Studies Of The Mechanisms Of Microcystin-LR-induced Apoptosis In Mouse Testis And Microcystin-LR Toxicity To Hypothalamus-pituitary-testis Axis

Microcystins(MCs)are secreted by freshwater cyanobacteria in the process of bloom,which have strong liver and kidney toxicity.In previous studies of our laboratory,it was discovered for the first time that MC-LR could significantly reduce serum testosterone level and induce spermatogenic dysfunction in male mice.Spermatogenic dysfunction seriously affects human health.Testosterone deficiency leads to many diseases,such as spermatogenic dysfunction,cardiovascular disease,diabetes and osteoporosis.Therefore,to explore the damage of MC-LR to male reproductive system,especially the mechanism of downregulation of testosterone induced by MC-LR,has great significance for assessing male health risks of MC-LR.In this thesis,the mechanism of MC-LR-induced apoptosis in mouse testis was studied.Then we assessed the toxic effect of MC-LR on hypothalamus-pituitary-testis axis,discovered the target cells of MC-LR in hypothalamus-pituitary-testis axis.Finally,we explored the cytotoxicity and effect of MC-LR to GnRH synthesis and secretion of its target cells:GnRH neurons.The thesis comprises of three parts:Part I Exploration of the mechanism of MC-LR-induced apoptosis in mouse testisObjectiveApoptosis was detected after acute exposure to MC-LR of mouse testis.The mechanism of MC-LR-induced apoptosis in mouse testis was explored.A model of MC-LR-exposed rat Ley dig cells was established to study the effects of MC-LR.Methods1.Male adult Balb/c mice were exposed to MC-LR(0,3.75,7.5,15 and 30μg/kg.b.w)by intraperitoneal injection for 1 and 4 days.Then mice were killed by cervical dislocation.Testicular tissue injuries were displayed by paraffin-section method.TUNEL staining was used to detect apoptosis of testes.2.Expressions of pp2Ay p53,Bcl-2,Bax,Cyt-c,c-jun,c-fos,c-mycs Caspase 3 and Caspase 8 were determined by RT-PCR.3.Protein level of PP2A/p-PP2A,p53/p-p53,Bcl-2/p-Bcl-2,Bax,Cyt-c and Caspase 3 were determined by Western Blot.4.After exposure to MC-LR(0,1,10,100,250,500,750 and 1000nM)for 48h,culture supernatant of Leydig cells was collected for the test of testosterone level.CCK-8 method was used to determine the viability of Leydig cells.FDA-PI staining was used to determine the survival rate of Leydig cells.Immunocytochemistry method was used to detect whether MC-LR could enter into Leydig cell.Results 1.After exposure to MC-LR for 1 day,the spermatogenic epithelium presented a slightly loosened appearance in its organization at 15 and 30μg/kg.b.w.After exposure to MC-LR for 4 days,spermatogenic epithelium also presented a slightly loosened appearance at 7.5μg/kg.b.w.Significant reduction of sperm was observed in seminiferous tubules at 30μg/kg.b.w.2.Tunel straning results showed a significant increase of apoptosis testicular cells along with the increasing dose and exposure time of MC-LR,suggesting that MC-LR induced testicular cells apoptosis with a dose-time depent characteristic in vivo.3.Results of RT-PCR showed that MC-LR significantly upregulated the expression of apoptosis related factors including Bax,caspase 3 and caspase 8 and proliferation related factors lincluding c-Myc,c-Jun and c-Fos.Western Blot results showed,MC-LR significantly upregulated the level of p-P53 and p-Bcl-2 but had no effect of the expression of Bcl-2.4.After exposure to MC-LR,results showed no significant changes in either the concentration of testosterone of cell supernatant or the viability&survival rate of Leidig cells.Immunocytochemistry results showed that MC-LR could not enter Leydig cells.Conclusion1.After entrance into the testis,MC-LR induced testicular cells apoptosis and structural damage,and then caused spermatogenic dysfunction.After entrance into target cells,MC-LR bound with its substrate PP2A,upregulated phosphorylation level of p53 and Bcl-2,via the Bax-Caspase pathway finally induced cell apoptosis.2.MC-LR had no cytotoxicity to Leydig cells and did not change the synthesis of testosteronePart