Extraction And Characterization Of The Sperm Membrane Proteins In Eriocheir Sinensis

Mature sperms have many kinds of proteins in it's plasma membrane. Some of them are sperm-specific, others bind to the sperm surface during the period of sperm maturation. These sperm membrane proteins(SMPs) play important role in the maintenance of sperm's shape and structure, the metabolism of sperms and the reproduction, especially in the fertilization.With Eriocheir sinensis as the experiment material, SMPs were studied in such respects as extraction, isolation and biochemical characteristics analysis. Firstly, the Protein compositions of various parts of the spermatophore were studied through the methods of fractionation and comparing PAGE. And a method for spermatophore fractionation was established, the feasibility of this method was examined by the means of PAGE and transmission electronic microscopy(TEM). Then, the sperm membrane was purified with the method of sucrose density gradients centrifugation, and the effect on it by isolation was observed in the transmission electronic microscope. Finally, SMPs were extracted from the surface of E. sinensis sperms by the use of decontaminant, and characterization of SMPs from E. sinensis were conducted.The result of SDS-PAGE showed that different parts in spermatophore, including spermatophore matrix, seminal plasma and the whole sperm, had different characteristic bands in PAGE maps, and the result could be repeated, so the method for spermatophore fractionation was reliable. In the Staining PAGE map of glycoproteins from seminal plasma and spermatophore matrix, there were 3 kinds of glycoproteins in seminal plasma, and 2 in spermatophore matrix. So it could be inferred that seminal plasma and spermatophore matrix had nutritional effect onsperms. But it would need further study to clarify the real function of spermatophore matrix. Under TEM ,most of the sperms kept intact after fractionation ,which proved the feasibility of the method on microstructure level and made the basis of the following experiment. Furthermore, with the method of ultrasonic wave homogenization and sucrose density gradients centrifugation, sperm membrane of E. sinensis was purified with good homogeneity successfully.Extraction buffers with 1% TritonX-100 and low concentration of SDS was applied to dissolve membrane proteins off E. sinensis's sperms. The membrane Protein composition was analyzed by SDS-PAGE. As SDS-PAGE maps showed , there were at least 11 kinds of SMPs in E. sinensis, the composition of SMPs is obviously different from that of the demembranated sperms. Some biochemical characteristics of SMPs in E. sinensis were studied in this paper: SMPs in E. sinensis were a group of glycoproteins, molecular weights of which between 38ku and 83ku, isoelectric points (PI) of which are all about 4.8.