POMC neurons control fertility through differential signaling of MC4R in Kisspeptin neurons

  1. Harvard Medical School, Boston, United States
  2. Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Boston, United States
  3. Division of Neuroscience, Oregon National Primate Research Center, Beaverton, United States
  4. Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, United States
  5. Harvard Program in Neuroscience, Boston, United States

Peer review process

Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers.

Read more about eLife’s peer review process.

Editors

  • Reviewing Editor
    Ashley Webb
    Buck Institute for Research on Aging, Novato, United States of America
  • Senior Editor
    Ma-Li Wong
    State University of New York Upstate Medical University, Syracuse, United States of America

Reviewer #1 (Public review):

Summary:

The authors investigate the role of the melanocortin system in puberty onset. They conclude that POMC neurons within the arcuate nucleus of the hypothalamus provide important but differing input to kisspeptin neurons in the arcuate or rostral hypothalamus.

Strengths:

Innovative and novel
Technically sound
Well-designed
Thorough

Weaknesses:

There were no major weaknesses identified.

Reviewer #2 (Public review):

Summary:

This interesting manuscript describes a study investigating the role of MC4R signalling on kisspeptin neurons. The initial question is a good one. Infertility associated with MC4 mutations in humans has typically been ascribed to the consequent obesity and impaired metabolic regulation. Whether there is a direct role for MC4 in regulating the HPG axis has not been thoroughly examined. Here, the researchers have assembled an elegant combination of targetted loss of function and gain of function in vivo experiments, specifically targetting MC4 expression in kisspeptin neurons. This excellent experimental design should provide compelling evidence for whether melanocortin signalling dirently affects arcuate kisspeptin neurons to support normal reproductive function. There were definite effects on reproductive function (irregular estrous cycle, reduced magnitude of LH surge induced by exogenous estradiol). However, the magnitude of these responses and the overall effect on fertility were relatively minor. The mice lacking MC4R in kisspeptin neurons remained fertile despite these irregularities. The second part of the manuscript describes a series of electrophysiological studies evaluating the pharmacological effects of melanocortin signalling in kisspeptin cells in ex-vivo brain slides. These studies characterised interesting differential actions of melanocortins in two different populations of kisspeptin neurons. Collectively, the study provides some novel insights into how direct actions of melanocortin signalling via the MC4 receptor in kisspeptin neurons contribute to the metabolic regulation of the reproductive system. Importantly, however, it is clear that other mechanisms are also at play.

Strengths:

The loss of function/gain of function experiments provides a conceptually simple but hugely informative experimental design. This is the key strength of the current paper - especially the knock-in study that showed improved reproductive function even in the presence of ongoing obesity. This is a very convincing result that documents that reproductive deficits in MC4R knockout animals (and humans with deleterious MC4R gene variants) can be ascribed to impaired signalling in the hypothalamic kisspeptin neurons and not necessarily caused as a consequence of obesity. As concluded by the authors: "reproductive impairments observed in MC4R deficient mice, which replicate many of the conditions described in humans, are largely mediated by the direct action of melanocortins via MC4R on Kiss1 neurons and not to their obese phenotype." This is important, as it might change how such fertility problems are treated.

I would like to see the validation experiments for the genetic manipulation studies given greater prominence in the manuscript because they are critical to interpretation. Presently, only single unquantified images are shown, and a much more comprehensive analysis should be provided.

Weaknesses:

(1) Given that mice lacking MC4R in kisspeptin neurons remained fertile despite some reproductive irregularities, this can be described as a contributing pathway, but other mechanisms must also be involved in conveying metabolic information to the reproductive system. This is now appropriately covered in the discussion.

(2) The mechanistic studies evaluating melanocortin signalling in kisspeptin neurons were all completed in ovariectomised animals (with and without exogenous hormones) that do not experience cyclical hormone changes. Such cyclical changes are fundamental to how these neurons function in vivo and may dynamically alter how they respond to hormones and neuropeptides. Eliminating this variable makes interpretation difficult, but the authors have justified this as a reductionist approach to evaluate estradiol actions specifically. However, this does not reflect the actual complexity of reproductive function.