Ⅱ Effect of MC-LR on hypothalamus-pituitary-testis axis and the target ObjectiveModels of MC-LR exposed Balb/c mice and SD rats were established to study the effects of MC-LR on hypothalamus-pituitary-testis axis and to assess the male endocrine toxicity of MC-LR.Distribution of MC-LR in hypothalamus and pituitary was determined.The target of MC-LR in hypothalamus-pituitary-testosterones axis was confirmed trough adding GnRH inhibitors or GuRH.Methods1.Male adult Balb/c mice were exposed to MC-LR(0,3,75,7.5,15 and 30 p.g/kg.b.w)by intraperitoneal injection for 1,4,7 and 14 days.Then the blood of mice was collected by removing an eye.Mice were killed by cervical dislocation.The weights before and after the experiment were detected.Serum LH,FSH and testosterone was determined.The expression of main factors included in hypothalamus-pituitary-testis axis like Kiss-1,GPR54,GnRH,GnRHr,LHβ,FSHβ,LHr and FSHr were tested by RT-PCR.2.Median lethal dose(LD50)of MC-LR to SD rats was determined.Based on the LD50,adult SD rats were exposed to MC-LR by continuous intraperitoneal injection with the dose of 30μg/kg.b.w for 1,3,5,7 and 14 days.Then the blood of mice was collected by removing an eye.Mice were killed by cervical dislocation.Radioimmunoassay method was used to test serum GnRH,LH,FSH and testosterone.TUNEL staining was used to detect the apoptosis of hypothalamus.3.Male adult SD rats were exposed to MC-LR(100 μg/kg.b.w)by intraperitoneal injection and killed by cervical dislocation before toxicity caused death.Western Blot was used to confirm the uptake of MC-LR into hypothalamus and pituitary.Immunofluorescence colocalization confirmed whether MC-LR could enter into GnRH neurons.4.GT1-7 cell line was cultured in vitro and was indentified by GPR54 and GnRH with immuncytochemistry method.GT1-7 cells were exposed to 1000nM MC-LR for 24h.Immunofluorescence staining was used to confirm whether MC-LR entered GT1-7 cells.5.Male adult SD rats were exposed to 0.313,0.625 andl.25mg/kg.b.w.cetrorelix by subcutaneous injection for 12h.Expression of LHβ and FSHβ and concentration of serum testosterone were detected.Best duration of cetrorelix was chosen.6.Based on the above experiments,male adult SD rats were exposed to 30μg/kg.b.w MC-LR for 5 days by intraperitoneal injection and supplementary 0.625 mg/kg.b.w.cetrorelix after the last time of MC-LR treatment.Male adult SD rats were exposed to 30μg/kg.b.w MC-LR for 14 days by intraperitoneal injection and supplementary GnRH after the last time of MC-LR treatment.All rats were killed and blood was collected.Serum LH,FSH and testosterone were tested to indentify whether the change of GnRH was the reason of the changes in LH,FSH and testosterone after MC-LR exposure.Results1.After exposure to MC-LR(0,3.75,7.5,15 and 30 μg/kg.b.w.)for 1,4 and 7 days,the weight of mice were not changed.But after 14 days treatment,the weight of mice at 30μg/kg.b.w significantly decreased.After 1 day injection,serum LH and testosterone increased at 30μg/kg.b.w;after 4 days injection,serum LH,FSH and testosterone increased at both 15 and 30μg/kg.b.w;after 7 days injection,serum LH and testosterone decreased a little;after 14 days injection,serum LH and testosterone decreased significantly in all MC-LR treated groups.RT-PCR results showed MC-LR did not change the expression of Kiss-1,GPR54,GnRHr,LHr or FSHr.Expressions of LHβ and FSHβ showed the same tendency of serum LH and FSH.But the expression of GnRH significantly decreased with the increasing concentration and duration of MC-LR exposure.2.After exposure to 30μg/kg.b.w MC-LR for 1,3 and 5 days,serum GnRH,LH,FSH and testosterone were all upregulated and up to the peak on the fifth day.After exposed to MC-LR for 7 and 14 days,serum GnRH,LH,FSH and testosterone were all downregulated quickly and shared the same tendency.TUNEL staining results showed no significant changes in the 1,3 and 5 days groups but significant changes in both 7 and 14 days groups.3.After exposure to 100μg/kg.b.w MC-LR,Western Blot results showed that MC-LR entered hypothalamus but not