Fig 2: Prenatal PrP exposure induced transgenerational transmission of diminished ovarian reserve to multiple generations.a Schematic illustration of the animal experimental design. b AMH concentrations in the serum of F1–F3 female offspring mice in control (Con) and prenatal PrP exposure (PrP) at 12 w (F1 n = 8, F2 n = 7, F3 n = 7 biological replicates). Data are presented as mean ± SEM (unpaired two-tailed t-test). c Representative ovarian HE staining images of F1–F3 offspring mice in Con and PrP groups at 12 w. Red arrow: atretic follicles. d The number of follicles of F1–F3 female offspring mice in Con and PrP groups at 12 w (n = 5 biological replicates). Data are presented as mean ± SEM (unpaired two-tailed t-test). e The proportion of regular and irregular estrous cycles in Con and PrP groups of F1–F3 offspring mice at 12 w (F1 n = 36, F2 n = 20, F3 n = 30 for each group; Chi-squared test, two-sided). f Representative estrous cycles of F3 offspring mice in Con and PrP groups at 12 w. g Levels of serum E2 in F1–F3 offspring mice with or without prenatal PrP exposure (F1 n = 5, F2 n = 6, F3 n = 7 biological replicates). Data are presented as mean ± SEM (unpaired two-tailed t-test). h Levels of serum P4 in F1–F3 offspring mice with or without prenatal PrP exposure (F1 n = 5, F2 n = 8, F3 n = 7 biological replicates). Data are presented as mean ± SEM (unpaired two-tailed t-test). PMF: primordial follicles, PF: primary follicles, SF: secondary follicles, ANF: antral follicles, ATF: atretic follicles. Source data are provided as a Source Data file. Figure 1a is created in BioRender. Wang, S. (2025) https://BioRender.com/jnngb84.

Fig 3: The hypomethylation and overexpression of Rhobtb1 led to DOR by inducing GC apoptosis.a The methylation levels of CpG site −1555 to TSS in the promoter region of Rhobtb1 following PrP exposure (n = 5 biological replicates). Data are presented as mean ± SEM (one-way ANOVA). b, c Protein levels of RhoBTB1 in KGN cells following PrP exposure. Data from (c) represent three independent experiments (mean ± SEM; one-way ANOVA). d AMH levels in KGN cells following PrP exposure (n = 3 biological replicates). Data are presented as mean ± SEM (one-way ANOVA). e E2 levels in KGN cells following PrP exposure (n = 3 biological replicates). Data are presented as mean ± SEM (one-way ANOVA). f, g Violin plot showing the apoptosis ratio of KGN cells after PrP exposure (n = 3 biological replicates; one-way ANOVA). The black dotted line represents median ratio. h, i The protein levels of BAX, BCL-2, and active-Caspase3 in KGN cells tested by western blotting following PrP exposure. Data from (i) represent three independent experiments (mean ± SEM; one-way ANOVA). j Representative images showing the infection of adenovirus with and without encoding Rhobtb1 in KGN cells. The experiment was repeated three times. k, l Protein levels of RhoBTB1 in GCs detected by western blotting following RhoBTB1 overexpression. Data from (l) represent three independent experiments (mean ± SEM; unpaired two-tailed t-test). m AMH levels in GCs following RhoBTB1 overexpression (n = 5 biological replicates). Data are presented as mean ± SEM (unpaired two-tailed t-test). n E2 levels in GCs following RhoBTB1 overexpression (n = 5 biological replicates). Data are presented as mean ± SEM (unpaired two-tailed t-test). o, p Violin plot showing the apoptosis ratio of GCs after RhoBTB1 overexpression (n = 3 biological replicates; unpaired two-tailed t-test). The black dotted line represents median ratio. q, r The protein levels of BAX, BCL-2, and active-Caspase3 in GCs detected by western blotting following RhoBTB1 overexpression. Data from (r) represent three independent experiments (mean ± SEM; unpaired two-tailed t-test). Source data are provided as a Source Data file.

Fig 4: Epigenetic therapy ameliorated DOR induced by prenatal PrP exposure in F1–F3 offspring mice.a Scheme for the animal experiment. b The percentage of mice with normal and irregular estrous cycles at 12 weeks (w) (F1 Con n = 8, PrP n = 10, PrP+MD n = 13, MD n = 13; F2 Con n = 9, PrP n = 12, PrP+MD n = 12, MD n = 11; F3 Con n = 19, PrP n = 23, PrP+MD n = 22, MD n = 18; Fisher’s exact test). c AMH concentrations in the serum of F1–F3 female offspring mice with or without PrP/methyl-donor diet (MD) treatment at 12 w (n = 6 biological replicates). Data are presented as mean ± SEM (one-way ANOVA). d–f The number of follicles in F1–F3 offspring mice at 12 w with or without PrP/MD treatment (n = 5 biological replicates). Data are presented as mean ± SEM (one-way ANOVA). g Representative ovarian images for TUNEL staining of F1–F3 offspring mice with or without PrP/MD treatment at 12 w. h The number of TUNEL-positive cells per 1 mm2 in ovarian sections of F1–F3 offspring mice with or without PrP/MD treatment at 12 w (n = 5 biological replicates). Data are presented as mean ± SEM (one-way ANOVA). i Representative IHC images showing immunoreactivity against RhoBTB1 in F1–F3 offspring ovaries at 12 w with or without PrP/MD treatment. j IHC scores based on IOD/Area of anti-RhoBTB1 in F1–F3 offspring ovaries at 12 w with or without PrP/MD treatment (n = 5 biological replicates). Data are presented as mean ± SEM (one-way ANOVA). Source data are provided as a Source Data file. Figure 8a is created in BioRender. Wang, S. (2025) https://BioRender.com/aaoydng.