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| Gene Synthesis | Cultured for12 hours and 24 hours, sperm was collected, and RNA, cAMP and protein were isolated for real-time PCR, ELISA and Western blot analyses, respectively. Still the rat hyperactivation-related genes, including CatSper2, LDHC, SLO3 and PKA, were analyzed using real-time PCR to detect the changes in the transfected sperm. GAPDH was the internal control in real-time PCR, and β-actin was the internal control in western blot experiment. All the primers sequences (Table 2) were analyzed by Genscript Company (Nanjing, China). | Get A Quote |
Hyperactivation is one of the most critical parts for fertilization. cAMP generated by soluble adenylyl cyclase (sAC) is necessary to activate sperm and is a prerequisite for sperm hyperactivation. The aim of this study is to investigate the function of sAC in hyperactivation in male rats. Four siRNAs of sAC gene were designed and were separately transformed into rat sperm using electrotransformation method. Cultured for 12 and 24 h, physiological and biochemical indexes of these sperm were analyzed, and the expressions of some hyperactivation-related genes were detected using real-time PCR. We demonstrated 26.3-30.8% and 49.1-50.5% reduction in sAC at the protein by Western blot and mRNA levels by real-time PC... More