pituitary.Immunofluorescence staining the hypothalamus tissue sections showed the double positive cells of MC-LR and GnRH,suggesting MC-LR entered.GnRH neurons.4.Immunocytochemistry results showed GPR54 and GnRH double positive cells,suggesting the cells were GT1-7 cells and had the ability to synthesize GnRH.Immunocytochemistry results showed that MC-LR entered GT1-7 cells.5.By testing the expression of LHβ and FSHβ and concentration of seru:m testosterone,0.625mg/kg.b.w cetrorelix had the strongest inhibitory effect on LH,FSH and testosterone.6.After exposed to 30μg/kg.b.w.MC-LR for 5 days,supplementary cetrorelix treatment could recover the serum LH,FSH and testosterone.7.After exposed to 30μg/kg.b.w.MC-LR for 14 days,supplementary GnRH treatment could recover the serum LH,FSH and testosterone.ConclusionsThe target of MC-LR in hypothalamus-pituitary-testosterone axis was GnRH neurons.MC-LR caused the downregulation of mRNA level of GnRH and first upregulation followed by downregulation of serum GnRH,which further caused the synchronization of first increased and then decreased level of serum LH,FSH and testosterone.Part Ⅲ Molecular mechanism of the cytotoxicity of MC-LR to GT 1-7 cells and GnRH synthesis and releaseObjectiveModel of MC-LR exposed GT1-7 cells was established to study effects of MC-LR on GT1-7 cells,the molecular mechanism of the cytotoxicity effects of MC-LR on GT1-7 cells and the mechanism of the effects of MC-LR on GnRH synthesis and release in GT1-7 cells.Methods1.GT1-7 cells were exposed to 0,1,10,100,1000 and 10000nM MC-LR for 48h.CCK-8 method was used to test the viability of GT1-7 cells,LDH method was used to determine the membrane leakage rate of GT1-7 cells.TUNEL method was used to determine apoptotic cells.2.GT1-7 cells were exposed to1000nM MC-LR for 48h.Exposed cells were tested on Apoptosis Antibody Arrays to find out apoptosis proteins with significant changes in their expression level.Genes with positive results were verified using Q-PCR.3.GT1-7 cells were exposed to 0,1,10,100,1000 and 10000nM MC-LR for 48h.Cell culture medium was harvested within 2 hours,in which GnRH concentration was examined.GnRH mRNA level was determined using Q-PCR.cAMP and Calcium ion were tested by ELISA.Activity of AC was tested by ELISA.Expression levels of transcription factors regulating GnRH expression including Oct-1、Otx2、Pbx1α、Dlx2、Fos and Jun were examined by Q-PCR.Results1.In CCK-8 and LDH tests,we found that cytoactivity significantly decreased as MC-LR concentration increased.We found using TUNEL tests that MC-LR induced GT1-7 cell apoptosis significantly.2.Analyzing the Apoptosis Antibody Array results,we found the expression of some apoptosis related proteins including Bax and Bid were upregulated,while that of Bcl-2,Bcl-w,p53,IGF-2,IGFbp-2,IGFbp-4,IcIAP-2,sTNF-R1 and sTNF-R2 were downregulated.mRNA level of Bcl-2,p53,IGF-2,IGFbp-2 and IGFbp-4 were examined by Q-PCR and were found consistent with array result.3.After exposure of GT1-7 to MC-LR,we found as MC-LR concentration increased,GnRH level in the cell culture medium exhibited an increase-followed-by-decrease trend.Q-PCR indicated GnRH transcription level decreased significantly as MC-LR concentration increased.Intracellular cAMP and Calcium ion level increased significantly.The activity of AC increased significantly.The expression level of transcriptional activators Oct-1,Otx2,Pbx1a,Dlx2 significantly decreased,and that of transcriptional repressor Fos increased and Jun decreased.Conclusions1.MC-LR showed significant toxicity on GT1-7 cells.Apoptosis was induced by MC-LR through downregulation of Bcl-2,Bcl-w,p53,IGF-2,IGFbp-2,IGFbp-4,IcIAP-2,sTNF-R1 and sTNF-R2 and upregulation of Bax and Bid.2.MC-LR induced increase of intracellular cAMP and calcium ion,downregulated GnRH transcriptional activators and upregulated GnRH transcriptional repressors.3.MC-LR activated AC,which induced significant increase in cAMP and calcium ion.After that,as GnRH transcriptional level decreased dramatically,GnRH release increased shortly and then decreased eventually.