Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review)>
Summary:
This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males and females is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.
We thank the reviewer for this very positive evaluation of our work and greatly appreciate their helpful comments and suggestions for improving the manuscript. We agree with the comment that the term “neuroestrogens” is misleading. Therefore, we have replaced “neuroestrogens” with “brain-derived estrogens” or “brain estrogens” throughout the manuscript, including the title.
In the following sections, “neuroestrogens” has been revised to align with the surrounding context.
Line 21: “in the brain, also known as neuroestrogens,” → “in the brain.”
Line 28: “neuroestrogens” → “these estrogens.”
Line 30: “mechanism of action of neuroestrogens” → “mode of action of brain-derived estrogens.”
Line 43: “brain-derived estrogens, also called neuroestrogens,” → “estrogens.”
Line 74: “neuroestrogen synthesis is selectively impaired while gonadal estrogen synthesis remains intact” → “estrogen synthesis in the brain is selectively impaired while that in the gonads remains intact.”
Line 77: “neuroestrogens” → “these estrogens.”
Line 335: “levels of neuroestrogens” → “brain estrogen levels.”
Line 338: “neuroestrogens” → “these estrogens.”
Line 351: “neuroestrogens” → “these estrogens.”
Line 357: “neuroestrogen action” → “the action of brain-derived estrogens.”
Line 359: “neuroestrogens” → “estrogen synthesis in the brain.”
Line 390: “active synthesis of neuroestrogens” → “active estrogen synthesis in the brain.”
Line 431: “neuroestrogens” → “estrogens in the brain.”
Line 431: “neuroestrogen action” → “the action of brain-derived estrogens.”
Line 433: “neuroestrogen action” → “their action.”
Strengths:
Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones.
The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation.
Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors'.
Includes multiple follow-up experiments, which lead to tests of internal replication and an impactful mechanistic proposal.
Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionarily ancient.
We again thank the reviewer for their positive evaluation of our work.
Weaknesses:
(1) As stated in the summary, the authors attribute the estrogen source to neurons and there isn't evidence this is the case. The impact of the findings doesn't rest on this either.
As noted in Response to reviewer #1’s summary comment, we have replaced “neuroestrogens” with “brain-derived estrogens” or “brain estrogens” throughout the manuscript.
Line 63: We have also added the text “In teleost brains, including those of medaka, aromatase is exclusively localized in radial glial cells, in contrast to its neuronal localization in rodent brains (18– 20).” Following this addition, “This observation suggests” in the subsequent sentence has been replaced with “These observations suggest.”
The following references (#18–20), cited in the newly added text above, have been included in the reference list, with other references renumbered accordingly:
P. M. Forlano, D. L. Deitcher, D. A. Myers, A. H. Bass, Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source. J. Neurosci. 21, 8943–8955 (2001).
N. Diotel, Y. Le Page, K. Mouriec, S. K. Tong, E. Pellegrini, C. Vaillant, I. Anglade, F. Brion, F. Pakdel, B. C. Chung, O. Kah, Aromatase in the brain of teleost fish: expression, regulation and putative functions. Front. Neuroendocrinol. 31, 172–192 (2010).
A. Takeuchi, K. Okubo, Post-proliferative immature radial glial cells female-specifically express aromatase in the medaka optic tectum. PLoS One 8, e73663 (2013).
(2) The d4 versus d8 esr2a mutants showed different results for aggression. The meaning and implications of this finding are not discussed, leaving the reader wondering.
Line 282: As the reviewer correctly noted, circles were significantly reduced in mutant males of the Δ8 line, whereas no significant reduction was observed in those of the Δ4 line. However, a tendency toward reduction was evident in the Δ4 line (P = 0.1512), and both lines showed significant differences in fin displays. Based on these findings, we believe our conclusion that esr2a<sup>−/−</sup> males exhibit reduced aggression remains valid. To clarify this point and address potential reader concerns, we have revised the text as follows: “esr2a<sup>−/−</sup> males from both the Δ8 and Δ4 lines exhibited significantly fewer fin displays than their wildtype siblings (P = 0.0461 and 0.0293, respectively). Circles followed a similar pattern, with a significant reduction in the Δ8 line (P = 0.0446) and a comparable but non-significant decrease in the Δ4 line (P = 0.1512) (Fig. 5L; Fig. S8E), showing less aggression.”
(3) Lack of attribution of previously published work from other research groups that would provide the proper context of the present study.
In response to this and other comments from this reviewer, we have revised the Introduction and Discussion sections as follows.
Line 56: “solely responsible” in the Introduction has been modified to “largely responsible”.
Line 57: “This is consistent with the recent finding in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in male-typical courtship (10)” has been revised to “This is consistent with recent observations in a few teleost species that genetic ablation of AR severely impairs male-typical behaviors (13–16) and with findings in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in maletypical courtship (12)” to include previous studies on the behavior of AR mutant fish (Yong et al., 2017; Alward et al., 2020; Ogino et al., 2023; Nishiike and Okubo, 2024) in the Introduction.
Line 65: “It is worth mentioning that systemic administration of estrogens and an aromatase inhibitor increased and decreased male aggression, respectively, in several teleost species, potentially reflecting the behavioral effects of brain-derived estrogens (21–24)” has been added to the Introduction. This addition provides an overview of previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015).
Line 367: “treatment of males with an aromatase inhibitor reduces their male-typical behaviors (31– 33)” has been edited to read “treatment of males with an aromatase inhibitor reduces their male-typical behaviors, while estrogens exert the opposite effect (21–24).”
After the revisions described above, the following references (#13, 14, and 22) have been added to the reference list, with other references renumbered accordingly:
L. Yong, Z. Thet, Y. Zhu, Genetic editing of the androgen receptor contributes to impaired male courtship behavior in zebrafish. J. Exp. Biol. 220, 3017–3021 (2017).
B. A. Alward, V. A. Laud, C. J. Skalnik, R. A. York, S. A. Juntti, R. D. Fernald, Modular genetic control of social status in a cichlid fish. Proc. Natl. Acad. Sci. U.S.A. 117, 28167–28174 (2020).
L. A. O’Connell, H. A. Hofmann, Social status predicts how sex steroid receptors regulate complex behavior across levels of biological organization. Endocrinology 153, 1341–1351 (2012).
(4) There are a surprising number of citations not included; some of the ones not included argue against the authors' claims that their findings were "contrary to expectation".
Line 68: As detailed in Response to reviewer #1’s comment 3 on weaknesses, we have cited previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015) in the Introduction.
The following revisions have also been made to avoid phrases such as “contrary to expectation” and “unexpected.”
Line 76: “Contrary to our expectations” → “Remarkably.”
Line 109: “Contrary to this expectation, however” → “Nevertheless.”
Line 135: “Again, contrary to our expectation, cyp19a1b<sup>−/−</sup> males” → “cyp19a1b<sup>−/−</sup> males.”
Line 333: “unexpected” → “noteworthy.”
Line 337: “unexpected” → “notable.”
(5) The experimental design for studying aggression in males has flaws. A standard test like a resident intruder test should be used.
We agree that the resident-intruder test is the most commonly used method for assessing aggression. However, medaka form shoals and lack strong territoriality, and even slight dominance differences between the resident and the intruder can increase variability in the results, compromising data consistency. Therefore, in this study, we adopted an alternative approach: placing four unfamiliar males together in a tank and quantifying aggressive interactions in total. This method allows for the assessment of aggression regardless of territorial tendencies, making it more appropriate for our investigation.
(6) While they investigate males and females, there are fewer experiments and explanations for the female results, making it feel like a small addition or an aside.
We agree that the data and discussion for females are less extensive than for males. However, we have previously elucidated the mechanism by which estrogen/Esr2b signaling promotes female mating behavior (Nishiike et al., 2021, Curr Biol, 1699–1710). Accordingly, it follows that the new insights into female behavior gained from the cyp19a1b knockout model are more limited than those for males. Nevertheless, when combined with our prior findings, the female data in this study offer valuable insights, and the overall mechanism through which estrogens promote female mating behavior is becoming clearer. Therefore, we do not consider the female data in this study to be incomplete or merely supplementary.
(7) The statistics comparing "experimental to experimental" and "control to experimental" aren't appropriate.
The reviewer raises concerns about the statistical analysis used for Figures 4C and 4E, suggesting that Bonferroni’s test should be used instead of Dunnett’s test. However, Dunnett’s test is commonly used to compare treatment groups to a reference group that receives no treatment, as in our study. Since we do not compare the treated groups with each other, we believe Dunnett’s test is the most appropriate choice.
Line 619: The reviewer’s concern may have arisen from the phrase “comparisons between control and experimental groups” in the Materials and Methods. We have revised it to “comparisons between untreated and E2-treated groups in Fig. 4, C and D” for clarity.
