Toxic Effects Of Chronic And Low-dose Exposure To Microcystin(-LR) On Male Reproductive System And The Mechanism Of Microcystin(-LR)-Induced Sertoli Cell Injury

Microcystins(MCs)are a family of naturally produced cyclicheptapeptide toxins produced by freshwater cyanobacteria.Among MCs,microcystin-LR(MC-LR)is the most prevalent and toxic class detected in natural blooms.The recent frequent outbreaks of cyanobacterial blooms have resulted in increasing numbers of lakes now facing the problem of MC pollution.The provisional guideline set by the World Health Organization(WHO)in 1998 for microcystin-LR(MC-LR)in drinking water is 1 ?g/L.Studies reported that the concentration of MCs in Lake Taihu in China was 15.6 ?g/L.MCs have been shown to invoke hepatotoxicity and neurotoxicity,kidney impairment and gastrointestinal disorders.Our previous studies about acute,sub-acute or chronic low-dose exposure to MC-LR were all demonstrated that they had toxic effect on male reproductive system in vivo in rat.It caused reduced testosterone levels,testicular atrophy,low sperm motility,and a high incidence of sperm abnormalities in a dose-time dependent manner.It is more likely that animals and humans ingest water that is contaminated with low dose of MC-LR every day.Based on these reasons,MC-LR was dissolved in the daily drinking water of mice in order to study the toxic effects of chronic and low-dose exposure to MCs on the male reproductive system.Our previous study proved that MC-LR caused apoptosis of cultured Sertoli cell.A recent study reported that autophagy,which is closely related to apoptosis induction,may be involved in the injury of a kidney cell line as a result of treatment by MC-LR.Therefore we chosed the autophagy and apoptosis in Sertoli cell to investigate the toxic mechanism of MC-LR in vitro.Part ? Effects of chronic and low-dose exposure to MC-LR on the male reproductionObjectiveTo study the toxic effects of chronic and low-dose exposure to MC-LR on the male reproductive system in vivo.Methods1.Male specific pathogen free(SPF)mice(n = 160)weighing from 15 to 25 g were randomly divided into two cohorts(3 months and 6 months).Each cohort had 4 groups:one control and three experimental with 20 mice in each group.In the experimental groups,mice were exposed orally at 1?g/L,3.2 ?g/L,and 10 ?g/L MC-LR for consecutive 3 and 6 months.The control animals received the same water only.After three-month and six-month treatment,blood samples were collected from carotid,respectively.2.The weight of body and drinking water was weighed.Serum testosterone(T),Luteinizing Hormone(LH)and Follicle-Stimulating Hormone(FSH)were measured by Radioimmunoassay(RIA).Sperm quality was checked.The paraffin sections of testis with H&E staining were prepared,and observed under light-microscopic.Testicular apoptosis was examined by TUNEL assay kit.Results1.After treatment with MC-LR for three months,no significant differences in body,testes or drinking water weights were noted between control and MC-LR treated mice.Testosterone levels declined at the dose of MC-LR(10?g/L),whereas serum FSH and LH levels showed a slightly increasing trend.Epididymal sperm qulity was significantly lower(P<0.05)in mice treated with MC-LR at the concentration of 3.2 or 10 ?g/L than in the control mice.In the group treated with 10 ?g/L MC-LR,the spermatogenic epithelium presented a slightly loosened appearance in its organization and the numbers of apoptotic cells increased slightly.2.After treatment with MC-LR for six months,no significant differences in body,testes or drinking water weights were noted between control and MC-LR treated mice.The level of testosterone was significantly decreased(P<0.01)in the 3.2 and 10 ?g/L MC-LR groups.At 10 ?g/L group,FSH levels increased substantially.The LH level increased dose-dependently in response to treatment with 3.2 and 10 ?g/L MC-LR.Epididymal sperm qulity was dramatically lower(P<0.01)in mice treated with MC-LR at the concentration of 3.2 or 10 ?g/L than in the control mice.The percentages of abnormal sperm showed a dose and time dependence with MC-LR dosage.Slight testicular atrophy in 3.2 ?g/L group was associated with sparse appearance of the seminiferous tubules.This effect was more pronounced in the 10 ?g/L group.In addition,the 10 ?