Review Reports - Gonadal Sex and Sex-Chromosome Complement Interact to Affect Ethanol Consumption in Adolescent Four Core Genotypes Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors present an interesting study with a smart study design.

The understandabilty of the paper is hampered by the use of - for the reader - unusual abreviations. Especially in abstract abbreviations XXM, XYM, XYF, XXF are not explained at all - this needs to be added. Also GS = gonadal sex is introduced ~5 times in the paper as abbreviation but only used ~50 times in the text - also abbreviation GS is combined ~20/50 times with abbreviation SCC, an abbreviation also introduced several times in the text.

It looks like the main message of the study is the new and smart study design. Results obtained only support that the study design is well suited to approach/ get insight kinto adolescent binge drinking and to highlight the gender differences in mice (and human).

Accordingly, part 4.3 of discussion should be deleted. Authors have no data on KDM6A and donot mention this gene in introduction. So it is not comprehensible why this part is included here.

The pure technical nature of this paper needs to be highlighted as data obtained and presented is acc. to the discussion of the authors themselves not new - apart from the use of 'four genetic genders'.

Author Response

Comments 1: The understandabilty of the paper is hampered by the use of - for the reader - unusual abreviations. Especially in abstract abbreviations XXM, XYM, XYF, XXF are not explained at all - this needs to be added. Also GS = gonadal sex is introduced ~5 times in the paper as abbreviation but only used ~50 times in the text - also abbreviation GS is combined ~20/50 times with abbreviation SCC, an abbreviation also introduced several times in the text.

Response 1: Our apologies. We acknowledge that our use of abbreviations was inconsistent. We have now revised the text to eliminate redundant definitions of our major abbreviations (GS, SCC).

Comment 2:

Accordingly, part 4.3 of discussion should be deleted. Authors have no data on KDM6A and donot mention this gene in introduction. So it is not comprehensible why this part is included here.

Response 2: We acknowledge this concern but also note that Reviewer 2 asked for the opposite - an expanded discussion of potential X and Y candidate genes that could be of relevance to our findings. We have opted to compromise between the two critiques by editing/reducing the discussion of Kdma6a and Kdm6b in Section 4.3.1, while also adding some text that is responsive to Reviewer 2.

Comment 3: The pure technical nature of this paper needs to be highlighted as data obtained and presented is acc. to the discussion of the authors themselves not new - apart from the use of 'four genetic genders'.

Response 3: The specific suggestion here is not entirely clear. The reviewer is correct that the application of the FCG model here to uncover the interactive roles of various sex-biasing factors is the novel contribution of this manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Jentsch et al aims to examine the contributions of sex-chromosome complement (SCC) and gonadal sex (GS) to ethanol consumption using a modified three‑bottle drinking‑in‑the‑dark (DID) paradigm with high‑resolution behavioral microstructural bout analysis. The authors reported that adolescent gonadal males drink more ethanol than gonadal females, particularly in the presence of an XX karyotype, and gonadal XY females drink more ethanol than XX mice. The finding of a significant SCC×GS interaction on binge‑like consumption is intriguing and has the potential to move the field forward, particularly when considering that the study was performed during adolescence, a developmental period of high translational relevance. The approach is conceptually strong and fills gaps in our understanding of genetic vs hormonal influences on alcohol intake.

However, I have several methodological and statistical concerns that need to be addressed, along with areas that need clarification. Please see my detailed comments below.

Major comments:

- One of my major concerns is that the authors do not report whether the three bottles (20%, 10%, 0%) were counterbalanced across left/center/right positions within cages. Without counterbalancing, apparent “preferences” or SCC×GS differences could be influenced by side bias rather than ethanol concentration. Please add details about bottle position randomization across sessions. If not performed, this represents a serious design limitation. The authors should either conduct a validation experiment with proper counterbalancing or acknowledge that side bias could influence outcomes and temper interpretations accordingly.

- Please add in the Methods if a leak test (empty cage measurements across the same time intervals) was performed. Further, describe load‑cell calibration (mass standards used, frequency, acceptable drift) and indicate whether leak values were used for correction or quality‑control filtering.

- To help the readers, please clarify if within-bout rates reflect instantaneous rates normalized over very short durations, not an hourly achievable intake. Please consider adding a sentence explaining the computational derivation and the session‑averaged rates for context.

- Similarly, please consider including BEC distributions by genotype.

- Although the manuscript acknowledges that the chow contains soy-derived phytoestrogens, could the authors justify using this diet in a sex‑difference study, or include it as a limitation?

