Effects of Benzo(a)Pyrene on the GnRH neuron function in the in vivo zebrafish model

Among the polycyclic aromatic hydrocarbons (PAHs), Benzo[a]pyrene (Bap) is considered a common endocrine disrupting chemical (EDC) with mutagenic and carcinogenic effects. The so-called “endocrine disruption hypothesis” suggests that exposure to EDC during fetal, neonatal and adult life may interfere with the development of hypothalamo-pituitary-gonadal (HPG) axis and function of reproductive organs, altering semen quality and reproductive hormone production. Recently, studies performed in different animal models, including zebrafish, described the adverse EDC effects on reproductive outcomes, but the effects of Bap on human GnRH (gonadotrophin-releasing hormone) system are far to be clarified. In this work, we take advantage of the vertebrate zebrafish model to investigate the effect of the exposure of Bap on HPG-axis function, taking advantage of the zebrafish line tg(GnRH3:EGFP). The transgenic embryos express the green fluorescence under the control of the GnRH3 promoter thus allowing the study of the entire development of the GnRH3 neurons, starting from their early proliferation at the level of the olfactory placode (OP) at 36 hours post fertilization (hpf), and migration at 48 hpf reaching the pre-optic area (POA) and the hypothalamus (Hy) at 72 hpf. To evaluate the effects of short-term exposure to Bap, we treated zebrafish embryos with increasing doses of Bap from 2.5 to 72 hpf comparing with the control vehicle (1.25% DMSO) and no significant changes in the survival rate neither morphological defects have been observed in embryos treated with 5 and 50nM of Bap. Regarding the analysis of GnRH3 network, we observed a dose-dependent reduction of GFP signal at the level of olphactory bulb (OB), anterior commissure (AC), Hy innervation, POA, and retina. In particular, we noticed with 5nM of Bap a mild reduction of GFP signal at the level of AC and retina, a severe reduction at the level of Hy and POA ad a signal comparable to control at the level of OP; regarding 50nM of Bap we observed a severe reduction of GFP signals in all these regions except for the OB and are ongoing FACS analysis for better characterize this phenotype quantifying the number of GnRH3 neurons. After the immunofluorescence using the anti-caspase3 antibody we excluded that the observed defects are the results of an increased apoptosis. According to recent hypothesis, GnRH3 neurons might differentiate from neural crest cells and ectodermal progenitors in a niche at the border between the respiratory epithelium and the vomeronasal/olfactory epithelium. Once in the vomeronasal organ GnRH3 neurons migrate out to the brain and reach their final destination in the hypothalamus. Therefore, we will investigate the effects of BAP exposure in olfactory neurogenesis, in particular in neural crest cells and in olfactory sensory neurons taking advantage of embryos of the tg (neurog1: EGFP) , tg (OMP: CFP) and tg (TRPC2:YFP) zebrafish lines.