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家族性疾病的成人发病和进展中起着关键作用。然而，目前尚不清楚在子宫内接触邻苯二甲酸盐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。已知邻苯二甲酸盐可引起睾丸发育不良综合征，其特征是出生后异常，如睾丸功能障碍增加、肛门生殖器距离缩短、精子发生异常和源于胎儿睾丸发育受损的睾丸癌。在我们的初步研究中，我们发现最丰富的邻苯二甲酸二（2-乙基己基）邻苯二甲酸二酯（DEHP）引起精小管中生殖细胞的紊乱，生精阶段分布的改变，细胞凋亡增加，多核性腺细胞数量增加，精子数量减少，这些都表明精子发生受损。有趣的是，这些影响在dehp处理的F0坝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，比较DEHP和车用油处理的F0雄性雄鼠F3后代精子DNA的DNA甲基化模式，发现许多显著的甲基化差异区域，表明DNA甲基化改变可能是跨代效应的潜在表观遗传机制之一。因此，我们试图验证一种假设，即在子宫内将F1生殖细胞暴露于DEHP会以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DNA甲基化模式的改变，这种改变在后代中持续存在。我们计划确定(1)对睾丸的剂量依赖性跨代效应；(2)精子干细胞（SSC）潜能的改变是否是跨代效应的潜在细胞机制；(3) DNA甲基化模式的改变是否是跨代效应的潜在表观遗传机制。这项剂量依赖性研究与确定细胞和分子机制的研究相结合，是评估邻苯二甲酸盐是否可能对动物和人类产生跨代效应以及这些跨代效应的机制的最佳方法。该建议的完成有可能影响我们对胎儿暴露于环境毒物和遗传性人类疾病的看法，以及邻苯二甲酸盐在靶向SSC相关基因，改变SSC增殖和分化程序中的作用。

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.