1.Epididymis And Testicular Hypofunction Caused By Activating P53 Expression Induced By ROS In Varicocele Rats 2.The Relationship Between Cation Channel Of Sperm(CatSper) And Asthenospermia

BackgroundVaricocel is considered as the most common cause of male infertility by the World Health Organization,and the global incidence of the disease is 15%.The pathophysiological mechanism of varicocele is thought to be that blood flow occlusion in the spermatic vein or hypofunction of its valves causes the blood stasis,which makes the spermatic vein coiling and expansion.However,its pathomechanism is not clearly understood.Hypoxia and oxidant stress in epididymis and testicle induced by varicocele are considered as important reasons for asthenospermia.It has been reported that hypoxia inducible factor-la(HIF-la)protein remains stable under hypoxia environment.There is a significant positive correlation between its protein expression and the level of hypoxia,which means HIF-1 a can be used as an internal signal for tissue hypoxia.Reactive oxygen species(ROS)is a metabolite produced by oxidation-reduction reactions.The excessive production of ROS or the deficiency of antioxidant system for the protection of sperm leads to sperm dysfunctions.The tumor suppressor protein p53(p53),first found in tumor tissues,can regulate the cell cycle as a kind of apoptosis-inducing protein.The excessive expression of p53 will induce the apoptosis under some pathological process.ObjectiveIn this study,we observed the varicocele conditions in the rats and precisely weighted the body mass index(BMI)of epididymis and testicle of rats with varicocele,measured the sperm concentration and motility by computer assisted sperm analysis(CASA),observed the changes in epididymal epithelial tissue and seminiferous tubules by hematoxylin-easin staining(HE staining),measured sialic acid(SA),carnitine,ROS in epididymis and testicular tissues by enzyme-linked immono sorbent assay(ELISA),and tested the expressions of HIF-1α and p 53 in epididymis and testicular tissues by immunohistochemistry(IHC)and Western Blot.It would be used to research the relation between hypoxia marked by the increasing expression of HIF-la and the changes of epididymal epithelial tissue and seminiferous tubules and even the asthenospermia was investigated,so as the possible mechanism of asthenospermia induced by epididymis and testicular hypofunctions of rats with varicocele.MethodNinety male Sprague-Dawley(SD)rats weighed 250g to 280g were supplied by Department of Laboratory Animal Science of Peking University Health Science Center.All rats were allowed to acclimate for 1 week,and then randomly divided into the following 3 equal groups:surgery(Surgery),sham-operated(Sham)and control groups(Control).A model of varicocele was surgically established with 30 rats in surgery group using the method described by Turner.The rats were anesthetized and fixed.An abdominal midline incision was made,and the left renal vein behind peritoneum was separated.A rigid hydrophilic 0.8mm guide wire was placed on the left renal vein.The suture was tied around the vein over the top of the guide wire.The guide wire was then withdrawn and the vein was allowed to expand to the limits of the ligature.The midline incision was closed in two layers using 4/0 silk sutures without obvious ischemia in left kidney.The same procedure was performed on the 30 rats in sham-operated group,with sutures placed on the same position but not tied down.The 30 rats in control group were fed on a normal diet/without any procedure.All rats were sacrificed 49 days after the operation.The body weight,left epididymis and testicle were weighted and the left spermatic vein diameter was measured precisely by vernier caliper after laparotomy.The sperms in left cauda epididymis were collected by diffusion method.The left cauda epididymis tissues were cut into pieces and put into normal saline 2 ml in a 37℃ constant temperature water bath for 2 minutes(not too long).About lOul suspending liquid was dropped on preheated blood cell counting plate.The sperm concentration,the rate of sperm motility(progressive + non-progressive,PR+NP)and progressive sperms were measured by CASA according to the fifth version of the WHO laboratory manual for the examination and processing of human semen.The contents of SA,carnitine and ROS in the homogenate of left epididymis tissues were separately measured by ELISA.The contents of ROS in the homogenate of left testicular tissues were separately measured by dichlorofluorescin diacetate(DCFDA).The values mentioned above were tested by ELISA(BIO-RAD imark,USA).The Optical Density(O.D.)values of SA and carnitine were measured at the wavelength of 570nm.The O.D.values of ROS were measured at the wavelength of 510 nm.HE staining of epididymis and testicular tissues:Fixed with 4%paraformaldehyde,dehydrated sequentially with ethanol,the tissues were embedded in paraffin,and