| Objective:This study was performed to research the reproductive toxicity on BALB/c male mice infected with toxoplasma gondii:①To establish mice reproductive toxicity model by intraperitoneal injection of toxoplasma gondi tachyzoites;To monitor male mice's general condition:the weight of testis,epididymis,change of the mount and shape of sperm,definite the features of this model and what studies it can be done;②To detect the injury of cells after toxoplasma gondi infection,the effect of apoptosis proteins in this process and to find if there is selectivity of injury to cell of different ploid;③To detect how the hormone(serum T,LH,testis local T)and their receptors change after toxoplasma gondi infection and analysis the influence of toxoplasma gondii on the signal pathway.To explore the mechanism of male fertility descent caused by toxoplasma gondii,and provided the theory basis fo preventing and controlling strategy to male sterility.Methods:(1)To monitor male mice's general condition:the weight of testis,epididymis,the change of mount and shape of sperm:Take health BALB/cmale mice,divided into 6 groups in random,8 per group.A group:the normal control group(0.2ml PBS);B groups:the infected groups (B1-4group were injected 2.5×10~3,5×10~3,1×10~4,2×10~4 toxoplasma gondii tachyzoites individely);C group:cyclophosphamide control group(40 mg/kg).All were excuted 6 days after infection,take out and weight testis and epididymis;,prepared sperm suspension, smeared,stained with 1%eosin and Giemsa,then we observed sperm's shape and counted the sperm in epididymis and seminiferous tubule.(2)To detect apoptosis of all cells of different ploid after toxoplasma gondi infection:take 60 health BALB/c male mice,divided into 6 groups in random,10 per group.:A group:the normal control group(0.2ml PBS);B groups:the infected groups(B1-4group were injected 2.5×10~3,5×10~3,1×10~4,2×10~4 toxoplasma gondii tachyzoites individely);C group:cyclophosphamide control group(40 mg/kg).All mice were excuted after 6 days infected and take out testis:one was prepared as spermatogenic cell suspension,then was fixed by 70%ice alcohol,dyed and was detected used flow cytometry in the next day;another testis went through alcoholic dehydration and paraffin imbedding,then was sliced 5μm,which were used to do pathologic observation and detect apoptosis protein Bax,Bcl-2 using SP method. (3)To detect the hormone(serum T,LH,testis local T)change after toxoplasma gondi infection.Grouped just like what did above After infected for 6 days,getting their blood through picking eyeball,then getting blood serum through 8000rpm,4℃centrifugalization and being conserved in -20℃condition,the blood serum was used to detect serum T,LH;Besides,the testis were made homogenate and the be centrifuged to get the supernatant,which was used to detect T of local tissue of testis.The testis went through alcoholic dehydration and paraffin imbedding,then was sliced 5μm,which were used to do pathologic observation and detect apoptosis protein LHR,AR using sABC method.Results:(1)Compare to normal control group,the body weight of infected groups(B1-4)and cyclophosphamide group ligntened,but have no significant difference(P＞0.05);the testis weight of B1-B4 group and cyclophosphamide group decreased,compared to normal control P＜0.05,but the epididymis weight was no notable change;the mount of sperm of infected and cyclophosphamide group reduced and there was significant difference compared to normal group(P＞0.05);malformation rate and acrosome damage rate of sperms of infected and cyclophosphamide group increased a little,but no significant difference(P＞0.05).(2)In testis pathological section of infected(B1-4)mice,there existed condensation of nuclear and emerged of multinucleated giant cell,cell layers of seminiferous tubule confused, intercellular space broadened,the mount of sperms decreased or even vanished;detected through flow cytometry,the diploid cell of testis of infected mice existed apoptosis obviously,meanwhile tetraploid cell decreased notably(P＜0.01),haploid cell changed a little;immunohistochemistry detection:the expression of Bcl-2 had no significant difference between normal control group and B1-B4 groups(P＞0.05),while Bax expression enhanced significantly in B1-B4 groups (P＜0.01),especially in spermatocytes,while it changed little in spermatospore,spermatid, interstitial tissue of testis.(3)LH of B1-B4 groups mice decreased a little compared to normal control group,but was not significant difference(P＞0.05).LH level was lowest in 2.5×10~3group among all groups,but the level setted up along with the infected dose increased.LH of cyclophosphamide injected mice heightened slightly(P＞0.05);serum T of infected mice and cyclophosphamide B1-B4 groups mice decreased obviously,local T of testis decreased more notablely,compared to normal control group P＜0.01.The ecpression of LHR was no significant deviation among groups (A,B1-B4,C),while AR expression enhanced significantly in spermatospore.Conclusions:1.The model of toxoplasma gondii acute infection was fit to research on toxoplasma gondii 2.Txoplasma gondii acute infection caused dysgenesia was because the injury of testis,that leaded to spermatogenesis disturbance,aspermatism,then fertility descent.3.The apoptosis of spermatogenic cell induced by Infection of toxoplasma gondii was diploid cell mainly,while induce decrease of tetraploid cell;apoptosis may be related to enhanced expression of Bax,expecially for spermatocytes,while do little to Bcl-2.4.Infection of toxoplasma gondii could reduce serum T,testis tissue T notabely,besides influenced the feedback of LH and5.After toxoplasma gondii infection,AR expressed higher in spermatocytes to compensate the descent level of T,while there was no obvious influence to LHR of Leydig cell after txoplasma gondii infection. |
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