For example, the authors focus on a reduced LH response to exogenous estradiol in ovariectomised mice as evidence that there might be a sub-optimal preovulatory LH surge. However, the preovulatory LH sure (in intact animals) was not measured.

They have not assessed why some follicles ovulated, but most did not. They have focused on the possibility that the ovulation signal (LH surge) was insufficient rather than asking why some follicles responded and others did not. This suggests some issue with follicular development, likely due to changes in gonadotropin secretion during the cycle and not simply due to an insufficient LH surge.

Reviewer #3 (Public review):

The manuscript by Talbi R et al. generated transgenic mice to assess the reproduction function of MC4R in Kiss1 neurons in vivo and used electrophysiology to test how MC4R activation regulated Kiss1 neuronal firing in ARH and AVPV/PeN. This timely study is highly significant in neuroendocrinology research for the following reasons.

(1) The authors' findings are significant in the field of reproductive research. Despite the known presence of MC4R signaling in Kiss1 neurons, the exact mechanisms of how MC4R signaling regulates different Kiss1 neuronal populations in the context of sex hormone fluctuations are not entirely understood. The authors reported that knocking out Mc4r from Kiss1 neurons replicates the reproductive impairment of MC4RKO mice, and Mc4r expression in Kiss1 neurons in the MC4R null background partially restored the reproductive impairment. MC4R activation excites Kiss1 ARH neurons and inhibits Kiss1 AVPV/PeN neurons (except for elevated estradiol).

(2) Reproduction dysfunction is one of obesity comorbidities. MC4R loss-of-function mutations cause obesity phenotype and impaired reproduction. However, it is hard to determine the causality. The authors carefully measured the body weight of the different mouse models (Figure 1C, Figure 2A, Figure 3B). For example, the Kiss1-MC4RKO females showed no body weight difference at puberty onset. This clearly demonstrated the direct function of MC4R signaling in reproduction but was not a consequence of excessive adiposity.

(3) Gene expression findings in the "KNDy" system align with the reproduction phenotype.

(4) The electrophysiology results reported in this manuscript are innovative and provide more details of MC4R activation and Kiss1 neuronal activation.

Overall, the authors have presented sufficient background in a clear, logical, and organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, and made a justified conclusion.

Comments on revisions:

The authors have addressed my comments.

Author response:

The following is the authors’ response to the original reviews.

Public reviews:

We are grateful to the reviewers and the editorial team for their feedback and thorough revisions of our paper. We also appreciate their acknowledgement that this study represents a significant advancement in the field of reproductive neuroendocrinology and offers insights on the contribution of obesity vs melanocortin signaling in women’s fertility. In the revised version, we will provide a more detailed clarification of the data and methodology and adhere to the reviewers’ suggestions.

Please find below our answers to specific concerns in the public review:

Given the fact that mice lacking MC4R in Kiss1 neurons remained fertile despite some reproductive irregularities, the overall tone and some of the conclusions of the manuscript (e.g., from the abstract: "... Mc4r expressed in Kiss1 neurons is required for fertility in females") were overstated. Perhaps this can be described as a contributing pathway, but other mechanisms must also be involved in conveying metabolic information to the reproductive system.

We will tone down these statements throughout the manuscript to indicate that MC4R in Kiss1 neurons plays a role in the metabolic control of fertility (rather than “…is required for fertility”)

The mechanistic studies evaluating melanocortin signalling in Kiss1 neurons were all completed in ovariectomised animals (with and without exogenous hormones) that do not experience cyclical hormone changes. Such cyclical changes are fundamental to how these neurons function in vivo and may dynamically alter the way they respond to neuropeptides. Therefore, eliminating this variable makes interpretation difficult.

Mice lack true follicular and luteal phases and therefore it is impossible to separate estrogen-mediated changes from progesterone-mediated changes (e.g., in a proestrous female). Therefore, we use an ovariectomized female model in which we can generate a LH surge with an E2-replacement regimen [1]. This model enables us to focus on estrogen effects, exclude progesterone effects, and minimize variability. Inclusion of cycling females would make interpretation much more difficult.

