Proteomics Study Of Sperm Protein Of Infertile Patients With Varicocele

Objective:(1) To establish a set of two dimensional gel electrophoresis(2-DE) techniques for proteomic research of sperm protein of infertilepatients with varicocele(VC);(2) Separated sperm protein of infertilepatients with VC through2-DE, analyze the profiles to get the differentiallyexpressed sperm proteins by using software.(3) Identified differentiallyexpressed proteins by the method of MS, confirmed the sperm proteinassociated with VC by PMFs．Methods:(1) Extracted the sperm protein of infertile patients with VC,treated the sperm protein with the techniques of acetone and clean-up kitthen, ran the2-DE with different IPG gels(pH3-10L, pH3-10NL and pH5-8),different amounts of protein loads(200ug,250ug and300ug) and differentrehydration(passive rehydration and active rehydration) methods;(2)Extracted the sperm protein of infertile patients with VC before and aftersurgery, pretreated the protein with the methods of clean-up kit and ran the2-DE with pH5-8gel,250ug protein loads and initiative rehydration method;(3) Got the differentially expressed sperm proteins via comparing theprofiles of two groups with software;(4) differentially expressed proteinswere identified with MALDI-TOF-TOF-MS, search in database with theobtained PMFs and bioinformation analysis. Results:(1) The method of acetone lost the small molecular weightprotein partially compared to the method of clean-up kit;(2) the proteinsspots of pH5-8were well-distributed with less streaks;(3) The backgroundof the2-DE profile of200ug was clear with only526proteins spots, thebackground of the map of250ug was clear and high resolution, and thebackground of the map of300ug was dirty with many streaks.(4) Comparedwith active rehydration, the background of the map of passive rehydrationwas nonuniformity with many streaks;(5) Compared the profiles of twogroups, we found20differentially expressed protein spots,15of which wereidentified successfully.Conclusion:(1) After the sperm sample was treated with thetechniques of clean-up kit, a high-resolution and clear2-DE map for spermprotein of VC patients was obtained with pH5-8gel,250ug protein loads andinitiative rehydration method;(2) There are different proteins expression ofsperm protein of infertile patients with VC before and after surgery. Thefurther study of these proteins could pave the way for the molecularmechanism of infertility caused by VC.