Reviewer #2 (Public Review):
Summary:
The novelty of this study stems from the observations that neuro-estrogens appear to interact with brain androgen receptors to support male-typical behaviors. The study provides a step forward in clarifying the somewhat contradictory findings that, in teleosts and unlike other vertebrates, androgens regulate male-typical behaviors without requiring aromatization, but at the same time estrogens appear to also be involved in regulating male-typical behaviors. They manipulate the expression of one aromatase isoform, cyp19a1b, that is purported to be brain-specific in teleosts. Their findings are important in that brain estrogen content is sensitive to the brain-specific cyp19a1b deficiency, leading to alterations in both sexual behavior and aggressive behavior. Interestingly, these males have relatively intact fertility rates, despite the effects on the brain.
We thank this reviewer for their positive evaluation of our work and constructive comments, which we found very helpful in improving the manuscript.
That said, the framing of the study, the relevant context, and several aspects of the methods and results raise concerns. Two interpretations need to be addressed/tempered:
(1) that the rescue of cyp19a1b deficiency by tank-applied estradiol is not necessarily a brain/neuroestrogen mode of action, and
Line 155: cyp19a1b-deficient males exhibited a severe reduction in brain E2 levels, yet their peripheral E2 levels remained comparable to those in wild-type males. Given this hormonal milieu and the lack of behavioral change in wild-type males following E2 treatment, the observed recovery of mating behavior in cyp19a1b-deficient males following E2 treatment can be best explained by the restoration of brain E2 levels. However, as the reviewer pointed out, we cannot rule out the possibility that bath-immersed E2 influenced behavior through an indirect peripheral mechanism. To address this concern, we have modified the text as follows: “These results suggest that reduced E2 in the brain is the primary cause of the mating defects, highlighting a pivotal role of brain-derived estrogens in male mating behavior. However, caution is warranted, as an indirect peripheral effect of bath-immersed E2 on behavior cannot be ruled out, although this is unlikely given the comparable peripheral E2 levels in cyp19a1b-deficient and wild-type males. In contrast to mating.”
(2) the large increases in peripheral and brain androgen levels in the cyp19a1b deficient animals imply some indirect/compensatory effects of lifelong cyp19a1b deficiency.
As stated in line 151, androgen/AR signaling has a strong facilitative effect on male-typical behaviors in teleosts. If increased androgen levels in the periphery and brain affected behavior, the expected effect would be facilitative. However, cyp19a1b-deficient males exhibited impaired male-typical behaviors, suggesting that elevated androgen levels were unlikely to be responsible. Although chronic androgen elevation could cause androgen receptor desensitization, which could lead to behavioral suppression, our long-term androgen treatments have consistently promoted, rather than inhibited, male-typical behaviors (e.g., Nishiike et al., Proc Natl Acad Sci USA 121:e2316459121). Hence, this possibility is also highly unlikely.
Reviewer #3 (Public Review):
Summary:
Taking advantage of the existence in fish of two genes coding for estrogen synthase, the enzyme aromatase, one mostly expressed in the brain (Cyp19a1b) and the other mostly found in the gonads (Cyp19a1a), this study investigates the role of neuro-estrogens in the control of sexual and aggressive behavior in teleost fish. The constitutive deletion of Cyp19a1b reduced brain estrogen content by 87% in males and about 50% in females. It led to reduced sexual and aggressive behavior in males and reduced sexual behavior in females. These effects are reversed by adult treatment with estradiol thus indicating that they are activational in nature. The deletion of Cyp19a1b is associated with a reduced expression of the genes coding for the two androgen receptors, ara, and arb, in brain regions involved in the regulation of social behavior. The analysis of the gene expression and behavior of mutants of estrogen receptors indicates that these effects are likely mediated by the activation of the esr1 and esr2a isoforms. These results provide valuable insight into the role of neuro-estrogens in social behavior in the most abundant vertebrate taxa. While estrogens are involved in the organization of the brain and behavior of some birds and rodents, neuro-estrogens appear to play an activational role in fish through a facilitatory action of androgen signaling.
We thank this reviewer for their positive evaluation of our work and comments that have improved the manuscript.
Strengths:
Evaluation of the role of brain "specific" Cyp19a1 in male teleost fish, which as a taxa are more abundant and yet proportionally less studied than the most common birds and rodents. Therefore, evaluating the generalizability of results from higher vertebrates is important. This approach also offers great potential to study the role of brain estrogen production in females, an understudied question in all taxa.
Results obtained from multiple mutant lines converge to show that estrogen signaling drives aspects of male sexual behavior.
The comparative discussion of the age-dependent abundance of brain aromatase in fish vs mammals and its role in organization vs activation is important beyond the study of the targeted species.
We again thank the reviewer for their positive evaluation of our work.
Weaknesses:
(1) The new transgenic lines are under-characterized. There is no evaluation of the mRNA and protein products of Cyp19a1b and ESR2a.
We did not directly assess the function of cyp19a1b and esr2a in our mutant fish. However, the observed reduction in brain E2 levels, with no change in peripheral E2 levels, in cyp19a1b-deficient fish strongly supports the loss of cyp19a1b function. This is stated in the Results section (line 97) as follows: “These results show that cyp19a1b-deficient fish have reduced estrogen levels coupled with increased androgen levels in the brain, confirming the loss of cyp19a1b function.”
Line 473: A previous study reported that female medaka lacking esr2a fail to release eggs due to oviduct atresia (Kayo et al., 2019, Sci Rep 9:8868). Similarly, in this study, some esr2a-deficient females exhibited spawning behavior but were unable to release eggs, although the sample size was limited (Δ8 line: 2/3; Δ4 line: 1/1). In contrast, this was not observed in wild-type females (Δ8 line: 0/12; Δ4 line: 0/11). These results support the effective loss of esr2a function. To incorporate this information into the manuscript, the following text has been added to the Materials and Methods: “A previous study reported that esr2a-deficient female medaka cannot release eggs due to oviduct atresia (59). Likewise, some esr2a-deficient females generated in this study, despite the limited sample size, exhibited spawning behavior but were unable to release eggs (Δ8 line: 2/3; Δ4 line: 1/1), while such failure was not observed in wild-type females (Δ8 line: 0/12; Δ4 line: 0/11). These results support the effective loss of esr2a function.”
The following reference (#59), cited in the newly added text above, have been included in the reference list:
D. Kayo, B. Zempo, S. Tomihara, Y. Oka, S. Kanda, Gene knockout analysis reveals essentiality of estrogen receptor β1 (Esr2a) for female reproduction in medaka. Sci. Rep. 9, 8868 (2019).
(2) The stereotypic sequence of sexual behavior is poorly described, in particular, the part played by the two sexual partners, such that the conclusions are not easily understandable, notably with regards to the distinction between motivation and performance.
Line 103: To provide a more detailed description of medaka mating behavior, we have revised the text from “The mating behavior of medaka follows a stereotypical pattern, wherein a series of followings, courtship displays, and wrappings by the male leads to spawning” to “The mating behavior of medaka follows a stereotypical sequence. It begins with the male approaching and closely following the female (following). The male then performs a courtship display, rapidly swimming in a circular pattern in front of the female. If the female is receptive, the male grasps her with his fins (wrapping), culminating in the simultaneous release of eggs and sperm (spawning).”
(3) The behavior of females is only assessed from the perspective of the male, which raises questions about the interpretation of the reduced behavior of the males.
In medaka, female mating behavior is largely passive, except for rejecting courtship attempts and releasing eggs. Therefore, its analysis relies on measuring the latency to receive following, courtship displays, or wrappings from the male and the frequency of courtship rejection or wrapping refusal. We understand the reviewer’s perspective that cyp19a1b-deficient females might not be less receptive but instead less attractive to males, potentially leading to reduced male mating efforts. However, since these females are approached and followed by males at levels comparable to wild-type females, this possibility appears unlikely. Moreover, cyp19a1b-deficient females tend to avoid males and exhibit a slightly female-oriented sexual preference. While these traits are closely associated with reduced sexual receptivity, they do not readily align with reduced sexual attractiveness. Therefore, it is more plausible to conclude that these females have decreased receptivity rather than being less attractive to males.
(4) At no point do the authors seem to consider that a reduced behavior of one sex could result from a reduced sensory perception from this sex or a reduced attractivity or sensory communication from the other sex.
Line 112: As noted above, the impaired mating behavior of cyp19a1b-deficient females is unlikely to be due to reduced attractiveness to males. Similarly, mating behavior tests using esr2b-deficient females as stimulus females suggest that the impaired mating behavior of cyp19a1b-deficient males cannot be attributed to reduced attractiveness to females. However, the possibility that their impaired mating behavior could be attributed to altered cognition or sexual preference cannot be ruled out. To reflect this in the manuscript, we have revised the text “, suggesting that they are less motivated to mate” to “. These results suggest that they are less motivated to mate, though an alternative interpretation that their cognition or sexual preference may be altered cannot be dismissed.”
(5) Aspects of the methods are not detailed enough to allow proper evaluation of their quality or replication of the data.
In response to this and other specific comments from this reviewer, we have revised the Materials and Methods section to include more detailed descriptions of the methods.
