (Sox9a, Dmrt1) also require rigorous validation.
(4) Most of the imaging data (immunofluorescence) is inconclusive. Immunofluorescence panels are small and lack monochrome channels, which severely limits interpretability. Larger, better-contrasted images (showing the merge and the monochrome of important channels) and quantification would enhance the clarity of these findings.
(B) Other comments about the science:
(1) In S3A, sox9a expression is not dose-responsive to Trpv4 modulation, weakening the causal inference.
(2) An antibody against Kdm6b (if available) should be used to confirm protein-level changes.
In sum, the interpretations are limited by the above concerns regarding data presentation and reagent specificity.
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Reviewer #2 (Public review):
Summary:
This study presents valuable findings on the molecular mechanisms driving the female-to-male transformation in the ricefield eel (Monopterus albus) during aging. The authors explore the role of temperature-activated TRPV4 signaling in promoting testicular differentiation, proposing a TRPV4-Ca²⁺-pSTAT3-Kdm6b axis that facilitates this gonadal shift.
Strengths:
The manuscript describes an interesting mechanism potentially underlying sex differentiation in M. albus.
Weaknesses:
The current data are insufficient to fully support the central claims, and the study would benefit from more rigorous experimental approaches.
(1) Overstated Title and Claims:
The title "TRPV4 mediates temperature-induced sex change" overstates the evidence. No histological confirmation of gonadal transformation (e.g., formation …
Reviewer #2 (Public review):
Summary:
This study presents valuable findings on the molecular mechanisms driving the female-to-male transformation in the ricefield eel (Monopterus albus) during aging. The authors explore the role of temperature-activated TRPV4 signaling in promoting testicular differentiation, proposing a TRPV4-Ca²⁺-pSTAT3-Kdm6b axis that facilitates this gonadal shift.
Strengths:
The manuscript describes an interesting mechanism potentially underlying sex differentiation in M. albus.
Weaknesses:
The current data are insufficient to fully support the central claims, and the study would benefit from more rigorous experimental approaches.
(1) Overstated Title and Claims:
The title "TRPV4 mediates temperature-induced sex change" overstates the evidence. No histological confirmation of gonadal transformation (e.g., formation of testicular structures) is presented. Conclusions are based solely on molecular markers such as dmrt1 and sox9a, which, although suggestive, are not definitive indicators of functional sex reversal.
(2) Temperature vs Growth Rate Confounding (Figure 1E):
The conclusion that warm temperature directly induces gonadal transformation is confounded by potential growth rate effects. The authors state that body size was "comparable" between 25{degree sign}C and 33{degree sign}C groups, but fail to provide supporting data. In ectotherms, growth is intrinsically temperature-dependent. Given the known correlation between size and sex change in M. albus, growth rate-rather than temperature per se-may underlie the observed sex ratio shifts. Controlled growth-matched comparisons or inclusion of growth rate metrics are needed.
(3) TRPV4 as a Thermosensor-Insufficient Evidence:
The characterisation of TRPV4 as a direct thermosensor lacks biophysical validation. The observed transcriptional upregulation of Trpv4 under heat (Figure 2) reflects downstream responses rather than primary sensor function. Functional thermosensors, including TRPV4, respond to heat via immediate ion channel activity-typically measurable within seconds-not mRNA expression over hours. No patch-clamp or electrophysiological data are provided to confirm TRPV4 activation thresholds in eel gonadal cells. Additionally, the Ca²⁺ imaging assay (Figure 2F) lacks essential details: the timing of GSK1016790A/RN1734 administration relative to imaging is unclear, making it difficult to distinguish direct channel activity from indirect transcriptional effects.
(4) Cellular Context of TRPV4 Activity Is Unclear:
In situ hybridisation suggests TRPV4 expression shifts from interstitial to somatic domains under heat (Figures. 2H, S2C), implying potential cell-type-specific roles. However, the study does not clarify: (i) whether TRPV4 plays the same role across these cell types, (ii) why somatic cells show stronger signal amplification, or (iii) the cellular composition of explants used in in vitro assays. Without this resolution, conclusions from pharmacological manipulation (e.g., GSK1016790A effects) cannot be definitively linked to specific cell populations.
(5) Rapid Trpv4 mRNA Elevation and Channel Function:
The authors report a dramatic increase in Trpv4 mRNA within one day of heat exposure (Figures 4D, S2B). Given that TRPV4 is a membrane channel, not a transcription factor, its rapid transcriptional sensitivity to temperature raises mechanistic questions. This finding, while intriguing, seems more correlational than functional. A clearer explanation of how TRPV4 senses temperature at the molecular level is needed.
(6) Inconclusive Evidence for the Ca2+ -pSTAT3-Kdm6b Axis:
Although the authors propose a TRPV4-Ca2+ -pSTAT3-Kdm6b-dmrt1 pathway, intermediate steps remain poorly supported. For example, western blot data (Figures 3C, 4B) do not convincingly demonstrate significant pSTAT3 elevation at 34{degree sign}C. Higher-resolution and properly quantified blots are essential. The inferred signalling cascade is based largely on temporal correlation and pharmacological inhibition, which are insufficient to establish direct regulatory relationships.
(7) Species-Specific STAT3-Kdm6b Regulation Is Unresolved:
The proposed activation of Kdm6b by pSTAT3 contrasts with findings in the red-eared slider turtle (Trachemys scripta), where pSTAT3 represses Kdm6b. This divergence in regulatory direction between the two TSD species is surprising and demands further justification. Cross-species differences in binding motifs or epigenetic context should be explored. Additional evidence, such as luciferase reporter assays (using wild-type and mutant pSTAT3 binding motifs in the Kdm6b promoter) is needed to confirm direct activation. A rescue experiment-testing whether Kdm6b overexpression can compensate for pSTAT3 inhibition-would also greatly strengthen the model.
(8) Immunofluorescence-Lack of Structural Markers:
All immunofluorescence images should include structural markers to delineate gonadal boundaries. Furthermore, image descriptions in the figure legends and main text lack detail and should be significantly expanded for clarity.
(9) Pharmacological Reagents-Mechanisms and References:
The manuscript lacks proper references and mechanistic descriptions for the pharmacological agents used (e.g., GSK1016790A, RN1734, Stattic). Established literature on their specificity and usage context should be cited to support their application and interpretation in this study.
(10) Efficiency of Experimental Interventions:
The percentage of gonads exhibiting sex reversal following pharmacological or RNAi treatments should be reported in the Results. This is critical for evaluating the strength and reproducibility of the interventions.
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