Evaluating the Effects of Neburon Exposure on Ovarian Folliculogenesis Using Zebrafish and Mouse Granulosa Cell Line

BACKGROUND: Ovarian folliculogenesis is crucial for female reproduction. This can be disrupted by various factors, including pollutants with aryl hydrocarbon receptor (AHR) agonistic activity, but the underlying mechanisms remain unclear. OBJECTIVES: Using the herbicide neburon, a moderate AHR agonist among current pesticides, we investigated its effects on ovarian folliculogenesis in zebrafish through life-cycle exposure at environmentally relevant concentrations to elucidate the associated mechanisms. METHODS: Wild-type (WT) and three different genotypes of female zebrafish (amh ^±;bmpr2a ^+/+, amh ^+/+;bmpr2a ^±, amh ^±;bmpr2a ^±) were exposed to neburon for 150 days. Neburon and its metabolites in fish were quantified using LC-MS/MS. Ovarian pathology was assessed by H&E and TUNEL staining. The differentially expressed pathways were identified by transcriptome analysis, followed by validation using RT-qPCR, WB, IHC, and ELISA. Finally, AHR antagonist, ChIP-RT-qPCR, and other methods were used to further elucidate the mechanism in mouse granulosa cell line (KK1). RESULTS: After neburon exposure, only four metabolites of neburon were detected but not the metabolite itself, and all these metabolites had AHR agonistic activity, indicating the persistent toxicity of neburon. Neburon exposure altered the follicle-stage distribution and poorer oocyte quality in WT zebrafish. Further experiments found that neburon exposure induced greater secretion of anti-Mullerian hormone (Amh), greater expression of genes in the Amh/Bmpr2a pathway, accelerated follicular development, and lower expression of insulin-like growth factors, which was associated with oocyte atresia. Notably, amh ^±;bmpr2a ^± zebrafish showed a rescued phenotype with regard to these neburon-associated outcomes. Moreover, AHR exhibited specific binding to the Amh promoter in KK1 cells, and neburon treatment enhanced their interaction. DISCUSSION: In zebrafish, AMH was identified as a critical target for reproductive disorders following neburon exposure. Despite the short half-lives of the currently used pesticides, their metabolites might still have significant toxicological risks. This study provides novel insight into how one AHR agonistic pollutant affects female zebrafish reproduction.