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
Roy, Sambit; Gandra, Divya; Seger, Christina et al. (2018) Oocyte-Derived Factors (GDF9 and BMP15) and FSH Regulate AMH Expression Via Modulation of H3K27AC in Granulosa Cells. Endocrinology 159:3433-3445
Kumar, T Rajendra (2018) Fshb Knockout Mouse Model, Two Decades Later and Into the Future. Endocrinology 159:1941-1949
Das, Nandana; Kumar, T Rajendra (2018) Molecular regulation of follicle-stimulating hormone synthesis, secretion and action. J Mol Endocrinol 60:R131-R155
Gilbert, Sara Babcock; Roof, Allyson K; Rajendra Kumar, T (2018) Mouse models for the analysis of gonadotropin secretion and action. Best Pract Res Clin Endocrinol Metab 32:219-239
Kumar, T Rajendra (2018) Extragonadal Actions of FSH: A Critical Need for Novel Genetic Models. Endocrinology 159:2-8
Romereim, Sarah M; Summers, Adam F; Pohlmeier, William E et al. (2017) Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions. Mol Cell Endocrinol 439:379-394
Liu, Peng; Ji, Yaoting; Yuen, Tony et al. (2017) Blocking FSH induces thermogenic adipose tissue and reduces body fat. Nature 546:107-112
Kumar, T Rajendra (2017) The SO(H)L(H) ""O"" drivers of oocyte growth and survival but not meiosis I. J Clin Invest 127:2044-2047
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- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in Fshb null mice. Mol Cell Endocrinol 437:224-236

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