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.