The Effects Of C-type Natriuretic Peptide (CNP) On Human Sperm And Fertilization Of Mice In Vitro

Objectives: To investigate the expression of CNP/NPR-B in human sperm and analysis thecorrelation with the sperm function.Methods: Using indirect immunofluorescence to detect the distribution of CNP/NPR-B inhuman ejaculated spermatozoa and observe the results by confocal laser scanningmicroscope.Results: The specifc receptor (NPR-B) of CNP is localized in the acrosomal region of thehead and the membrane of the front-end tail of human sperm, while there is no signal ofCNP in human sperm.Conclusions: The specific receptor (NPR-B) of CNP presented in the acrosomal region ofthe head and the membrane of the front-end tail of sperm suggests that CNP/NPR-B mayaffect the sperm motor function, acrosome reaction and fertilization Objectives: To determinate the influence of CNP upon human sperm vitality, motility and acrosome reaction.Methods: Normal human semen samples were collected from consenting donors at Hubeihuman sperm bank of China. Separated sperm were cultured in vitro. Every sample wasdivided into three groups by the concentration of CNP: the negative control group; the CNP10-7mol/L group; the CNP10-6mol/L group. The vitality and motility of sperm wereanalysed at5min,30min,60min,120min,240min for each groups respectively. Thesperm motility was determined by eosin staining, and all tests were repeated at least3times.The acrosome reaction of sperm was analysed by using Coomassie brilliant blue stainingtechniques at0h,3h,6h for each groups respectively, and all tests were repeated at least3times.Results:1. Compared with the control group at same time point, the vitality and motility ofCNP treated groups were higher at30min after CNP added in sperm suspension.Especially the changes were showed as dose-dependent, the group of CNP10-6mol/L wasmore significant difference (P<0.01) than CNP10-7mol/L (P<0.05).2. After co-incubatedwith CNP for6h, compared with the control group, the percentage of acrosome reactionsperm was significantly increased, especially the changes were showed as dose-dependent,the group of CNP10-6mol/L was more significant difference (P<0.01) than CNP10-7mol/L (P<0.05).Conclusions: CNP could enhance the vitality, motility and acrosome reaction of spem invitro cultured. These indicate that CNP might play an important role in sperm capacitation,fertilization. Objectives: To observe the influence of CNP upon fertilization and embryo developmentof mice in vitro and analysis the relationship of dose-response.Methods: All of the materials used in this study were divided into three groups based on the final concentration of CNP added (0,10-7and10-6mol/L). Take kunming mice femaleand male respectively18and6. The female mice were superovulated and promoted eggmaturation. Collected mature oocytes were placed in cultre medium randomly. Then takethe sperm from epididymal tail of the male mice and add the sperm in the cultre mediumdroplet (on average5-10eggs were present in each cultre medium droplet). Put the disk in37°C,5%CO2incubator for4-6h. Then wash the oocytes and replace them in the newculture medium. Observations: fertility was considered to be the percentage of two-cellembryos produced by IVF scored24h after insemination. According to the development offertilized eggs in the mice in vivo, we statisticed the blastocyst formation rate after96h.Results: The fertilization rate of the control was62.7%; the CNP10-7mol/L group was66%; the CNP10-6mol/L group was73.8%. Compared with the control, the fertilizationrate of CNP treated groups were higher, but only the difference between the control andCNP10-6mol/L group is statistical (P<0.05) by using χ2-test. The blastocyst formation rateof the control was30.4%; the CNP10-7mol/L group was45.7%; the CNP10-6mol/L groupwas35.5%. Compared with the control, the blastocyst formation rate of CNP10-6mol/Lgroup was slightly higher, the difference has no statistical significance by using χ2-test, butthere was a significant difference between the control and CNP10-7mol/L group (P<0.01).Conclusions: CNP can increase the fertilization and blastocyst formation of mice in vitro.The possible reasons are: The increase of fertilization is likely related to the effect of CNPon sperm function, such as increasing the vitality, motility and the rate of·acrosomereaction of sperm. Moreover, The increased blastocyst formation rate of mice in vitroindicates that CNP is an important factor of early embryonic development, particularly at10-7mol/L.