The Xenopus tropicalis model for developmental endocrine and reproductive toxicity : Histological and molecular endpoints for disrupted spermatogenesis

Abstract: Endocrine disrupting chemicals (EDCs) are linked to adverse effects in both humans and wildlife. There are however, large knowledge gaps regarding cause-effect and dose-response relationships between the interference with endocrine pathways and adverse effects in the organism, especially at puberty and in subsequent generations. Using the frog model Xenopus tropicalis, endocrine and adverse effects were determined for environmentally relevant concentrations of imazalil, propiconazole and linuron. Flutamide was used as an anti-androgenic model substance. Sexual development was analysed at three juvenile ages and the pubertal onset in males was determined to five weeks post metamorphosis. Pale and dark spermatogonial stem cells (SSCs) were histologically characterized, these sperm stages are not previously described in Xenopus, to the best of my knowledge. Dark SSC, secondary spermatogonia and their ratio were shown to be sensitive endpoints for both flutamide and imazalil juvenile exposure. Imazalil decreased mRNA levels of ID4, increased DDX4 and decreased their ratio. These genes were associated with the germ cell stages affected and are suggested as new endpoints for disrupted spermatogenesis. The number of early spermatogonia is linked to fertility, hence the histological effects of imazalil can be seen as adverse outcomes. In the testes of the imazalil males, the levels of CYP19 and 3β-HSD mRNA were decreased and these changes were associated with the altered numbers of SSCs and secondary spermatogonia. Juvenile propiconazole exposure resulted in increased Aldh1a2 mRNA levels in the testes, but no histological effects on spermatogenesis were observed. These results demonstrate different modes of action of propiconazole and imazalil in the juvenile gonads. Chronic tadpole propiconazole exposure increased brain CYP19 activity at metamorphosis and decreased the time to metamorphosis. Two months after, the testis and Müllerian ducts were smaller and less mature compared with the controls, indicating that propiconazole can induce endocrine and adverse effects, but differently depending on timing of exposure (life stage) and exposure duration. Male offspring of fathers developmentally exposed to linuron demonstrated altered growth and were less fertile, whereas the main findings in the grand-offspring were altered growth and metabolism demonstrating transgenerational effects after developmental exposure to a pesticide. The findings in this thesis contribute with methods and knowledge on how EDCs induces adverse effects via endocrine pathways in juvenile animals and in subsequent generations. The results from this thesis thereby further increase the understanding how EDC exposure can affect humans and wildlife.