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GnRH pulse frequency and irregularity play a role in male aging

An Author Correction to this article was published on 20 October 2021

This article has been updated

Abstract

Gonadotropin-releasing hormone (GnRH) has a role in hypothalamic control of aging, but the underlying patterns and relationship with downstream reproductive hormones are still unclear. Here we report that hypothalamic GnRH pulse frequency and irregularity increase before GnRH pulse amplitude slowly decreases during aging. GnRH is inhibited by nuclear factor (NF)-κB, and GnRH pulses were controlled by oscillations in the transcriptional activity of NF-κB. Exposure to testosterone under pro-inflammatory conditions stimulated both NF-κB oscillations and GnRH pulses. While castration of middle-aged mice induced short-term anti-aging effects, preventing elevation of luteinizing hormone (LH) levels after castration led to long-term anti-aging effects and lifespan extension, indicating that high-frequency GnRH pulses and high-magnitude LH levels coordinately mediate aging. Reprogramming the endogenous GnRH pulses of middle-aged male mice via an optogenetic approach revealed that increasing GnRH pulses frequency causes LH excess and aging acceleration, while lowering the frequency of and stabilizing GnRH pulses can slow down aging. In conclusion, GnRH pulses are important for aging in male mice.

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Fig. 1: GnRH and LH pulses change during mouse aging.
Fig. 2: Inverse relationship between NF-κB signaling oscillation and GnRH pulses.
Fig. 3: Effects of testosterone on NF-κB cytoplasmic–nuclear oscillation.
Fig. 4: Effects of castration on GnRH pulses.
Fig. 5: Effects of castration on aging physiology.
Fig. 6: Healthspan and lifespan extension by castration in middle age in combination with LH inhibition.
Fig. 7: Optogenetic resetting of GnRH activation frequency in vivo.
Fig. 8: Effects on healthspan and lifespan of GnRH optogenetic stimulation in middle-aged mice.

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Source data are provided with this paper. There is no restriction on data availability for this manuscript.

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Acknowledgements

We thank the Cai laboratory members for technical support, A. Wolfe (Johns Hopkins University) for the Gnrh-Cre mouse model, P. Mellon (University of California, San Diego) for GT1-7 and LβT4 cells, and the IVIS facility at Einstein for in vivo imaging assistance. This study was supported directly or indirectly through Einstein resources and NIH grants R01 AG031774, DK121435 and HL147477 (all to D.C.).

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Z.W. co-designed and performed experiments, including GnRH pulse imaging, optogenetic models and physiological assays, castration model follow-up, cell culture models, immunostaining, histology and biochemistry, performed data analysis, contributed to interpretation of the data and prepared the figures for the paper. W.W. co-designed and performed experiments, including castration, LH knockdown, physiology and biochemistry experiments, and performed data analysis and interpretation. M.K. contributed to the castration model, GnRH knockdown and physiology studies. D.C. conceived the idea and hypothesis, conceptualized the project, designed the whole study and specific aims, constructed the experimental framework, supervised the study, led data analysis and interpretation, and wrote the paper.

Corresponding author

Correspondence to Dongsheng Cai.

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The authors declare no competing interests.

Additional information

Peer review information Nature Aging thanks Cláudia Cavadas and Han Kyoung Choe for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Pulses of blood FSH concentrations in male mice of different ages.

Blood FSH concentrations were measured during 180 mins in young (3 months), early middle-aged (11–12 months) and late middle-aged (16–17 months) mice. a: Representative blood FSH concentrations. b–d: Quantitative analysis of pulse frequency (b), peak concentration (c), and AUC value (d) of blood FSH concentrations. Comparisons of all groups were performed with one-way ANOVA but without statistical significance, n = 5 mice per group (b–d). Bar graphs reflect mean ± s.e.m.

Source data

Extended Data Fig. 2 A schematic model of oscillated GnRH mRNA production and NF-κB signaling.

This schematic diagram through drawing illustrative curves (which were not experimental data) is to generalize the concept for the relationship among oscillations of NF-κB activation and the production of IκBα protein, IκBα mRNA and GnRH mRNA in GnRH cells. Based on this conceptual model, it can be deduced that when NF-κB is chronically up-activated during aging, it leads to an equilibrium of fast-frequency NF-κB nuclear oscillation and fast-frequency pulsatile GnRH mRNA production.

