| It is nearly a century ago that the first document attempts to immunized with male gametephomogenats from heterospecies produced sperm reacting antibodies in animals. Mature spermhave many kinds of proteins in its plasma membrane. Some of them are sperm-spcific, othersbind to sperm surface during the period of sperm maturation. These sperm membrane proteinsplay important role in the maintenance of sperm shape and structure, the metabolism of spermand the reproduction, especially in the fertilization. Since then it has been amply documentedthat deliberated immunization of female animals of numerous species with preparations ofisologous sperm or mature testis results in infertility due to either fertilization failure orpremplantation embryo mortality or to both.With sperm of wapiti (Cervus elaphus) as the experiment material, The sperm membraneproteins were studied in such respects as extraction, isolation, purification and physiochemicalcharacteristics analysis. To raised polyclonal antibodies preparation with purified spermmembrane proteins of wapiti as antigens, and study on localization of the sperm membraneproteins of wapiti on sperm, the effect on the ability of sperm. The main experimental result ofthe present work were shown as follows:1. Isolation, purification and identification of the sperm membrane proteins of wapiti:The sperm membrane proteins were extracted from the surface of wapiti sperms by the use.ofdecontaminant, base on the result of western-blot and SDS-PAGE, the condition of isolationand purification were estabilished. 4 sperm membrane proteins were purification by SDS-PAGE, staining 0.3mol/L KCl, repeated freeze-thaw and centrifugation. The purified spermmembrane proteins migrated as a single homogeneous band when analyzed by SDS-PAGEunder reducing and nonreducing conditions, respectively. This result reveals that the spermmembrane proteins has no inter-chain disulfide bond. The purity was attested to thehomogeneity.Physicochemical properties of the sperm membrane proteins were determined. Theestimated molecular weight of the sperm membrane proteins were 98.4kDa, 67.6kDa,47.3kDa, 29.1kDa, and the PI were 4-7(pH). The concentration of sperm membrane proteinsof wapiti were 95.67μg/ml, 184.23μg/ml, 47.68μg/ml, 58.32μg/ml; the concentration ofthe total protein was 468.12μg/ml by BCA. 3 sperm membrane proteins of wapiti wereglycoprotein in 4 purified proteins.2.2-D gel electrophoresis: Our results have been shown, there were 823 point obtainde inthe map of 2-DE of the sperm membrane proteins of wapiti. The purity molecular weight was10-100 kDa and the PI was 4-7(pH). 3. Polyclonal antibodies prepareation: In order to study the function of the spermmembrane proteins, polyclonal antibodies were raised in BALB/C mouse. The titer of theantisera was about 1-2×10~3 assayed by ELISA. The result of Western-blot has been indiciatedthat the immunity reactive of the sperm membrane proteins of wapiti, and it was a kind ofspecific antigen.4. The effct of polyclonal antibodies on sperm activity: The effct of the polyclonalantibodies was not significant.on the activity of wapiti sperm before capacitation in vitro. Itwas not effct on mouse; but the effct on the activity of wapiti s.perm after capacitationmarkedly, The effects mentioned above were dose-dependent5. Localization of the sperm membrane proteins of wapiti on sperm: The result ofconfocal laser scaning microscope showed that the purified sperm membrane proteins werelocated in the acrosomal of wapiti sperm, Location of sperm of mouse was the same as.6. identification of polyclonal antibodies of sperm membrane proteins: an agar geldiffusion method showed that the sperm membrane proteins were specific antigen, Theantibodies antiserum was heart. Liver. Kidney.and lung, the purity was reacted with testist andvasdeferens. It was a kind of tissue specificity.
|
PDF Full Text Request