HORMONAL CONTROL OF OVIDUCTAL SECRETORY PROTEINS

这项研究的长期目标是增加我们的知识 输卵管功能和了解复杂的生化 以及发生在精子、卵子和 植入前胚胎与母体输卵管及其分泌物 允许受精，早期卵裂阶段的胚胎 妊娠的发展和确立。这样做的目的是 研究建议是：1)完成鉴定， De-nevo的表征、分布和纯化 合成输卵管分泌蛋白(OSP)，特别是 雌激素诱导的酸性115,000和85,000 mR蛋白；2) 测试OSP的功能；3)研究OSP的结构和 重组DNA技术对OSP的调控作用我们会 全程定量生产从头合成的OSP 玛瑙类固醇处理母猪的发情周期、妊娠早期和 分别观察壶腹和峡部的贡献。 它们合成A4e的时间跨度。我们将净化 发情前期出现的主要OSP(115Kd、85Kd、Piless Than 4) 并通过离子交换、亲和等方法从外植体培养基中发情 和/或凝胶过滤层析。多克隆生物的生产 主要OSP的抗体将允许免疫细胞化学检测 在输卵管上皮、精子、未受精卵和早期 卵裂期的胚胎，可能暗示有功能。超微结构 分析和组织学改变将与OSP相关 综合与分配。使用体外测试，我们将检查 OSP是否具有免疫抑制活性。此外，我们还将 确定单独或组合的OSP是否具有补充性 抑制活性和调节母体体液免疫 系统。我们将确定精子、卵子或胚胎 特异性结合OSP或卵子是否与早期卵裂 胚胎通过内吞作用摄取OSP。将使用体外培养 以检验输卵管是否能刺激胚胎发育。 我们将提纯主要的OSP(115Kd和 85Kd)，检测有无微粒体的mRNA翻译 并构建了主要基因的cdna文库。 包括编码115K和蛋白质的那些。特异性cDNAs 将被克隆和鉴定，推导出氨基酸序列 和蛋白质数据库进行身份验证。CDNAs将是 用于探针，以确定特定OSP nRNA的量，并 研究发情周期的调节或激素的作用 治疗。这些研究可以确定和定义机制和 受精和胚胎发育的控制。

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.