Line 469: The following text has been added to describe the method for domain identification in medaka Esr2a: “The DNA- and ligand-binding domains of medaka Esr2a were identified by sequence alignment with yellow perch (Perca flavescens) Esr2a, for which these domain locations have been reported (58).”
The following reference (#58), cited in the newly added text above, have been included in the reference list:
S. G. Lynn, W. J. Birge, B. S. Shepherd, Molecular characterization and sex-specific tissue expression of estrogen receptor α (esr1), estrogen receptor βa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 149, 126–147 (2008).
Line 540: The text “, and the total area of signal in each brain nucleus was calculated using Olyvia software (Olympus)” has been revised to include additional details on the single ISH method as follows: “. The total area of signal across all relevant sections, including both hemispheres, was calculated for each brain nucleus using Olyvia software (Olympus). Images were converted to a 256-level intensity scale, and pixels with intensities from 161 to 256 were considered signals. All sections used for comparison were processed in the same batch, without corrections between samples.”
Line 596: The following text has been added to include additional details on the double ISH method: “Cells were identified as coexpressing the two genes when Alexa Fluor 555 and fluorescein signals were clearly observed in the cytoplasm surrounding DAPI-stained nuclei, with intensities markedly stronger than the background noise.”
(6) It seems very dangerous to use the response to a mutant abnormal behavior (ESR2-KO females) as a test, given that it is not clear what is the cause of the disrupted behavior.
esr2b-deficient females have fully developed ovaries, a normal sex steroid milieu, and sexual attractiveness to males comparable to wild-type females, yet they are completely unreceptive to male courtship (Nishiike et al., 2021, Curr Biol, 1699–1710). Although, as the reviewer noted, the detailed mechanisms underlying this phenotype remain unclear, it is evident that the loss of estrogen/Esr2b signaling in the brain severely impairs sexual receptivity. Therefore, using esr2b-deficient females as stimulus females in the mating behavior test eliminates the influence of female sexual receptivity and male attractiveness to females, enabling the exclusive assessment of male mating motivation. This rationale is already presented in the Results section (lines 116–120), and we believe this experimental design offers a robust framework for assessing male mating motivation.
Additionally, the mating behavior test with esr2b-deficient females complemented the test with wildtype females, and its results were not the sole basis for our discussion of the male mating behavior phenotype. The results of both tests were largely concordant, and we believe that the conclusions drawn from them are highly reliable.
Meanwhile, in the test with esr2b-deficient females, cyp19a1b-deficient males were courted more frequently by these females than wild-type males. As the reviewer noted, this may suggest an anomaly in the test. Accordingly, we have confined our discussion to the possibility that “Perhaps cyp19a1b<sup>−/−</sup> males are misidentified as females by esr2b-deficient females because they are reluctant to court or they exhibit some female-like behavior” (line 131).
(7) Most experiments are weakly powered (low sample size) and analyzed by multiple T-tests while 2 way ANOVA could have been used in several instances. No mention of T or F values, or degrees of freedom.
Histological analysis was conducted with a relatively small sample size, as our previous experience suggested that interindividual variability in the results would not be substantial. As significant differences were detected in many analyses, further increasing the sample size is unnecessary.
Although two-way ANOVA could be used instead of multiple T-tests for analyzing the data in Figures 4D, 4F, 6D, S4A, and S4B, we applied the Bonferroni–Dunn correction to control for multiple pairwise comparisons in multiple T-tests. As this comparison method is equivalent to the post hoc test following two-way ANOVA, the statistical results are identical regardless of whether T-tests or two-way ANOVA are used.
For the data in Figures 4D, 4F, S4A, and S4B, the primary focus is on whether relative luciferase activity differs between E2-treated and untreated conditions for each mutant construct. Therefore, two-way ANOVA is not particularly relevant, as assessing the main effect of construct type or its interaction with E2 treatment does not provide meaningful insights. Similarly, in Figure 6D, the focus is solely on whether wild-type and mutant females differ in time spent at each distance. Given this, two-way ANOVA is unnecessary, as analyzing the main effect of distance is not meaningful.
Accordingly, two-way ANOVA was not employed in this study, and therefore, its corresponding F values were not included. As the figure legends specify the sample sizes for all analyses, specifying degrees of freedom separately was deemed unnecessary.
(8) The variability of the mRNA content for the same target gene between experiments (genotype comparison vs E2 treatment comparison) raises questions about the reproducibility of the data (apparent disappearance of genotype effect).
As the reviewer pointed out, the overall area of ara expression is larger in Figure 2J than in Figure 2F. However, the relative area ratios of ara expression among brain nuclei are consistent between the two figures, indicating the reproducibility of the results. Thus, this difference is unlikely to affect the conclusions of this study.
Additionally, the differences in ara expression in pPPp and arb expression in aPPp between wild-type and cyp19a1b-deficient males appear less pronounced in Figures 2J and 2K than in Figures 2F and 2H. This is likely attributable to the smaller sample size used in the experiments for Figures 2J and 2K, resulting in less distinct differences. However, as the same genotype-dependent trends are observed in both sets of figures, the conclusion that ara and arb expression is reduced in cyp19a1b-deficient male brains remains valid.
(9) The discussion confuses the effects of estrogens on sexual differentiation (developmental programming = permanent) and activation (= reversible activation of brain circuits in adulthood) of the brain and behavior. Whether sex differences in the circuits underlying social behaviors exist is not clear.
We recognize that the effects of adult steroids are sometimes not considered to be sexual differentiation, as they do not differentiate the neural substrate, but rather transiently activate the already masculinized or feminized substrate. Arnold (2017, J Neurosci Res 95:291–300) contends that all factors that cause sex differences, including the transient effects of adult steroids, should be incorporated into a theory of sexual differentiation, and indeed, these effects may be the most potent proximate factors that make males and females different. We concur with this perspective and have adopted it as a foundation for our manuscript.
In teleosts, early developmental exposure to steroids has minimal impact, and sexual differentiation relies primarily on steroid action in adulthood (Okubo et al., 2022, Spectrum of Sex, pp. 111–133). This is evidenced by the effective reversal of sex-typical behaviors through experimental hormonal manipulation in adult teleosts and the absence of transient early-life steroid surges observed in mammals and birds. Accordingly, our discussion on brain sexual differentiation, including the statement in line 347, “This variation among species may represent the activation of neuroestrogen synthesis at life stages critical for sexual differentiation of behavior that are unique to each species”, remains well-supported. Additionally, given these considerations, while sex differences in neural circuit activation are evident in teleosts, substantial structural differences in these circuits are unlikely.
(10) Overall, the claims regarding the activational role of neuro-estrogens on male sexual behavior are supported by converging evidence from multiple mutant lines. The role of neuroestrogens on gene expression in the brain is mostly solid too. The data for females are comparatively weaker. Conclusions regarding sexual differentiation should be considered carefully.
We agree that the data for females are less extensive than for males. However, we have previously elucidated the mechanism by which estrogen/Esr2b signaling promotes female mating behavior (Nishiike et al., 2021). Accordingly, it follows that the new insights into female behavior gained from the cyp19a1b knockout model are more limited than those for males. Nevertheless, when integrated with our prior findings, the data on females in this study provide significant insights, and the overall mechanism through which estrogens promote female mating behavior is becoming clearer. Therefore, we do not consider the female data in this study to be incomplete or merely supplementary.
Recommendations For The Authors:
Reviewer #1 (Recommendations For The Authors):
The authors set out to answer an intriguing question regarding the hormonal control of innate social behaviors in medaka. Specifically, they wanted to test the effects of cyp19a1b mutation on mating and aggression in males. They also test these effects in females. Their approach takes them down several distinct experimental pathways, including one investigating how cyp19a1a function is related to androgen receptor expression and how estrogens themselves may act on the androgen receptor to modulate its expression, as well as how different esr genes may be involved. The study and its results are valuable and a clear, general conclusion of a pathway from brain aromatase>estrogens>esr genes> androgen receptor can be made. This is important, novel, and impactful. However, there are issues with how the study logic is set up, the approach for assessing certain behaviors, the statistics used, the interpretation of findings, and placing the findings in the proper context based on previous work, which manifests as a general issue where previous work is not properly attributed to.
Thank you for your thoughtful review. We have carefully addressed each specific comment, as detailed below.
Major comments:
(1) The background for the rationale of the current study is misleading and lacks proper context. The authors root the logic of their experiment in determining whether estrogens regulate male-typical behaviors because the current assumption is androgens are "solely responsible" for male-typical behaviors in teleosts. This is not the case. Previous studies have shown aromatase/estrogens are involved in male-typical aggression in teleosts. For example, to name a couple:
Huffman, L. S., O'Connell, L. A., & Hofmann, H. A. (2013). Aromatase regulates aggression in the African cichlid fish Astatotilapia burtoni. Physiology & behavior, 112, 77-83.
O'Connell, L. A., & Hofmann, H. A. (2012). Social status predicts how sex steroid receptors regulate complex behavior across levels of biological organization. Endocrinology, 153(3), 1341-1351.
