STUDY QUESTION Does direct kisspeptin signaling in the oocyte have a role in the control of follicular dynamics and ovulation? SUMMARY ANSWER Kisspeptin signaling in the oocyte plays a relevant physiological role in the direct control of ovulation; oocyte-specific ablation of kisspeptin receptor, Gpr54, induces a state of premature ovulatory failure in mice that recapitulates some features of premature ovarian insufficiency (POI). WHAT IS KNOWN ALREADY Kisspeptins, encoded by the Kiss1 gene, are essential for the control of ovulation and fertility, acting primarily on hypothalamic GnRH neurons to stimulate gonadotropin secretion. However, kisspeptins and their receptor, Gpr54, are also expressed in the ovary of different mammalian species, including humans, where their physiological roles remain contentious and poorly characterized. STUDY DESIGN, SIZE, DURATION A novel mouse line with conditional ablation of Gpr54 in oocytes, named OoGpr54−/−, was generated and studied in terms of follicular and ovulatory dynamics at different age-points of postnatal maturation. A total of 59 OoGpr54−/− mice and 47 corresponding controls were analyzed. In addition, direct RNA sequencing was applied to ovarian samples from 8 OoGpr54−/− and 7 control mice at 6 months of age, and gonadotropin priming for ovulatory induction was conducted in mice (N = 7) from both genotypes. PARTICIPANTS/MATERIALS, SETTING, METHODS Oocyte-selective ablation of Gpr54 in the oocyte was achieved in vivo by crossing a Gdf9-driven Cre-expressing transgenic mouse line with a Gpr54 LoxP mouse line. The resulting OoGpr54−/− mouse line was subjected to phenotypic, histological, hormonal and molecular analyses at different age-points of postnatal maturation (Day 45, and 2, 4, 6 and 10–11 months of age), in order to characterize the timing of puberty, ovarian follicular dynamics and ovulation, with particular attention to identification of features reminiscent of POI. The molecular signature of ovaries from OoGpr54−/− mice was defined by direct RNA sequencing. Ovulatory responses to gonadotropin priming were also assessed in OoGpr54−/− mice. MAIN RESULTS AND THE ROLE OF CHANCE Oocyte-specific ablation of Gpr54 caused premature ovulatory failure, with some POI-like features. OoGpr54−/− mice had preserved puberty onset, without signs of hypogonadism. However, already at 2 months of age, 40% of OoGpr54−/− females showed histological features reminiscent of ovarian failure and anovulation. Penetrance of the phenotype progressed with age, with >80% and 100% of OoGpr54−/− females displaying complete ovulatory failure by 6- and 10 months, respectively. This occurred despite unaltered hypothalamic Gpr54 expression and gonadotropin levels. Yet, OoGpr54−/− mice had decreased sex steroid levels. While the RNA signature of OoGpr54−/− ovaries was dominated by the anovulatory state, oocyte-specific ablation of Gpr54 significantly up- or downregulated of a set of 21 genes, including those encoding pituitary adenylate cyclase-activating polypeptide, Wnt-10B, matrix-metalloprotease-12, vitamin A-related factors and calcium-activated chloride channel-2, which might contribute to the POI-like state. Notably, the anovulatory state of young OoGpr54−/− mice could be rescued by gonadotropin priming. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Conditional ablation of Gpr54 in oocytes unambiguously caused premature ovulatory failure in mice; yet, the ultimate molecular mechanisms for such state of POI can be only inferred on the basis of RNAseq data and need further elucidation, since some of the molecular changes observed in OoGpr54−/− ovaries were secondary to the anovulatory state. Direct translation of mouse findings to human disease should be made with caution since, despite the conserved expression of Kiss1/kisspeptin and Gpr54 in rodents and humans, our mouse model does not recapitulate all features of common forms of POI. WIDER IMPLICATIONS OF THE FINDINGS Deregulation of kisspeptin signaling in the oocyte might be an underlying, and previously unnoticed, cause for some forms of POI in women. STUDY FUNDING/COMPETING INTEREST(S) This work was primarily supported by a grant to M.P. and M.T.-S. from the FiDiPro (Finnish Distinguished Professor) Program of the Academy of Finland. Additional financial support came from grant BFU2017-83934-P (M.T.-S.; Ministerio de Economía y Competitividad, Spain; co-funded with EU funds/FEDER Program), research funds from the IVIRMA International Award in Reproductive Medicine (M.T.-S.), and EFSD Albert Renold Fellowship Programme (S.T.R.). The authors have no conflicts of interest to declare in relation to the contents of this work. TRIAL REGISTRATION NUMBER N/A.
