Molecular mechanisms by which estrogens affect male germ cell programming during development

哺乳动物生殖细胞的发育是一个独特而复杂的过程。胎儿和产后早期的发育窗口是至关重要的。事实上，在这段时间内，生殖细胞（性腺细胞）经历了表观遗传重编程、增殖、静止和分化。最近的证据表明，成人生殖功能障碍可能源于生殖细胞发育在这一特定时期的失调。我们已经证明，这一发育时期对雌激素样化合物的内分泌干扰特别敏感。然而，围产期卵泡细胞编程和异种雌激素靶点的信号机制和分子基础仍然知之甚少。我的研究项目旨在阐明哺乳动物胎儿和新生儿雄性生殖细胞发育的潜在分子基础，并确定这些过程的错误调节如何改变成年期的生殖功能。核心假设是，在生殖细胞发育过程中发生的表观遗传重编程，通过从头DNA甲基化和组蛋白翻译后修饰，调节生殖细胞编程所需的关键调节基因的表达，并且这种机制可以通过雌激素信号通路靶向。利用仅生殖细胞表达绿色荧光蛋白的转基因大鼠模型，我们将通过体内和体外模型阐明雌激素信号如何影响性腺细胞的基因表达和表观遗传机制。在目的1中，我们将描述当生殖细胞增殖和新生DNA甲基化发生时，体内妊娠暴露于乙炔雌二醇和染料木素的短期影响。两种治疗方法之间的比较将区分两种雌激素受体途径。对基因表达的影响将使用全基因组转录组分析进行评估。进一步分析处理对表观遗传重编程和细胞分化的影响。最后，深入了解受雌激素治疗影响的基因的表观遗传调控。在目的2中，我们将使用两种体外模型和药物抑制剂来破坏参与DNA甲基化和组蛋白修饰的酶，我们将揭示雌激素信号传导和表观遗传机制之间的串扰，以调节性腺细胞的基因表达。我们将使用永生化的性腺细胞细胞系和器官培养来确定表观遗传机制的失调如何调节性腺细胞增殖、分化或细胞死亡，以及雌激素如何改变这种调节。总之，这些研究将更好地理解雌激素如何通过靶向表观遗传重编程影响男性生殖细胞发育。胎儿发育是细胞编程过程中一个关键而独特的时期，细胞在此期间获得终生的信息。了解男性生殖细胞编程的分子机制以及外源药物如何破坏这些机制对于确定生殖结果对胎儿编程的依赖性至关重要。

Germ cell development in mammals represents a unique and complex process. The fetal and early postnatal windows of development are critical. Indeed, during this time window, germ cells (gonocytes) undergo epigenetic reprogramming, proliferation, quiescence and differentiation. Recent evidence have indicated that adult reproductive dysfunction can originate from misregulation of germ cell development during this specific period of development. We have demonstrated that this period of development is particularly sensitive to endocrine disruption by estrogen-like compounds. Yet, the signaling mechanisms and molecular basis involved in perinatal gonocyte programming and the targets of xenoestrogens remain poorly understood. My research program aims to elucidate the underlying molecular basis for fetal and neonatal male germ cell development in mammals and determining how the misregulation of these processes might alter reproductive function in adulthood. The central hypothesis is that the epigenetic reprogramming that occurs during gonocyte development, through de novo DNA methylation and histones post-translational modifications, regulates the expression of key regulators genes that are necessary for germ cell programming, and that such mechanism can be targeted by the estrogen signalling pathway. Taking advantages of a transgenic rat model in which only germ cells express the green fluorescent protein, we will elucidate how the estrogen signaling can affect gene expression and the epigenetic machinery in gonocytes by using both in vivo and in vitro models. In Objective 1, we will characterize the short-term impact of in vivo gestational exposure to ethinyl estradiol and genistein when gonocytes proliferate and de novo DNA methylation occurs. Comparison between treatments will discriminate between the two estrogen receptors pathways. The impact on gene expression will be evaluated using genome wide transcriptome analysis. Further analysis of the effect of treatment on epigenetic reprogramming and cell differentiation will be done. Finally, in depth comprehension of epigenetic regulation of genes which expression is affected by estrogenic treatment will be obtained. In Objective 2, we will use two in vitro models and pharmaceutical inhibitors to disrupt the enzymes involved in DNA methylation and histone modifications, we will reveal the crosstalk between the estrogen signaling and the epigenetic machinery in regulating gene expression in gonocytes. We will use an immortalized gonocyte cell line and organ culture to determine how the misregulation of epigenetic mechanisms regulates gonocytes proliferation, differentiation or cell death, and how estrogens alter such regulation. Altogether, these studies will provide a better understanding of how estrogens affect male germ cell development by targeting the epigenetic reprogramming. Fetal development represents a key and unique period in cell programming when cells acquire information for a lifetime. Understanding the molecular mechanisms underlying male germ cell programming and how xenobiotics can disrupt these mechanisms is essential to establish the dependency of reproductive outcomes on fetal programming.