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，尚不清楚子宫内邻苯二甲酸酯（普遍存在的环境毒素）的暴露是否具有遗传给下一代的表观遗传效应。已知邻苯二甲酸酯会导致睾丸发育不全综合征，其特征为出生后异常，如隐睾增加、肛门与生殖器距离缩短、精子发生异常和睾丸癌，这些都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）（含量最高的邻苯二甲酸酯）可导致生精小管中生殖细胞的解体、生精阶段分布的变化、细胞凋亡增加、多核生殖母细胞数量增加和精子计数减少，这些共同表明精子发生受损。有趣的是，在DEHP处理的F0代母鼠的F1至F4代（F1-F4）小鼠睾丸中观察到这些效应。我们的初步结果还显示了一个新的发现，这些睾丸的跨代效应部分源于精原干细胞（SSCs）的功能受损。此外，DEHP和溶剂油处理的F0代母鼠的F3代后代精子DNA的DNA甲基化模式的比较显示了许多显著强的差异甲基化区域，表明DNA甲基化改变可能是跨代效应的潜在表观遗传机制之一。因此，我们试图检验以下假设，即在子宫内将F1生殖细胞暴露于DEHP会以剂量依赖性方式引起跨代效应，并且这种跨代结果（包括精原干细胞功能受损）是由于生殖细胞中DNA甲基化模式的改变在后代中持续存在。我们计划确定（1）睾丸的剂量依赖性跨代效应;（2）精原干细胞（SSC）潜能改变是否是跨代效应的潜在细胞机制;（3）DNA甲基化模式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，是评估邻苯二甲酸酯是否可能对动物和人类产生跨代效应以及导致这些跨代效应的机制的最佳方法。该提案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思考，以及邻苯二甲酸酯在靶向SSC相关基因、改变SSC增殖和分化编程方面的作用。

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.