HORMONAL CONTROL OF OVIDUCTAL SECRETORY PROTEINS

输卵管分泌蛋白的激素控制

• 批准号: 3318739
• 负责人: WILLIAM C BUHI
• 金额: $ 10.77万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3318739
• 关键词: antibody formation; complementary DNA; early embryonic stage; egg /ovum; estrogens; estrus; fallopian tubes; fertilization; gel electrophoresis; gel filtration chromatography; genetic library; genetic manipulation; genetic transcription; genetic translation; genital secretion; glycoproteins; histopathology; hormone regulation /control mechanism; humoral immunity; immunochemistry; immunosuppression; laboratory rabbit; macromolecule; membrane proteins; messenger RNA; molecular cloning; monoclonal antibody; nucleic acid probes; ovariectomy; peptide hormone; progesterone; protein biosynthesis; protein sequence; proteins; sex hormones; sperm; swine

The long term goals of this research are to increase our knowledge of oviductal functions and to understand the complex biochemical and molecular interactions that occur between sperm, ova, and the preimplantation embryo and the maternal oviduct and its secretions that allow fertilization, early cleavage-stage embryonic development and establishment of pregnancy. The goal of this research proposal is: 1) To complete the identification, characterization, distribution and purification of de novo synthesized oviductal secretory proteins (OSP), in particular the estrogen-induced acidic 115,000 Mr and 85,000 Mr proteins; 2) To test for function of OSP; and 3) To study the structure and regulation of OSP by recombinant DNA techniques. We will quantitate production of de novo synthesized OSP throughout the estrous cycle, early pregnancy, in onyx steroid-treated gilts and examine the contribution of the ampulla and isthmus in each and the time span during which they a4e synthesized. We will purify the major OSP (115Kd, 85Kd, pIless than 4) that appear at proestrus and estrus from explant culture medium by ion-exchange, affinity and/or gel filtration chromatography. Production of polyclonal antibodies to the major OSP will allow innunocytochemical detection on oviductal epithelium, sperm, unfertilized ova, and early cleavage-stage embryos and may suggest function. Ultrastructural analysis and histological changes will be correlated with OSP synthesis and distribution. Using in vitro tests we will examine whether OSP have immunosuppressive activity. Further, we will determine whether individual or combined OSP have complement inhibitory activity and can regulate the maternal humoral immune system. We will determine whether sperm, ova, or embryos specifically bind OSP or whether ova and early cleavage-stage embryos take up OSP by endocytosis. In vitro cultures will be used to examine whether the oviduct can stimulate embryonic development. We will purify polyadenylated mRNA for the major OSP (115Kd and 85Kd), examine mRNA translation with and without microsomal membranes, and construct a cDNA library of major mRNA species including those encoding the 115K and proteins. Specific cDNA's will be cloned and characterized, the amino acid sequences derived and protein data base examined for identity. The cDNA's will be used for probes to determine amounts of specific OSP nRNA and to study regulation during the estrous cycle or with hormone treatment. These studies may identify and define mechanisms and controls for fertilization and embryonic development.

这项研究的长期目标是增加我们的知识 输卵管功能并了解复杂的生化 以及精子、卵子和精子之间发生的分子相互作用 植入前胚胎和母体输卵管及其分泌物 允许受精，早期卵裂阶段胚胎 妊娠的发育和建立。 此举的目标 研究建议是：1）完成鉴定， de novo 的表征、分布和纯化 合成的输卵管分泌蛋白（OSP），特别是 雌激素诱导的酸性 115,000 Mr 和 85,000 Mr 蛋白； 2) 至 测试OSP的功能； 3）研究结构和 通过重组 DNA 技术调节 OSP。 我们将 整个过程中从头合成的 OSP 的定量生产 发情周期、妊娠早期、缟玛瑙类固醇处理后备母猪和 检查壶腹部和峡部的贡献 它们合成的时间跨度。 我们会净化 发情前期出现的主要 OSP（115Kd、85Kd、pI 小于 4） 通过离子交换、亲和力从外植体培养基中分离和发情 和/或凝胶过滤色谱法。 多克隆的生产 主要 OSP 的抗体将允许免疫细胞化学检测 对输卵管上皮、精子、未受精卵和早期 卵裂期胚胎并可能提示功能。 超微结构 分析和组织学变化将与 OSP 相关 合成和分配。 我们将使用体外测试来检查 OSP是否具有免疫抑制活性。 此外，我们将 确定单个或组合 OSP 是否具有补体 抑制活性并可调节母体体液免疫 系统。 我们将确定是精子、卵子还是胚胎 特异性结合 OSP 或是否卵子和早期卵裂阶段 胚胎通过内吞作用吸收 OSP。 将使用体外培养物 检查输卵管是否能刺激胚胎发育。 我们将纯化主要 OSP（115Kd 和 85Kd)，检查有或没有微粒体的 mRNA 翻译 膜，并构建主要 mRNA 种类的 cDNA 文库 包括编码 115K 和蛋白质的基因。 特定 cDNA 将被克隆和表征，衍生的氨基酸序列 和蛋白质数据库检查身份。 cDNA 将是 用于探针测定特定 OSP nRNA 的量并 研究发情周期或激素的调节 治疗。 这些研究可以确定和定义机制 控制受精和胚胎发育。