sliced to a thickness of 5 microns.Some slices were stained with hematoxylin and eosin,then dehydrated by ethanol,and made transparent with xylene.After that,they were placed on a neutral resin sheet and examined microscopically.The expressions of HIF-1α and p 53 in left epididymis and testicular tissues were performed by the streptavidin-peroxidase method.Briefly,after tissue antigen recovery,endogenous peroxidase was inactivated by incubating sections with 3%hydrogen peroxide.Sections were incubated with primary antibody overnight at 4℃.HIF-1α expression was assessed with mouse monoclonal to HIF-1α(Mouse monoclonal to HIF-1-alpha,Abcam,abl),(dilution 1:400)and p53 expression was assessed using Anti-p53(Mouse monoclonal to p53,Abcam,ab26),(dilution 1:1000).The samples were incubated in refrigerator at 4℃ for the night.Then,the Anti-mouse IgG(dilution 1:1000)were added with incubation for 30 minutes at 37℃ and then with streptavidin conjugated to horseradish peroxidase for 30 minutes at 37℃.Lastly the slices were colored with DAB/H202.After rinsing fully,the slices were re-stained with hematoxylin and processed in dehydration,transparency,drying and mounting.Five perspectives of the immunohistochemistry slices were selected with the high power microscope.The HIF-la and p53 expression were judged according to the proportion of positive cells and staining intensity to develop immunohistochemistry semiquantitative scoring standards:positive cells score:0 point:<1%;lpoint:1%to 10%;2 points:11%to 50%;3 points:51%to 80%;4 points:>80%.Staining intensity score:1 point:weak;2 points:medium;3 points:strong.The above 2 scores were added to make the final judgment result.The protein densities of epididymis and testicular tissues were measured by the BCA method.Protein SDS-PAGE electrophoresis(100μg,10%)was performed and the protein was transferred.The Anti-HIFla(Mouse monoclonal to HIF-1-alpha,Abeam,ab1)at 1:400 and Anti-p53(Mouse monoclonal to p53,Abeam,ab26)at 1:1000 and Anti-β-actin at 1:3000 were all incubated at 4℃ overnight.The PVDF membranes after the action mentioned above were waved and washed with TBST by 10 min ×3 times.And the secondary antibody was diluted in different proportion:Anti-mouse IgG at 1:1000 was incubated at room temperature for 90 minutes.The The membranes were waved and washed with TBST by 10 min ×3 times.The photograph of the protein bands was scanned and the gray level of protein band was analyzed by the Quantity one software.SPSS 17.0 software(IBM,Chicago,USA)was used for statistical analysis.All values of measurement data are expressed as the mean ± SE.The t test was applied in the intergroup comparison.The one-way ANOVA was used to compare the differences among the three groups.Correlation between ROS and p53 used linear regression analysis.And P-value<0.05 was considered statistically significant.ResultsThe symbol for the successful model of rats with varicocele is the left spermatic vein diameter is higher than 1 mm without significant difference in the weight between two kidneys.Successful models were created as follows:27 rats for surgery group(There were 3 rejected rats:one was nephrarctia on the left,the second one was orchiatrophy and epididymis atrophy on the left and the third one was nephrarctia,orchiatrophy and epididymis atrphy on the left.),30 for sham-operated group and 30 for control group.The left spermatic vein external diameters of the rats in surgery group were significantly larger than that in the other two groups(P=0.000<0.001),and there is no difference between sham-operated and control groups.There is no significant difference in body weight among three groups.The testis weight in surgery group was significantly decreasing than that in the other two groups(P=0.0012<0.01).Compared to the sham-operated and control groups,the testis mass index in surgery group was significantly lower(testis mass index=left testis weight/rat body weight×100%,P=0.001<0.01).By One-way ANOVA,there was no significant difference in sperm concentration in all three groups.And the percentage of progressively(PR)moving sperm and sperm motile rate(progressive + non-progressive,PR+NP)in surgery group were significantly lower than those in the other two groups(PR:P=0.000<0.001;PR+NP:P=0.046<0.05.There is no statistical difference in PR and sperm motile rate between sham-operated and control groups.By HE staining,the epididymal epithelial cells were larger in number and closer in arrangement in sham-operated and control groups.The epididymal epithelial cells were arranged loosely and many physalides appeared in surgery group.By immunohistochemistry staining,the positive HIF-la and p53 expressions in rat epididymis and testicular tissues were significantly higher in surgery group than those in the other two groups(HIF-1α:P=0.000<0.0001,p53:P=0.000<0.0001).There was no statistical difference in HIF-1 a or p53 between sham-operated and control group.By Western Blot,it’s