HORMONAL CONTROL OF OVIDUCTAL SECRETORY PROTEINS

这项研究的长期目标是增加我们的知识 了解输卵管功能的复杂的生物化学 以及精子、卵子和精子之间的分子相互作用。 着床前胚胎和母体输卵管及其分泌物 允许受精，早期卵裂阶段的胚胎 怀孕的发展和建立。 这个目标 研究建议是：1）完成鉴定， 从头鉴定、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蛋白质; 2） OSP的功能测试; 3）研究OSP的结构和功能， 通过重组DNA技术调节OSP。 我们将 在整个过程中定量从头合成的OSP的产生 玛瑙类固醇处理后备母猪的动情周期、早期妊娠和 检查壶腹和峡部在每一个中的贡献， 他们合成的时间跨度。 我们将净化 在发情前期出现的主要OSP（115 Kd，85 Kd，pI <4 通过离子交换、亲和层析、 和/或凝胶过滤色谱法。 产生多克隆 主要OSP的抗体将允许免疫细胞化学检测 输卵管上皮、精子、未受精卵和早期 卵裂阶段的胚胎，并可能暗示功能。 超微结构 分析和组织学变化将与OSP相关 合成和分配。 我们将通过体外试验 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，可调节母体体液免疫 系统 我们将确定精子卵子或胚胎 特异性结合OSP或卵和早期卵裂阶段 胚胎通过内吞作用摄取OSP。 将使用体外培养物 以检验输卵管是否能刺激胚胎发育。 我们将纯化主要OSP的多聚腺苷酸化mRNA（115 Kd和125 Kd）。 85 Kd），检测有和无微粒体的mRNA翻译 膜，并构建主要mRNA种类的cDNA文库 包括编码115 K和蛋白质的那些。 特异性cDNA 将被克隆和表征，所衍生的氨基酸序列 并检查蛋白质数据库的同一性。 cDNA将是 用于探针，以确定特定OSP nRNA的量， 研究发情周期或激素的调节作用 治疗 这些研究可以确定和定义机制， 控制受精和胚胎发育。

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.