Extended Data Fig. 3 Late age physiology and lifespan of mice with castration at the age of 8 months.

(a) Schematic diagram of the experimental procedure. (b–i) Mice were assigned into subgroups for different behavioral assays at the age of 18–19 months for locomotion (b), treadmill (c), coordination (d), novel recognition (e), sociality (f), learning and memory (g), adhesive removal (h), and odor smelling (i). (j–k) Subgroups of mice were profiled for pulse frequencies (j) and peak concentrations (k) of blood LH. (l) Lifespan follow-up of these mice. P values are shown in figures for comparisons between groups as indicated (k, l) or labeled as non-significant (NS) (b–j), two-tailed Student’s t-test (b–k), log-rank (Mantel-Cox) test (l); n = 8 per group (b–i), n = 5 per group (j, k); n = 22 mice per group (l). Data are mean ± s.e.m.

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Extended Data Fig. 4 Effects of middle-age castration plus LH inhibition on histology.

Middle-aged mice with castration and LH knockdown (KD) were described in Fig. 4, and subgroups were used for examining skeletal muscle (a) and skin (b) histology. The results present the representative H&E staining. The statistical analyses are shown in Fig. 4n–p. Scale bars: 100 μm.

Extended Data Fig. 5 GnRH-Cre in subpopulation of Sox2-positive hypothalamic cells.

GnRH-Cre mice were crossed with Rosa 26-lox-STOP-lox-GFP mice leading to the offspring of GnRHCre:Rosa GFP mice, and the hypothalamic sections of these offspring were made for co-immunostaining of Sox2 and GnRH-Cre-dependent GFP expression (labelled as GnRH-GFP). White arrows point to representative cells which are co-positive for Sox2 and GFP. Images represent 4 independent repeats per condition. Scare bar: 100 μm.

Extended Data Fig. 6 Fast optogenetic stimulation prevents NF-κB inhibition from slowing LH pluses.

GnRH-Cre mice (12-month-old male) received hypothalamic injection of AAV together with a lentivirus containing GnRH promoter-driven DN-IκBα (labelled as DNIκBα) vs. control (labeled as Control), and subsequently these animals were treated with fast optogenetic stimulation vs. sham procedure as described in Fig. 5 and profiled for blood concentrations of LH (a–d) and FSH (e–h). (a, e) Representative blood LH (a) and FSH (e) concentrations. (b–d, f–h) Quantitative analysis of pulse frequency (b, f), peak concentration (c, g) and AUC value (d, h) of blood LH (b-d) and FSH (f–h). P values are shown in figures for comparisons between groups as indicated, one-way ANOVA with Tukey’s post hoc test (b–d), n = 5 mice per group (b–d). Comparisons of all groups with one-way ANOVA for lacking statistical significance (f–h), n = 5 mice per group (f–h). Data are mean ± s.e.m.

Source data

Extended Data Fig. 7 Fast optogenetic stimulation reduces the anti-aging effect of NF-κB inhibition.

GnRH-Cre male mice (12 months old) received hypothalamic injection of AAV together with a lentivirus containing GnRH promoter-driven DNIκBα vs. Control, and subsequently these animals were treated with fast optogenetic stimulation vs. sham as described in Fig. 5 and profiled for physiology. (a–c) Longitudinal body weight (a) and food intake (b) follow-up and body composition measured at the age of about 18 months (c). (d–m) Aging-related physiology including behavioral functions assessed when 18–19 months old (d–j) and tissue histology assessed when 24 months old (k–m). P values are shown in figures for comparisons between groups as indicated, one-way ANOVA with Tukey’s post hoc test (d–m); n = 10 mice per group (a–b), n = 7 mice per group (c), n = 8 mice per group (d–m). Data are mean ± s.e.m.

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Wang, Z., Wu, W., Kim, M.S. et al. GnRH pulse frequency and irregularity play a role in male aging. Nat Aging 1, 904–918 (2021). https://doi.org/10.1038/s43587-021-00116-5

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