And even a recent paper sheds light on a possible AR>aromatase.estradiol hypothesis of male typical behaviors:
Lopez, M. S., & Alward, B. A. (2024). Androgen receptor deficiency is associated with reduced aromatase expression in the ventromedial hypothalamus of male cichlids. Annals of the New York Academy of Sciences.
Interestingly, the authors cite Hufmann et al in the discussion, so I don't understand why they make the claims they do about estrogens and male-typical behavior.
Related to this, is an issue of proper attribution to published work. Indeed, missing are key references from lab groups using AR mutant teleosts. Here are a couple:
Yong, L., Thet, Z., & Zhu, Y. (2017). Genetic editing of the androgen receptor contributes to impaired male courtship behavior in zebrafish. Journal of Experimental Biology, 220(17), 3017-3021.
Alward, B. A., Laud, V. A., Skalnik, C. J., York, R. A., Juntti, S. A., & Fernald, R. D. (2020). Modular genetic control of social status in a cichlid fish. Proceedings of the National Academy of Sciences, 117(45), 28167-28174.
Ogino, Y., Ansai, S., Watanabe, E., Yasugi, M., Katayama, Y., Sakamoto, H., ... & Iguchi, T. (2023). Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish. Nature communications, 14(1), 1428.
As noted in Response to reviewer #1’s comment 3 on weaknesses, we have revised the Introduction and Discussion sections as follows.
Line 56: “solely responsible” in the Introduction has been modified to “largely responsible”.
Line 57: The text “This is consistent with the recent finding in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in male-typical courtship (10)” has been revised to “This is consistent with recent observations in a few teleost species that genetic ablation of AR severely impairs male-typical behaviors (13–16) and with findings in medaka fish (Oryzias latipes) that estrogens act through the ESR subtype Esr2b to prevent females from engaging in male-typical courtship (12)” to include previous studies on the behavior of AR mutant fish (Yong et al., 2017; Alward et al., 2020; Ogino et al., 2023; Nishiike and Okubo, 2024) in the Introduction.
Line 65: “It is worth mentioning that systemic administration of estrogens and an aromatase inhibitor increased and decreased male aggression, respectively, in several teleost species, potentially reflecting the behavioral effects of brain-derived estrogens (21–24)” has been added to the Introduction, providing an overview of previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015).
Line 367: “treatment of males with an aromatase inhibitor reduces their male-typical behaviors (31– 33)” has been edited to read “treatment of males with an aromatase inhibitor reduces their male-typical behaviors, while estrogens exert the opposite effect (21–24).”
After the revisions described above, the following references (#13, 14, and 22) have been added to the reference list:
L. Yong, Z. Thet, Y. Zhu, Genetic editing of the androgen receptor contributes to impaired male courtship behavior in zebrafish. J. Exp. Biol. 220, 3017–3021 (2017).
B. A. Alward, V. A. Laud, C. J. Skalnik, R. A. York, S. A. Juntti, R. D. Fernald, Modular genetic control of social status in a cichlid fish. Proc. Natl. Acad. Sci. U.S.A. 117, 28167–28174 (2020).
L. A. O’Connell, H. A. Hofmann, Social status predicts how sex steroid receptors regulate complex behavior across levels of biological organization. Endocrinology 153, 1341–1351 (2012).
While Lopez and Alward (2024) provide valuable insights into the regulation of cyp19a1b expression by androgens, our study focuses specifically on the functional aspects of cyp19a1b. Expanding the discussion to include expression regulation would divert from the primary focus of our manuscript. For this reason, we have opted not to cite this reference.
(2) As it is now, the authors are only citing a book chapter/review from their own group. This is a serious issue as it does not provide the proper context for the work. The authors need to fix their issues of attribution to previously published work and the proper interpretation of the work that they are aware of as it pertains to ideas proposed on the roles of androgens and estrogens in the control of male-typical behaviors. This is also important to get the citations right because the common use of "contrary to expectations" when describing their results is actually not correct. Many of the observations are expected to a degree. However, this doesn't take away from a generally stellar experimental design and mostly clear results. The authors do not need to rely on enhancing the impact of their paper by making false claims of unexpected findings. The depth and clarity of your findings are where the impact of your work is.
As detailed in Response to reviewer #1’s comment 3 on weaknesses, we have cited previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015) in the Introduction.
Additionally, as noted in Response to reviewer #1’s comment 4 on weaknesses, we have made the
following revisions to avoid phrases such as “contrary to expectation” and “unexpected.”
Line 76: “Contrary to our expectations” → “Remarkably.”
Line 109: “Contrary to this expectation, however” → “Nevertheless.”
Line 135: “Again, contrary to our expectation, cyp19a1b<sup>−/−</sup> males” → “cyp19a1b<sup>−/−</sup> males.”
Line 333: “unexpected” → “noteworthy.”
Line 337: “unexpected” → “notable.”
(3) The experimental design for studying aggression in males has flaws. A standard test like a residentintruder test should be used. An assay in which only male mutants are housed together? I do not understand the logic there and the logic for the approach isn't even explained. Too many confounds that are not controlled for. It makes it seem like an aspect of the study that was thrown in as an aside.
As noted in Response to reviewer #1’s comment 5 on weaknesses, medaka form shoals and lack strong territoriality. As a result, even slight differences in dominance between the resident and intruder can substantially impact the outcomes of the resident-intruder test. Therefore, we adopted an alternative approach in this study.
(4) Hormonal differences in the mutants seem to vary based on sex, and the meaning of these differences, or how they affect interpreting the findings, wasn't discussed. There was no acknowledegment of the fact that female central E2 was still at 50%, meaning the "rescue" experiments using peripheral injections are not given the proper context. For example, this is different than giving a fish with only 16% of their normal central E2 an E2 injection. Missing as well is a clear hypothesis for why E2 injections did not rescue aggression deficits in cyp19a1b mutant males.
Line 385: As the reviewer pointed out, the degree of brain estrogen reduction in cyp19a1b-deficient fish differs greatly between males and females. This is likely because females receive a large supply of estrogens from the ovaries. Given that estrogen levels in cyp19a1b-deficient females were 50% of those in wild-type females, it can be inferred that half of their brain estrogens are synthesized locally, while the other half originates from the ovaries. This is an important finding, and we have already noted in the Discussion that “females have higher brain levels of estrogens, half of which are synthesized locally in the brain (i.e., neuroestrogens)” However, as this explanation was not sufficiently clear, we have revised it to “females have higher brain levels of estrogens, with half being synthesized locally and the other half supplied by the ovaries.”
The reviewer raised a concern that conducting the estrogen rescue experiment in females, where 50% of brain estrogens remain, might be inappropriate. However, as this experiment was conducted exclusively in males, this concern is not applicable.
Line 377: As noted in the reviewer’s subsequent comment, the failure of aggression recovery in E2treated cyp19a1b-deficient males could be due to insufficient induction of ara/arb expression in aggression-relevant brain regions. To address this concern, we have inserted the following statement into the Discussion after “the development of male behaviors may require moderate neuroestrogen levels that are sufficient to induce the expression of ara and arb, but not esr2b, in the underlying neural circuitry”: “This may account for the lack of aggression recovery in E2-treated cyp19a1b-deficient males in this study.”
(5) In relation to that, the "null" results may have some of the most interesting implications, but they are barely discussed. For example, what does it mean that E2 didn't restore aggression in male cyp19 mutants? Is this a brain region factor? Could this relate to findings from Lopez et al NYAS, where male and female Ara mutants show different effects on brain-region-specific aromatase expression? And maybe this relates to the different impact of estrogens on ar expression. Were the different effects impacted in aggression areas? Maybe this is why E2 injection didn't retore aggression in males. You could make the argument that: (1) E2 doesn't restore ar expression in aggression regions and that's why there was no rescue. Or (2) that the circuits in adulthood that regulate aggression are NOT dependent on aggression but in early development they are. Another null finding not expanded on is why the two esr2a mutant lines showed differences. There is no reason to trust one line over the other, meaning we still don't know whether esr2a is required for latency to follow.
As stated in our response to the previous comment, we have added the following text to the Discussion (line 377): “This may account for the lack of aggression recovery in E2-treated cyp19a1b-deficient males in this study.” Meanwhile, as discussed in lines 341–342, it is highly unlikely that the neural circuits regulating aggression are primarily influenced by early-life estrogen exposure, because androgen administration in adulthood alone is sufficient to induce high levels of aggression in both sexes. This notion is further supported by previous observations that cyp19a1b expression in the brain is minimal during embryonic development (Okubo et al., 2011, J Neuroendocrinol, 23:412–423).
The findings of Lopez and Alward (2024) pertain to the regulation of cyp19a1b expression by androgen receptors. While this represents an important aspect of neuroendocrine regulation, it does not appear to be directly relevant to our discussion on cyp19a1b-mediated regulation of androgen receptor expression.
