| CHAPTER 1 The Expression of Sperm Specific Calcium Channel Cat Sper1 in Patients with VaricoceleObjective: To investigate the relationship between varicocele and sperm specific calcium channel(Cat Sper1)via examining the expression of Cat Sper1 in sperm of patients with varicocele,so as to provid a new research direction for the pathogenesis of varicocele induced infertility.Methods: 20 healthy volunteers and 110 patients with varicocele were included in this study.The semen was available by masturbation,and the semen parameters were measured by computer-assisted semen analysis(CASA).Additionally,optimized separation of semen using percoll discontinuous gradient centrifugation.Furthermore,the expression of Cat Sper1 m RNA and protein were measured by semi-quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)and Western Blot separately.Results: When compared with control group,the difference for semen volume and liquefaction time were not statistically significant in varicocele group(P>0.05).While the a+b-level motility of sperm and sperm viability in varicocele group were significantly decreased compared with control group(P<0.01).The expression of Cat Sper1 m RNA and protein in low motility(group3)was remarkably lower than that in normal motility group(group 2)and control group(group 1)(P<0.01).The expression of Cat Sper1 m RNA and protein in group 2 was slightly lower than group 1,but the difference between 2 groups was not statistically significant(P>0.05).There were no remarkably statistically difference in the expression of Cat Sper1 m RNA and protein among the 3 groups of Clinical degree I,II and III with varicocele.Likewise,the expression of Cat Sper1 m RNA and protein between the unilateral varicocele group and bilateral varicocele group was not significant difference.The expression of Cat Sper1 m RNA and protein in venous reflux group was remarkably lower than that in group without venous reflux and control group(P<0.01).However,there were no statistically difference in the expression of Cat Sper1 between the no venous reflux group and control group(P>0.05).Conclusion: The expression of Cat Sper1 in sperm of patients with varicocele decreased,which may be the secondary attack target of varicocele induced infertility.The low sperm vitality and venous blood reflux is associated with the low expression of Cat Sper1,which will offer a new method for the diagnosis and treatment of varicocele.CHAPTER 2 The Expression of CatSper1 In a Rat Model of Varicocele and The Protection of L-carnitine on ItObjective: To exam the expression of sperm specific calcium channel Cat Sper1 in epididymal sperm and observe the improvement of Cat Sper1 by L-carnitine on sperm in a rat model of varicocele.Methods: Seventy male rats were randomly divided into Seven groups(n =10 in each group): Control group(group A),Varicocele group(group B),Varicocele+normal saline group(group C),and Varicocele+small-,middle-,large-dose levocarnitine groups(group D,E,F).The experimental model of varicocele was established by partial ligation of left renal vein.Twelve weeks later,the animals in group C were gavaged with normal saline(1m L·kg-1·d-1),while the animals in groups D,E,F were respectively gavaged with levocarnitine(0.05,0.1,0.2 g·kg-1·d-1)for consecutive 35 days.group G were gavaged more 14 days at the same dose with group F.We measured the semen parameters by computer-assisted semen analysis(CASA)and Cat Sper1 in sperms by RT-PCR and Western Blot.Results: The percentage of sperm motility with grade a+b,sperm viability and sperm density were imparity lower in groups B than that in group A(P<0.01).The expression of Cat Sper1 m RNA and protein was remarkably lower than that in group A(1.44±0.67 VS 0.71±0.38;1.87±0.67 VS0.84±0.42,P<0.01).Compared with group C,the percentage of sperm motility with grade a+b,sperm viability and the expression of Cat Sper1 in sperms were significantly increased in group D,E,F and G,especially in group,F,G which increased more remarkably(P<0.01).But,there were no difference in sperm density among the groups above.There were no remarkably statistically difference in the expression of Cat Sper1 between the group G and F.Conclusion: The expression of Cat Sper1 decreased in a rat model of varicocele.levocarnitine might increase the sperm density,viability through and the expression of Cat Sper1,suggesting that levocarnitine might be beneficial to asthenozoo spermia caused by varicocele.CHAPTER 3 The Effects of Cat Sper1 on Mitochondrial Respiration and Energy Metabolism in SpermObjective: To determine the effects of Cat Sper1 on sperm mitochondrial respiratory and energy metabolism by detecting mitochondrial respiratory control rate(RCR)and ATP after the inhibition of Cat Sper1 by Cat Sper1 monoclonal antibodies.Methods: The semen were collected by masturbation from 20 healthy volunteers,and semen parameters was assessed according to the standard of WHO,and were available by masturbation.Each sample were divided into two groups(group A,B)after using a swim-up procedure.The sample in group A were incubated with Earles,while the sample in group B were incubated with anti Cat Sper1 monoclonal antibodies at a concentration of 50?g/ml.The semen parameters,Cells mitochondrial RCR and ATP content of sperm were detected at 1h,2h and 4h,respectively.Results: Compared with group A,the a+b-level motility of sperm was significantly decreased in group B at all time point(P<0.01),especially in a-level motility of sperm.The a-level motility of sperm significantly decreased at 1h,while the rate declined slowly at 2h and4 h,and there were no difference among 3 time point in group B(P>0.05).The state III,RCR and ATP content in sperm were significantly decreased in group B than that in group A(P<0.01).However,there were no difference in state III,RCR and ATP content in sperm among the time point of 1h,2h and 4h in group B(P>0.05).Conclusion: Inhibition of Cat Sper1 can decrease the respiratory function of cells mitochondrial and reduce the ability to generate ATP in sperm that make semen vigor decreased.Our finding might offer a new theory for varicocele induced the low expression of Cat Sper1 which leading to infertility. |
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