higher in HIF-1α expressions in epididymis and testicular tissues in surgery group,compared to the other two groups(P=0.000<0.001),measured by gray specific value between HIF-1α and internal reference.It’s higher in p53 expression in epididymis and testicular tissues in surgery group,compared to the other two groups(P=0.000<0.001).There is no statistical difference HIF-1α and p53 expressions in rat epididymis and testicular tissues between sham-operated and control groups.By linear regression analysis,there is a significantly positive relationship between ROS content and relative expression level of p53 band in the left testicular tissues of surgery group(P=0.041<0.05,r=0.387).And the same trend is also observed in the left testicular tissues of the three groups(P:=0.000<0.001,r=0.618).Conclusions1.Varicocele induces hypoxia in epididymis and testicular tissues marked by the increasing expression of HIF-1α.2.Varicocele leads to the epididymis dysfunctions marked by the decreasing SA and carnitine.3.The accumulation of ROS in epididymis and testicular tissues induced by hypoxia improves the abnormal expressions of p53.4.The abnormally increasing expressions of p53 in epididymis and testicular tissues causes marked apoptosis,resulting in the dysfunctions of epididymis and testicle.The decreasing functions of epididymis and testicle further impact on the spermatogenesis and spermioteleosis,leading to the decreasing sperm motility.BackgroundWeak sperm disease is a common cause of male infertility.The etiology and pathogenesis of weak sperm disease is unclear.Asthenospermia mainly reduced to sperm motility and motor ability.Previous known cause of asthenospermia concluded:infection,abnormal semen,varicocele,endocrine disorders,autoimmune abnormalities and sperm flagellar structure defect.But these are environmental factors causing asthenospermia.With the in-depth study to sperm as the object,founding that ion transmembrane transport plays an important role in the physiological activities of sperm.The current study shows that,Ca2+ plays an important role in sperm motility,capacitation,hyperactivation,acrosome reaction and sperm egg binding process.A recent study found that,in the main section of the sperm flagellum and top body,a cation channel of a sperm specific(cation channel of sperm,CatSper)protein family,exercise capacity regulation of sperm mediated through Ca2+ flow,and play an important role in the process of hyperactivation and acrosome reaction.The expression or function problems of CatSper channel protein,may be one of the important pathogenesis of asthenospermia.The effect of carnitine drugs in the treatment of male infertility has been confirmed in many clinical studies.Carnitine has left-handed,right-handed isoforms.In mammals,only L-carnitine exists and plays an important physiological role.The animal model and clinical research confirmed that,L-carnitine has certain protective effect on spermatogenic impairment caused by varicocele,electromagnetic radiation and cryptorchidism,L-carnitine can enhance the vitality of the sperm and improve asthenospermia.ObjectiveIn this study we use the evidence-based medicine method:random,double blind,controlled.Applicate computer assisted sperm analysis system(computer assisted sperm analysis,CASA)to observe the effect of finished product of L-carnitine on sperm concentration and motility in patients with weak sperm disease.Using western blot test method to observe if spermatozoa CatSper channel protein of asthenospermia patients decreased compared with the normal population.If there is downward,whether L-carnitine drug treatment can upregulate the expression of CatSper channel,so as to strengthen exercise force,sperm hyperactivation ability,and becomes an effective method in the treatment of asthenospermia.MethodFrom 2011 June to 2012 April,we collected semen samples in Reproductive Medicine Center of the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine and No.3 Hospital of Peking University.According to the standard,we choose 40 normal semen samples based on newly diagnosed semen analysis;according to the inclusion and exclusion criteria,the initial screening of asthenospermia patients qualified in 120 cases,diagnosed asthenospermia by CASA semen analysis.120 patients were divided into 3 groups using the random,double blind and controlled method,respectively 40 cases in L-carnitine treatment group,40 patients in the placebo group,and blank control group with 40 cases.L-carnitine treatment group given levocarnitine oral solution,1.0g,bid,po.The placebo group received oral placebo,1.0g,bid,po.The control group was given life intervention for 1 months,not the medication.The three groups are in a month for a course of treatment.Semen samples were collected before and after treatment in each group.Patients collected semen with masturbation in sterile with cover take fine cup,and placed in a constant temperature of 37 ℃ box,observe