Research ArticleIMMUNOLOGY

Inhibition of the NLRP3 inflammasome prevents ovarian aging

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Science Advances  01 Jan 2021:
Vol. 7, no. 1, eabc7409
DOI: 10.1126/sciadv.abc7409
  • Fig. 1 NLRP3 signaling is associated with ovarian aging.

    (A) Body weight progression of female C57BL6/J mice evaluated per month. (B) Mean serum AMH levels to evaluate the progression of ovarian reserve during aging. N = 8 per group. (C and D) Western blot analysis with representative blots including NLRP3, caspase-1, IL-1β, LC3, and p62 levels in the ovary of WT mice at different ages. Densitometric analysis is shown as mean ± SD, n = 10 mice; *P < 0.05, **P < 0.005, and ***P < 0.001; 2 months old versus other ages. N = 6 to 8 per group. (E) Immunofluorescence (IF) visualization of NLRP3 (green) and nuclei (blue) in ovary and uterus tissues from young and old WT mice. Oocyte (asterisk), GCs (arrows), corpus luteum (CL), and follicular atresia (FA). DAPI, 4′,6-diamidino-2-phenylindole. (F) Correlation of NLRP3 expression versus serum AMH levels during aging. The correlation was established by calculating correlation coefficients. (G) Human NLRP3 transcript expression levels were determined in GCs by real-time quantitative reverse transcription polymerase chain reaction (PCR); n = 20 for control and n = 20 for DOR groups. (H) Western blot analysis with representative blots including NLRP3, caspase-1, and IL-1β levels in GCs from four representative patients with DOR compared to four representative age-matched controls. Densitometric analysis is shown as mean ± SD. *P < 0.05, **P < 0.005, and ***P < 0.001; control versus patients.

  • Fig. 2 NLRP3 signaling suppression in mice extends life span and improves hormonal status in female mice.

    (A) Kaplan-Meier graph showing a significant increase in n that pertains to the maximum life span in WT mice compared with Nlrp3−/− mice. (B) Body weights of the groups over time. (C) Representative photographs of 28-month-old mice. (D) Mean serum AMH levels by enzyme-linked immunosorbent assay (ELISA) and ovarian AMH protein levels (top) measured by Western blot (WB) to evaluate the progression of ovarian reserve during aging in WT mice compared with Nlrp3−/− mice. On top, ovarian AMH protein levels. (E and F) Analysis of serum concentrations of FSH and E2 measured by ELISA. N = 8 per group. (G) Representative images showing comparative size and weights of uterine horns in WT and Nlrp3−/− mice. (H and I) Representative micrographs of 4- and 12-month-old WT and Nlrp3−/− ovarian sections. The number of ovarian follicles at developmental stages [primordial follicle (PMF), primary follicle (PF), secondary follicle (SF), and antral follicle (AF); asterisks in the image, PMF and arrowhead, atretic follicle] was assessed in every fifth serial section of WT and Nlrp3−/−. N = 15 to 20 per group. Data are shown as means ± SD. **P < 0.005, ***P < 0.001, aaaP < 0.001, Nlrp3−/− versus WT mice; aaP < 0.01, 4-month-old versus 12-month-old mice. Photo credit: Beatriz Castejón-Vega, Institute of Molecular, Cell and Systems Biology, University of Glasgow (C); Mario D. Cordero, Cátedra de Reproducción y Genética Humana del Instituto para el Estudio de la Biología de la Reproducción Humana (INEBIR)–Universidad Europea del Atlántico (UNEATLANTICO)–Fundación Universitaria Iberoamericana (FUNIBER) (G).

  • Fig. 3 Nlrp3 deletion improves reproductive rate and autophagy pathway.

    Pregnancy rate (A) and mean litter size (B) in aged WT and Nlrp3−/− mice. Data are shown as means ± SD. ***P < 0.001, Nlrp3−/− versus WT mice; aaaP < 0.001, 4-month-old versus 12-month-old mice. (C) Western blot analysis with representative blot including BECLIN 1, ATG12, LC3, p62, BAX, and Bcl-2 levels in the ovary of 4- and 12-month-old WT and Nlrp3−/−. Densitometric analysis is shown as means ± SD, n = 10 mice per group and age. **P < 0.005 and ***P < 0.001; WT versus Nlrp3−/−; aaaP < 0.001, aaP < 0.01, aP < 0.05, young versus old mice.

  • Fig. 4 Transcriptional changes in ovaries from old WT and Nlrp3−/− mice.

    (A) The most enriched KEGG pathways are shown for both overexpressed (691) and underexpressed genes (762). Differentially expressed genes were extracted with fold change > 2 and P < 0.05 (Bea). GeneRatio shows the relative frequency of genes that belong to that pathway. (B) Heatmap that shows the expression values (z scores) of differently expressed protein-coding genes involved in the following pathways: cell cycle, oocyte meiosis, and progesterone-mediated oocyte maturation, separated by the three replicates of aged WT and Nlrp3−/−.

  • Fig. 5 NLRP3 pharmacological inhibition improves female fertility.

    (A) Body weight chart of 8-month-old female mice treated either with vehicle or MCC950. Mice were fed with the standard diet for 12 weeks, and their body weights were monitored weekly. Hormonal status in female mice given MCC950 treatment was determined by (B) AMH, (C) FSH, and (D) E2 serum levels in female compared with vehicle treated mice (E and F). N = 8 per group. Representative micrographs of vehicle and MCC950 ovarian sections and quantification of the number of ovarian follicles (PMF, PF, SF, and AF; and arrowhead, atretic follicle). N = 10 per group. Pregnancy rate (G) and mean litter size (H) in vehicle- and MCC950-treated mice. N = 15 per group. Data are presented as means ± SD. ***P < 0.001, **P < 0.01, *P < 0.05, MCC950-treated versus vehicle-treated mice.

Supplementary Materials

  • Supplementary Materials

    Inhibition of the NLRP3 inflammasome prevents ovarian aging

    José M. Navarro-Pando, Elísabet Alcocer-Gómez, Beatriz Castejón-Vega, Elena Navarro-Villarán, Mónica Condés-Hervás, María Mundi-Roldan, Jordi Muntané, Antonio J. Pérez-Pulido, Pedro Bullon, Chun Wang, Hal M. Hoffman, Alberto Sanz, Gabriel Mbalaviele, Bernhard Ryffel, Mario D. Cordero

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