Fig 2: Paternal exposure to BPA accelerated hepatic senescence in the offspring. A–F) Representative images of the immunofluorescence staining for F4/80 (F4/80+ is red and Dapi is blue) and Masson's trichrome staining (blue is collagen), and the TEM images in the liver of male (A–C) and female offspring (D–F). The quantitation data were shown in the right panel. G,I) Detection of the serum inflammatory factors in the male (G) and female (I) offspring. H,J) The protein expression of several cellular senescence markers in the male (H) and female (J) offspring. β‐actin was used as an internal reference. The quantification of protein bands was shown in Figure S7I,J of the Supporting Information. K–N) AML12 cells were transfected for 48 h with either a single miR149‐5p, miR150‐5p, miR700‐5p mimic, a mixture of mimics (IAL‐miRs), or a control mimic (miRNC). O–Q) AML12 cells were cotransfected for 48 h with the mixture‐mimics (IAL‐miRs) and pEGFP‐Lepr (pLepr) or their corresponding controls. (K,O) The mRNA expression of Ccnd1 in AML12 cells. β‐actin was served as a reference gene. (L,P) The protein expression of cellular senescence markers in the AML12 cells. β‐actin was used as an internal reference. β‐actin was used as an internal reference. The quantification of protein bands was shown in Figures S8H and S9D of the Supporting Information. (M,N,Q) The senescence‐associated SA‐β‐gal and EdU fluorescence staining for proliferation. Quantification of data was shown in the 5N (Figure S9E,F, Supporting Information). R–T) An in vitro transwell coculture system to study the effect of IAL‐miRs–Lepr pathway‐regulated hepatocytes senescent on adipocyte differentiation. (R) Schema of the coculture model. The transfected AML12 cells were seeded as donor cells and cocultured with recipient 3T3‐L1 adipocytes in a transwell system. (S,T) The fat storage evaluated using Oil Red O staining in adipocytes cocultured with AML12 cells transfected with either IAL‐miRs mimics (S) or both IAL‐miRs mimics and pLepr (T). (A–J) n = 3–4 mice per group as indicated; (K–T) n = 3 independent experiments/group. Data were presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, versus corresponding control offspring, or AML12 cells transfected with control mimic. # p < 0.05, ## p < 0.01, ### p < 0.001, AML12 cells cotransfected IAL‐miRs mimics and pEGFP‐N1 versus AML12 cells cotransfected IAL‐miRs mimics and pLepr.

Fig 3: The IAL‐miRs–Lepr axis regulated hepatic lipid homeostasis and insulin signal in the paternal BPA‐exposed offspring. A) Lepr‐focused regulatory network. The node size indicated the closeness centrality in the network, and node color represented the degree of connectivity. B,E) The mRNA expression of hub genes in the Lepr‐focused regulatory network. β‐actin served as a reference gene. C,F) The expression of the downstream effector proteins involved in Lepr‐mediated regulation of glucose and lipid metabolism. The Akt phosphorylation was detected in the treated AML12 cells stimulated with 100 nm insulin for 15 min before harvesting. β‐actin served as a reference gene. The quantification protein bands were shown in Figures S8E and S9B of the Supporting Information. D,G) Lipid droplets labeled with BODIPY. The quantification of BODIPY‐positive cells was shown in Figures S8F and S9C of the Supporting Information. (B–D) The AML12 cells were transfected with the single miR149‐5p mimic, miR150‐5p mimic, miR700‐5p mimic, mixture‐mimics (IAL‐miRs), or control mimic (miRNC) for 48 h; and (E–G) the AML12 cells were cotransfected with the mixture‐mimics (IAL‐miRs), pEGFP‐Lepr (pLepr) or corresponding controls for 48 h as indicated. H,J) Representative images of HE‐ and Oil Red O‐stained liver sections in the male (H) and female offspring (J). I,K) The TC and TG contents in the liver of male (J) and female offspring (K). L–O) Validation of the protein (L,N) and mRNA (M,O) expression of genes regulated by Lepr in the liver of male (L,M) and female offspring (N,O). β‐actin was used as an internal reference. The quantification data of protein expression were shown in Figure S7C,D of the Supporting Information. P,Q) Analysis of insulin signal pathway in the liver of male (P) and female offspring (Q). The mice were fasted overnight, given an intraperitoneal insulin injection (1.0 IU kg−1), and sacrificed after 10 min to prepare liver protein extracts. β‐actin served as a reference gene. (B–G) n = 3 independent experiments/group; (L–Q) n = 3–4 mice per group as indicated. Data were presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, versus corresponding control offspring, or AML12 cells transfected with miRNC. # p < 0.05, ## p < 0.01, ### p < 0.001, AML12 cells cotransfected IAL‐miRs mimics and pEGFP‐N1 versus AML12 cells cotransfected IAL‐miRs mimics and pLepr.