To ensure the reliability of behavioral analyses in mutant fish, we consider a phenotype valid only when it is consistently observed in two independent mutant lines. In the mating behavior test examining esr2adeficient males using esr2b-deficient females as stimulus females, Δ8 line males exhibited a shorter latency to initiate following than wild-type males, whereas Δ4 line males did not. This discrepancy led us to refrain from drawing conclusions about the role of esr2a in mating behavior, even though the mating behavior test using wild-type females as stimulus females yielded consistent results in the Δ8 and Δ4 lines. Therefore, we do not consider the reviewer’s concern to be a significant issue.
(6) Not sure what's going on with the statistics, but it is not appropriate here to treat a "control" group as special. All groups are "experimental" groups. There is nothing special about the control group in this context. all should be Bonferroni post-hoc tests.
Line 619: As detailed in Response to reviewer #1’s comment 7 on weaknesses, we consider Dunnett’s test the most appropriate choice for the experiments presented in Figures 4C and 4E. We acknowledge that the reviewer’s concern may stem from the phrase “comparisons between control and experimental groups” in the Materials and Methods section. To clarify this point, we have revised it to “comparisons between untreated and E2-treated groups in Fig. 4, C and D” for clarity.
Minor comments:
Line 47: then how can you say the aromatization hypothesis is "correct"? it only applies to a few species so far. Need to change the framing, not state so strongly such a vague thing as a hypothesis being "correct".
Line 45: To address this concern, we have modified “widely accepted as correct” to “widely acknowledged”, ensuring a more precise characterization.
Figure 1: looks like a dosage effect in males but not females. this should be discussed at some point, even if just to mention a dosage effect exists and put it in context.
Line 91: We have revised the sentence “In males, brain E2 in heterozygotes (cyp19a1b+/−) was also reduced to 45% of the level in wild-type siblings (P = 0.0284) (Fig. 1A)” by adding “, indicating a dosage effect of cyp19a1b mutation” to make this point explicit.
Were male cyp19 KO aggressive towards females?
We have not observed cyp19a1b-deficient males exhibiting aggressive behavior towards females in our experiments. Therefore, we do not consider them aggressive toward females.
Please explain how infertility would lead to reduced mating.
Line 142: As the reviewer has questioned, even if cyp19a1b-deficient males exhibit infertility due to efferent duct obstruction, it is difficult to imagine that this directly leads to reduced mating. However, the inability to release sperm could indirectly affect behavior. To address this, we have added “, possibly due to the perception of impaired sperm release” after “If this is also the case in medaka, the observed behavioral defects might be secondary to infertility.”
Describe something about the timing of the treatment here. How can peripheral E2 injections restore it when peripheral levels are normal? Did these injections restore central levels? This needs to be shown experimentally.
Line 517: As described in the Materials and Methods, E2 treatment was conducted by immersing fish in E2-containing water for 4 days. However, we had not explicitly stated that the water was changed daily to maintain the nominal concentration. To clarify this and address reviewer #2’s comment 9, we have revised “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan) or vehicle (ethanol) alone by immersion in water for 4 days” to “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan), which was first dissolved in 100% ethanol (vehicle), or with the vehicle alone by immersion in water for 4 days, with daily water changes to maintain the nominal concentration.”
Line 522: The treatment effectively restored mating activity and ara/arb expression in the brain, suggesting a sufficient increase in brain E2 levels. However, we did not measure the actual increase, and its extent remains uncertain. To reflect this in the manuscript, we have now added the following sentence: “Although the exact increase in brain E2 levels following E2 treatment was not quantified, the observed positive effects on behavior and gene expression suggest that it was sufficient.”
I know the nomenclature differs among those who study teleosts, but it's ARa and then gene is ar1 (as an example; arb would be ar2). You're recommended the following citation to remain consistent:
Munley, K. M., Hoadley, A. P., & Alward, B. A. (2023). A phylogenetics-based nomenclature system for steroid receptors in teleost fishes. General and Comparative Endocrinology, 114436.
Paralogous genes resulting from the third round of whole-genome duplication in teleosts are typically designated by adding the suffixes “a” and “b” to their gene symbols. This convention also applies to the two androgen receptor genes, commonly referred to as ara and arb. While the alternative names ar1 and ar2 may gain broader acceptance in the future, ara and arb remain more widely used at present. Therefore, we have chosen to retain ara and arb in this manuscript.
Line 268: how is this "suggesting" less aggression? They literally showed fewer aggressive displays, so it doesn't suggest it - it literally shows it.
Line 285: Following this thoughtful suggestion, we have changed “suggesting less aggression” to “showing less aggression.”
Line 317: how can you still call it the primary driver?
The stimulatory effects of aromatase/estrogens on male-typical behaviors are exerted through the potentiation of androgen/AR signaling. Thus, we still believe that androgens—specifically 11KT in teleosts—serve as the primary drivers of these behaviors.
Line 318: not all deficits, like aggression, were rescued.
Line 334: To address this comment, “These behavioral deficits were rescued by estrogen administration, indicating that reduced levels of neuroestrogens are the primary cause of the observed phenotypes: in other words, neuroestrogens are pivotal for male-typical behaviors in teleosts” has been modified and now reads “Deficits in mating were rescued by estrogen administration, indicating that reduced brain estrogen levels are the primary cause of the observed mating impairment; in other words, brain-derived estrogens are pivotal at least for male-typical mating behaviors in teleosts.”
Line 324: what do you mean by "sufficient"? To show that, you'd have to castrate the male and only give estrogen back. the authors continue to overstate virtually every aspect of their study, seemingly in an unnecessary manner.
Line 341: Our intention was to convey that brain-derived estrogens early in life are not essential for the expression of male-typical behaviors in teleosts. However, we recognize that the term “sufficient” could be misinterpreted as implying that estrogens alone are adequate, without contributions from other factors such as androgens. To clarify this, we have revised the text from “neuroestrogen activity in adulthood is sufficient for the execution of male-typical behaviors, while that in early in life is not requisite. Thus, while” to “brain-derived estrogens early in life is not essential for the execution of male-typical behaviors. While.”
Line 329: so? in adult mice, amygdala aromatase neurons still regulate aggression. The amount in adulthood seems less important compared to site-specific functions.
Line 346: We do not intend to suggest that brain aromatase activity in adulthood plays a negligible role in male behaviors in rodents, as we have already acknowledged its necessity in the Introduction (lines 42–43). To enhance clarity and prevent misinterpretation, we have added “, although it remains important for male behavior in adulthood” to the end of the sentence: “brain aromatase activity in rodents reaches its peak during the perinatal period and thereafter declines with age.”
Line 351: This contradicts what you all have been saying.
Line 65: As mentioned in Response to reviewer #1’s comment 3 on weaknesses, the following text has been added to the Introduction: “It is worth mentioning that systemic administration of estrogens and an aromatase inhibitor increased and decreased male aggression, respectively, in several teleost species, potentially reflecting the behavioral effects of brain-derived estrogens (21–24)”, providing an overview of previous studies on the effects of estrogens and aromatase on male fish aggression (Hallgren et al., 2006; O’Connell and Hofmann, 2012; Huffman et al., 2013; Jalabert et al., 2015). With this revision, we believe the inconsistency has been addressed.
Line 367: Additionally, we have revised the sentence from “treatment of males with an aromatase inhibitor reduces their male-typical behaviors (31–33)” to “treatment of males with an aromatase inhibitor reduces their male-typical behaviors, while estrogens exert the opposite effect (21–24).”
Line 360: change to "...possibility that is not mutually exclusive,"
Line 378: We have revised the phrase as suggested from “Another possibility, not mutually exclusive,” to “Another possibility that is not mutually exclusive.”
Line 363: but it didn't rescue aggression
Line 381: In response, we have revised the sentence from “This possibility is supported by the present observation that estrogen treatment facilitated mating behavior in cyp19a1b-deficient males but not in their wild-type siblings” to “This possibility is at least likely for mating behavior, as estrogen treatment facilitated mating behavior in cyp19a1b-deficient males but not in their wild-type siblings.”
Line 367: on average
To explain the sex differences in the role of aromatase, what about the downstream molecular or neural targets? In mammals, hodology is related to sex differences. there could be convergent sex differences in regulating the same type of behaviors as well.
Our findings demonstrate that brain-derived estrogens promote the expression of ara, arb, and their downstream target genes vt and gal in males, while enhancing the expression of npba, a downstream target of Esr2b signaling, in females. The identity of additional target genes and their roles in specific neural circuits remain to be elucidated, and we aim to address these in future research.
Lines 378-382: this doesn't logically follow. pgf2a could be the target of estrogens which in the intact animal do regulate female sexual receptivity. And how can you say this given that your lab has shown in esr2b mutants females don't mate?
We agree that PGF2α signaling may be activated by estrogen signaling, as stated in lines 404–407: “the present finding provides a likely explanation for this apparent contradiction, namely, that neuroestrogens, rather than or in addition to ovarian-derived circulating estrogens, may function upstream of PGF2α signaling to mediate female receptivity.” The observation that esr2b-deficient females do not accept male courtship is also stated in lines 401–403: “we recently challenged it by showing that female medaka deficient for esr2b are completely unreceptive to males, and thus estrogens play a critical role in female receptivity.”