The kinase IKKβ controls pro-inflammatory gene expression, and its activity in the liver and leukocytes was shown to drive metabolic inflammation and insulin resistance in obesity. However, it was also proposed that liver IKKβ signaling protects obese mice from insulin resistance and endoplasmic reticulum (ER) stress by increasing XBP1s protein stability. Furthermore, mice lacking IKKβ in leukocytes display increased lethality to lipopolysaccharides. This study aims at improving our understanding of the role of IKKβ signaling in obesity. We induced IKKβ deletion in hematopoietic cells and liver of obese mice by Cre-LoxP recombination, using an INF-inducible system, or a liver-specific IKKβ deletion in obese mice by adenovirus delivery of the Cre recombinase. The histopathological, immune, and metabolic phenotype of the mice was characterized. IKKβ deletion in the liver and hematopoietic cells was not tolerated in mice with established obesity exposed to the TLR3 agonist poly(I:C) and exacerbated liver damage and ER-stress despite elevated XBP1s. By contrast, liver-specific ablation of IKKβ in obese mice reduced steatosis and improved insulin sensitivity in association with increased XBP1s protein abundance and reduced expression of denovo lipogenesis genes. We conclude that IKKβ blockage in liver and leukocytes
Kisspeptins, encoded by the Kiss1 gene, are crucial brain factors for the control of reproduction, with an essential role in the generation of the pre‐ovulatory surge of gonadotropins and, hence, ovulation. Yet, kisspeptins are also found in peripheral reproductive organs and their receptor, Gpr54, is expressed in various target tissues, including the ovary, where its physiological role remains contentious. Global Gpr54−/− mice are hypogonadotropic and infertile; female null mice display disrupted folliculogenesis and anovulation. However, despite the predominant brain actions of kisspeptins, how much of this phenotype is due to local, and not central, kisspeptin signaling deficiency remains unknown. To address this, we generated a conditional oocyte‐specific Gpr54 deficient mouse line (OoGpr54−/−), by crossing Gdf9‐Cre+ mice with Gpr54loxP/loxP mice. The efficacy of Gdf9‐promoter to drive the expression of Cre in oocytes was verified by a breeding test, and the Gpr54 depletion from oocytes was confirmed with RT‐qPCR. At the time of pubertal transition, OoGpr54−/− females were indistinguishable from their wiltype controls with preserved puberty onset. However, already at 2 months of age, 45% of OoGpr54−/− females showed histological features reminiscent of ovarian failure, with presence of large atretic antral follicles and absence of corpora lutea as an index of anovulation. These features were also associated with disrupted estrous cyclicity characterized by an increase in the length of the estrus phase of the uterine cycle. Penetrance of this phenotype progressed with age, with 55% of OoGpr54−/− females at 3–4 months, 80% at 6 months and 100% at 10 months displaying features of complete premature ovarian insufficiency (POI). In addition, 4‐month‐old OoGpr54−/− female mice displaying POI showed decreased serum estradiol and progesterone levels, yet sustained Gpr54 expression in the hypothalamus and unaltered basal gonadotropin levels. Ovarian RNASeq analyses revealed global changes in expression profiles in OoGpr54−/− mice, including genes involved in cellular communication, steroidogenesis and intracellular signaling pathways. Finally, the anovulatory state of OoGpr54−/− mice was rescued by exogenously administered gonadotropin priming, and was largely prevented by early onset consecutive pregnancies, although multiparous OoGpr54−/− dams showed a slight decrease in fecundity. Our results pinpoint the important role of oocyte kisspeptin signaling in the direct control of ovulation, whose deregulation may also be an underlying cause for POI in women.Support or Funding InformationFiDiPro programme of Academy of Finland and Sigrid Juselius foundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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