(1) Bosch et al., 2013 Mol & Cell Endo; https://doi.org/10.1016/j.mce.2012.12.021

Use of the POMC-Cre to target ontogenetic inputs to Kiss1 neurons might have targeted a wider population of cells than intended.

POMC is transiently expressed during embryonic development in a portion of cells fated to be Kiss1 or NPY/AgRP neurons [1-2]. Therefore, this is a valid concern when crossing with a floxed mouse. However, use of AAVs in adult animals avoids this issue and leads to specific expression in POMC neurons [3]. This POMC-Cre mouse has been used extensively with AAVs to drive specific expression in POMC neurons by other laboratories [4-7]. Therefore, we are confident that our optogenetic studies have narrowly targeted POMC inputs.

(1) Padilla et al., 2010 Nat Med; https://doi.org/10.1038/nm.2126

(2) Lam et al., 2017 Mol Metab; https://doi.org/10.1016/j.molmet.2017.02.007

(3) Stincic et al., 2018 eNeuro; https://doi.org/10.1523/eneuro.0103-18.2018

(4) Fenselau et al., 2017 Nat Neuro; https://doi.org/10.1038/nn.4442

(5) Rau & Hentges, 2019 J Neuro; https://doi.org/10.1523/jneurosci.3193-18.2019

(6) Fortin et al., 2021 Nutrients; https://doi.org/10.3390/nu13051642

(7) Villa et al., 2024 J Neuro; https://doi.org/10.1523/jneurosci.0222-24.2024

Recommendations for Authors

We thank the reviewers and the editorial team for their comments and thorough revisions of our paper. We have now addressed their comments and edited the manuscript accordingly:

Reviewer #1 (Recommendations For The Authors):

L80 -This is an awkward sentence; it isn't an inverse agonist of the AgRP; this may read better just to say that the inverse agonist, AgRP.

Thank you for this comment. This has now been changed in the text (L80).

L86 - This text reads as if mice have an inherent obesity issue.

This has also now been addressed in the text (L86).

L131 - The numbers of digits past the decimal point should match for both mean and SEM.

This has also now been addressed throughout the text.

Figure 1D: Revise the bar graphs with distinct SEM bars, as these data are not generated within the same mice.

The graphs are now changed, and they include distinct SEM and individual data points.

Figure 2I-L - An n of 3 for controls is pretty minimal, though the clustering of data points is tight.

We thank the reviewer for this comment, and we emphasize that while we agree that an n=3 for controls is minimal, the mRNA level values of this group are close, therefore the clustering of the data points is tight. We are happy to provide the raw data value for these groups if the reviewer wishes to.

L159 - The role of reduced dynorphin mRNA is pretty speculative with regard to basal levels of LH, especially since no other indices of LH secretion were affected. It should also be recognized that mRNA levels do not always equate to activity.

We agree with the reviewer that our explanation of the role of the reduced dynorphin with regards to the elevated basal LH is speculative, however, we only report that the higher LH levels correlates with the lower expression of the Pdyn gene expression, which is in line with the well documented role of Dynorphin on inhibiting LH secretion. We also recognize that mRNA levels don’t necessarily reflect activity. We have now added this statement to the text (L159).

L164 - Given the ovary data, it seems that the increase seen in KO mice isn't quite sufficient, but is it known how much of a surge is necessary for ovulation in mice?

We agree with the reviewer’s comment that the LH surge in Kiss1MC4RKO group is not enough to consistently induce ovulation, which is supported by the decrease in the numbers of corpora lutea data (Figure 2, O).

According to literature, an LH surge in the female mice is estimated by a LH value >4 ng/ml (Bahougne et al., 2020). According to this rule, our data show that only two females out of six had LH surge in the KO group, while four females out of five had LH surge in the control group.

L211 - According to the figure, LH pulses were not recovered and remained similar to KO levels. Looking at the LH secretory patterns presented, it seems like the pulse frequency data should be interpreted with some caution, given that some of the pulses identified are tenuous at best.

