GONADOTROPIN RECEPTOR REGULATION IN VIVO AND IN VITRO

在上一个赠款期间，我们的实验室确定了几个 卵巢中的转化生长因子-β（TGF-β）亚型， 证明了TGF-B和卵泡之间的显著相互关系， 刺激激素（FSH）调节卵巢细胞功能。 我们 在条件培养物中也检测到生长抑制活性 猪颗粒细胞（GC）的培养基，其不是TGF-β， 分子量估计值并不代表任何已知的增长 因子或其结合蛋白。 具体而言，目前的结果 表明存在推定的小分子量多肽 抑制3 H-胸苷摄取/细胞增殖， 在这些细胞中诱导凋亡（程序性细胞死亡）。 由于我们 了解局部作用的生化因子调节 卵巢卵泡发育的停止实际上是不存在的， 分离和鉴定新的/新的抑制因子， 卵泡细胞生长和/或诱导程序性细胞死亡将是非常重要的 这一领域重大研究进展。 因此，具体 该建议的目的是在分子水平上阐明 （1）确定因果效应子的特征： 同一因素或不同因素导致增长 抑制和凋亡活性; 2）分离推定的生长 抑制性多肽并克隆其基因- a）通过 制备型和分析型HPLC，B）测定氨基酸序列，c） 产生抗体，d）用抗体筛选cDNA文库或标记 寡核苷酸，和e）确定核苷酸序列; 3）分析 基因组DNA和mRNA相对于物种和细胞分布- a） 制备标记的cDNA探针，B）基因组DNA的Southern印迹分析， c）mRNA的北方印迹分析，包括来自人卵巢的mRNA 细胞，以确定转录本的大小和细胞的模式， 表情 这些研究的长期目标是提供一个基本的 了解卵巢细胞功能，最终将是 适用于生育调节问题。

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.