Levonorgestrel-induced anal fin masculinization in mosquitofish: Do TGF-β/BMP signaling and osteogenic remodeling underpin endocrine disruption in aquatic ecosystem?

Abstract

Levonorgestrel (LNG), a common synthetic progestogen, has emerged as an endocrine-disrupting contaminant in aquatic ecosystems. Although recent studies have recognized its androgenic effects, there exist critical gaps in understanding its influence on growth and associated molecular mechanisms. The processes driving anal fin elongation in mosquitofish (Gambusia affinis) following exposure to progestogens, such as LNG, remain poorly characterized. To address these knowledge gaps, we investigated LNG-induced androgenic effects on growth and the molecular basis of anal fin masculinization in adult female mosquitofish exposed to environmentally relevant LNG concentrations (500 ng/L) over a four-week period. Comprehensive physiological and morphological assessments (e.g., Fulton's condition factor, gonadosomatic index, and anal fin/skeletal analyses) revealed significant masculinization in LNG-exposed females. Transcriptomic profiling of anal fin tissue demonstrated that LNG-mediated masculinization and elongation at 500 ng/L were associated with the activation of the transforming growth factor-beta (TGF-β) and bone morphogenetic protein (BMP) signaling pathways. The exposure altered the transcriptional levels of key osteoblast- and osteoclast-related genes (i.e., sp7, col10a1, and nfatc1), implicating dysregulated bone remodeling in fin structural changes. Androgen receptor (ar) expression in anal fin tissue remained unchanged, suggesting that LNG's androgenic effects occurred independently of direct ar transcriptional modulation in this tissue. This study provides the first evidence that LNG disrupts bone morphogenetic and sex-related gene transcription, driving anal fin masculinization in female mosquitofish. These findings advance our understanding of LNG's androgenic impacts and propose the mosquitofish anal fin as a potential biomarker for endocrine disruption. By elucidating molecular pathways linking LNG exposure to morphological changes, this study paves the way for assessing the ecological risks of progestogen pollution in aquatic ecosystems.