We agree that the LH pulses identified by our software (criteria described in the methods) are variable in shape (LH pulses are difficult to detect clearly in gonad intact females) and did not differ in number between groups; however, the reinsertion of Mc4r within Kiss1 neurons restored LH basal levels, amplitude and total secretory mass, which are clear indicatives of a significant improvement in the ability of these mice to release LH.

L218 - Is there a reason why the surge was not looked at in these groups?

Ovarian histology is the best indicator of ovulation. In these mice, corpora lutea were absent, indicating impaired ovulation, thus, we did not consider performing an LH surge protocol was necessary.

L244 - This would also fit with previous findings in sheep that not all Kiss neurons express MC receptors

We agree with this comment.

L329 - Given the rapidity of its actions, how would this membrane ER function during a normal surge?

Rapid estrogen signaling can act to ease transitions between states. Membrane delimited E2 actions can quickly attenuate or enhance coupling between receptors and signaling cascades. These effects will precede E2-driven changes in gene expression that produce more stable alterations in signaling. This combination of mechanisms will reduce any lag between rises in serum E2 and physiological effects. Considering the abbreviated mouse reproductive cycle, parallel mechanisms acting on different timescales are particularly important.

L365 - I'm a little confused as to how this particular work sheds light on a role for MC3R. Is the relative distribution of the two isoforms within Kiss neurons known?

In the present study, we report that hypothalamic Mc3r expression decreases leading up to the age of puberty onset (p30), in line with the profile of expression of Mc4r and a recent publication involving Mc3r in puberty onset (Lam et al., 2021), suggesting that both receptors may be involved in the control of reproductive function, potentially through the direct regulation of Kiss1 neurons as characterized in our present study.

L422 - While I understand the nature of this statement, the receptor may simply reflect the activity of what binds to it, i.e., AgRP vs. alpha-MSH, suggesting that maybe the prepubertal period is more AgRP-dominated.

We agree with this statement, and this needs to be further investigated.

L495 - Reinsertion of Mc4R in Kiss1 neurons

Thank you for this comment. This is now corrected in the text (L501).

L524 - Bilateral ovariectomy of 6-month

Thank you for this comment. This is now corrected in the text (L530).

L538 - Is it known what stage of the cycle these mice were in when samples were collected?

Yes, the samples were collected in diestrus. This is now mentioned in the text (L548)

L556 - Pulse amplitude is usually measured relative to the preceding nadir.

The method that we have been consistently using in our lab is the average of the 4 highest LH values in the samples collection period for each animal. We have found this to be consistent and representative of the overall amplitude (McCarthy et al., 2021; Talbi et al., 2021).

L594 - This is a little confusing - the whole MBH would contain the ARH, but only the ARH was collected from the KO mice. If the whole MBH, dynorphin and Tac3, and Tac3 are expressed outside of the ARC, making interpretation of changes specifically within the ARH is difficult.

Here (L592), we describe two different experiments, as mentioned by i) and ii).

For experiment 1 (i): MBH was used in the WT mice at ages P10, P15, P22 and P30 to investigate the expression of the melanocortin genes (Agrp, Pomc, Mc3r and Mc4r).

For experiment 2 (ii): In both KO and control groups, only the micro-dissected ARH was used to investigate genes expressions of Pdyn, Kiss1, Tac2, Tacr3.

Reviewer #2 (Recommendations For The Authors):

The validation experiments for the various manipulations are currently presented in the supplementary data. Still, in my opinion, these are critically important for interpreting the data, and it should be considered to present these more comprehensively in the main body of the manuscript. In Figure S1, it seems that the exposure of the two images is not the same, with a higher background in the control. Has this image been adjusted to highlight the staining, while the other has not? It looks like there remains a low level of expression still present in at least some of the KO cells - this may reflect difficulties using RNAscope (with its extreme amplification) to detect the absence of a signal, or it could also be that the knockout is incomplete. A percentage of cells still express MC4R. I think this should be acknowledged or discussed.

We thank the reviewer for the feedback. While we agree that the validation of the mouse model is critical, we would like to keep it in the supplemental data.

