| Part 1 Preparation of OY-TES-1-N/-C truncated protein polyclonal antibodyObjective: Preparation of OY-TES-1-N/-C truncated proteins polyclonal antibodies,to make up for lack of commercialization of antibodies.Methods:(1)OY-TES-1-N and OY-TES-1-C fusion proteins were expressed from recombinant plasmids constructed in the previous work.(2)The two fusion proteins were purified with Amylose Resin afinity chromatograph.(3)The two purified fusion proteins were used to immunize the New Zealand white rabbits for generating polyclonal antibodies.(4)The anti-OY-TES-1-N and anti-OY-TES-1-C polyclonal antibodies were purified with protein A afinity chromatograph.(5)The titer and specificity of the two antibodies were detected by indirect ELISA and Western blotting,respectively.Result :(1)OY-TES-1-N and OY-TES-1-C fusion proteins were successfully acquired.(2)The two high purity fusion proteins were obtained.(3)Humoral immunity reaction of New Zealand rabbit was induced by fusion proteins immuned.(4)The two high purity with were obtained.(5)The titer of OY-TES-1-N and OY-TES-1-C polyclonal antibodies were about 1 :128000.And the antibodies could specifically react to the fusion proteins,respectively.Conclusion: The OY-TES-1-N and OY-TES-1-C polyclonal antibodies with high specificity and sensitivity had been successfully prepared.Part 2 Expression of OY-TES-1 in human normal tissueObjective: To understand the expression profile of OY-TES-1 in fetal organs and adult testis,epididymal ductu and ejaculated spermatozoa.Methods:(1)Immunohistochemistry was performed with anti-OY-TES-1-N antibody in fetal organs including esophagus,stomach,duodenum,colon,liver,pancreas,heart,aorta,lung,trachea,adrenal gland,adenohypophysis,thymus,spleen,bladder,kidney,testis,ovary,spinal cord,sciatic nerve,skin.(2)Immunohistochemistry was performed with anti-OY-TES-1-N/-C antibodies in adult testis and epididymal duct.(3)Immunocytochemistry was performed with anti-OY-TES-1-N/-C antibodies in ejaculated spermatozoa.Result:(1)OY-TES-1-N was expressed in fetal organs investigated and exhibited different expression levels in different tissues,including epithelium,muscular tissue and nervous tissue.(2)OY-TES-1-N/-C were expressed in adult testis and epididymal duct.(3)Only the OY-TES-1-N expressed in acrosome ejaculated spermatozoa.Conclusion:(1)The expression of OY-TES-1-N in fetus exhibites tissue specificity.(2)The expression of OY-TES-1-N/-C in adult testis and epididymal duct exhibites cell-and region-specific patterns,which suggests that they may participate in spermatogenesis and spermiogenesis,as well as epididymal maturation,storage and transmission of sperm.(3)Hominine mature OY-TES-1,OY-TES-1-N,is posttranslationally produced by removal of the C-terminal half of the precursor OY-TES-1 during spermatogenesis and/or epididymal maturation of sperm.It suggests that OY-TES-1-C may play an important role in sperm capacitaion,acrosome recation and/ or sperm-oocyte membrane fusion.Part 3 The research of OY-TES-1-N antibody to sperm capaciation in virto and its mechanismObjective: To understand the effect of OY-TES-1-N antibody to sperm capaciation and the mechanism.Methods:(1)The percentage of capacitaion live spermatozoa was analysised by co-staining of CTC and H33258.(2)The expression of OY-TES-1-N was detected by co-staining of CTC and immunonuorescence.(3)Spermatozoa were cultured in various OY-TES-1-N antidody coneenrtation(50?g/ml,100?g/ml and 200?g/ml).(4)The motility rate of spermatozoa was detected by eosin-nigrosin staning.(5)The percentage of capacitaion spermatozoa was detedcted by CTC staning.(6)The vitality of spermatozoa was evaluated by computer assisted sperm analysis.(7)Mitochondrial membrane potential of permatozoa was detected by flow cytometry.Result:(1)Spermatozoa were incubated in the BWW medium for 5h for capacitation.(2)The location of OY-TES-1-N expression was transformed during spermatozoa capacitation.(3)Spermatozoa of different treated collected after incubation 5h.(4)No significant effect of OY-TES-1-N polyclonal antibody on sperm viability.