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. 2024 Jun 17;14(9):3760-3776.
doi: 10.7150/thno.95197. eCollection 2024.

Sleep deprivation causes gut dysbiosis impacting on systemic metabolomics leading to premature ovarian insufficiency in adolescent mice

Jiamao Yan  1   2 , Xiaoyuan Zhang  1   2 , Kexin Zhu  1 , Mubin Yu  1 , Qingchun Liu  1 , Massimo De Felici  3 , Teng Zhang  2 , Junjie Wang  1 , Wei Shen  1
Affiliations

Sleep deprivation causes gut dysbiosis impacting on systemic metabolomics leading to premature ovarian insufficiency in adolescent mice

Jiamao Yan et al. Theranostics. .

Abstract

Rationale: Currently, there are occasional reports of health problems caused by sleep deprivation (SD). However, to date, there remains a lack of in-depth research regarding the effects of SD on the growth and development of oocytes in females. The present work aimed to investigate whether SD influences ovarian folliculogenesis in adolescent female mice. Methods: Using a dedicated device, SD conditions were established in 3-week old female mice (a critical stage of follicular development) for 6 weeks and gut microbiota and systemic metabolomics were analyzed. Analyses were related to parameters of folliculogenesis and reproductive performance of SD females. Results: We found that the gut microbiota and systemic metabolomics were severely altered in SD females and that these were associated with parameters of premature ovarian insufficiency (POI). These included increased granulosa cell apoptosis, reduced numbers of primordial follicles (PmFs), correlation with decreased AMH, E2, and increased LH in blood serum, and a parallel increased number of growing follicles and changes in protein expression compatible with PmF activation. SD also reduced oocyte maturation and reproductive performance. Notably, fecal microbial transplantation from SD females into normal females induced POI parameters in the latter while niacinamide (NAM) supplementation alleviated such symptoms in SD females. Conclusion: Gut microbiota and alterations in systemic metabolomics caused by SD induced POI features in juvenile females that could be counteracted with NAM supplementation.