Line 396-397: or the remaining estrogens are enough to activate esr2b-dependent female-typical mating behaviors.
We agree that cyp19a1b deficiency did not completely preclude female mating behavior, most likely because residual estrogens in the brains of cyp19a1b-deficient females enable weak activation of Esr2b signaling. However, the relevant section in the Discussion is not focused on examining why mating behavior persisted, but rather on considering the implications of this finding for the neural circuits regulating mating behavior. Therefore, incorporating the suggested explanation here would shift the focus and would not be appropriate.
Line 420-421: this is a lot of variation. Was age controlled for?
The time required for medaka to reach sexual maturity varies with rearing density and food availability. Due to space constraints, we adjust these parameters as needed, which led to variation in the ages of the experimental fish. However, since all experiments were conducted using sibling fish of the same age that had just reached sexual maturity, we believe this does not affect our conclusions.
Line 457: have these kits been validated in medaka?
Although we have not directly validated its applicability in medaka, its extensive use in this species suggests that it us unlikely to pose any issues (e.g., Ussery et al., 2018, Aquat Toxicol, 205:58–65; Lee et al., 2019, Ecotoxicol Environ Saf, 173:174–181; Kayo et al., 2020, Gen Comp Endocrinol, 285:113272; Fischer et al., 2021, Aquat Toxicol, 236:105873; Royan et al., 2023, Endocrinology, 164:bqad030).
Line 589, re fish that spawned: how many times did this happen? Please note it is based on genotype and experiment. This could be important.
Line 627: In response to this comment, we have added the following details: “Specifically, 7/18 cyp19a1b<sup>+/+</sup>, 11/18 cyp19a1b<sup>+/−</sup>, and 6/18 cyp19a1b<sup>−/−</sup> males were excluded in Fig. 1D; 6/10 cyp19a1b<sup>+/+</sup>, 3/10 cyp19a1b<sup>+/−</sup>, and 6/10 cyp19a1b<sup>−/−</sup> females were excluded in Fig. 6B; 2/23 esr1+/+ and 5/24 esr1−/− males were excluded in Fig. S7; 2/24 esr2a+/+ and 3/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8A; 0/23 esr2a+/+ and 0/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8B.”
Reviewer #2 (Recommendations For The Authors):
Abstract:
(A1) The framing of neuroestrogens being important for male-typical rodents, and not for other vertebrate lineages, does not account for other groups (birds) in which this is true (the authors can consult their cited work by Balthazart (Reference 6) for extensive accounting of this). This makes the novelty clause in the abstract "indicating that neuro-estrogens are pivotal for male-typical behaviors even in nonrodents" less surprising and should be acknowledged by the authors by amending or omitting this novelty clause. The findings regarding androgen receptor transcription (next sentence) are more important and pertinent.
Line 27: We recognize that the aromatization hypothesis applies to some birds, including zebra finches, as stated in the Introduction (lines 48–49) and Discussion (lines 432–433). However, this was not reflected in the Abstract. Following the reviewer’s suggestion, we have changed “in non-rodents” to “in teleosts.”
(A2) The medaka line that has been engineered to have aromatase absent in the brain is presented briefly in the abstract, but can be misinterpreted as naturally occurring. This should be amended, by including something like "engineered" or "directed mutant" before 'male medaka fish'.
Line 24: We have added “mutagenesis-derived” before “male medaka fish” in response to this comment.
Introduction:
(I1) The paragraph on teleost brain aromatase should acknowledge that while the capacity for estrogen synthesis in the brain is 100-1000 fold higher in teleosts as compared to rodents and other vertebrates, the majority of this derives from glial and not neural sources. This can be confusing for readers since the term 'neuroestrogens' often refers to the neuronal origin and signalling. And this observation includes the exclusive radial glial expression of cyp19a1b in medaka (Diotel et al., 2010), and first discovered in midshipman (Forlano et al., 2001), each of which should also be cited here. In addition, the authors expend much text comparing teleosts and rodents, but it is worth expanding these kinds of comparisons, especially by pointing out that parts of the primate brain are found to densely express aromatase (see work by Ei Terasawa and others).
In response to this comment and a similar comment from reviewer #1, we have replaced “neuroestrogens” with “brain-derived estrogens” or “brain estrogens” throughout the manuscript.
Line 63: We have also added the text “In teleost brains, including those of medaka, aromatase is exclusively localized in radial glial cells, in contrast to its neuronal localization in rodent brains (18– 20).” As a result of this addition, we have changed “This observation suggests” to “These observations suggest” in the subsequent sentence.
Line 51: Additionally, to include information on aromatase in the primate brain, we have added the following text: “In primates, the hypothalamic aromatization of androgens to estrogens plays a central role in female gametogenesis (10) but is not essential for male behaviors (7, 8).”
The following references (#10 and 18–20), cited in the newly added text above, have been included in the reference list, with other references renumbered accordingly:
E. Terasawa, Neuroestradiol in regulation of GnRH release. Horm. Behav. 104, 138–145 (2018).
P. M. Forlano, D. L. Deitcher, D. A. Myers, A. H. Bass, Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source. J. Neurosci. 21, 8943–8955 (2001).
N. Diotel, Y. Le Page, K. Mouriec, S. K. Tong, E. Pellegrini, C. Vaillant, I. Anglade, F. Brion, F. Pakdel, B. C. Chung, O. Kah, Aromatase in the brain of teleost fish: expression, regulation and putative functions. Front. Neuroendocrinol. 31, 172–192 (2010).
A. Takeuchi, K. Okubo, Post-proliferative immature radial glial cells female-specifically express aromatase in the medaka optic tectum. PLoS One 8, e73663 (2013).
(I2) It is difficult to resolve from the introduction and work cited how restricted cyp19a1b is to the medaka brain. Important for the results of this study, it is not clear whether it is more of a bias in the brain vs other tissues, or if the cyp19a1b deficiency is restricted to the brain, and gonadal/peripheral cyp19 expression persists. The authors need to improve their consideration of the alternatives, i.e., that this manipulation is not somehow affecting: 1) peripheral aromatase expression (either cyp19a1a or cyp19a1b) in the gonad or elsewhere, 2) compensatory processes, such as other steroidogenic genes (are androgen synthesizing enzymes increasing?).
Our previous study demonstrated that cyp19a1b is expressed in the gonads, but at levels tens to hundreds of times lower than those in the brain (Okubo et al., 2011, J Neuroendocrinol 23:412–423). Additionally, a separate study in medaka reported that cyp19a1b expression in the ovary is considerably lower than that of cyp19a1a (Nakamoto et al., 2018, Mol Cell Endocrinol 460:104–122). Given these observations, any potential effect of cyp19a1b knockout on peripheral estrogen synthesis is likely negligible. Indeed, Figures S1C and S1D confirm that cyp19a1b knockout does not alter peripheral E2 levels.
Line 72: To incorporate this information into the Introduction and address the following comment, we have added the following text: “In medaka, cyp19a1b is also expressed in the gonads, but only at a level tens to hundreds of times lower than in the brain and substantially lower than that of cyp19a1a (26, 27).”
The following references (#26 and 27), cited in the newly added text above, have been included in the reference list, with other references renumbered accordingly:
K. Okubo, A. Takeuchi, R. Chaube, B. Paul-Prasanth, S. Kanda, Y. Oka, Y. Nagahama, Sex differences in aromatase gene expression in the medaka brain. J. Neuroendocrinol. 23, 412–423 (2011).
M. Nakamoto, Y. Shibata, K. Ohno, T. Usami, Y. Kamei, Y. Taniguchi, T. Todo, T. Sakamoto, G. Young, P. Swanson, K. Naruse, Y. Nagahama, Ovarian aromatase loss-of-function mutant medaka undergo ovary degeneration and partial female-to-male sex reversal after puberty. Mol. Cell. Endocrinol. 460, 104–122 (2018).
We have not assessed whether the expression of other steroidogenic enzymes is altered in cyp19a1bdeficient fish, and this may be investigated in future studies.
(I3) Related, there are documented sex differences in the brain expression of cyp19a1b especially in adulthood (Okubo et al 2011) and this study should be cited here for context.
Line 72: As stated in our previous response, we have cited Okubo et al. (2011) by adding the following sentence: “In medaka, cyp19a1b is also expressed in the gonads, but only at a level tens to hundreds of times lower than in the brain and substantially lower than that of cyp19a1a (26, 27).”
Methods
(M1) The rationale is unclear as presented for using mutagen screening for cype19a1b while using CRISPR for esr2a. Are there methodological/biochemical reasons why the authors chose to not use the same method for both?
At the time we generated the cyp19a1b knockouts, genome editing was not yet available, and the TILLING-based screening was the only method for obtaining mutants in medaka. In contrast, by the time we generated the esr2a knockouts, CRISPR/Cas9 had become available, enabling a more efficient and convenient generation of knockout lines. This is why the two knockout lines were generated using different methods.