(5)The percentage of capacitaion sperm was induced by treated with different concentration of OY-TES-1-N antibody.(6)The vitality of spermatozoa was induced by treated with different concentration of OY-TES-1-N antibody.(7)The mitochondrial membrane potential of sperm was induced by treated with different concentration of OY-TES-1-N antibody.Conclusion: Proteins involved in process of capacitation and inhibit sperm vitality in vitro by cutting the mitochondrial membrane potential.Part 4 The research of OY-TES-1-N antibody to sperm acrosome reaction in virto and its mechanismObjective: To understand the effect of OY-TES-1-N antibody to sperm capaciation and the mechanism.Methods:(1)The percentage of live spermatozoa acrosome reaction was analysised by co-staining of FITC-PSA and H33258.(2)The expression of OY-TES-1-N was detected by co-staining of FITC-PSA and immunonuorescence.(3)Spermatozoa were cultured in various OY-TES-1-N antidody coneenrtation(50?g/ml,100?g/ml and 200?g/ml)with 2.5 ? M A23187.(4)The motility rate of spermatozoa was detected eosin-nigrosin staning.(5)The percentage of spermatozoa acrosome reaction was detedcted by Coomassie brilliant blue staning.(6)The fertilization potential of spermatozoa was evaluated by sperm penetration of zone-free hamster egg test assay.(7)The level of cAMP,PKA and PKC of permatozoa was detected,respectively.Result:(1)Spermatozoa were incubated in BWW medium with 2.5?M A23187 for 2 h for induce acrosome reaction.(2)The fluorescence signal of OY-TES-1-N protein in head of sperm decreased or diminished after acrosome reaction.(3)Spermatozoa of different treated collected after incubation 2h.(4)No significant effect of OY-TES-1-N polyclonal antibody on sperm viability.(5)The percentage of sperm acrosome reaction was induced by treated with different concentration of OY-TES-1-N antibody.(6)The pencnration rate and penetration index of spermatozoa was induced by treated with different concentration of OY-TES-1-N antibody.(7)The level of cAMP,PKA and PKC of permatozoa was induced by treated with different concentration of OY-TES-1-N antibody.Conclusion: Human sperm acrosome reaction requires at least activates both the cAMP/PKA and PKC signaling pathways in spermatozoa and facilitates sperm-oocyte fusion.These clues suggested that the OY-TES-1-N may play a vital role for sperm during fertilization.Part 5 The effects of active immunization OY-TES-1 on reproduction performance of rabbitObjective: To understand the effect of passive immunization OY-TES-1 on reproduction performance of rabbit.Methods:(1)The expression of OY-TES-1 was detected in testis,epididymal ductu of rabbit.(2)One group was immunized by OY-TES-1 fusion protein,another group was served as untreated contro1.(3)Body weight and sexual behaviour were investigated.(4)The density of ejaculated spermatozoa was detected.(5)The deformity rate of ejaculated spermatozoa was detected.(6)The motility rate of ejaculated spermatozoa was detected by eosin-nigrosin staning.(7)The vitality of spermatozoa was evaluated by optical microscope.(8)The organ coefficient of testis was calculated.(6)The histological analysis of the testes was performed by HE staining.Result:(1)OY-TES-1-N/-C were expressed in rabbit testis and epididymal duct.(2)Humoral immunity reaction of New Zealand rabbit was induced by OY-TES-1 full long fusion protein immuned.(3)The body weight and sexual behavior of immunized rabbits was similar to untreated ones.(4)The density of ejaculated spermatozoa was induced by immunization against OY-TES-1.(5)No significant effect of OY-TES-1-N polyclonal antibody on the deformity rate of ejaculated spermatozoa.(6)The motility rate of spermatozoa was induced by immunization against OY-TES-1.(7)The vitality of spermatozoa was induced by immunization against OY-TES-1.(8)The organ coefficient of testis was induced.(9)The obvious pathological changes of testes histology were detected in immunizated rabbits.Conclusion: The immunization of OY-TES-1 fusion protein extremely resulted in obvious descent of fertility potential. |
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