Transgenerational Inheritance Of Gestational TCDD Exposure On Imprinted Gene And Spatial Learning And Memory

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin can be transmitted through placenta and breast milk, causing severely abnormal physiological and functional changes to the directly exposed ones and their offspring. Many studies have confirmed that environmental stimuli during fetal development could induce epigenetic modifications, resulting in transgenerational inheritance of phenotype. The effect of gestational TCDD exposure on the transgenerational inheritance of imprinted gene alteration in somatic tissues have not yet been reported; whether gestational TCDD exposure could induce spatial learning and memory deficits and if these effects could be transmitted across generations have also not yet been studied.Objective The aim of our study was to explore effects of gestational TCDD exposure on growth and development of the F1-F3 generations, as well as the effects on the imprinted gene Igf2 in male liver in the F1 and F3 generation, and further investigate the epigenetic transgenerational effects; gestational TCDD exposure induced spatial learning and memory deficits in adult rats in the F1-F3 generations was also studied.Methods Transgenerational animal model was established. The F0 pregnant SD rats was exposed to TCDD during gonadal sex determination period(Gestational day 8-14, GD 8-14) in gavage with doses of 0, 200, 800 ng/kg bw. Crown-rump length and tail length were measured in perinatal pups of F1-F3 generation, while AGD and weight gain were monitored through adulthood. Bisulfite Genomic Sequencing was used to determine the methylation status of ICR and DMR2 in the Igf2/H19 locus in male liver of the F1 and F3 generation. Real-time PCR was used to measure m RNA expression levels of Igf2, DNMT1, DNMT3 A and DNMT3 B. Morris water maze was used to test the spatial learning and memory ability in the F1-F3 generations of adult rats.Results 1 Gestational exposure to TCDD during gonadal sex determination did not affect the litter size or the sex ratio in the F1-F3 generations, but cause a significant decrease insurvival rate of PND 4 and PND 21 in the F1 generation. Crown-rump length and tail length during perinatal days were shortened, and so was AGD in PND 21 and PND 63 in the F1 generation. These effects in the F2 and F3 generation were gradually weakened across generations; 2 Gestational exposure to TCDD during gonadal sex determination cause lower birth weight and slow weight gain during perinatal days. High-dose(800 ng/kg bw) TCDD exposure in utero can cause sustained lower weight than the control group in the F1 generation and this phenomenon could be still manifested in the F3 generation; while in the low-dose(200 ng/kg bw) TCDD group, the offspring appeared weight gain higher than the control group to some extent, and continued to the F3 generation; 3 Gestational TCDD exposure can cause changes in the weight of reproductive organs and organ coefficients in the F1-F3 generations, and the effects were attenuated in the F2 and F3 generation; long-term effects on the general organs(spleen, kidney, heart, liver) and organ coefficients in the offspring of F1-F3 generation were also observed. 4 Gestational TCDD exposure caused decreased m RNA expression level of Igf2 in male liver of the F1 and F3 generation. In the F1 generation, the expression of Igf2 in the 200 and 800 ng/kg bw TCDD group were down-regulated 0.81-fold and 0.39-fold(P < 0.05); After two two generations’ transmission, the Igf2 expression in the F3 generation still shows a clear tendency to reduce, with the expression of Igf2 in the 200 and 800 ng/kg bw TCDD group were 0.25-fold and 0.33-fold(P < 0.05) compared to the control group; 5 Analysis of the methylation status of the differentially methylated regions of Igf2 showed that the methylation level of ICR in the F1 generation were elevated along with TCDD increases, while methylation level of DMR2 region decreased in a dose-dependent manner. After two generations’ transmission, similar trends were still existed in the F3 generation; 6 Along with the methylation changes of Igf2, the DNMTs were also affected by gestational TCDD exposure: m RNA expression of DNMT1, DNMT3 A and DNMT3 B were changed in a non-monotonic manner in the F1 generation, and these effects are still prominent in the F3 generation; 7 In the acquisition training phase of Morris Water Maze, adult rats in the F1-F3generations after gestational TCDD exposure showed longer latency and longer path length to reach the hidden platform compared to the control group, and the effects remained slightly different in the F3 generation; 8 In the probe test of Morris Water Maze, the times of crossing over previous platform in the male rat of 800 ng/kg bw TCDD group in the F1 generation was significantly reduced, but the effect did not manifest in the F2, F3 generation, neither in the females in the F1-F3 generations. Time spent in the target quadrant of male and female adult rats in the 800 ng/kg bw TCDD group was longer than the control group; 9 In the acquisition training phase of Morris Water Maze, a significant sex difference was observed. The male of TCDD group in the F2 generation spent longer time to reach the hidden platform than the females in the same dose group; while in the F1 and F2 generation, the males in the TCDD group performed worse even than the control females, showing a certain degree of feminization. Gender differences were attenuated after two generations’ transmission.Conclusions 1 Gestational TCDD exposure during gonadal sex determination could induce changes in the general indicators of growth and development, as well as AGD. These effects were gradually attenuated across generations. 2 The effects on weight gain after gestational TCDD exposure may show "inverted U" phenomenon: weight gain of the offspring could be stimulated at a low dose, while that was directly suppressed at a high dose. The phenomenon may be transmitted across generations. 3 Gestational TCDD exposure during critical window may disrupt organogenesis, which manifested a wide range of developmental toxic effects. The reproductive system, the immune system, as well as kidney, heart and liver were all affected. Some of the effects could also be lasted through multiple generations, promoting risk of diseases; 4 Growth trends combined with the organ weight changes, we believe gestational TCDD exposure may not directly lead to adverse pregnancy outcome, on the contrary, it may have long-lasting but subtle effects or damage to the growth and development of the offspring. Besides, the transgenerational effects observed in this study may alsobe involved with the epigenetic effects, which have implications in future studies; 5 Gestational TCDD exposure during gonadal sex determination could disrupt the epigenetic modifications of imprinted gene Igf2, resulting changes in the expression level. Abnormal methylation patterns of imprinted genes could escape the genome-wide re-methylation and be inherited transgenerationally; DNA methyltransferase may play a role in TCDD induced transgenerational inheritance of methylation patterns of imprinted gene; 6 Gestational TCDD exposure could cause spatial learning and memory deficits in the F1-F3 generations with transgenerational effects; Gestational TCDD exposure-induced spatial learning and memory deficits showed gender differences. Male offspring in the TCDD group showed "feminized" performance, suggesting that TCDD may cause the "demasculinization" of the male offspring by disrupting hormone homeostasis, but that effect was attenuated after two generations’ transmission.