(M2) Measurement of steroids in biological matrices is not straightforward, and it is good that the authors use multiple extraction steps (organic followed by C18 columns) before loading samples on the ELISA plates, which are notoriously sensitive. Even though these methods have been published before by this group of authors previously, the quality control and ELISA performance values (recovery, parallelism, etc.) should be presented for readers to evaluate.
Thank you for appreciating our sample purification method. Unfortunately, we have not evaluated the recovery rate or parallelism, but we recognize this a subject for future studies.
(M3) Mating behavior - E2 treated males were not co-housed with social partners for the full 24 hr before testing, but instead a few hours (?) prior to testing. The rationale for this should be spelled out explicitly.
Line 494: In response to this comment, we have added “to ensure the efficacy of E2 treatment” to the end of the sentence “The set-up was modified for E2-treated males, which were kept on E2 treatment and not introduced to the test tanks until the day of testing.”
(M4) The E2 treatment is listed as 1ng/ml vs. vehicle (ethanol). Is the E2 dissolved in 100% ethanol for administration to the tank water? Clarification is needed.
Line 517: As the reviewer correctly assumed, E2 was first dissolved in 100% ethanol before being added to the tank water. To provide this information and address reviewer #1’s minor comment 5, we have revised “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan) or vehicle (ethanol) alone by immersion in water for 4 days” to “males were treated with 1 ng/ml of E2 (Fujifilm Wako Pure Chemical, Osaka, Japan), which was first dissolved in 100% ethanol (vehicle), or with the vehicle alone by immersion in water for 4 days, with daily water changes to maintain the nominal concentration.”
(M5) The authors exclude fish from the analysis of courtship display behavior for those individuals that spawned immediately at the start of the testing (and therefore it was impossible to register courtship display behaviors). How often did fish in the various treatment groups exhibit this "fast spawning" behavior? Was the occurrence rate different by treatment group? It is unlikely that these omissions from the data set drove large-scale patterns, but an indication of how often this occurred would be reassuring.
Line 627: In response to this comment, we have included the following details: “Specifically, 7/18 cyp19a1b<sup>+/+</sup>, 11/18 cyp19a1b<sup+/−</sup>, and 6/18 cyp19a1b<sup>−/−</sup> males were excluded in Fig. 1D; 6/10 cyp19a1b+/+, 3/10 cyp19a1b+/−, and 6/10 cyp19a1b<sup>−/−</sup> females were excluded in Fig. 6B; 2/23 esr1+/+ and 5/24 esr1−/− males were excluded in Fig. S7; 2/24 esr2a+/+ and 3/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8A; 0/23 esr2a+/+ and 0/23 esr2a<sup>−/−</sup> males were excluded in Fig. S8B.” These data indicate that the proportion of excluded males is nearly constant within each trial and is independent of the genotype of the focal fish.
Results
(R1) It is striking to see the genetic-'dose' dependent suppression of brain E2 content by heterozygous and homozygous cyp19a1b deficiency, indicating that, as the authors point out, the majority of E2 in the male medaka brain (and 1/2 in the female brain) have a brain-derived origin. It is important also for the interpretation that there are large compensatory increases in brain levels of androgens, when E2 levels drop in the cyp19a1b mutant homozygotes. This latter point should receive more attention.
Also, there are large increases in peripheral androgen levels in the homozygote mutants for cyp19a1b in both males and females. This indicates a peripheral effect in addition to the clear brain knockdown of E2 synthesis. These nuances need to be addressed.
In response to this comment, we have revised the Results section as follows:
Line 91: “, indicating a dosage effect of cyp19a1b mutation” has been added to the end of the sentence “In males, brain E2 in heterozygotes (cyp19a1b<sup>+/−</sup>) was also reduced to 45% of the level in wild-type siblings (P = 0.0284) (Fig. 1A).”
Line 94: To draw more attention to the increase in brain androgen levels caused by cyp19a1b deficiency, “Brain levels of testosterone” has been modified to “Strikingly, brain levels of testosterone.”
Line 100: “Their peripheral 11KT levels also increased 3.7- and 1.8-fold, respectively (P = 0.0789, males; P = 0.0118, females) (Fig. S1, C and D)” has been modified and now reads “In addition, peripheral 11KT levels in cyp19a1b<sup>−/−</sup> males and females increased 3.7- and 1.8-fold, respectively (P = 0.0789, males; P = 0.0118, females) (Fig. S1, C and D), indicating peripheral influence in addition to central effects.”
(R2) The interpretation on page 4 that cyp19a1b deficient males are 'less motivated' to mate is premature, given the behavioral measures used in this study. There are several competing explanations for these findings (e.g., alterations in motivation, sensory discrimination, preference, etc.) that could be followed up in future work, but the current results are not able to distinguish among these possibilities.
Line 112: We agree that the possibility of altered cognition or sexual preference cannot be dismissed. To incorporate this perspective, we have revised the text “, suggesting that they are less motivated to mate” to “These results suggest that they are less motivated to mate, though an alternative interpretation that their cognition or sexual preference may be altered cannot be dismissed.”
(R3) On page 5, the authors present that peripheral E2 manipulation (delivery to the fish tank) restores courtship behavior in males, and then go on to erroneously conclude that this demonstrates "that reduced E2 in the brain was the primary cause of the mating defects, indicating a pivotal role of neuroestrogens in male mating behavior." Because this is a peripheral E2 treatment, there can be manifold effects on gonadal physiology or other endocrine events that can have indirect effects on the brain and behavior. Without manipulation of E2 directly to the brain to 'rescue' the cyp19a1b deficiency, the authors cannot conclude that these effects are directly on the central nervous system. Tellingly, the tank E2 treatment did not rescue aggressive behavior, suggestive of the potential for indirect effects.
Line 155: As detailed in Response to reviewer #2’s specific comment 1, we have revised the text from “These results demonstrated that reduced E2 in the brain was the primary cause of the mating defects, indicating a pivotal role of neuroestrogens in male mating behavior. In contrast” to “These results suggest that reduced E2 in the brain is the primary cause of the mating defects, highlighting a pivotal role of brain-derived estrogens in male mating behavior. However, caution is warranted, as an indirect peripheral effect of bath-immersed E2 on behavior cannot be ruled out, although this is unlikely given the comparable peripheral E2 levels in cyp19a1b-deficient and wild-type males. In contrast to mating.”
(R4) The downregulation of androgen-dependent gene expression (vasotocin in pNVT and galanin in pPMp) in the cyp19a1b deficient males (Figure 3) could be due to exceedingly high levels of brain androgens in the cyp19a1b deficient males. The best way to test the idea that estrogens can restore the expression to be more wild-type directly (like what is happening for ara and arb) is to look at these same markers (vasotocin and galanin) in these same brain areas in the brains of E2-treated males. The authors should have these brains from Figure 2. Unless I missed something, those experiments were not performed/reported here. It is clear that the ara and arb receptors have EREs and are 'rescued' by E2 treatment, but in principle, there could be indirect actions for reasons stated above for the behavior due to the peripheral E2 tank application.
Thank you for your insightful comment. We agree that the current results cannot exclude the possibility that excessive androgen levels caused the downregulation of vt and gal. However, our previous studies showed that excessive 11KT administration to gonadectomized males and females increased the expression of these genes to levels comparable to wild-type males (Yamashita et al., 2020, eLife, 9:e59470; Kawabata-Sakata et al., 2024, Mol Cell Endocrinol 580:112101), making this scenario unlikely. That said, testing whether estrogen treatment restores vt and gal expression in cyp19a1bdeficient males would be informative, and we see this as an important direction for future research.
Discussion
(D1) The authors need to clarify whether EREs are found in other vertebrate AR introns, or is this unique to the teleost genome duplication?
We have identified multiple ERE-like sequences within intron 1 of the mouse AR gene. However, sequence data alone do not provide sufficient evidence of their functionality, rendering this information of limited relevance. Therefore, we have chosen not to include this discussion in the current paper.
Reviewer #3 (Recommendations For The Authors):
(1) The authors are strongly encouraged to report information regarding the effect of Cyp19a1b deletion on the brain content of aromatase protein (ideally both isoforms investigated separately) as the two isoforms are mostly but not completely brain vs gonad specific. The analysis of other tissues would also strengthen the characterization of this model.
We agree that measuring aromatase protein levels in the brain of our fish would be valuable for confirming the loss of cyp19a1b function. However, as no suitable method is currently available, this issue will need to be addressed in future studies. While this constitutes indirect evidence, the observed reduction in brain E2 levels, with no change in peripheral E2 levels, in cyp19a1b-deficient fish strongly suggests the loss of cyp19a1b function, as noted in Response to reviewer #3’s comment 1 on weaknesses.
(2) As presented, this study reads as niche work. A better description of the behavior and reproductive significance of the different aspects of the behavioral sequence would allow a better understanding of the results and would thus allow the non-specialist to appreciate the significance of the observations.