We also agree that the exposure looks different between the KO and WT controls, and we thank the reviewer for this comment. The quality of the photograph decreased when transferring to the manuscript. This has now been improved in the revised figure.

As for the MC4R expression in some of the KO cells, we believe that MC4R is expressed in non Kiss1 cells as shown in the merged figure. Therefore, we believe that the Knockout of Mc4r in Kiss1 neurons is complete in these mice.

The clear difference from the PVN's lack of effect is convincing and indicates that a specific knockout has been achieved. Is equivalent data also available for the AVPV population of cells that are examined later in the manuscript? Do those Kiss1 neurons also express the MC4R? The same question applies to the knock-in experiment: Was the expression of MC4R also driven in the AVPV population using this approach

Yes, Kiss1 neurons in the AVPV also express MC4R as indicated in this study, and thus Mc4r is removed/reinserted in the AVPV as well in this mouse model.

The quantitative RT-qPCR data on developmental changes in metabolic signaling molecules are really peripheral to the paper's main question. Relative to the validation experiments (as discussed above), I think these are less important data and could be placed into a supplementary figure. The discussion of these data becomes problematic, e.g., on line 359, the changes are described as "a low melanocortin tone..." but this seems problematic when referring to reduced expression of AgRP, an inverse agonist at the MC4R. If you are going to present these data, individual data points should be shown. Similarly, the question about whether this is a PCOS-like phenotype is perhaps worth asking. Still, the simple assessment of T and AMH could also be reported in a sentence without necessarily showing the data (or placing it in a supplementary figure). Better to focus on the key question - which is the role of MC4R signaling in Kiss1 neurons.

We understand this reviewer’s concerns, however, due to the impact of MC4R signaling (particularly in the context of AgRP) on puberty, we strongly believe that the reader will benefit from expression profile across ages so we will respectfully disagree and keep in the main figure.

Per this reviewer’s comment, we have now added individual data points to Figure 1D.

We also agree with the reviewer that the T and AMH data are not in the main scope of the paper, but since we uncovered a PCOS-like phenotype in female mice with specific deletion of Mc4r from Kiss1 neurons, it is important to keep these data in the main figure to show that the phenotype does not fully resemble a PCOS model.

Having praised the experimental design, I think it is fair to acknowledge that the reproductive data from these experiments remain difficult to interpret. I understand that it is difficult to illustrate estrous cycles, but the "quantitative" data on percentages of time spent in any one stage are not as informative as seeing the actual individual patterns in Figure 2B. Were all of the animals consistently like the one illustrated, with persistent diestrus and only occasional evidence of ovulation?

We agree that Figure 2C may be difficult to interpret but it is the best way to capture the all the data points for each group.

All the 5 Kiss1MC4RKO females had persistent diestrus phases with only one or two estrus phases over 15 days (except for one female who had 4 estrous days), compared to control females who had 7 to 9 days of estrous, as shown in the graph (except for one female who had 5 days of estrus over 15 days period).

Given that LH pulses appear to be normal, does this, in fact, suggest an ovarian problem? Is that possible? Are MC4R and Kiss1 co-expressed in the ovary? Or do you think this suggests an ovulation problem, perhaps driven by the impaired LH surge?

This reviewer is correct in that our findings suggest a central defect in ovulation based on the deficit observed in the preovulatory LH surge. Thus, it is possible to have normal LH pulses, which are driven by one population of Kiss1 neurons (ARH) and the LH surge, driven by a distinct population of Kiss1 neurons (AVPV).

Similarly, the response to the "LH surge induction protocol" is impaired (why not look at endogenous LH surges?). It seems that ovulation should be an all-or-none phenomenon in that if the LH surge is sufficient to induce ovulation, then all available follicles would be ovulated. If it is not, then no follicles will be ovulated. Why fewer follicles are ovulated in the gene-targeted animals seems more likely to be due to impaired follicular development rather than a subthreshold LH surge. So, this again points back to the ovary. Or perhaps we need a more thorough assessment of the pattern of LH pulses throughout the cycles in these animals.

