Transgenerational effects of Bisphenol A on embryogenesis, GnRH3 neural systems, and locomotor behavior in Japanese medaka,

Accumulating evidence indicates that chronic exposure to Bisphenol A (BPA), a xenoestrogen, may disrupt normal brain function and behavior mediated by gonadotropin-releasing hormone (GnRH) pathways. In a previous study, we showed that chronic developmental exposure to BPA (200 ng/ml), exerted neurobehavioral effects in Japanese medaka with extra-hypothalamic GnRH3 neurons tagged with green fluorescent protein (GFP). Yet transgenerational consequences of low-dose BPA exposure on GnRH3 neural systems and animal behavior are not well known. Here we examine whether BPA effects are carried over to the next generation (F2).

F1 medaka were exposed to vehicle or BPA (200 ng/ml) for different durations in their life span: i) G1- throughout life; ii) G2- only the first 2 weeks (1-14 day post fertilization or dpf); iii) G3- only during neurogenesis (1-6 dpf); and iv) G4- only during sex differentiation (6-14 dpf). Following exposure, F1 medaka were raised to adulthood and fertilized F2 eggs were collected daily and kept in the incubator maintained at 26-28 C°. To assess transgenerational effects of BPA on F2 GnRH3-GFP neurons, brain images were acquired from live embryos at 3 and 4 dpf and fluorescence intensity of GnRH3-GFP neurons at the terminal nerve (TN) and the trigeminal nerve (TG) were analyzed. GnRH-GFP fluorescence has been positively correlated with GnRH mRNA expression. Brain-growth, body sizes, embryonic survival rates, and times to hatch were also measured. At 3 dpf, TG GnRH3-GFP neurons in G4 groups showed 58.9% decreased fluorescence intensity (p<0.01). At 4 dpf, TN and TG-GnRH3-GFP intensity of G3 embryos were reduced 43% and 28.9%, respectively, while TG-GnRH3-GFP neurons showed 105% increase in the G1 group. In addition, G2 and G3 embryos had smaller brain sizes at 4 dpf. Embryonic survival rates in G1 (63.3 %) were significantly lower (p<0.01) than those of other groups, which was accompanied with the delayed hatching time (p<0.001). Locomotor activity of F2 larvae was tested at 20 dpf. We found that larvae in G1, G2 and G3 have significantly decreased locomotor activity in distance covered (110±18.3 mm, 92.36±19 mm, 70.66±19.4 mm, respectively, vs. 193.5±31.2 mm control, p<0.01), and velocity of movement in G2 and G3 (1.74±0.5 mm/s, 1.56±0.4 mm/s, respectively, vs. 3.23±0.52 mm/s control, p<0.05). The sizes of individual TN-GnRH3 neurons and synaptic connectivity were assessed by measuring the diameter of cell bodies and quantifying the pre-synaptic marker SV2 expression. The results will be reported. These data provide evidence of that BPA exposure during critical periods in embryonic development may have transgenerational effects.