Inagaki T, Smith N L, Sherva K M, Ramakrishnan S
Department of Biology, University of Puget Sound, Tacoma, WA, USA; Neuroscience Program, University of Puget Sound, Tacoma, WA, USA.
Department of Chemistry/Biochemistry, University of Puget Sound, Tacoma, WA 98416, USA.
Neurotoxicology. 2016 Dec;57:163-173. doi: 10.1016/j.neuro.2016.09.021. Epub 2016 Oct 3.
Growing evidence indicates that chronic exposure to Bisphenol A (BPA) may disrupt normal brain function and behavior mediated by gonadotropin-releasing hormone (GnRH) pathways. Previous studies have shown that low dose BPA (200ng/ml) exposure during embryogenesis altered development of extra-hypothalamic GnRH3 systems and non-reproductive locomotor behavior in medaka. Effects of parental low-dose BPA exposure on the development of GnRH3 systems and locomotor behavior of offspring are not well known. This study examines whether the neurophysiological and behavioral effects of BPA in parents (F0 generation) are carried over to their offspring (F1 generation) using stable transgenic medaka embryos/larvae with GnRH3 neurons tagged with green fluorescent protein (GFP). Parental fish were exposed to BPA (200ng/ml) for either life-long or different developmental time windows. Fertilized F1 eggs were collected and raised in egg/fish water with no environmental exposure to BPA. All experiments were performed on F1 embryos/larvae, which were grouped based on the following parental (F0) BPA exposure conditions - (i) Group 1 (G1): through life; (ii) G2: during embryogenesis and early larval development [1-14days post fertilization (dpf)]; (iii) G3: during neurogenesis (1-5dpf); and (iv) G4: during sex differentiation (5-14dpf). Embryos from unexposed vehicle treated parents served as controls (G0). G1 embryos showed significantly reduced survival rates and delayed hatching time compared to other groups, while G4 embryos hatched significantly earlier than all other groups. At 3 dpf, the GnRH3-GFP intensity was increased by 47% in G3 embryos and decreased in G4 embryos by 59% compared to controls. At 4dpf, G1 fish showed 42% increased intensity, while GFP intensity was reduced by 44% in G3 subjects. In addition, the mean brain size of G1, G3 and G4 embryos were smaller than that of control at 4dpf. At 20dpf, all larvae from BPA-treated parents showed significantly decreased total movement (distance covered) compared with controls, with G2 and G3 fish showing reduced velocity of movement. While at 20 dpf no group differences were seen in the soma diameter of GnRH3-GFP neurons, a 34% decrease in SV2 expression, a marker for synaptic transmission, in G1 larvae was observed. These data suggest that parental BPA exposure during critical windows of embryonic development or chronic treatment affects next-generation offspring both in embryonic and larval brain development as well as larval behavior.
越来越多的证据表明，长期接触双酚A（BPA）可能会扰乱由促性腺激素释放激素（GnRH）途径介导的正常脑功能和行为。先前的研究表明，胚胎发育期间低剂量双酚A（200ng/ml）暴露会改变青鳉下丘脑外GnRH3系统的发育以及非生殖运动行为。亲代低剂量双酚A暴露对后代GnRH3系统发育和运动行为的影响尚不清楚。本研究使用稳定的转基因青鳉胚胎/幼虫，其GnRH3神经元用绿色荧光蛋白（GFP）标记，以研究亲代（F0代）双酚A的神经生理和行为效应是否会遗传给它们的后代（F1代）。亲代鱼在整个生命周期或不同的发育时间窗口暴露于双酚A（200ng/ml）。收集受精的F1卵，并在无双酚A环境暴露的鱼卵/鱼水中饲养。所有实验均在F1胚胎/幼虫上进行，根据亲代（F0）双酚A暴露条件将其分组 - （i）第1组（G1）：终生暴露；（ii）G2：胚胎发生期和幼体早期发育阶段[受精后1 - 14天（dpf）]；（iii）G3：神经发生期（1 - 5dpf）；（iv）G4：性别分化期（5 - 14dpf）。未暴露于载体的亲代处理的胚胎作为对照（G0）。与其他组相比，G1胚胎的存活率显著降低，孵化时间延迟，而G4胚胎的孵化时间明显早于所有其他组。在3 dpf时，与对照组相比，G3胚胎的GnRH3 - GFP强度增加了47%，G4胚胎的GnRH3 - GFP强度降低了59%。在4dpf时，G1鱼的强度增加了42%，而G3组的GFP强度降低了44%。此外，在4dpf时，G1、G3和G4胚胎的平均脑尺寸小于对照组。在20dpf时，与对照组相比，所有来自双酚A处理亲代的幼虫的总运动量（移动距离）显著降低，G2和G3鱼的移动速度降低。虽然在20 dpf时，GnRH3 - GFP神经元的胞体直径在各实验组之间没有差异，但在G1幼虫中观察到突触传递标记物SV2表达下降了34%。这些数据表明，胚胎发育关键窗口期间的亲代双酚A暴露或长期处理会影响下一代后代的胚胎和幼体脑发育以及幼体行为。
