The follicle constitutes a microenvironment in the ovary in which factors produced by the oocyte and somatic cells act in a paracrine and/or autocrine manner to regulate numerous processes including follicular growth and ovulation. To date, individual intrafollicular factors differentially expressed within the preovulatory follicle have been identified and investigated on a case-by-case basis. Therefore, studies are designed using rhesus monkeys and women to determine the feasibility of utilizing a proteomics approach to systematically identify proteins that are differentially expressed through the periovulatory interval (Specific Aim #1) and abnormal preovulatory follicle development (Specific Aim #2), respectively. Follicular fluid will be collected from rhesus monkeys undergoing a controlled ovarian stimulation (COS) protocol such that material can be retrieved prior to (Oh) and at specific times after (12 and 36h) the administration of an ovulatory stimulus (hCG). The macaque follicular fluid collected at the different time points of the periovulatory interval will be separated by 2-dimensional (2D) electrophoresis and analyzed via a fluorescence-based detection system. The gels corresponding to the different stages of the ovulatory process will be analyzed by the Specialized Cooperative Centers Program in Reproduction Research (SCCPRR) proteomics core facility located at the University of North Carolina at Chapel Hill. Differentially expressed proteins will be localized, removed, and digested with trypsin via a fully automated robotic workstation. Proteins isolated from the individual gels will be identified through the use of either a matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF/MS) or an electron spray ionization-mass spectrometer (ESI-MS/MS) located at the SCCPRR core facility. Follicular fluid will also be collected from women seeking infertility treatment for a male factor or those donating oocytes (controls), as well as from women undergoing infertility treatments due to polycystic ovary syndrome (PCOS). The follicular fluid from PCOS patients will be compared to controls using the previously described proteomics methodology (2D electrophoresis/MS) to identify proteins that are differentially expressed through the course of abnormal follicle development (i.e. PCOS). The proposed pilot studies will establish the utility of a systematic proteomics approach to identify novel or previously unappreciated intrafollicular mediators of normal and abnormal ovarian processes.
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