Line 103: In response to this comment and Reviewer #3’s comment 2 on weaknesses, we have revised the sentence from “The mating behavior of medaka follows a stereotypical pattern, wherein a series of followings, courtship displays, and wrappings by the male leads to spawning” to “The mating behavior of medaka follows a stereotypical sequence. It begins with the male approaching and closely following the female (following). The male then performs a courtship display, rapidly swimming in a circular pattern in front of the female. If the female is receptive, the male grasps her with his fins (wrapping), culminating in the simultaneous release of eggs and sperm (spawning)” in order to provide a more detailed description of medaka mating behavior.
(3) The data regarding female behavior are limited and incomplete. It is suggested to keep this for another manuscript unless data on the behavior of the female herself is added. Indeed, analyzing female's behavior from the male's perspective complicates the interpretation of the results while a description of what the females do would provide valuable and interpretable information.
We thank the reviewer for this thoughtful suggestion and agree that the data and discussion for females are less extensive than for males. However, we have previously elucidated the mechanism by which estrogen/Esr2b signaling promotes female mating behavior (Nishiike et al., 2021). Accordingly, it follows that the new insights into female behavior gained from the cyp19a1b knockout model are more limited than those for males. Nevertheless, when combined with our prior findings, the female data in this study offer valuable insights, and the overall mechanism through which estrogens promote female mating behavior is becoming clearer. Therefore, we do not consider the female data in this study to be incomplete or merely supplementary.
(4) In Figure 2, the validity to run multiple T-tests rather than a two-way ANOVA comparing TRT and genotype is questionable. Moreover, why are the absolute values in CTL higher than in the initial experiment comparing genotypes for ara in PPa, pPPp, and NVT as well as for arb in aPPp. More importantly, these graphs do not seem to reproduce the genotype effects for ara in pPPp and NVT and for arb in aPPp.
The data in Figures 2J and 2K were analyzed with an exclusive focus on the difference between vehicletreated and E2-treated males, without considering genotype differences. Therefore, the use of T-tests for significance testing is appropriate.
As the reviewer noted, the overall ara expression area is larger in Figure 2J than in Figure 2F. However, as detailed in Response to reviewer #3’s comment 8 on weaknesses, the relative area ratios of ara expression among brain nuclei are consistent between the two figures, indicating the reproducibility of the results. Thus, we consider this difference unlikely to affect the conclusions of this study.
Additionally, the differences in ara expression in pPPp and arb expression in aPPp between wild-type and cyp19a1b-deficient males appear smaller in Figures 2J and 2K compared to Figures 2F and 2H. This is likely due to the smaller sample size used in the experiments for Figures 2J and 2K, which makes the differences less distinct. However, since the same genotype-dependent trends are observed in both sets of figures, the conclusion that ara and arb expression is reduced in cyp19a1b-deficient male brains remains valid.
(5) More information is required regarding the analysis of single ISH - How was the positive signal selected from the background in the single ISH analyses? How was this measure standardized across animals? How many sections were imaged per region? Do the values represent unilateral or bilateral analysis?
Line 540: Following this comment, we have provided additional details on the single ISH method in the manuscript. Specifically, “, and the total area of signal in each brain nucleus was calculated using Olyvia software (Olympus)” has been revised to “The total area of signal across all relevant sections, including both hemispheres, was calculated for each brain nucleus using Olyvia software (Olympus). Images were converted to a 256-level intensity scale, and pixels with intensities from 161 to 256 were considered signals. All sections used for comparison were processed in the same batch, without corrections between samples.”
(6) More information should be provided in the methods regarding the image analysis of double ISH. In particular, what were the criteria to consider a cell as labeled are not clear. This is not clear either from the representative images.
Line 596: To provide additional details on the single ISH method in the manuscript, we have added the following sentence: “Cells were identified as coexpressing the two genes when Alexa Fluor 555 and fluorescein signals were clearly observed in the cytoplasm surrounding DAPI-stained nuclei, with intensities markedly stronger than the background noise.”
(7) There is no description of the in silico analyses run on ESR2a in the methods.
The method for identifying estrogen-responsive element-like sequences in the esr2a locus is described in line 549: “Each nucleotide sequence of the 5′-flanking region of ara and arb was retrieved from the Ensembl medaka genome assembly and analyzed for potential canonical ERE-like sequences using Jaspar (version 5.0_alpha) and Match (public version 1.0) with default settings.”
However, the method for domain identification in Esr2a was not described. Therefore, we have added the following text in line 469: “The DNA- and ligand-binding domains of medaka Esr2a were identified by sequence alignment with yellow perch (Perca flavescens) Esr2a, for which these domain locations have been reported (58).”
The following reference (#58), cited in the newly added text above, have been included in the reference: S. G. Lynn, W. J. Birge, B. S. Shepherd, Molecular characterization and sex-specific tissue expression of estrogen receptor α (esr1), estrogen receptor βa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 149, 126–147 (2008).
(8) Information about the validation steps of the EIA that were carried out as well as the specificity of the antibody the steroids and the extraction efficacy should be provided.
We have not directly validated the applicability of the EIA kit, but its extensive use in medaka suggests that it us unlikely to pose any issues (e.g., Ussery et al., 2018, Aquat Toxicol, 205:58–65; Lee et al., 2019, Ecotoxicol Environ Saf, 173:174–181; Kayo et al., 2020, Gen Comp Endocrinol, 285:113272; Fischer et al., 2021, Aquat Toxicol, 236:105873; Royan et al., 2023, Endocrinology, 164:bqad030).
The specificity (cross-reactivity) of the antibodies is detailed as follows.
(1) Estradiol ELISA kits: estradiol, 100%; estrone, 1.38%; estriol, 1.0%; 5α-dihydrotestosterone, 0.04%; androstenediol, 0.03%; testosterone, 0.03%; aldosterone, <0.01%; cortisol, <0.01%; progesterone, <0.01%.
(2) Testosterone ELISA kits: testosterone, 100%; 5α-dihydrotestosterone, 27.4%; androstenedione, 3.7%; 11-ketotestosterone, 2.2%; androstenediol, 0.51%; progesterone, 0.14%; androsterone, 0.05%; estradiol, <0.01%.
(3) 11-Keto Testosterone ELISA kits: 11-ketotestosterone, 100%; adrenosterone, 2.9%; testosterone, <0.01%.
As this information is publicly available on the manufacturer’s website, we deemed it unnecessary to include it in the manuscript.
Unfortunately, we have not evaluated the extraction efficacy of the samples, but we recognize this a subject for future studies.
(9) I wonder whether the evaluation of the impact of the mutation by comparing the behavior of a group of wild-type males to a group of mutated males is the most appropriate. Justifying this approach against testing the behavior of one mutated male facing one or several wild-type males would be appreciated.
We agree that the resident-intruder test, in which a single focal resident is confronted with one or more stimulus intruders, is the most commonly used method for assessing aggression. However, medaka form shoals and lack strong territoriality, and even slight dominance differences between the resident and the intruder can increase variability in the results, compromising data consistency. Therefore, in this study, we adopted an alternative approach: placing four unfamiliar males together in a tank and quantifying aggressive interactions in total. This method allows for the assessment of aggression regardless of territorial tendencies, making it more appropriate for our investigation.
(10) Lines 329-331: this sentence should be rephrased as it contributes to the confusion between sexual differentiation and activation of circuits. The restoration of sexual behavior by adult estrogen treatment pleads in favor of an activational role of neuro-estrogens on behavior rather than an organizational role. Therefore, referring to sexual differentiation is misleading, even more so that the study never compares sexes.
As detailed in Response to reviewer #3’s comment 9 on weaknesses, we consider that all factors that cause sex differences, including the transient effects of adult steroids, need to be incorporated into a theory of sexual differentiation. In teleosts, since steroids during early development have little effect and sexual differentiation primarily relies on steroid action in adulthood, our discussion on brain sexual differentiation remains valid, including the statement in line 347: “This variation among species may represent the activation of neuroestrogen synthesis at life stages critical for sexual differentiation of behavior that are unique to each species.”
(11) Lines 384-386: I may have missed something but I do not see data supporting the notion that neuroestrogens may function upstream of PGF2a signaling to mediate female receptivity.
Line 403: We acknowledge that our explanation was insufficient and apologize for any confusion. To clarify this point, “Given that estrogen/Esr2b signaling feminizes the neural substrates that mediate mating behavior, while PGF2α signaling triggers female sexual receptivity,” has been added before the sentence “The present finding provides a likely explanation for this apparent contradiction, namely, that neuroestrogens, rather than or in addition to ovarian-derived circulating estrogens, may function upstream of PGF2α signaling to mediate female receptivity.”
Additional alteration
Reference list (line 682): a preprint article has now been published in a peer-reviewed journal, and the information has been updated accordingly as follows: “bioRxiv doi: 10.1101/2024.01.10.574747 (2024)” to “Proc. Natl. Acad. Sci. U.S.A. 121, e2316459121 (2024).”