An LH surge induction protocol allows us to submit all female mice to the same conditions and expect a similar response, which is then optimal to compare with animals with an expected ovulation deficit, as it eliminates external factors. We disagree in that ovulation is an all-or-none phenomenon because in mice numerous follicles mature at the same time and thus a decrease in the number of ovulated oocytes may be significant between groups even if the animals are not completely infertile.

Collectively, my assessment of these data is that there are effects on reproduction, but they are actually relatively subtle. There were abnormal cycles and impaired LH surge in response to exogenous estrogen. But the animals are not actually infertile, so can ovulate and express normal reproductive behavior. So while there is a role for MC4R signalling in Kiss1 neurons, it may be a contributing modulatory role rather than a major regulatory mechanism. I think the tone of the descriptions should reflect this. I like the way it is framed in some parts of the discussion ("reproductive impairments...mediated by MC4R in Kiss1 neurons and not by their obese phenotype"), but the overall significance of this is overstated in some places, such as the abstract and in other parts of the discussion ("this population is tightly controlled by melanocortins").

As mentioned in previous responses, ovulation in mice is not all-or nothing, so while the mice can reproduce, the disruption in the central mechanisms that control ovulation and irregular estrous cycles are a significant advancement in the field with strong translational potential to species where only one oocyte is usually ovulated, like in humans, where reproductive disorders in MC4R patients had been attributed to the obesity phenotype rather than to a central action of MC4R (as the reviewer captured in their comment). This is one of the main findings of this study.

The overstatement has been now addressed throughout the text.

For in vitro studies, all mice were ovariectomized and given estradiol "replacement." What was the rationale for this? Wouldn't this suppress the basal activity of these neurons? Then it appears that some of the animals were studied as ovariectomised (for an unspecified time but apparently ">7 days", without hormone replacement. The basal activity of these cells would be dramatically different. I think these artificial manipulations make these data quite difficult to interpret. How does this reflect the situation in a normal (or abnormal) estrous cycle? My understanding is that the brain slice approach already compromises the ability of this population of cells to function as a coordinated network (i.e., coordinated episodes of activity that are seen in vivo have not been observed in vitro in brain slices). Ovariectomizing and providing exogenous hormones also removes the additional regulatory elements of the cyclical changes in hormone inputs, so the cells may or may not behave like they would in vivo. Perhaps the authors could justify their choice of experimental model.

We have clarified that the mice were ovariectomized for 7-10 days. A group of 3 mice are OVXed at once and then used on subsequent days a week later. This delay is both for the recovery of the animal and to allow for “washout” of endogenous ovarian hormones. For optogenetic studies, we were not measuring basal activity. Rather, we prioritized the ability to detect a postsynaptic response. While E2 decreases the networked activity of Kiss1- ARH neurons, the Hcn channels, calcium channels, and Vglut2 expression are all increased. This leads to increased excitability and more glutamate release. Mice lack true follicular and luteal phases and therefore it is impossible to separate estrogen-mediated changes from progesterone-mediated changes (e.g., in a proestrous female). Therefore, we use an ovariectomized female model in which we can generate a LH surge with an E2-replacement regimen (Bosch et al., J Mol Cell Endocrinology 2013). This model enables us to focus on estrogen effects, exclude progesterone effects, and minimize variability. Finally, we have documented that Kiss1ARH neurons retain the synchronization of their neuronal firing in the hypothalamic slice preparation (Qiu et al., eLife 2016).

Figure 4E shows neurons' staining after expressing a Cre-dependent channel rhodopsin vector into POMC-Cre mice. The number of labelled cells looks markedly larger than expected for adult POMC neurons. Was the specificity of this approach to neurons expressing POMC checked? I understand that the POMC-Cre mice have been criticised for ectopic expression of Cre during development in other populations of neurons in the arcuate nucleus that does not express POMC, such as the AgRP neurons (e.g., PMID: 22166984). Is it possible that this is not a problem in adult animals? Has that been validated in these animals? The description of the method suggests that it is acknowledged that some of the expression driven in these animals might be in AgRP neurons. Still, optogenetic activation of these cells will include all cells expressing Cre at the time of AAV administration.

