Regulation of early embryo development by nitric oxide

描述（由申请人提供）：在胚胎发育的围着床期，大量潜在妊娠丢失。据国家研究理事会生命科学委员会估计，我国每年有20多万名婴儿在胚胎发育期间出现畸形。 其他婴儿出生时体重明显偏低，可能导致出生后死亡或残疾。这些异常的大部分可能是由于正常胚胎发育的改变。我们的研究表明，一氧化氮（NO）是必需的植入前胚胎发育。此外，暴露于雌激素，诱导植入，增加生产的NO在休眠囊胚10倍。然而，关于NO对围植入期胚胎基因表达的影响知之甚少。 本实验的目的是阐明NO在早期胚胎发育和着床启动中的调控机制。这将通过进行以下实验来完成：（a）比较妊娠第1-4天（植入前）胚胎中一氧化氮合酶（NOS）基因表达的定位。 结果将表明NO的产生是否需要多于一种NOS基因的诱导，以及在植入前胚胎期间是否存在这些基因的差异表达;（B）确定妊娠第1-4天胚胎中NO产生的信号级联的性质。具体而言，我们将研究细胞周期基因的作用，时间过程和细胞特异性，包括：早期直接基因：c-los，c-jun和e-myc（mRNA和蛋白质），细胞周期蛋白（D、E、A和B）和视网膜母细胞瘤基因。结果将确定NO是否通过立即早期基因活化为调节细胞周期的基因起作用：（c）确定NO产生的抑制是否改变细胞周期检查点基因的表达。确定抑制NO对妊娠第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NO的抑制是否通过改变检查点基因的表达而停止正常的有丝分裂。 这项研究将确定胚胎中NO产生的状态，其对胚胎基因表达的影响将产生有关正常和异常生殖功能的重要和有意义的信息。 此外，这些信息应该对人类生育治疗产生影响，作为选择更健康的IVF衍生胚胎的工具，增加成功怀孕的可能性。

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.