Fig 4: Inheritance testing and target gene screening of BPA‐upregulated miRNAs in paternal spermatozoa. A) The expression of sperm miRNAs in the metabolic organs (liver, skeletal muscle, and adipose tissue) in offspring. U6 was used as a loading control. B–D) Transcriptome analysis of hepatic gene expression profile in the male offspring. The PCA plot, the volcano plots, and the cluster heat map of the differential genes were shown in Figure S5A–C of the Supporting Information. B) The GO‐BP pathway related to glucose and lipid metabolism based on GO analysis in the liver transcriptome data. The top 25 enriched GO‐BP, GO‐CC, and GO‐MF terms were shown in Figure S5D–F of the Supporting Information. Color bars, the log (q‐value) of enrichment; Gene count, differential gene count in the indicated pathway; GeneRatio, differential gene count in the indicated pathway versus total differential gene count. C) The KEGG pathway enrichment analysis of the liver transcriptome data. Pathways involved in glucose and lipid metabolism were considered as hub pathways and bolded. D) The interaction network of differentially expressed genes mapped on the hub KEGG pathways. The square represented the hub KEGG pathways and the circle represented the differentially expressed genes. E) Venn diagram depicting the number of genes that overlap among miR700‐5p target genes, miR150‐5p target genes, and the downregulated genes (defined by a fold change ≤ −2 and an adjusted p‐value ≤0.05) identified in the liver transcriptomic analysis. The name and fold changes in the expression of 29 overlapped genes were summarized in the table on the right. F) The putative miRs binding sites in the 3′UTR of Lepr. G–I) AML12 cells were transfected with the single miR149‐5p mimic, miR150‐5p mimic, miR700‐5p mimic, mixture‐mimics (IAL‐miRs), or control mimic for 48 h. (G,H) The mRNA (G) and protein (H) expression of Lepr. Expression levels were normalized to β‐actin. (I) The relative luciferase activity in AML12 cells transfected with reporter vector containing wild (SMITE‐WT) or mutated (SMITE‐MT) “SMITE” binding sites. J–P) The impact of IAL‐AgomiRs overexpression in zygotes on offspring. (J) Schematic diagram of microinjection and the experimental procedure. IAL‐AgomiRs and corresponding Ctrl‐AgomiRs were microinjected into zygotes, which were subsequently transferred to surrogate mothers. The resulting offspring were kept for 14 days after birth for the further experiments (K–P). (K) The body weight in offspring. (L) The random blood glucose in offspring. (M) The liver weight in offspring. (N) The expression of miR149‐5p, miR150‐5p, and miR700‐5p in the liver of offspring. U6 RNA served as the loading control. (O,P) The mRNA (O) and protein (P) expression of Lepr and Dicer1 in the liver of offspring. β‐actin was used as an internal reference. (A–E) n = 3–6 mice per group as indicated; (G–I) n = 3 independent experiments per group; and (J–P) n = 3–6 mice from different litters/group as indicated. Data were presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, versus corresponding controls.