Peer review process
Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.
Read more about eLife’s peer review process.Editors
- Reviewing EditorAshley WebbBuck Institute for Research on Aging, Novato, United States of America
- Senior EditorMa-Li WongState 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 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.
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.
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.
