Based On The Support Of Cytoskeleton Research, The Effect And Mechanism Of Tonifying Kidney And Filling The Sperm To Treat Male Infertility And Oligozoospermia

The incidence of male infertility is continuously rising because of change of living environment and modern life style,the postponement of childbearing age.It is very difficult to identify the cause of male infertile for most of patients who only displays a reduced number of spermatozoa(oligospermia)and do not have obvious symptom.oligospermia is the leading cause of male infertile.Up to now,the pathogenesis of oligospermia is incompletely understood,the apoptosis of spermatogenic cells and sperm led to dyszoospermia.Traditional chinese medical holds that kidney-jing deficiency is the main pathogenesis of oligospermia and nourishing kidney-essence is oligospermia's basic treatment.It has been proved that tonifying kidney and replenishing essence rescription represented by Wuzi Yanzong can inhibit apoptosis of spennatogenic cells and spenn,while the injury of sertoli cell cytoskeleton may lead to apoptosis and injury of spermatogenic cells.The prework we did mainfested that,after co-culturede GTW and TM4 cells,fracture of sertoli cell cytoskeleton and down-expression of vimentin was found,while Wuzi Yanzong could improve the damagement of spermatogenic cells.So we hypothesis that"tonifying kidney and replenishing essence rescription represented by Wuzi Yanzong can improve spermatogenesis by repairing sertoli cell cytoskeleton and inhibiting apoptosis",to improve this idea we design the study:Part one:The clinical researchObjective:To explore effect and mechanism of tonifying kidney and essence method,represented by Wuzi Yanzong pill,as a treatment of oligospermia with kidney deficiency.Methods:This study did not set up control group on account of there is lack of the evidence-based drug confessed by the whole medical world and the advantages of Wuzi Yanzong pill had been demonstrated.Treatment period was 90 days,50 patients with oligospermia of kidney deficiency type were given Wuzi Yanzong liquids(10ml,2 times a day).spermatozoon quality were measured before and after treatment(the patients whose spermatozoon quality examined for twice in one week met the demands of oligospermia could be included and the better result of sperm count would be recorded),the apoptosis of sperm was detected by Western blot.After the start of research,patients should be follow-up every 30 days with TCM syndrome socre;at the same time adverse events,pregnant or not were recorded.Results:Medication during the observation period,4 patients'mate were pregnant.The whole number of sperm was obviously improved in 76.7%patients.Compared with before treatment,sperm concentration,total sperm number,total sperm motility,a grade sperm percentage,the percentages of progressive sperm were improved significantly,there was statistically significant(P<0.05);although semen volume has been improved,there was not statistically significant(P>0.05).As a indicator of sperm apoptosis,caspase3 expression was significantly higher than before treatment(P<0.05).The score of kidney essence deficiency was associated with the number and the time of follow-up,which showed a downward trend,there was statistically significant(P<0.01).Besides,the most syndrome score also showed a downward trend,there was statistically significant(P<0.01).According to the result of group statistics,the total sperm number of 20?29 age-group,30?39 age-group and 40?age-group(the oldest patient was 50 years old)were on the rise after treatment,there was statistically significant(P<0.05).The total sperm number of the normal weight group and the over weight group were on the rise after treatment,there was statistically significant(P<0.01).But compared with the total sperm number of the over weight group before and after treatment,there was not statistically significant.The total sperm number of the microrchidia group and the normal testicular volume group were on the rise after treatment,there was statistically significant(P<0.01).The total spem number of the normal percentage of forward motile sperm group and the asthenospermia group were on the rise after treatment,there was statistically significant(P<0.01).During the study,there was not adverse reaction report.Conclusions:The tonifying kidney and essence method which represented by Wuzi Yanzong pill could increase the main indices of the sperm quality and male patients' fertility,ease the symptoms of kidney essence deficiency.The mechanism of tonifying kidney and essence method improving sperm count may due to inhibit the apoptosis of sperm.Part two:Animal experimentsObjective:To investigate the effects and mechanisms of Wuzi Yanzong Pills(WYP)on improving sperm quality in rats with kidney essence deficiency.Methods:Five groups of male Sprague-Dawley rats including the control group,the model group,the low-dose WYP group,the medium-dose WYP group and the high-dose WYP group(5 in each group)were treated daily with vehicle,Tripterygium Wilfordil glycosides(GTW)either alone(20 mg/kg)or followed by WYP(0.5,1,or 2.0 g/kg daily)respectively for 30 days.The epididymal tissues were taken to assess the sperm count,the percentage of sperm forward movement and the sperm motility.Western Blot and immunhistochemistry were employed to detect the expressions of vimentin and a tubulin.Immunohistochemistry and Tunel method were used to detect the expression of PCNA and the apoptosis of spermatogenic cells respectively.Results:Compared with the model group,the sperm count,the percentage of sperm forward movement and the sperm motility of the medium-dose WYP group and the high-dose WYP group were significantly higher than those of the model group(P<0.01);The expression of vimentin in the testes of the high-dose WYP group was increased(P<0.05),and the expressions of a-tubulin in the testes of the WYP groups were increased(P<0.05);The expressions of vimentin and in the Sertoli cells of the WYP groups were significantly increased(P<0.01),and the expressions of ?-tubulin in the Sertoli cells of the WYP groups were significantly increased(P<0.01);The apoptotic rates of spermatogenic cells in the WYP groups were decreased(P<0.05);The expressions of PCNA in the spennatogenic cells of the WYP groups were improved,but the differences had no statistical significance(P>0.05).Conclusions:Wuzi Yanzong Pills can improve the quality of spermatozoa in the rats with kidney essence deficiency,possibly through repairing the cytoskeletal proteins in the Sertoli cells and inhibiting the apoptosis of spennatogenic cells.