Keywords: Gut microbiota; Mouse; Niacinamide metabolism; Premature ovarian insufficiency; Sleep deprivation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Body weight and gut microbiota changes caused by sleep deprivation. (A) Schematic diagram of the sleep deprivation (SD) experiment. (B) Changes of body weight in control (CTRL) and SD females during the experimental time (n=12). (C) Body weight of CTRL and SD females at the end of the experimental time (n=12). (D) ANOSIM similarity analysis of gut microbiota obtained from CTRL and SD females at the end of the experimental time. (E) PCA of the data shown in D. (F) α-Diversity indexes, including Shannon, Simpson, Chao1, and ACE of the data shown in D. (G) Relative abundance of the bacterial phyla in the gut microbiota of CTRL and SD females. (H) Relative abundance of bacterial genera in the gut microbiota of CTRL and SD females. (I) LDA algorithm of CTRL vs SD gut microbiota bacterial genera with an LDA score threshold of 2.0. (J) Cladogram of the main taxa of microbiota in CTRL and SD females based on LEfSe analysis.
Figure 2
Figure 2
Intestinal barrier disturbance caused by sleep deprivation. (A) Representative H&E histological sections of small intestine samples collected from CTRL and SD females. (B-D) Comparison of villi height, crypt depth, and villi height/crypt depth of small intestine samples of CTRL and SD females. (E) Fluorescent immunostaining (IF) for the indicated tight junction proteins on small intestinal sections of CTRL and SD females. (F-G) Representative WB and relative densitometric evaluation of the amount of the indicated tight junction proteins and inflammatory cytokines in the small intestine of CTRL and SD females. (H-I) IF quantitative statistics of CD4 and CD68 proteins associated with intestinal immune cells.
Figure 3
Figure 3
Impact of sleep deprivation on the metabolome. (A) PLS-DA discriminant analysis of the metabolome in blood serum from CTRL and SD females. (B) Volcano map of metabolite changes caused by SD. (C) KEGG metabolic pathway enrichment analysis. (D) Heatmap of metabolites in niacinamide (NAM) metabolic pathways. (E-F) Comparison of the relative abundance of NAM and 1-Methylnicotinamide in the metabolome of CTRL and SD females. (G) Spearman correlation analysis between NAM and different bacterial genera. The red font represents the bacteria genera with significant differences shown between CTRL and SD groups.
Figure 4
Figure 4
Effects of sleep deprivation on ovary index and follicle dynamics. (A) Blood serum concentrations of AMH, E2, FSH, and LH hormones at the end of the experimental time. (B) Representative picture of the CTRL and SD ovary at the end of the experimental time, bar =1 mm. (C) Ovary and body weight ratio (ovary index) in CTRL and SD females at the end of the experimental time (n=12). (D) Representative IHC for the oocyte-specific MVH protein on CTRL and SD ovary sections, bar = 200 μm. (E) Number of different classes of follicles in the 9 sections of CTRL and SD ovaries at the end of the experimental time. (F) Percent of primordial follicles and growing follicles (all other follicle classes, see below) for sections of CTRL and SD ovaries at the end of the experimental time. (G-I) Representative WB and relative densitometric evaluation of the amount of the indicated proteins from CTRL and SD ovaries at the end of the experimental time. PmF: Primordial follicle, PF: Primary follicle, SF: Secondary follicle, AF: Antral follicle.
Figure 5
Figure 5
Sleep deprivation affected oocyte maturation, spindle assembly, and mitochondrial function. (A) Representative pictures of GV, GVBD, and PB1. Scale bar = 100 μm. (B) Percent of GVBD and PB1 in CTRL and SD oocytes after in vitro culture for 4-6 h and 12-16 h (n>90). (C) Representative pictures of normal and abnormal spindle morphologies and chromosome alignment. (D) Percent of aberrant spindles and misaligned chromosomes in CTRL and SD oocytes after in vitro culture for 8 h (n>90). (E) Volcano map of gene transcript differences in CTRL and SD oocytes. (F) Enrichment terms of down-regulated genes. (G) Gene expression levels of the indicated genes related to mitochondrial function. (H) RT-qPCR of the same genes showed in G. (I) Enrichment terms of up-regulated genes.
Figure 6
Figure 6
Fecal microbiota transplantation from SD females reproduces the effects of SD in normal host females. (A) Study design of fecal microbiota transplantation (FMT). (B-C) Body and ovary weights of females under the indicated experimental conditions. (D) Concentration of niacinamide (NAM) in the blood serum obtained from females under the indicated experimental conditions. (E) Total number and different follicle class number for sections of ovaries from females under the indicated experimental conditions. (F) Percent of PmF and growing follicles in the ovaries of females under the indicated experimental conditions. (G) Representative WB and relative densitometric evaluation of the amount of the indicated proteins in the ovaries of females under the indicated experimental conditions.
Figure 7
Figure 7
Dietary niacinamide supplementation alleviated the effects of sleep deprivation on the ovarian follicle reserve. (A) Study design of niacinamide (NAM) supplementation. (B) Total number and different follicle class number for sections of ovaries of females under the indicated experimental conditions. (C) Percent of PmF and growing follicles in the ovaries of females under the indicated experimental conditions. (D) Representative WB and relative densitometric evaluation of the amounts of the indicated proteins in the ovaries of females under the indicated experimental conditions. (E) Representative pictures of oocytes at GV, GVBD, and PB1 stages in SD and SD+NAM groups. Scale bar = 100 μm. (F) Percentage of oocytes at GVBD and PB1 stages in SD, and SD+NAM groups after in vitro culture (n>90). (G) The statistical effect of vaginal plug rate in CTRL, SD, and SD+NAM groups, also including false pregnancies or miscarriage rate (H, number of female mice with vaginal plugs in each group >29), pup numbers (I), and average birth weight of offspring (J).
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