The PCOS–NAFLD Multidisease Phenotype Occurred in Medaka Fish Four Generations after the Removal of Bisphenol A Exposure

As a heterogeneous reproductive disorder, polycystic ovary syndrome (PCOS) can be caused by genetic, diet, and environmental factors. Bisphenol A (BPA) can induce PCOS and nonalcoholic fatty liver disease (NAFLD) due to direct exposure; however, whether these phenotypes persist in future unexposed generations is not currently understood. In a previous study, we observed that transgenerational NAFLD persisted in female medaka for five generations (F4) after exposure to an environmentally relevant concentration (10 μg/L) of BPA. Here, we demonstrate PCOS in the same F4 generation female medaka that developed NAFLD. The ovaries contained immature follicles, restricted follicular progression, and degenerated follicles, which are characteristics of PCOS. Untargeted metabolomic analysis revealed 17 biomarkers in the ovary of BPA lineage fish, whereas transcriptomic analysis revealed 292 genes abnormally expressed, which were similar to human patients with PCOS. Metabolomic–transcriptomic joint pathway analysis revealed activation of the cancerous pathway, arginine–proline metabolism, insulin signaling, AMPK, and HOTAIR regulatory pathways, as well as upstream regulators esr1 and tgf signaling in the ovary. The present results suggest that ancestral BPA exposure can lead to PCOS phenotypes in the subsequent unexposed generations and warrant further investigations into potential health risks in future generations caused by initial exposure to EDCs.


Description of supporting information
Information provided in the supporting material section shows mechanisms associated with PCOS in the ovary of medaka fish whose ancestors were exposed to BPA four generations ago.
The analysis was performed using the Qiagen IPA software, KEGG pathway analysis (opensource software), GSEA and metaboanalyst 5.0 software.Results are provided as disease network and pathways suggested by transcriptional and metabolomic analysis.

Contents
Table 1.Primers used in quantitative real-time PCR.

Figures Figure S1.
Figure S1.A global alteration in gene expression in the ovary of the BPA lineage.Heatmap

Figure S2 .
Figure S2.Gene Ontology analysis in the ovary of BPA lineage. A. Cellular component, B.

Figure S3 .
Figure S3.DEGs enriched in Insulin signaling pathway in the ovary of BPA lineage.

Figure S4 .
Figure S4.DEGs found in MAPK signaling pathway in ovary of BPA lineage.

Figure S5 .
Figure S5.DEGs enriched in AMPK signaling pathway in ovary of BPA lineage.

Figure S6 .
Figure S6.DEGs found in Rap1 signaling pathway in ovary of BPA lineage.

Figure S7 .
Figure S7.DEGs found in cAMP signaling pathway in ovary of BPA lineage.

Figure S8 .
Figure S8.Standard GSEA analysis and heatmap of enriched DEGs associated with

Figure S9 .
Figure S9.Standard GSEA analysis and heatmap of enriched DEGs.A) cell cycle B) catabolic

Figure S11 .
Figure S11.Molecular mechanism of cancer triggering pathway in the ovary of the BPA lineage

Figure S12 .
Figure S12.Molecular mechanism of autophagy triggering pathway in the ovary of the BPA

Figure S13 .
Figure S13.Molecular mechanism of HOTAIR mechanism in the ovary of the BPA lineage fish.

Figure S14 .
Figure S14.Gene disease network via ingenuity Pathway analysis (IPA) showing activation of

Figure S15 .
Figure S15.Predicted top 15 upstream regulator determined by IPA.Upstream regulator

Figure S16 .
Figure S16.Score plot showing significant difference in metabolites in biological replicates of

Figure S17 .
Figure S17.Categorization of significant metabolites (VIP>1) found in the ovary of BPA

Figure S18 .
Figure S18.The enrichment of metabolites is associated with several metabolic pathways.

Figure S19 .
Figure S19.Gene metabolite interaction network in the ovary of BPA lineage showing positive

Figure S20 .
Figure S20.Mapping of differential metabolites (round blue circle) and genes (red triangle)

Figure S1 .Figure S3 .
Figure S1.A global alteration in gene expression in the ovary of the BPA lineage.Heatmap

Figure S4 .
Figure S4.DEGs found in MAPK signaling pathway in ovary of BPA lineage.

Figure S5 .
Figure S5.DEGs enriched in AMPK signaling pathway in ovary of BPA lineage.

Figure S8 .
Figure S8.Standard GSEA analysis and heatmap of enriched DEGs associated with pathogenesis in the

Figure S11 .
Figure S11.Molecular mechanism of cancer triggering pathway in the ovary of the BPA lineage fish.

Figure S12 .
Figure S12.Molecular mechanism of autophagy triggering pathway in the ovary of the BPA lineage fish.

Figure S13 .
Figure S13.Molecular mechanism of HOTAIR mechanism in the ovary of the BPA lineage fish.

Figure S14 .
Figure S14.Gene disease network via ingenuity Pathway analysis (IPA) showing activation of akt1, tnf, and ifng associated with activation disease specific pathways.

Figure S15 .
Figure S15.Predicted top 15 upstream regulator determined by IPA.Upstream regulator mediated active target molecules and mechanistic network indicating potential involvement of upstream regulator in ovarian pathogenesis triggered by ancestral BPA exposure effect.

Figure S16 .
Figure S16.Score plot showing significant difference in metabolites in biological replicates of BPA lineage and control lineage fish.

Figure S17 .
Figure S17.Categorization of significant metabolites (VIP>1) found in the ovary of BPA lineage.

Figure S18 .
Figure S18.The enrichment of metabolites is associated with several metabolic pathways.

Figure S19 .
Figure S19.Gene metabolite interaction network in the ovary of BPA lineage showing positive association of metabolites with gene expression.

Figure S20 .
Figure S20.Mapping of differential metabolites (round blue circle) and genes (red triangle) found in carbon metabolism of cancer.

Table S1 .
Primers used in quantitative real-time PCR.