GONADOTROPIN RECEPTOR REGULATION IN VIVO AND IN VITRO

在之前的资助期间，我们的实验室发现了几个 卵巢和卵巢中的转化生长因子-β亚型 研究表明，转化生长因子-β与卵泡- 促性腺激素(FSH)对卵巢细胞功能的调节我们 还检测了条件培养物中的生长抑制活性 非转化生长因子-β的猪颗粒细胞(GC)的培养液 分子量估计并不代表任何已知的增长 因子或它们的结合蛋白。具体地说，当前结果 表明可能存在一种小分子多肽 对~3H-胸腺嘧啶核苷摄取/细胞增殖和活性的抑制 诱导这些细胞的凋亡(程序性细胞死亡)。因为我们的 对局部作用的生物化学因子调节的知识 停止卵泡发育实际上是不存在的， 新/新抑制因子的分离与鉴定 毛囊细胞生长和/或诱导细胞程序性死亡 这一领域的重大研究进展。相应地，具体的 这一提议的目的是在分子水平上阐明 致因效应者的特征(S)：1)判断致因效应者是否 同一因素或不同因素对增长起作用 抑制和凋亡活性；2)分离假定的生长 抑制性多肽基因的克隆与鉴定 制备和分析高效液相色谱法，b)测定氨基酸序列，c) 产生抗体，d)用抗体或标记筛选cdna文库 寡核苷酸，e)确定核苷酸序列；3)分析 基因组DNA和mRNA与物种和细胞分布的关系--a) 制备标记的cDNA探针，b)基因组DNA的Southern杂交分析， C)Northern印迹分析信使核糖核酸，包括来自人卵巢的信使核糖核酸 细胞，以确定转录本(S)的大小和细胞模式 表情。 这些研究的长期目标是提供一个基本的 对卵巢细胞功能的了解，最终将是 适用于生育调控问题。

During the previous grant period our laboratory identified several Transforming Growth Factor-Beta (TGF-beta) sub-types in the ovary and demonstrated significant interrelationships between TGF-B and Follicle- Stimulating Hormone (FSH) in regulating ovarian cellular function. We also detected growth inhibitory activity in the conditioned culture medium of porcine granulosa cells (GC) which was not TGF-beta and by molecular weight estimate does not represent any of the known growth factors or their binding proteins. Specifically, current results indicate the presence of a putative small-molecular-weight polypeptide inhibitory to 3H-thymidine uptake/cell proliferation and activity which induces apoptosis (programmed cell death) in these cells. Since our knowledge of the locally acting biochemical factors regulating the cessation of ovarian follicular development is virtually non-existent, the isolation and identification of new/novel factors which inhibit follicle cell growth and/or induce programmed cell death would be very significant research advance in this area. Accordingly, the specific aims of this proposal are to elucidate at the molecular level the characteristics of the causative effector(s): 1) to determine whether the same factor or different factors are responsible for the growth inhibitory and apoptotic activities; 2) to isolate the putative growth inhibitory polypeptide and clone the gene for it - a) isolate by preparative and analytical HPLC, b) determine amino acid sequence, c) generate antibodies, d) screen cDNA libraries with antibodies or labelled oligonucleotides, and e) determine nucleotide sequence; 3) to analyze genomic DNA and mRNA with respect to species and cell distribution - a) prepare labelled cDNA probes, b) Southern blot analysis of genomic DNA, c) Northern blot analysis of mRNA, including that from human ovarian cells, to determine transcript(s) size and patterns of cellular expression. The long term objective of these studies is to provide a basic understanding of ovarian cellular function which will eventually be applicable to the problem of fertility regulation.