POMC is transiently expressed during embryonic development in a portion of cells fated to be Kiss1 or NPY/AgRP neurons. Therefore, this is a valid concern when crossing with a floxed mouse. However, use of AAVs in adult animals avoids this issue and leads to specific expression in POMC neurons. This POMC-Cre mouse has been used extensively with AAVs to drive specific expression in POMC neurons by other laboratories (Padilla et al., Nat Med 2010; Lam et al., Mol Metab 2017; Stincic et al., eNeuro 2018 eNeuro; Fenselau et al., Nat Neuro 2017). We have previously shown that AAV-driven mCherry expression is limited to cells labeled with a beta-endorphin antibody (Stincic et al., 2018 eNeuro). Therefore, we are confident that our optogenetic studies have narrowly targeted POMC inputs.

Some additional explanation of the electrophysiology result may be required. For example, on Line 292, I'm confused by Fig 4M. Why is the response to 20Hz stimulation different in this cell (compared to the one in 4L) before administering naloxone? What proportion of cells showed this opposite response? On line 307: Is 5 cells sufficient for testing the POMC inputs onto AVPV and PeN Kiss1 neurons? How many slices/animals are included in collecting these 5 cells? The rapid action of STX illustrates the ability to modulate the response to MTII, but I am struggling to understand the implications of this in a physiological context. Suppose this response is desensitized by longer-term treatment with E2 (as indicated in the manuscript). Is it relevant to normal regulation during the cycle (particularly in the AVPV, where the key regulatory step seems to be the prolonged exposure to high estradiol as part of the preovulatory signals leading up to the LH surge)?

As stated in the text, E2 has been shown to increase POMC expression and beta-Endorphin immunostaining. We do not know the effects of E2 on aMSH expression and release. E2 also tends to attenuate the coupling between inhibitory postsynaptic metabotropic (Gi,o-coupled) receptors and signaling cascades. So, there is likely a combination of pre- and post-synaptic mechanisms contributing to these responses. However, the focus of the current studies was on the predominant melanocortin signaling and, as such, we chose to eliminate the influence of opioid signaling. We have added two more cells to this group, both of which were successfully rescued for a total of 5 of 6 cells (6 slices, 5 animals). Between the labeling of b-endorphin fibers and high rate of rescue, we do believe that this is sufficient evidence to support a direct POMC input to Kiss1AVP/PeN neurons.

Line 52: "Here, we show that Mc4r expressed in Kiss1 neurons is required for fertility in females." The knockout animals remain fertile, so this conclusion needs to be re-worded.

Thank you for this comment. This has now been changed (L52).

Line 80: "The melanocortin 4 receptor (MC4R) binds α-melanocyte stimulating hormone (αMSH), an agonist product of the pro-opiomelanocortin (Pomc) gene, and the inverse agonist of the agouti-related peptide (AgRP) to regulate food intake and energy expenditure" Is this the correct wording? I think it should be stated that AgRP is an inverse agonist at the MC4R, not that αMSH is the inverse agonist of AgRP. Re-work this sentence.

Thank you for this comment. This has now been changed (L79-80).

Line 88: "... however, conflicting reports exist". Describe what these conflicting reports show. Many MC4 variants ("mutations") are expressed in humans, but few will fully inactivate signalling like the mouse knockout.

We thank the reviewer for this comment. By conflicting data, we refer to the studies that report no reproductive impairments in women with MC4R mutations. Either because the metabolic impairments (obesity, hyperphagia, hyperinsulinemia, hyperleptinemia, etc) are so strong that the focus is skewed to these issues, without a full reproductive assessment in these women, or simply because the reviewer mentioned, not all MC4R mutations fully inactivate its signaling in humans - as opposed to mouse models where reproductive disruption has been described previously in full body MC4RKOs.

Line 91: "...that largely affects females". Is this a genuine sex difference, or are reproductive deficits simply more overt in female rodents? I think the Coss paper (reference 19 in the manuscript) showed a greater effect of diet-induced obesity in males than in females.

