The Role Of C Type Natriuretic Peptide(CNP)on Sperm Maturation In Rat Epididymis

The first section: the distribution of CNP and NPR-B in the tissue and the epididymis of ratsObjective :(1)to detect the expression of CNP and its receptor NPR-B in all tissues of rats.(2)to detect the expression of CNP and its receptor NPR-B in the caput,corpus and cauda of the epididymis of rats.(3)the expression of CNP and its receptor NPR-B in the epididymis of rats at different time periods after birth.(4)the protein concentration of CNP and c GMP in the epididymal fluid of rats was detected.Methods(1)The expression levels of CNP and NPR-B in rat heart,liver,spleen,lung,kidney,testis,prostate,seminal vesicle and epididymis were detected by real-time quantitative PCR.(2)the expression of CNP and NPR-B in the caput,corpus and cauda of epididymis was detected by fluorescence quantitative PCR,immunohistochemistry and Western blot.(3)the epididymal tissues of rats with different ages after birth were used to detect the expression of CNP and NPR-B by fluorescence quantitative PCR.(4)Elisa was used to detect the protein concentration of CNP and c GMP in the epididymal fluid of rats.Results(1)CNP and NPR-B m RNA were expressed in all tissues and organs of rats.CNP expressed the highest in the seminal vesicle,followed by in the prostate and epididymis.The expression of NPR-B in the epididymis was the highest,followed by in the prostate and seminal vesicle.(2)The CNP and NPR-B were expressed in all the epididymis tissues.In the caput the expression is highest,and the cauda is least.(3)the expression of CNP and NPR-B was the highest at birth and fifth weeks after birth.(4)CNP,c GMP expressed the largest amount in the epididymal caput liquid.Conclusion CNP and its receptor NPR-B expressed mainly in the rat epididymis,prostate,seminal vesicle.In the epididymis,they mainly expressed in the caput.The concentration of CNP and c GMP was most in the caput of the epididymal fluid.CNP and NPR-B in rats at birth and fifth weeks after birth was at he highest expression level.Second section: the effect of CNP on epididymal spermObjective :(1)The effect of the androgen concentration on the production of CNP and its receptor NPR-B in rats。(2)The expression of NPR-B in the the epididymal sperm of rats.(3)the effect of incubating CNP on the sperm motility in the epididymal caput,corpus and cauda of rats in vitro.Methods(1)After castration of 10-12 w SD rats,it was divided into 9 groups(4 in each group).The first five groups were killed after being castrated at 0,1,3,5,and 7 days after being castrated.The latter four groups were injected with testosterone(3mg/Kg/d)10 days after being castrated.At the 1,3,5,and 7 days after injection of testosterone,the SD rats were sacrificed.Each group took RNA from epididymal tissue to make fluorescent quantitative PCR,and collected serum to detect testosterone content in serum by Elisa.(2)The expression of NPR-B in the spermatozoa of rats was detected by immunofluorescence.(3)10-7mol/LCNP incubated the epididymal sperm in vitro,and the sperm motility was detected in 0,2,4,6,and 8h respectively.Results(1)compared with the 0th day,the serum testosterone content decreased significantly,and the expression of CNP and NPR-B decreased as well.After the testosterone supplementation,the expression of CNP and NPR-B recovered.(2)NPR-B is expressed in all section of the epididymal sperm,mainly expressed in the middle of the sperm head and tail.(3)the incubation of CNP in vitro can increase the sperm motility of epididymal sperm.Conclusion The CNP and its receptor NPR-B in the epididymis of rats are probably synthesized and secreted under the regulation of testosterone secreted by the testis,and have a certain dependence on androgens.The spermatozoa in epididymis of rats all express CNP specific receptor NPR-B,and CNP can increase the activity of epididymal sperm in vitro.