Effect Of Transient Scrotal Hyperthermia On Spermatogenesis

Part ?.Effect of transient scrotal hyperthermia on spermatogenesis in men:a randomized clinical studyObjectives:In this prospective randomized clinical study,we aimed to evidence the reproductive impairment of frequent scrotal heat exposure,from the perspective of sperm parameters,sperm function,semen plasma biochemical markers,sperm DNA structure and protein levels,to evaluate whether different frequencies of heat exposure cause different degrees of damage to spermatogenesis.Methods:A total of 20 normozoospermic subjects were randomly divided into 2 groups to undergo testicular warming in a 43 ? water bath 10 times,for 30 min each time;the subjects in group 1 underwent testicular warming for 10 consecutive days and those in group 2 once every 3 days.Sperm parameters,total acrosin activity,epididymis and accessory sex gland function,sperm chromatin structure assay(SCSA),sperm mitochondrial membrane potential(MMP),apoptosis,and seminal plasma soluble Fas(sFas)were analyzed before treatment and every 2 weeks after,for a total of 10 times.In group 1,some critical proteins involved in heat stress,hypoxia,structure and function of sperm mitochondria and flagella were evaluated before hyperthermia and 2,6,10,and 16 weeks after hyperthermia.Results:We found an obvious reversible decrease in sperm concentration(p=0.005 for group 1 and p=0.008 for group 2 when the minimums were compared with baseline levels,the same below),motility(p=0.009 and 0.021,respectively),the hypoosmotic swelling test score(p=0.007 and 0.008,respectively),total acrosin activity(p=0.018 and 0.009,respectively),and an increase in the seminal plasma malondialdehyde concentration(p=0.005 and 0.017,respectively).The decrease of spenn concentration was greater for group 2 than for group 1(p=0.031).Both groups showed a reversible increase in the proportion of sperm with a disrupted MMP(both p<0.05 when the minimums were compared with baseline levels,the same below),sperm apoptosis(both p<0.01)and high DNA stainability(both p<0.05).The sFas concentration in both groups showed no obvious changes except one:the value at week 2 was significantly increased over baseline in group 1(p= 0.036).The level of Bcl-2 decreased significantly at week 6 and 10(p= 0.017 and 0.05,respectively)and recovered to baseline at week 16.Proteins involved in heat stress and mitochondria functions were up-regulated,whereas in flagella structure and function was down-regulated(all p<0.05).Conclusions:Transient scrotal hyperthermia seriously,but reversibly,negatively affected spermatogenesis,causing the decreases in sperm concentration,sperm motility,viability and acrosin activity,whereas no obvious effect was observed on reproductive hormone levels,epididymis and accessory sex gland function.Intermittent heat exposure more seriously suppresses spermatogenesis compared to consecutive heat exposure.Scrotal hyperthermia also caused sperm DNA damage and apoptosis,which were more severe in consecutive heat treatment group.And the apoptosis was mainly based on mitochondria dependent pathway.Additionally,transient scrotal hyperthermia induced a series of molecule response involving in heat shock,mitochondria function as well as the structure and function related to flagellum.Those proteins may be critical in the heat induced spermatogenic impairment.Part ?.iTRAQ-based proteomic analysis to identify differentially expressed proteins in rhesus monkey testis after transient scrotal hyperthermiaObjectives:To investigate the difference of protein expressions before and after scrotal hyperthermia in rhesus monkey testis.And to find protein targets which played critical roles in heat induced spermatogenic impairment.Methods:A total of eight male adult rhesus monkeys received scrotal hyperthermia for 30 min daily,6 consecutive days.Venous blood and tissues from testicular biopsies were collected 2 weeks before hyperthermia and once every 2 weeks after.Routine sperm parameters,reproductive hormones,testicular morphology and apoptosis were evaluated.Equal amount of proteins from testicular tissues collected before hyperthermia(day 0),day 8 and day 60 were pooled together separately for differential proteomics analysis.Bioinformatics analysis was performed to investigate the biological process,molecular function,cellular component as well as the potential signaling pathways of those differentially expressed proteins.Finally,some of those proteins were selected for further validation by using immunochemistry and western blot techniques.Results:Sperm concentration was 66.5±13.0×106/ml before hyperthermia and decreased obviously from day 15,and reached 0.5±0.3×106/ml and almost 0 at day 30 and 45,respectively(p<0.01 when compared with baseline).However,the value recovered to baseline level at day 60(81.0±20.2×106/ml).Progressive sperm motility showed a similar pattern of variation with sperm concentration.At day 8,morphology of seminiferous tubules became disrupted and apoptotic germ cell were much more than the baseline.A total of 105 differentially expressed proteins were identified at day 8 in comparison with day 0.Of which 36 proteins were up-regulated and 69 were down-regulated(Fold-change<0.67 or>1.5,p<0.05).The main biological functions of these proteins were nucleic acid binding and unfolded protein binding etc.Additionally,a total of 14 KEGG