Fig 2: Acetyl-CoA oversupply accelerates ovarian aging.a Cell cycle distribution is shown by flow cytometry in the different groups. b Representative images of SA-β-gal staining in GCs with different groups. Scale bars, 200 μm. c Schematic showing continuous feeding of 4-month-old female mice with water and exogenous sodium acetate trihydrate (SAT, 200 mM) for 30 days. d–f Images of the ovaries of female mice in the control and SAT group were taken (d), and the ovary weight (e) and ovary index (ovary weight/body weight) (f) were measured. g-i Levels of acetyl-CoA (g), succinyl-CoA (h), and succinate (i) in ovarian tissues from mice with or without SAT treatment. j, k Immunoblot analyses of global lysine acetylation (j) and H4K8ac (k) levels in ovaries from control and SAT female mice. l Representative IHC images of ovaries from control and SAT female mice stained with anti-p21 antibody, with similar results were obtained in three independent experiments. Scale bars, 100 μm. m Protein levels of FSHR and AMH in the ovaries from control and SAT female mice. n Representative H&E-stained sections of ovaries from control and SAT female mice. Scale bars, 500 and 100 μm. o Quantification of total follicle per ovary in the control and SAT female mice. p Quantification of the number of follicles per ovary from control and SAT female mice. Follicles were classified into primordial (Pm), activated primordial (APF: oocyte diameter greater than 20 μm without cuboidal granulosa cells), primary (Pr), or secondary-antral (Sec-Ant) stages. q Mean pup numbers of control and sat female mice. Quantitative data are presented as mean ± SD, and statistical analyses were performed using unpaired two-tailed Student’s t-test (a, b, e–i, o–q). For (a, b, j, k, m) n = 3 biologically independent experiments. For (e-i, o, p) n = 6 biologically independent experiments. For (q) n = 5 biologically independent experiments. For (m, n) n = 4 biologically independent experiments. Exact P values are indicated in the figures.

Fig 3: SIRT5-dependent desuccinylation deficiency is associated with ovarian aging.a–f Immunoblot analyses of ovaries from young and aged female bovine. g,h Immunoblot analyses of granulosa cells isolated from young (YGCs) and aged (AGCs) ovaries. i, j Western blot analysis of SIRT5 (i) and succinylation (j) levels in GCs treated with or without Etoposide. k, l Western blot analysis of SIRT5 (k) and succinylation (l) levels in GCs with overexpressing SIRT5. m SIRT5 and succinylation levels in GCs with SIRT5 knockdown were examined by immunoblotting. n Protein levels of SIRT3 or SIRT5 relative to the mitochondrial marker COXⅣ. o PAS‑H staining of ovarian sections from wild-type (WT) and SIRT5 knockout mice. Scale bars: 50 μm. p Follicle numbers in ovarian sections were quantified. q SIRT5 protein expression levels in ovaries from WT and SIRT5 knockout mice. r, s Levels of AMH (r), and FSH (s) in serum of WT and SIRT5 knockout mice. t Representative offspring and litter size in WT and SIRT5 knockout mice. u Schematic showing the 30-day continuous treatment of 4-month-old female mice with drinking water or 100 mM sodium succinate (SUC). v Lysine succinylation levels were determined by immunoblotting in the ovaries of control and SUC female mice. w Representative immunohistochemical (IHC) images for p21 in the ovaries from control and SUC-treated female mice, with similar results were obtained in three independent experiments. Scale bars, 100 μm. x Representative offspring and litter size in control and SUC-treated mice. Quantitative data are presented as mean ± SD, and statistical analyses were performed using unpaired two-tailed Student’s t-test (a–f, h,j, l–n, p, r, t,v, x). For (a–n), n = 3 biologically independent experiments. For (q, v), n = 3 biologically independent mice per group. For (o, p, x), n = 5 biologically independent mice per group. For box plots r, s, center line = median, box bounds = 25th/75th percentiles, whiskers span data minima and maxima. For (r,t), n = 6 biologically independent experiments. Exact P values are indicated in the figures.