Epigenetic Reprogramming in Germline by Phthalates

描述(由申请人提供)：最近的研究结果表明，表观遗传学和胎儿环境在成人家族性疾病的发生和发展中起着关键作用。然而，目前尚不清楚在宫内暴露于邻苯二甲酸酯这种普遍存在的环境毒素是否具有遗传给下一代的表观遗传效应。已知邻苯二甲酸酯可导致睾丸发育不良综合征，其特征是出生后异常，如冷冻增加，肛门距离缩短，精子发生异常，以及睾丸癌，源于胎儿睾丸发育受损。在我们的初步研究中，我们发现邻苯二甲酸二(2-乙基己基)酯(DEHP)是最丰富的邻苯二甲酸二(2-乙基己基)酯(DEHP)，它引起生精小管生精细胞的紊乱，生精阶段分布的改变，细胞凋亡率增加，多核性腺细胞数量增加，精子数量减少，这些共同表明精子发生受损。有趣的是，这些效应在用DEHP处理的F0母鼠的F1至F4代(F1-F4)后代的小鼠睾丸中观察到。我们的初步结果还显示了一个新的发现，即这些睾丸跨代效应部分源于精原干细胞(SSCs)功能受损。此外，对DEHP和车用油处理的F0代母鼠F3代精子DNA甲基化模式的比较显示，有许多显著差异的甲基化区域，表明DNA甲基化改变可能是跨代效应的潜在表观遗传学机制之一。因此，我们试图测试这样一种假设，即在子宫内将F1生殖细胞暴露于DEHP会以一种剂量依赖的方式引起跨代效应，并且这种跨代结果--包括精原干细胞功能的损害--是由于生殖系中DNA甲基化模式的变化，这种模式在后续世代中持续存在。我们计划确定(1)对睾丸的剂量依赖的跨代效应；(2)精原干细胞(SSC)潜力改变是否是跨代效应的潜在细胞机制；以及(3)DNA甲基化模式改变是否是跨代效应的潜在表观遗传学机制。这项剂量依赖的研究，与确定细胞和分子机制的研究相结合，是评估邻苯二甲酸酯对动物和人类是否可能产生跨代影响以及这些跨代影响的机制是什么的最好方法。这项提议的完成可能会影响我们对胎儿暴露于环境毒物和可遗传人类疾病的思考，以及邻苯二甲酸盐在靶向SSC相关基因、改变SSC增殖和分化编程方面的作用。 公共卫生相关性：胎儿暴露在环境污染物邻苯二甲酸盐中，会导致小鼠睾丸出现类似疾病的情况，并会遗传给多代人。我们将研究邻苯二甲酸盐对精原干细胞的剂量依赖的跨代效应，并确定表观遗传学的改变是否是这些效应的机制。这项研究有可能影响我们对胎儿暴露于环境毒素和成人家族性疾病的发病和发展的看法。

DESCRIPTION (provided by applicant): Recent findings indicate that epigenetics and the fetal environment play a critical part in the adult onset and progression of familial diseases. However, it is not known whether in utero exposure of phthalates, ubiquitous environmental toxins, has epigenetic effects that are inherited to the next generation. Phthalates are known to cause testicular dysgenesis syndrome, characterized by postnatal anomalies such as increased crytorchidism, shortened anogenital distance, aberrant spermatogenesis, and testicular cancer, stemming from impaired fetal testicular development. In our preliminary studies, we found that di(2- ethylhexyl) phthalate (DEHP), the most abundant phthalate, caused disorganization of germ cells in seminiferous tubules, changes in the distribution of spermatogenic stages, increased apoptosis, increased number of multinucleated gonocytes, and decreased sperm counts, which are collectively indicative of impaired spermatogenesis. Interestingly, these effects were observed in the mouse testis of F1 to F4 generation (F1-F4) offspring of DEHP-treated F0 dams. Our preliminary results also showed a novel finding that these testicular transgenerational effects are in part originating from impaired function of spermatogonial stem cells (SSCs). Moreover, the comparison of DNA methylation patterns of sperm DNA from F3 offspring of DEHP- and vehicle oil-treated F0 dams showed a number of significantly strong differentially methylated regions, indicating that an altered DNA methylation may be one of the underlying epigenetic mechanisms for the transgenerational effects. Thus, we seek to test the hypothesis that exposing F1 germ cells to DEHP in utero causes transgenerational effects in a dose-dependent manner, and that this transgenerational outcome -- including impairment of spermatogonial stem cell function -- is due to alterations in the DNA methylation pattern in the germline that persists in subsequent generations. We plan to determine (1) dose-dependent transgenerational effects on the testis; (2) if altered spermatogonial stem cell (SSC) potential is the underlying cellular mechanism for transgenerational effects; and (3) if altered DNA methylation pattern is the underlying epigenetic mechanism of transgenerational effects. This dose-dependent study, in combination with studies to determine the cellular and molecular mechanisms, is the best way to evaluate if transgenerational effects of phthalates are possible for animals and humans and what mechanisms are responsible for these transgenerational effects. Completion of this proposal has the potential to impact our thinking on the fetal exposure of environmental toxicants and heritable human diseases, and the role of phthalates on targetting the SSC-related genes, altering the SSC proliferation and differentiation programming. PUBLIC HEALTH RELEVANCE: Fetal exposure of environmental contaminants, phthalates, caused disease-like condition in the mouse testis, which was passed to multiple generations. We will investigate the phthalate dose-dependent transgenerational effects on spermatogonial stem cells and determine whether altered epigenetics are the mechanism underlying these effects. This investigation has the potential to impact our thinking on the fetal exposure to environmental toxins and the adult onset and progression of human familial diseases.