if semen liquefied every 15min,semen liquefaction,peeled weighing,take semen liquefaction completely 10 ul to the blood cell counting plate(preheating).Using computer assisted sperm analysis system in human semen examination and treatment of "WHO" Laboratory Manual(Fifth Edition)for sperm concentration and sperm activity rate(forward movement + non forward motion,progressive + non-progressive,PR+NP)detection.Take 1,5ml semen after analysis,extraction and protein concentration was determined by BCA assay,protein SDS-PAGE electrophoresis(100 ug,10%)and protein transfer.After transfer using inhibition liquid to dilute the first antibody according to the following ratio:Anti-CatSper(H-300):1:100,4 degrees for the night;Anti-CatSper2(H-60):1:100,4 degrees for the night;Anti-CatSper3(M-201):1:100,4 degrees for the night;Anti-GAPDH:1,1000,1 hour at room temperature.Remove the PVDF film after anti reaction,TBST shake washing,10min*3 times.Using inhibition liquid to dilute the second antibody according to different proportion:Anti-mouse IgG,1:1000 dilution with inhibition liquid,incubation for 1 h in room temperature;Anti-rabbit IgG,using inhibiting liquid according to 1:1000 dilution,incubation for 1 h in room temperature.Two anti immersion after incubation,shaker membrane washing with TBST liquid,10min*3 times.Put the PVDF film in darkroom,tablet and develope fixing.Analysize protein bands of gray using Quantity one.SPSS 17.0 software(IBM,Chicago,USA)was used for statistical analysis.All values of measurement data are expressed as the mean ± SE.The t test was applied in the intergroup comparison.The one-way ANOVA was used to compare the differences among the three groups.And P-value<0.05 was considered statistically significant.ResultsThe age,average sterile time,abstinence days before treatment,sperm concentration,sperm activity ratio(PR+NP)and sperm forward movement(PR)indexes were not statistically significant(P>0.05)after statistical analysis in three groups of asthenospermia patients.The patients in the three groups after treatment were collected,37 cases of the control group(excluding blank lost follow-up of 2 cases,1 case with incomplete data),35 patients in the placebo group(excluding loss of follow-up in 3 cases,1 case of incomplete information,not prescribed medication in 1 case),L-carnitine group 34 cases(loss of follow-up in 3 cases,1 case with incomplete data,2 case not prescribed medication).By t test,the number of days of abstinence and sperm concentration in three groups were not statistically significant(P>0.05)before and after treatment.Based on one-way ANOVA test,there were no significant differences in abstinence days and sperm concentration among three groups of patients after the treatment(P>0.05).By t test,sperm progressive motility and sperm activity rate significantly increased in L-carnitine group,the difference was statistically significant(PR:P<0.0001,PR+NP:P<0.0001);according to the One-way ANOVA test,sperm progressive motility and sperm activity rate improved obviously in L-carnitine group than in the placebo group and blank control group after treatment,the difference was statistically significant(PR:P=0.0008<0.001,PR+NP:P<0.0001).Taking the ratio of CatSperl gray value and the corresponding reference gray value as the relative expression of CatSperl.Western blot results by One-way ANOVA test,shows that the asthenospermia sperm CatSperl protein decreased significantly compared with the normal group(P<0.0001).By t test,the expression of CatSperl protein increased significantly in L-carnitine group after treatment than before treatment,the difference was statistically significant(P<0.0001).Taking the ratio of CatSper2 gray value and the corresponding reference gray value as the relative expression of CatSper2.Western blot results by One-way ANOVA test,shows that the asthenospermia sperm CatSper2 protein decreased significantly compared with the normal group(P<0.0001).By t test,the expression of CatSper2 protein increased significantly in L-carnitine group after treatment than before treatment,the difference was statistically significant(P<0.0001).Taking the ratio of CatSper3 gray value and the corresponding reference gray value as the relative expression of CatSper3.Western blot results by One-way ANOVA test,shows that the asthenospermia sperm CatSper3 protein decreased significantly compared with the normal group(P<0.0001).By t test,the expression of CatSper3 protein increased significantly in L-carnitine group after treatment than before treatment,the difference was statistically significant(P<0.0001).Conclusions1.The expression or function obstacle of CatSper channel protein is one of the important pathogenesis of asthenospermia.2.Carnitine drugs treat asthenospermia through enhancing the expression of CatSper channel protein or improve the function of the channel,thereby enhancing the movement force,sperm hyperactivation and fertilization ability.