Regulation of early embryo development by nitric oxide

描述(由申请人提供)：在胚胎发育的围植入期阶段，有相当数量的潜在妊娠丢失。据国家研究理事会生命科学委员会估计，该国每年出生的婴儿有20多万在胚胎发育过程中出现异常。其他婴儿的出生体重明显较低，这可能导致出生后生活中的死亡或残疾。这些异常中的很大一部分可能是由于正常胚胎发育的改变造成的。我们的研究表明，一氧化氮(NO)是植入前胚胎发育所必需的。此外，暴露于雌激素会诱导着床，使休眠胚泡中NO的生成量增加10倍。然而，NO对围着床期胚胎基因表达的影响却知之甚少。 本实验的目的是阐明NO在早期胚胎发育和着床启动过程中的调控机制。这将通过进行以下实验来实现：(A)比较一氧化氮合酶(NOS)基因在怀孕1-4天(植入前)胚胎中的表达定位。结果将表明，NO的产生是否需要一个以上的NOS基因的诱导，以及这些基因在植入前胚胎中是否存在差异表达；(B)确定怀孕1-4天胚胎中NO产生的信号级联的性质。具体地说，我们将研究细胞周期基因的作用、时间进程和细胞特异性，包括：早期即时基因：c-los、c-jun和e-myc(mRNA和蛋白)，细胞周期蛋白(D、E、A和B)和视网膜母细胞瘤基因对NO的反应。结果将确定NO是否通过激活即刻早期基因作用于调节细胞周期的基因：(C)确定抑制NO产生是否改变了细胞周期检查点基因的表达。确定抑制NO对妊娠1-4天胚胎中细胞周期检查点基因ATM、ATR、MAD和BUB水平的时程和细胞特异性的影响。结果将表明，如果抑制NO通过改变检查点基因的表达来阻止正常的有丝分裂。 这项拟议的研究将确定胚胎中NO产生的状态及其对胚胎基因表达的影响，将产生关于正常和异常生殖功能的重要和有意义的信息。此外，这一信息应该对人类生育治疗有意义，作为选择更健康的体外受精胚胎的工具，成功怀孕的可能性更高。

DESCRIPTION (provided by applicant): A significant number of potential pregnancies are lost during the periimplantation stages of embryo development. It has been estimated by the Commission on Life Sciences of the National Research Council that more than 200,000 infants born each year in this country have abnormalities that arise during embryonic development. Other infants have markedly low birth weights which may result in death or disability later in postnatal life. A large portion of these abnormalities may result from alterations of normal embryonic development. Our studies have indicated that nitric oxide (NO) is required for preimplantation embryo development. In addition, exposure to estrogen, which induced implantation, increases the production of NO 10 fold in dormant blastocysts. However, little is known about the affect of NO on gene expression in periimplantation embryos. The purpose of the proposed experiments is to elucidate the integrated mechanisms regulated by NO in early embryonic development and initiation of implantation. This will be accomplished by conducting the following experiments: (a) Compare the localization of expression of the nitric oxide synthase (NOS) genes in embryos on days 1-4 of pregnancy (preimplantation). Results will indicate if induction of more than one NOS gene is required for NO production and if there is differential expression of these genes during preimplantation embryo; (b) Determine the nature of signaling cascades generated by NO in the embryo on days 1-4 of pregnancy. Specifically we will examine the effect, time course and cell specificity of cell cycle genes including: early immediate genes: c-los, c-jun and e-myc (mRNA and protein), cyclins (D,E, A and B) and retinoblastoma gene in response to NO. Results will determine if NO is acting through the activation of immediate early genes to genes regulating the cell cycle: (c) Determine if the inhibition of NO production alters expression of cell-cycle checkpoint genes. Determine the effect of inhibition of NO on the time course and cell specificity of levels of the cell-cycle checkpoint genes ATM, ATR, MAD and BUB in embryos on days 1-4 of pregnancy. Results will indicate if inhibition of NO stops normal mitotic division by altering expression of checkpoint genes. The proposed study will establish the status of NO production in the embryo and its effects on embryonic gene expression will generate important and meaningful information regarding normal and abnormal reproductive functions. Furthermore, this information should have implications in human fertility treatments, as a tool to select healthier IVF-derived embryos with increased probability for successful pregnancy.