1. Embryonic low dose BP A Exposure has transgenerational effects on GnRH neurons in Japanese medaka Natalie Smith 1, Tomoko Inagaki 2, Siddharth Ramakrishnan 2,3 Department of Chemistry 1, Neuroscience Program 2, Department of Biology 3, University of Puget Sound, WA INTRODUCTION This study examines the transgenerational effects of the xenoestrogen Bisphenol A (BPA) on the Gonadotropin-releasing hormone (GnRH) neural pathway. GnRH neurons responsible for reproductive and allied behaviors in most vertebrates. Previous studies in our lab on medaka embryonic BPA low-dose exposure showed a 23.7% increase in the terminal nerve and a 14.3% decrease in trigeminal region at 3dpf. Here we wanted to determine if these effects were carried into the next generation Parental medaka were exposed to a chronic low-dose of BPA ONLY during early embryonic development. First generation fertilized embryos in untreated water were then examined for changes in the GnRH neural system. A B 1.T erminal Nerve (A) 2.Trigeminal Region (B) METHODS Chronic Low Does BP A Exposure (200 ng/mL) Using GnRH3:GFP medaka (with GnRH3 neurons tagged with green fluorescent protein) Embryonic only exposure of parental groups Parental group then raised to maturity, fertilized embryos collected from non-treated tank water. Three Different Groups (Fig. 1): 1.G1: Parental treatment for from egg laying until 2 weeks post fertilization (14dpf) 2.G2: Parental treatment for 0-6 dpf 3.Control: Parental exposure to vehicle control (EtOH) only Fluorescence imaging of GnRH3-GFP neuronsat dpf 3-5 Two Regions of Interest examined: Fluorescence normalized per region area Fluorescent Intensity calculated as percentage from control Hatching rate of each group measured Brain size measured with eye-to-eye distance using bright-field microscopy Bright-field and fluorescent imagery of embryos 200 ng/mL BPA treatment Parental exposure during Embryonic development G1G2 2 week exposure from egg laying Exposure from 0-6 dpf Parents are raised until maturity in regular water Fertilized embryos are collected Figure 1. Experimental overview of BPA treatment of parental groups and fluorescent imagery of first generation untreated embryos. A B All results are reported with standard error of mean DPF stands for Days post fertilization. Significance is reported as a two-tail t-test Significant difference in brain size shows differential effect of BPA on first generation embryos Table 1. Fluorescent Intensity at DPF 3-5 DPF TN (% Control) TG (% Control) 3 G1: 38.96 G2: 43.04 G1: -32.39 G2: -28.92 G1: -3.21 G2: -16.08 G1: 4.57 G2: 11.28 G1: -28.04 G2: -43.03 G1: 69.49 G2: -20.69 4 5 Figure 2. Normalized Fluorescence at DPF 3 for the terminal nerve (TN) and trigeminal (TG) regions of interest (ROI). G1 followed by G2 data are reported at each ROI. For TN, G1 n=8; G2 n=12. For TG, G1 n=7; G2 n=11. Figure 3. Normalized Fluoreoscence at DPF 4 for the terminal nerve (TN) and trigeminal (TG) regions of interest (ROI). G1 followed by G2 data are reported at each ROI. For TN, G1 n=10; G2 n=17 (p<0.05). For TG, G1 n=11; G2 n=18 (p<0.04). Figure 4. Normalized Fluorescence at DPF 5 for the terminal nerve (TN) and trigeminal (TG) regions of interest (ROI). G1 followed by G2 data are reported at each ROI. For TN, G1 n=6; G2 n=4. For TG, G1 n=6; G2 n=4. DPF 3 Normalized Fluorescence % Control -35 0 35 70 DPF 5 Normalized Fluorescence % Control -50 0 50 100 TN TG DPF 4 Normalized Fluorescence % Control -50 -25 0 25 TN TG * * TN CONCLUSIONS & FURTHER STEPS Just parental exposure to chronic low dose BPA results significant changes in embryonic development of GnRH neurons in F1 embryos Brain sizes also seem to vary initially in the embryo, but go back to normalcy with time These changes are different from previous studies seen in GnRH development in BPA exposed parental embryos How does this affect behavior? Does it have impact on reproduction/development? How does it affect GnRH physiology? Study supported by NSF CAREER Award 1253126 to SR RESULTS Figure 6. Hatching rate as observed with collected embryos inventoried each day after fertilization. Number of Hatchings 0 4 8 12 16 9 DPF 781011 Hatching Rate Control G1 G2 DPF 3 DPF DPF 5 Normalized Brain Size DPF 3-5 % Control -30 -20 -10 0 10 Figure 5. Embryonic brain size as percentage from control on DPF 3-5. G1 data followed by G2 data are reported for each DPF. For DPF 3: G1 n=8 (p<0.01), G2 n=13. For DPF 4: G1 n=10 (p<0.03), G2 n=19 (p<0.01). For DPF 5: G1 n=6, G2 n=8 (p<0.03). * * * * G1- Two week (0-14dpf) embryonic parental exposure to 200ng/ml BPA G2: 0-6dpf embryonic parental exposure to 200ng/ml BPA *