We believe that sex differences exist with regards to the role of MC4R in the regulation of fertility, as we show that most of this effect is mediated by MC4R signaling in Kiss1 AVPV neurons, a neuronal population that is specific to the female brain.

As far as we can tell, the Coss paper (Villa et al., 2024) has only tested males but not females. Moreover, they investigated the effect of diet induced obesity in mice on their fertility (specifically LH secretion), while in this study we are specifically looking at the deletion of MC4R from Kiss1 neurons, and these mice were not obese (Figure 2A). While both these conditions induce impaired fertility, the mechanisms and signaling pathways are different (our mice lack MC4R signaling while the obese mice have a decrease in MC4R expression but the signaling is still functional).

Line 392: also Hessler et al. PMID: 32337804.

This reference is now added to the text (Line 393).

Line 433. The discussion of how advanced puberty onset (seen in the Kiss1-specific KO animals) might be caused by MC4R signalling in AVPV Kiss1 neurons, which are sexually dimorphic, which might explain sex differences in puberty timing in mammals seems extremely speculative and based on limited data. More targeted experiments would be needed to address this, and I think this speculation should be removed here.

This speculation has now been removed from the text.

Line 438: "Furthermore, our findings suggest that metabolic cues, through the regulation of the melanocortin output onto Kiss1AVPV/PeN neurons, are essential for the timing and magnitude of the GnRH/LH surge." Again, I think this is overstating the present data, which has only looked at an artificial hormone administration regime. The animals are fertile and, thus, must be able to mount a sufficient LH surge. The major effect, in fact, seems to be on their cycle, perhaps leading to impaired follicular development. Please acknowledge that this will be one of the multiple pathways by which metabolic information is fed into the HPG axis.

In addition to the effect on their cycles as mentioned by the reviewer, the Kiss1MC4RKO females also display impaired fertility (Figure 2, S-T) and fewer corpora lutea which is in line with the impaired mounting of LH surge (Figure 2, M). Even if the LH surge is induced by the hormone administration protocol, it only reflects the natural ability of the HPG axis to mount the surge, as this regimen is only there to mimic the endogenous hormonal changes leading to LH surge and therefore ovulation, in a controlled manner. Nonetheless, we agree with this reviewer that this is not the sole mechanism by which metabolism regulates reproductive function and this has been emphasized in the paper. (line 443)

Reviewer #3 (Recommendations For The Authors):

The decreased melanocortin tone drives puberty onset (Figure 1D), and this is correlative. The transgenic animals' hypothalamic expression of Agrp, Pomc, Mc4r, and Mc3r can be measured to strengthen the claim. Hprt expression should be demonstrated, as this housekeeping gene was used as a common denominator.

We thank the reviewer for this comment. While we think that indeed, measuring Agrp, Pomc, Mc4r, and Mc3r gene expressions in the transgenic mice will strengthen our claim and give more insights into the melanocortins tone during pubertal maturation, this is unfortunately not feasible as it will involve generating a lot of mice (at least n=40 pups for an n=5/group, KO and control littermates, females only -which will require setting up lots of breeding pairs-) during different ages throughout puberty.

As for the gene expression of Hprt, because we have 6 mice per age, 4 ages total, every gene (Agrp, Pomc, Mc4r, Mc3r) was run in a separate plate with Hprt as its own housekeeping gene. Samples were run in duplicates for each Hprt and melanocortin genes in a 96 well = 48 wells for Hprt and 48 wells for each of the melanocortin genes. Therefore, it won’t be possible to represent one Hprt expression for all the four genes, however every gene was normalized to the Hprt gene expression that was ran in the same plate).

In Figures 4 and 5, dot plots can be used (as opposed to the bar graphs) to better reflect the individual data points.

Figures 4 and 5 have been revised to include individual data points.

The electrophysiology experiment requires more details in the method section. In addition to the publication cited, a brief recap of the methodology used in this paper, such as the focal application of MTII (Figure 4B), is also needed.

We have added more details to the Methods.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation