Correlation Analysis And Mechanism Exploration Of C-type Natriuretic Peptide(CNP) And Asthenozoospermia

Part 1 Correlation analysis of CNP and sperm motility of asthenozoospermia[Purpose]To detect the concentration of CNP in seminal plasma of normal people and asthenozoospermia patients respectively,then to explore the effect of CNP on sperm motility of asthenozoospermia patients and its possible mechanism.[Methods]This section of the experiment is divided into four parts.1.According to the inclusion and exclusion criteria prepared in advance,semen samples from normal people and asthenozoospermia patients were collected,to conduct pretreatment and separation of sperm and seminal plasma,and use enzyme-linked immunosorbent assay?ELISA?to detect the concentration of CNP in seminal plasma;sperm were preserved for folow-up experiments.2.To treat sperm of asthenozoospermia samples with CNP?blank control group:0mol/L;experimental group:10-6mol/L?,sperm motility of each group was detected in 10 min,30 min?60 min,120 min and 240 min after the addition of CNP.3.Experimental groups were as follows:?1?blank control group;?2?the CNP group?10-6mol/L?;?3?8-Br-cGMP?cGMP analogue?group;?4?KT5823?PKG inhibitor?group;?5?CNP+KT5823 group;?6?CNP+NPR-B antagonist group;?7?8-Br-cGMP+KT5823.According to the above groups,the corresponding substances were added,and the sperm were treated in vitro.After 1 hour of treatment,the differences of sperm motility and cGMP level between the experimental group and the blank control group were compared respectively.4.Expression level of sperm CatSperl mRNA of normal people and asthenozoospermia patients were detected by RT-PCR.Then,sperm of asthenozoospermia patients were treated in vitro with CNP?blank control group:0 mol/L;experimental group:10-6 mol/L?,and expression level of CatSper1 mRNA was detected 1 h later.[Results]47 cases of normal semen samples and 45 cases of asthenospermia semen samples were collected up to now,and the concentrations of CNP of seminal plasma were 259.75±12.57pg/ml and 223.04±20.91pg/ml respectively,with significant statistical differences?p<0.05?.Sperm of asthenozoospermia samples were treated in vitro with CNP?Omol/L,10-6mol/L?,sperm motility of the CNP group was higher than that of the control group,and the difference was most significant at the 60th and 120th minutes?p<0.01?.After 1h of sperm treatment,sperm motility of each group was compared.The sperm motility of CNP group and 8-Br-cGMP group was higher than that of the control group?p<0.05?,and sperm motility of CNP+KT5823 group and NPR-B antagonist group were lower than that of CNP group?p<0.01?;the concentration of cGMP in each group was detected,and the results were consistent with the trend of sperm motility,that is,the cGMP concentration in the sperm of the CNP group and the 8-Br-cGMP group was higher than that of the control group?p<0.01?,the cGMP concentration in the spermatozoa of CNP+KT5823 group and NPR-B antagonist group was lower than that of CNP group?p<0.01?.The expression of CatSperl mRNA in spermatozoa was lower than that in normal people?p<0.01?.The expression of CatSperl mRNA was slightly higher than the control group after treatment of sperm and CNP?10-6mol/L?,however,the difference was not statistically significant.[Conclusions]The concentration of CNP in seminal plasma of asthenozoospermia patients was lower than that of normal group;the treatment of 10-6mol/L CNP with sperm can increase sperm motility,after 60min and 120min of treatment,the difference of sperm motility of experimental group and control group was the most significant;CNP can increase sperm motility through NPR-B/cGMP pathway;the expression of CatSperl mRNA in spermatozoa of asthenozoospermia patients was lower than that of normal group,and the effect of CNP on CatSperl mRNA expression needs further exploration.Part 2 Influence of CNP on body weight and reproductive system of asthenozoospermia mice[Purpose]To establish an animal model of asthenozoospermia and compare the effects of CNP on body weight and reproductive system of asthenozoospermia mice.[Methods]An animal model of asthenozoospermia was established by intraperitoneal injection of Cyclophosphamide?CTX?.Groups are as follows:?1?group A:blank control?PBS?;?2?group B:CTX?50mg/kg/d?;?3?group C:CTX?50mg/kg/d?+ CNP?25?g/kg/d?;?4?group D:CTX?50mg/kg/d?+ CNP?50?g/kg/d?,The drug injection protocol were as follows:mice of group A were given continuous injection of PBS for 10 days once a day;mice of group B were continuously injected with CTX?50 mg/kg/d?for 5 days,and then continuously injected with PBS for 5 days;mice of group C were continuously injected with CTX?50mg/kg/d?for 5 days,and continuous injection of CNP?25?g/kg/d?for 5 days afterwards;mice of group D were continuously injected with CTX?50mg/kg/d?for 5 days,and continuously injected with CNP?50?g/kg/d?for 5 days.The effects of CNP on body weight and reproductive system?semen quality,testosterone level,reproductive organ morphology?of each group were compared.[Results]The body weight of mice of group B?14.48±1.78g?was significantly lower than that of group A?24.2211.67g?.The weight of mice of group C?17.0410.99g?was higher than that of group B,and the differences were statistically significant.Compared with group A?2.20±0.25mg/g b.w.?,the epididymal gonad index of group B?1.45±0.38mg/g b.w.?was significantly lower;the epididymal gonad indexes of group C and group D were 2.03±0.34mg/g b.w.and 1.8710.29mg/g b.w.respectively,which were both higher than those of group B,and the differences were statistically significant?p<0.05?,however,there was no significant difference between the testicular gonad index of each group.In addition,the total sperm motility of group B was?18₅513₄2?%,which was significantly lower than that of group A??30.50±2.30?%?.Compared with group B,the total sperm motility of group C and group D were?31.2518.93?%and?29.4117.67?%respectively,which were higher than that of group B,with statistical differences?p<0.05?.After injection of cyclophosphamide,the sperm density of group B??2.09±0₄1?×106 cells/mL/two epi.?was lower than that of the control group??3.6110.35?×106cells/mL/two epi.?,sperm density of group C??2.99±0.48?×106cells/mL/two epi.?was higher than that of group B?p<0.01?.compared with blank control group,testosterone levels of group B?only injected CTX?were significantly lower,testosterone levels of group C and group D were higher than that of group B,both of which were statistically different.HE staining showed that the tissue morphology of testis and epididymis in group C was improved to a certain degree compared with group B,but there was no significant difference in seminal vesicles.[Conclusions]CNP can improve the growth of mice with asthenozoospermia to some extent.The gonad index of the epididymis and sperm motility of group C and group D were higher than those of group B,and low concentration of CNP was able to alleviate the damage of cyclophosphamide to reproductive system,of which effect is more pronounced.In addition,CNP has certain antagonistic effects on decrease of testosterone concentration induced by cyclophosphamide.Part 3 Mechanism Exploration of CNF on the regulation of sperm motility in vivo[Purpose]To explore the possible mechanism of CNP in regulating sperm motor function in vivo.[Methods]This section of experiment is divided into three parts.First,the effect of CNP on tissue oxidative stress:testes of the above 4 groups of mice were homogenized to detect glutathione peroxidase?GSH-Px?,superoxide dismutase?SOD?,catalase?CAT?,malondialdehyde?MDA?;ROS immunofluorescence staining of testes was used to analyze the effects of CNP on tissue oxidative stress levels.Second,the effect of CNP on expression of cGMP and CatSperl mRNA in sperm:the sperm in the epididymis of each group was collected to detect the concentration of cGMP of sperm;the sperm in the epididymal caput of each group was collected,and CatSperl mRNA expression levels of sperm were detected by RT-PCR.Third,the impact of CNP on immune function:ELISA method was used to detect the concentration of TNF-? and IL-6 in serum;the mRNA levels of TNF-? and IL-6 in different tissues of genital tract?testis and epididymis?were detected.[Results]Compared with group B?CTX?,the levels of GSH-Px,SOD and CAT of group C?CTX+25?g/kg/d CNP?were higher,while the levels of MDA in group C were lower than those in group B,the results were statistically significant?P<0.05?;the levels of GSH-Px and CAT of group D?CTX+ 50?g/kg/d CNP?were higher than those of group B,and group D had lower MDA level than group B,and there was no significant difference between its SOD level and that of group B.The cGMP levels of epididymal sperm in group C and group D were higher than those in group B,the differences were statistically significant.Immunofluorescence staining showed that ROS concentration of testes in group C and group D were lower than that in group B,while epididymial ROS concentrations showed no significant differences between groups.The levels of CatSper1 mRNA in group C and group D were higher than those in group B,but the difference was not statistically significant.Compared with group A,the levels of TNF-a and TL-6 in serum of group B were increased,while levels of TNF-? and IL-6 in serum of group C were lower than those of group B.Levels of TNF-? mRNA in testis and epididymis of group B were higher than that of group A,and levels of TNF-? mRNA in testis and epididymis of group C were lower than that of group B.Meanwhile,levels of IL-6 mRNA in testis and epididymis of group B were higher than that of group A,levels of IL-6 mRNA in testis and epididymis of group C were lower than that of group B,level of TL-6 mRNA in epididymis of group D was lower than that of group B.[Conclusions]CNP can regulate sperm motility by increasing cGMP levels of sperm in epididymis;CNP may also affect sperm motility by improving tissue oxidative stress levels and reducing the expression of immune factors such as TNF-? and IL-6.