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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Program Projects (P01)
Project #
5P01AG029531-05
Application #
8449610
Study Section
Special Emphasis Panel (ZAG1-ZIJ-5)
Project Start
Project End
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
5
Fiscal Year
2013
Total Cost
$342,339
Indirect Cost
$107,862
Name
Wichita State University
Department
Type
DUNS #
053078127
City
Wichita
State
KS
Country
United States
Zip Code
67260
Hua, G; Lv, X; He, C et al. (2016) YAP induces high-grade serous carcinoma in fallopian tube secretory epithelial cells. Oncogene 35:2247-65
Wang, Huizhen; Butnev, Vladimir; Bousfield, George R et al. (2016) A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Δ) Asn(24Δ)) FSHβ subunit fails to rescue Fshb null mice. Mol Cell Endocrinol 426:113-24
Wang, Huizhen; Hastings, Richard; Miller, William L et al. (2016) Fshb-iCre mice are efficient and specific Cre deleters for the gonadotrope lineage. Mol Cell Endocrinol 419:124-38
Wang, Huizhen; May, Jacob; Butnev, Viktor et al. (2016) Evaluation of in vivo bioactivities of recombinant hypo- (FSH(21/18)) and fully- (FSH(24)) glycosylated human FSH glycoforms in Fshb null mice. Mol Cell Endocrinol 437:224-236
Wang, Huizhen; Graham, Ian; Hastings, Richard et al. (2015) Gonadotrope-specific deletion of Dicer results in severely suppressed gonadotropins and fertility defects. J Biol Chem 290:2699-714
He, C; Lv, X; Hua, G et al. (2015) YAP forms autocrine loops with the ERBB pathway to regulate ovarian cancer initiation and progression. Oncogene 34:6040-54
He, Chunbo; Mao, Dagan; Hua, Guohua et al. (2015) The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression. EMBO Mol Med 7:1426-49
Butnev, Viktor Y; Butnev, Vladimir Y; May, Jeffrey V et al. (2015) Production, purification, and characterization of recombinant hFSH glycoforms for functional studies. Mol Cell Endocrinol 405:42-51
Jiang, Chao; Hou, Xiaoying; Wang, Cheng et al. (2015) Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells. J Clin Endocrinol Metab 100:E852-60
Bousfield, George R; Butnev, Vladimir Y; White, William K et al. (2015) Comparison of Follicle-Stimulating Hormone Glycosylation Microheterogenity by Quantitative Negative Mode Nano-Electrospray Mass Spectrometry of Peptide-N Glycanase-Released Oligosaccharides. J Glycomics Lipidomics 5:

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