pathways were enriched and the main pathways were spliceosome,apoptosis,protein processing in endoplasmic reticulum,HIF-1 signaling pathway and MAPK signaling pathway etc.We also identified 26 differentially expressed proteins at day 60 when compared with day 0,of which 24 proteins were up-regulated and 2 were down-regulated.The main biological functions of these proteins were enzyme,protein and polysaccharide binding so as to play a regulative role.A total of 5 KEGG pathways were enriched and the main pathways were TGF-? signaling pathway,PPAR signaling pathway,Complement and coagulation cascades,cell adhesion and oxygen transport.The results of immunochemistry and western blot showed that CIRBP,PSIP1 and sam68 were all obviously up-regulated and Decorin was obviously down-regulated after scrotal hyperthermia,which was consistent with the proteomics results.Nevertheless,both LDHC and CLGN showed very low expression levels and the expression of LDHC was not obviously changed,and CLGN showed an increasing trend after hyperthermia,which was not fully consistent with the proteomics results.Conclusions:Transient scrotal hyperthermia caused severe and reversible spermatogenic impairment in rhesus monkeys.A serious of protein expression levels were changed during the early and recovery stages.Some of those differentially expressed proteins like CIRBP,PSIP1,sam68 as well as Decorin may play critical roles in heat induced spermatogenic impairment.Part ?.Regulative effect of TGF-? on CIRBP in the mouse testis after transient scrotal hyperthermiaObjectives:To evaluate the expression levels of TGF-? before and after scrotal hyperthermia,and to investigate whether heat stress induced down-regulation of CIRBP was mediated by TGF-?.Methods:This study was conducted by using in vivo and in vitro models.For the in vivo study,ICR mice were anesthetized and the testis were immersed into the 43?water bath for 30 min,and then the testis were collected 24 hours after.Expression levels of TGF-?1 TGF-?2 and TGF-?3 were evaluated by using real-time PCR and western blot techniques.Expression level of CIRBP was also tested 24 hours after single scrotal hyperthermia and the combination treatment of scrotal hyperthermia and TGF-?antagonist injection.For the in vitro study,GC2-spd cells were cultured with different types of TGF-?(all final concentrations were 10 ng/ml).Expression level of CIRBP was examined 48 hours after TGF-? treatment to evaluate the regulative effect of TGF-? on CIRBP.Results:The real-time PCR and western blot results showed that both TGF-?2 and TGF-?3 were up-regulated 24 hours after scrotal hyperthermia,when compared with control group(p<0.05),whereas the expression of TGF-?1 in heat treatment group was not significantly different from control group.Scrotal heat tress obviously decreased the expression level of CIRBP(p=0.003),and a small trend to rescue was observed after testis injection with 5 ?g LY2109761.However,a significant increase of CIRBP expression was achieved when injected with a higher does of LY2109761(10 ?g/testis),when compared with single heat treatment group(p=0.038).No significant difference was observed between the combination of heat and testis injection with 10 ?g LY2109761 and control group(p>0.05).Gene expression level of Cirbp significantly decreased in the GC2-spd cells 48 hours after cell heat stress(p<0.01).Never-theless,the levels also decreased significantly when cultured with TGF-?2 and TGF-?3(both p<0.01),but not TGF-?1(p=0.09).The down-regulation of Cirbp was most obvious in cells cultured with all those three TGF-?s whereas the difference was not statistically significant from single TGF-(3 treatment group.Conclusions:Scrotal hyperthermia induced the up-regulation of TGF-? in mouse testis,mainly TGF-?2 and TGF-?3,which played a negative effect of regulation on the expression of CIRBP in spermatocytes.Heat-induced down-regulation of Cirbp may be mediated by the up-regulation of TGF-?.Part ?.Effect of CIRBP down regulation on germ cell proliferation and apoptosis Objectives:To evaluate the effect of CIRBP down regulation on germ cell proliferation and apoptosis.Methods:In order to knock down the expression of CIRBP in germ cells,we used Cirbp gene targeted siRNA to transfect GC2-spd cells.CCK8 based cell proliferation assay was performed to evaluate the proliferation levels in GC2-spd cells 48 hours after transfection.Apoptosis was also detected by using Annexin-V and Pyridine iodide(PI)staining and analyzed with flow cytometry.Results:Three Cirbp gene targeted siRNAs and another negative control oligo were used for GC2-spd cell transient transfection.RT-PCR and western blot techniques were applied to evaluate the interfering effect 48 hours after transfection and the results showed a much better interfering efficiency of siCirbp-277(54.5%)than other siRNAs.Cell proliferation activity in siCirbp-277 group was significantly lower than that in normal control(p<0.001)group and siCirbp-NC group(p=0.001).The mean ratio of apoptosis was 11.7%in siCirbp-277 group and the value was 4.7-fold and 3.8-fold higher than that in normal control(p<0.001)and siCirbp-NC group(p<0.001),respectively.Conclusions:Cell proliferation activity was obviously supressed,and apoptosis was significantly induced by the down regulation of CIRBP in GC2-spd cells,which may play a critical role in heat induced spermatogenic impairment.