The goal of the FSH process core laboratory (Core B) is to provide instumentation, support and training in protein chemistry techniques adapted to glycoprotein characterization. This core laboratory has extensive experience with gonadotropin purification and characterization. The gonadotropic hormones play a central role in regulating the reproductive system. They are released in response to episodic stimulation by hypothalamic neuronal activity to coordinate the later stages of follicle development initiated by endogenous gonadal cycles. In turn, gonadal hormones, particularly the steroid hormone estradiol feed back to the pituitary and alter gonadotropin synthesis and posttranslational processing. While steroid hormones are used therapeutically in contraceptive procedures, it is the gonadotropins that are used to treat infertility. The core will provide three support functions for the proposed project, as follows: Function #1. Produce hFSH glycofoms. Di-glycosylated, tetra-glycosylated and other hFSH glycoforms will be expressed by Dr. Bin Shuai in mammalian cell lines. Dr. Shuai will provide FSH-containng conditioned culture medium to Dr. Butnev. Function #2. Purify and chemically characterize hFSH glycoform preparations. Dr. Vladimir Butnev will purify and chemically characterize hFSH glycoforms. This will include isolating hFSH glycoforms from pituitary extracts and conditioned culture meduim, determining chemical purity, determining protein quantity, assessing protein integrity, as well as determining carbohydrate composition. FSH characterization is critical for interpreting the biological studies performed in Projects 1-3. FSH glycoform quality control data will be posted monthly on the Glycomics data-sharing page maintained by Core C. Function #3. Provide FSH assay and cell culture support for Project researchers. Dr. Jeffrey V. May will perform receptor-binding and steroidogenic assays in the well-characterized porcine granulosa cell primary culture system to confirm biological activity of purified hFSH'glycoforms. The preparations will then be released to project investigators. These assays will also be made available to project investigators as a service. Projects 1 and 2 will perform some experiments in a common cell model. Initially, this will be porcine granulosa cells. Dr. May will evaluate human granulosa cell lines that become available. For example project investigators wasted valuable time determining that KGN cells, a supposed human granulosa cell line, were unsuitable for our studies. If this project is funded, then Core B will evaluate cell lines and suitable lines will be made available to the projects. This core will also provide support for monoclonal antibody expression to support project 1. Dr. May will serve as overall Core Leader based upon his experience in primary cell culture as well as protein chemistry and molecular biology.
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