Steroid Receptors in Placental Tissue and Vasculature during Early Pregnancy

描述(申请人提供)：在怀孕早期发生的关键事件包括胚胎/胎儿的剧烈生长和发育，以及胎盘形成，包括胎盘的血管形成。事实上，包括人类在内的所有哺乳动物中，胚胎损失的大部分发生在怀孕早期，占受精卵母细胞的30%到50%。类固醇‘驱动’血流到怀孕的子宫，胎盘血管生成和血管生成因子的表达。近年来，我们发现早孕和早孕当天子宫内膜血管生成显著增加，辅助生殖技术(ART)不仅影响子宫内膜血管生成，而且影响子宫-胎盘早期雌激素受体和孕激素受体(ER和PR)的表达和定位。因此，为了更好地了解它们的生理作用，我们需要量化ER和PR在自然交配后和ART后妊娠的子宫-胎盘血管和其他组织中的表达和定位。我们还必须在体外和体内评估子宫胎盘类固醇受体激活改变的功能后果。因此，长期目标是 该项目旨在确定雌激素和孕激素及其受体在正常妊娠和不良妊娠的关键早期阶段对子宫-胎盘生长和发育，特别是血管形成的调节作用。为了达到这一目标，我们将评估自然交配和体内或体外胚胎移植后胎盘核膜ER和PR的表达。我们的总体假设是，在应用ART后，ER和PR的表达和/或功能在早孕期间发生变化，此外，在早孕期间子宫胎盘组织中的表达和/或功能受到损害。我们将使用免疫荧光(IF)和图像分析以及Western印迹来评估ER和PR蛋白的表达，并使用激光捕获显微切割和qPCR来评估mRNA的表达。我们将使用的子宫-胎盘组织已经从最近的其他研究中收集，包括自然交配(动物模型1，AIM 1)和移植体内或体外产生的胚胎(动物模型2，AIM 2)后在早期怀孕期间获得的组织。我们的具体假设是，随着正常妊娠的进展，ER和PR的表达和定位受到不同的调节(目标1，假设1)，并被ART的应用改变(目标2，假设2)。最后，在这些研究中，我们将使用我们已被证明不仅与农业相关，而且与人类健康相关的具有良好特征的家畜不良妊娠模型。由于性激素在妊娠早期调节子宫-胎盘血管功能和发育方面的重要性，这些研究的结果将对妊娠的建立产生重大影响，并将导致新的和知情的策略和治疗工具，以提高妊娠成功率，防止自然流产、着床失败和/或其他疾病，这对母婴的健康和福祉具有巨大的影响。

DESCRIPTION (provided by applicant): The critical events that occur during early pregnancy include dramatic growth and development of the embryo/fetus and also placentation, which includes vascularization of the placenta. In fact, the majority of embryonic loss, amounting to 30 to 50% of fertilized oocytes, occurs during early pregnancy in all mammals, including humans. Steroids 'drive' blood flow to the pregnant uterus, placental angiogenesis and angiogenic factor expression. Recently, we have shown dramatic increase in endometrial angiogenesis during early pregnancy and that day of early pregnancy as well as assisted reproductive techniques (ART) affect not only endometrial angiogenesis but also expression and localization of estrogen and progesterone receptors (ER and PR) in the early utero-placenta. Thus, to better understand their physiological role, we need to quantify expression and evaluate localization of ER and PR in the utero-placental vasculature and other tissue compartments in pregnancies after natural mating and also after ART. We also must evaluate the functional consequences of altered utero-placental steroid receptor activation in vitro and in vivo. Therefore, the Long- Term Goal of this project is to determine the role of estrogens and progesterone and their receptors in the regulation of utero-placental growth and development, and especially vascularization, during the critical early stages of normal and compromised pregnancies. To meet this goal, we will evaluate placental expression of nuclear and membrane ER and PR after natural mating and after transfer of embryos produced in vivo or in vitro. Our overall hypothesis is that expression and/or function of ER and PR changes during early pregnancy and, in addition, is compromised during early pregnancy in utero-placental tissues after application of ART. We will evaluate expression of ER and PR protein using immunofluorescence (IF) followed by image analysis as well as Western blot, and mRNA expression using laser capture micro-dissection followed by qPCR. The utero-placental tissues we will use were already collected from other recent studies and comprise tissues obtained during early pregnancy after natural mating (Animal Model 1, Aim 1) and after transfer of embryos produced in vivo or in vitro (Animal Model 2, Aim 2). Our specific hypotheses are that expression and localization of ER and PR is differentially regulated as normal pregnancy progresses (Aim 1, hypothesis 1), and is altered by application of ART (Aim 2, hypothesis 2). Lastly, for these studies we will use our well- characterized domestic animal models of compromised pregnancy that have previously been shown to have relevance not only to agriculture but also to human health. Because of the importance of sex steroids in regulating utero-placental vascular function and development during early pregnancy, the results of these studies will have major implications for pregnancy establishment and will lead to new and informed strategies and therapeutic tools to improve pregnancy success and prevent spontaneous abortion, implantation failure and/or other disorders, which has enormous implications for maternal and fetal health and well-being.