/L group also showed loss and derangement of spermatogenic cells,enlargement of the lumen of the seminiferous tubules,thinning of the spermatogenic epithelium,as well as depopulation of Leydig cells,Sertoli cells,and mature sperm.The number of apoptotic cells was significantly increased at MC-LR doses of 3.2 and 10 ?g/L.Conclusion1.Chronic and low-dose exposure to MC-LR dose not have significantly effect on the basic physiological index of male mice.2.MC-LR inhibits the synthesis of T and decreases levels of serum T to affect spermatogenesis.3.MC-LR can destroy the structure of testicular tissue and cause apoptosis of testicular cells,then making spermatogenesis suffered serious damage.Part ? Microcystin-LR induces autophagy and apoptosis in rat Sertoli cell in vitroObjectiveTo investigate the toxic effect of MC-LR on Sertoli cellin vitro.Based on autophagy and apoptosis in Sertoli cell,to preliminarily study the possible mechanisms of MC-LR on male reproduction.Methods1.Testes were isolated from 28d old SD rats,and digested with 0.25%Trpsin and 0.1%collagenase respectively,and then the mixture of Sertoli cells and Spermatocytes were obtained.After 48 h culturing,cells were washed with PBS,cultured for 24 h,the Sertoli cell with high purity were got.2.Sertoli cells were divided into 5 groups:0?0.5?5?50 and 500 nM MC-LR group.3.The entering of MC-LR by Sertoli cell was detected by immunofluorescence and Western blot.4.We use CCK-8 to examine the toxic effect of MC-LR on Sertoli cell viability and LDH assay kit to measure the integrity of the Sertoli cell membrane.5.We use transmission electron microscopy(TEM)and immunofluorescence to detected the presence of autophagy.6.We use polymerase chain reaction(PCR),Western blot and immunofluorescence to examine the effect of MC-LR on the autophagy in Sertoli cell.7.We use Western blot and immunofluorescence to detect the expression of autophagy and apoptosis related gene and protein after treatment with MC-LR in Sertoli cell.Results1.The uptake of MC-LR was proved by immunofluorescent staining and Western blot in Sertoli cell.2?Viability of Sertoli cells exposed to MC-LR for 24 or 48 h was detected by CCK-8.Cell viability was significantly decreased in cells exposed to high MC-LR conditions(50-500 nM)(P<0.05).The release rate of lactate dehydrogenase was significantly increased(P<0.01)after high MC-LR exposure(50,500 nM)for 24 h or 48 h.3.After starvation treatment,there was increased punctate pattern of LC3(LC3-?)distributed in the cytoplasm and in perinuclear regions in Sertoli cells.The presences of autophagic vacuoles were proved by transmission electron microscope.4.With the exposure dose increase,the mRNA level of LC3 was first increased under mild concentrations of MC-LR(0.5-5 nM)and 50 nM for 24 h or 48 h and then decreased under 50-500 nM MC-LR compared with control.The protein levels and immunofluorescence of LC3-? were up-regulated with MC-LR dose increase,and reached its peak at 50 nM MC-LR for 48 h.Especially,50-500 nM MC-LR caused the mRNA level of negative regulator of autophagy p62 declined,protein level increased.5.Western blot and immunofluorescence results demonstrated the level of autophagy in Sertoli cell treated with MC-LR exceeded the control group,nutrient starvation group and autophagy activation group exceeded the control group,in addtion,the level of autophagy in Sertoli cell exceeded nutrient starvation group and autophagy activation group.After being treated with autophagy inhibitor(CQ)which could inhibit the fusion of autophagosome with lysosome,the level of autophagy in 50-500 nM MC-LR was between nutrient starvation group and autophagy activation group.Meanwhile,lysosome-associated membrane protein(LAMP-1)significantly decreased with the dose of MC-LR increase and the exposure time elongation.6.Cyt C and caspase 3 within Sertoli cell were significantly increased(P<0.01)after exposure to high MC-LR exposure conditions(50,500 nM).The Bcl-2 expression was significantly decreased(P<0.01)after high MC-LR exposure(50,500 nM)for 48 h,but the expression of Bax was not changed compared with control.After treatment with 3-MA for 48 h,no parameters that were correlated to autophagy were found to be modulated upon MC-LR treatment,and these includethe cell viability,the release rate of LDH,the mRNA and protein levels of LC3,and the protein expression of Bax,Bcl-2,Cyt C and caspase 3.No significant accumulation was observed followingtreatment with increasing doses of MC-LR byimmunofluorescent staining.Conclusion1.MC-LR could enter Sertoli cell and caused the cell viability decreased as well as the integrity of membrane damaged.2.Autophagy was present in Sertoli cell.3.Autophagy was induced by low dose of MC-LR in Sertoli cell.Exposure to high dose of MC-LR(50-500 nM)caused the function of lysosome damaged which was showed by the decrease of LAMP-1,and then autophagosomes within the cell were accumulated(showed by the accumulation of p62).4.The accumulated autophagosomes promote the apoptotic process via the decreased expression of Bcl-2.