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

  1. Rajae Talbi
  2. Todd L Stincic
  3. Kaitlin Ferrari
  4. Choi Ji Hae
  5. Karol Walec
  6. Elizabeth Medve
  7. Achi Gerutshang
  8. Silvia León
  9. Elizabeth A McCarthy
  10. Oline K Rønnekleiv
  11. Martin J Kelly
  12. Víctor M Navarro

  • Curated by eLife

    eLife logo

    eLife assessment

    The study presents compelling evidence that the melanocortin system originating in the arcuate nucleus of the hypothalamus plays a crucial role in puberty onset, representing a significant advance in our understanding of reproductive biology. The work, which represents a fundamental advance, employs innovative approaches and benefits from the combined expertise of two respected laboratories, enhancing the robustness of the findings. Given the potential impact on human health and the strength of the evidence presented, this work will likely influence the field substantially and may inform future clinical applications.

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Inactivating mutations in the melanocortin 4 receptor ( MC4R ) gene cause monogenic obesity. Interestingly, female patients also display various degrees of reproductive disorders, in line with the subfertile phenotype of MC4RKO female mice. However, the cellular mechanisms by which MC4R regulates reproduction are unknown. Kiss1 neurons directly stimulate gonadotropin-releasing hormone (GnRH) release through two distinct populations; the Kiss1 ARH neurons, controlling GnRH pulses, and the sexually dimorphic Kiss1 AVPV/PeN neurons controlling the preovulatory LH surge. Here, we show that Mc4r expressed in Kiss1 neurons is required for fertility in females. In vivo , deletion of Mc4r from Kiss1 neurons in female mice replicates the reproductive impairments of MC4RKO mice without inducing obesity. Conversely, reinsertion of Mc4r in Kiss1 neurons of MC4R null mice restores estrous cyclicity and LH pulsatility without reducing their obese phenotype. In vitro , we dissect the specific action of MC4R on Kiss1 ARH vs Kiss1 AVPV/PeN neurons and show that MC4R activation excites Kiss1 ARH neurons through direct synaptic actions. In contrast, Kiss1 AVPV/PeN neurons are normally inhibited by MC4R activation except under elevated estradiol levels, thus facilitating the activation of Kiss1 AVPV/PeN neurons to induce the LH surge driving ovulation in females. Our findings demonstrate that POMC ARH neurons acting through MC4R, directly regulate reproductive function in females by stimulating the “pulse generator” activity of Kiss1 ARH neurons and restricting the activation of Kiss1 AVPV/PeN neurons to the time of the estradiol-dependent LH surge, and thus unveil a novel pathway of the metabolic regulation of fertility by the melanocortin system.

Article activity feed

  1. Version published to 10.7554/elife.100722.1 on eLife
  2. Version published to 10.7554/elife.100722 on eLife
  3. eLife

    eLife assessment

    The study presents compelling evidence that the melanocortin system originating in the arcuate nucleus of the hypothalamus plays a crucial role in puberty onset, representing a significant advance in our understanding of reproductive biology. The work, which represents a fundamental advance, employs innovative approaches and benefits from the combined expertise of two respected laboratories, enhancing the robustness of the findings. Given the potential impact on human health and the strength of the evidence presented, this work will likely influence the field substantially and may inform future clinical applications.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:

    The authors investigate the role of the melanocortin system in puberty onset. They conclude that proopiomelanocortin (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.

  5. eLife

    Reviewer #2 (Public Review):

    Summary:

    This interesting manuscript describes a study investigating the role of MC4R (melanocortin 4 receptor) signalling on kisspeptin (Kiss1) neurons. The initial question is a good one. Infertility in human MC4R mutations has typically been ascribed to the consequent obesity and impaired metabolic regulation. Whether MC4R directly regulates the hypothalamic-pituitary-gonadal (HPG) axis has not been thoroughly examined. Here, the researchers assembled an elegant combination of loss and gain of function in vivo experiments, specifically targeting MC4R expression in Kiss1 neurons. This is an excellent experimental design and one that should provide compelling evidence for whether there is a direct role for melanocortin signalling in arcuate Kiss1 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). Still, the magnitude of these responses and the overall effect on fertility were relatively minor, as mice lacking MC4R in Kiss1 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 Kiss1 neurons in ex-vivo brain slides. These studies characterised interesting differential actions of melanocortins in two different Kiss1 neuronal populations. The study provides some novel insights into how direct actions of melanocortin signalling via the MC4R in Kiss1 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 and gain of function experiments provide a conceptually simple but hugely informative experimental design, which 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 Kiss1 neurons and not necessarily simply caused as a consequence of obesity. Validation experiments for these studies are needed, given their great prominence in the manuscript, because these are critical to interpretation.

    Weaknesses:

    (1) 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.

    (2) 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.

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

  6. eLife

    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 the field of 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 completely 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's 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 the age of 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 are in line 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 and logically 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.

  7. eLife

    Author response:

    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

  8. Version published to 10.1101/2024.02.18.580873v2 on bioRxiv
  9. Version published to 10.1101/2024.02.18.580873v1 on bioRxiv