- The current discussion highlights KDM6A/KDM6B and broad epigenetic regulation, but this section reads more like a list of possible mechanisms than a coherent mechanistic model. Please rewrite to link SCC with epigenetic regulation, reward circuitry responsiveness, and binge intake. Other X‑escapee genes that have known roles in neurodevelopment and reward processing, and other Y‑linked factors (not just Sry) that might influence prefrontal- or striatal-related ethanol responsiveness. Discuss how SCC might shape dopaminergic signaling, stress reactivity, or corticostriatal maturation during adolescence.

- I commend the authors for the impressive microstructural data. However, the manuscript could benefit from addressing whether SCC×GS modulates the degree of front‑loading, not just total intake. Also, include if/how the ethanol consumption in a single bout aligns with known motivational or pharmacokinetic drivers of binge behavior, whether high within‑bout rates suggest reduced satiety/aversive feedback or altered interoceptive sensitivity across genotypes, and whether XX vs. XY mice differ in bout initiation thresholds, persistence, or reward sensitivity and how these microstructural data can corelate to human binge‑onset behavior.

- Please expand the discussion about why SCC modulation is strongest in gonadal females, but GS modulation is strongest in XX mice. Is this a consequence of dosage compensation/escape‑from‑X‑inactivation mechanisms, differential receptor expression, Y‑linked effects on neural circuit maturation, or genotype‑specific sensitivity to ethanol’s pharmacodynamic effects? A detailed discussion of possible mechanisms will considerably improve the interest in the manuscript.

- The DID sessions occur during early adolescence, when both males and females are undergoing rapid endocrine changes. The manuscript briefly mentions organizational vs. activational effects but does not integrate these concepts with the observed patterns. To improve the interest and relevance of the manuscript, please discuss whether the SCC×GS interaction observed reflects organizational effects occurring pre‑ or perinatally. If could be any acute activational effects of rising adolescent gonadal hormones during early adolescence. Include also if/how the adolescent endocrine environment differs in XX vs XY mice with matched gonadal types, and if/how changes in hormone receptor expression (ERα, ERβ, AR) could interact with SCC to affect the drinking patterns that the authors report in this study.

- Another major concern is the surprisingly minimal discussion about the translation of the findings. The discussion should be expanded to include integrating pubertal timing and endocrine transitions (particularly testosterone surge effects) as mechanistic parallels and differentiating behavioral/psychosocial and biological contributors to sex differences observed in humans. Further, a discussion about how SCC×GS interactions in mice may model specific subgroups (e.g., early vs. late pubertal adolescents, sex‑chromosome aneuploidies) needs to be included.

Author Response

Comment 1: - One of my major concerns is that the authors do not report whether the three bottles (20%, 10%, 0%) were counterbalanced across left/center/right positions within cages. Without counterbalancing, apparent “preferences” or SCC×GS differences could be influenced by side bias rather than ethanol concentration. Please add details about bottle position randomization across sessions. If not performed, this represents a serious design limitation. The authors should either conduct a validation experiment with proper counterbalancing or acknowledge that side bias could influence outcomes and temper interpretations accordingly.

Response 1: We appreciate the reviewer pointing this very important issue out. We always rotate the position of the solutions within and across animals, so this is now indicated in section 2.2.3.

Comment 2: - Please add in the Methods if a leak test (empty cage measurements across the same time intervals) was performed. Further, describe load‑cell calibration (mass standards used, frequency, acceptable drift) and indicate whether leak values were used for correction or quality‑control filtering.

Response 2: This is another very important point. We have addressed the use of leak tests and weight validations with edits to section 2.2.1.

Comment 3: - To help the readers, please clarify if within-bout rates reflect instantaneous rates normalized over very short durations, not an hourly achievable intake. Please consider adding a sentence explaining the computational derivation and the session‑averaged rates for context.

Response 3: Thank you for raising this issue. Bouts varied in amount of ethanol consumed (measured in g/kg body weight) and duration, therefore, the rate measure was calculated as amount/unit time. Individual bouts typically lasted under 1 minute. We chose to use hour (h) as the unit time, but could easily have chosen minutes or seconds as the time unit. The reader can easily derive these by dividing by the appropriate denominator. This is clarified in section 2.2.5.

Comment 4: Similarly, please consider including BEC distributions by genotype

Response 4: Values in Figure 3 are separated by genotype; please note the 4 different symbols in the legend for Figure 3.

Comment 5: - Although the manuscript acknowledges that the chow contains soy-derived phytoestrogens, could the authors justify using this diet in a sex‑difference study, or include it as a limitation?

Response 5: This is addressed as a limitation in the new section 4.4.

Comment 6: - The current discussion highlights KDM6A/KDM6B and broad epigenetic regulation, but this section reads more like a list of possible mechanisms than a coherent mechanistic model. Please rewrite to link SCC with epigenetic regulation, reward circuitry responsiveness, and binge intake. Other X‑escapee genes that have known roles in neurodevelopment and reward processing, and other Y‑linked factors (not just Sry) that might influence prefrontal- or striatal-related ethanol responsiveness. Discuss how SCC might shape dopaminergic signaling, stress reactivity, or corticostriatal maturation during adolescence.

Response 6:

Comment 7:

Response 7: This point is addressed in Figure 2 (and associated sections of the Results). Panel A shows the amount of ethanol consumed as a function of time (early, middle and late 40 min bin) in the session. These data reveal that SCC and gonadal sex both impact front loading (consumption in early, relative to late time bins).

Comment 8: - Please expand the discussion about why SCC modulation is strongest in gonadal females, but GS modulation is strongest in XX mice. Is this a consequence of dosage compensation/escape‑from‑X‑inactivation mechanisms, differential receptor expression, Y‑linked effects on neural circuit maturation, or genotype‑specific sensitivity to ethanol’s pharmacodynamic effects? A detailed discussion of possible mechanisms will considerably improve the interest in the manuscript.

Response 8: Much is unknown about the associated mechanisms contributing to this observed interaction. Section 4.2 has been expanded to address this point, but again, further speculation would be unsupportable with data at this time.

Comment 9: The DID sessions occur during early adolescence, when both males and females are undergoing rapid endocrine changes. The manuscript briefly mentions organizational vs. activational effects but does not integrate these concepts with the observed patterns. To improve the interest and relevance of the manuscript, please discuss whether the SCC×GS interaction observed reflects organizational effects occurring pre‑ or perinatally. If could be any acute activational effects of rising adolescent gonadal hormones during early adolescence. Include also if/how the adolescent endocrine environment differs in XX vs XY mice with matched gonadal types, and if/how changes in hormone receptor expression (ERα, ERβ, AR) could interact with SCC to affect the drinking patterns that the authors report in this study.

Response 9: This is an entirely fair point. A great deal remains unknown about how gonadal maturation and associated sex steroid secretions emerge as a function of XX and XY karotype in the FCG model. Section 4.2 has been edited to reflect that. Still, so much is unknown that additional speculation is unwarranted at this time.

Comment 10: Another major concern is the surprisingly minimal discussion about the translation of the findings. The discussion should be expanded to include integrating pubertal timing and endocrine transitions (particularly testosterone surge effects) as mechanistic parallels and differentiating behavioral/psychosocial and biological contributors to sex differences observed in humans. Further, a discussion about how SCC×GS interactions in mice may model specific subgroups (e.g., early vs. late pubertal adolescents, sex‑chromosome aneuploidies) needs to be included.

Response 10: In our view, the translational value of these studies is that they reveal how sex-biasing factors can act to impact ethanol responses in humans. Thus, it's biological information of value for understanding patterns of human behavior, without needing to be face-valid in any way.

Reviewer 3 Report

Comments and Suggestions for Authors

In this article the authors investigate whether there is an effect of sex chromosome complement (SCC), gonadal sex, or both on ethanol consumption. They use a 3 bottle choice paradigm with varied concentrations of ethanol and measure total consumption, as well as features of consumption such as bout number and length. They investigate effects of SCC and GS on individual sessions of DID as well as within session effects. They find that there is an interaction between SCC and GS wherein ethanol consumption is highest in XXM and lowest in XXF. Chromosome differences were biggest in gonadal females and gonadal sex effects were greatest in XX mice. This study is of high interest and emphasizes the importance of investigating the effects of SCC and GS, not just blanket sex differences, on ethanol consumption. Further, these results indicate the importance of using varied ethanol concentrations and measuring distinct features of consumption during DID.

Methods:

Could the authors please clarify that 4 daily, 2 hour sessions were used? There is some language about consecutive 2 hour sessions being used, but it is not always clear if these are back to back or daily.

Sample sizes are given as almost n=40. The authors should add some details about how many cohorts of mice were tested in order to obtain these sample sizes and how many individual litters were used. Does this n~40 include the mice used for BECs or were those separate?

Results/discussion:

Overall, results indicate that XYF consume more ethanol than XXF and XXM more than XXF. When looking at these data, for a lot of measures these effects seem specifically driven by the low consumption in XXF. The authors should briefly discuss this finding and if there are any specific mechanisms that might drive low ethanol consumption in XXF alone.

The authors present a strong synopsis of current datasets for DID and sex differences and focus on how age, concentration, session length, sex, bottle # etc can alter sex differences found during DID. It would be beneficial for the authors to discuss what they believe is the best experimental design moving forward in order to better understand sex differences in ethanol consumption. If so many factors can reverse or change these sex differences, how can we assign meaning or interpret any of these findings?

The authors discuss that it is unlikely that SCC effects pre-adult levels of hormones in order to affect behavior and provide citations for this. However, these studies all focus on specific hormone levels and don’t investigate receptor expression or other changes that could affect hormone signaling while maintaining baseline hormone levels. The authors should discuss whether receptor levels might be changed or other downstream mechanisms, even though hormone levels themselves might be spared. For example, one of these citations (115) discussed an increase in vasopressin fiber area in XX mice, while testosterone levels were unchanged.

Author Response

Comment 1: Could the authors please clarify that 4 daily, 2 hour sessions were used? There is some language about consecutive 2 hour sessions being used, but it is not always clear if these are back to back or daily.

Response 1: The reviewer is totally correct. We have clarified this point in edits to section 2.2.3.

Comment 2: Sample sizes are given as almost n=40. The authors should add some details about how many cohorts of mice were tested in order to obtain these sample sizes and how many individual litters were used. Does this n~40 include the mice used for BECs or were those separate?

Response 1: Details on the number of litters used was added to section 2.1. All mice involved in the BEC studies were also behaviorally studied, allowing for the correlations reported in Figure 3.

Comment 2: Overall, results indicate that XYF consume more ethanol than XXF and XXM more than XXF. When looking at these data, for a lot of measures these effects seem specifically driven by the low consumption in XXF. The authors should briefly discuss this finding and if there are any specific mechanisms that might drive low ethanol consumption in XXF alone.

Response 2: We have added a sentence to the Discussion indicating that XXF mice exhibit the lowest consumption. We surmise that a combination of GS and SCC effects cause this low response.

Comment 3: The authors present a strong synopsis of current datasets for DID and sex differences and focus on how age, concentration, session length, sex, bottle # etc can alter sex differences found during DID. It would be beneficial for the authors to discuss what they believe is the best experimental design moving forward in order to better understand sex differences in ethanol consumption. If so many factors can reverse or change these sex differences, how can we assign meaning or interpret any of these findings?

Response 3: We share the reviewer's uncertainty as to the methodological or experimental design factors that have contributed to a diverse set of findings. We cannot alone resolve them and feel that speculating on this in the manuscript would be unwise. We would argue that the use of FCG mice, which allows for a dissection of the role for typically confounded sex-biasing factors, could help to clarify the specific roles for these mechanisms in specific ethanol-related outcomes.

Comment 4: The authors discuss that it is unlikely that SCC effects pre-adult levels of hormones in order to affect behavior and provide citations for this. However, these studies all focus on specific hormone levels and don’t investigate receptor expression or other changes that could affect hormone signaling while maintaining baseline hormone levels. The authors should discuss whether receptor levels might be changed or other downstream mechanisms, even though hormone levels themselves might be spared. For example, one of these citations (115) discussed an increase in vasopressin fiber area in XX mice, while testosterone levels were unchanged.

Response 4: This is an important point, and we have added text to section 4.2 to acknowledge this.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors made some small changes.

They have now deleted all explanations for abbreviations in the text. They need to mention the full word the first time they use an abbreviation.

Apart from the use of 'four genetic genders', it is still hard to understand what is new in this paper.

As I understand it, the results obtained simply confirm previous data. If this is true, the authors need to rewrite the paper as a technical note about the model system they created. The title promises more insight into the reasons for the interaction between gonadal sex, sex chromosome complement, and ethanol consumption in mice. However, the model simply confirms what is already known, without offering anything new.

Author Response

Comment 1: They have now deleted all explanations for abbreviations in the text. They need to mention the full word the first time they use an abbreviation.

Response 1: We believe we have now corrected any discrepancies.

Comment 2:

Apart from the use of 'four genetic genders', it is still hard to understand what is new in this paper.

Response 2: We have tried to explain more clearly what is quite new about these studies in the final paragraph of the Introduction. Both the age at the time of testing (adolescent) is unique, addressing an important question in the field. Moreover, the method of assessment of ethanol intake is novel, allowing for a finer description of the behavior. Collectively, these aspects of our study add to the literature in an important way.