Low-dose Pb Exposure At The Onset Of Early Puberty Caused Spermatogenesis Impairment And The Intervention Effect Of Zn In Mice

Background Lead(Pb)is a toxic heavy metal that can be accumulated into the human body.Pb is considered to have no physiological function for human,and can cause toxicity effects to body even in trace amounts of exposure.Epidemiological studies showed that Pb could reduce semen quality and affect adult male fertility.Several animal studies also showed that Pb decreased sperm quality and fertilizing capacity.It was reported that the reproductive dysfunction that was observed in adult male animals might have originated from their early life stage,particularly at puberty.However,most of the animals started to expose to Pb at adulthood,but very few at puberty in current studies.Puberty is a key developmental period of mammalian testis,as well as is the onset period of spermatogenesis.Proliferation of Sertoli cells and germ cells are rapid at early and mid-term puberty,as well as the growth of testis.When coming to terminal puberty,cells proliferation become slow,and testicular volume is almost close to the size of that in early adulthood,thus the production of sperm do not increase with the ages.Therefore,pubertal testis may be more sensitive to Pb toxicity,which can continuously affect spermatogenesis even in low-dose exposure,and is only visible when coming to adulthood.The underlying mechanisms of spermatogenesis dysfunction in adult originating from low-dose Pb exposure at early puberty remain need to be disclosed.Here we established mice models with spermatogenesis dysfunction caused by low-dose Pb exposure for 1-3 months at the onset of early puberty.Pb exposure doses were selected by simulating the internal dose(blood Pb,[BPb])which were acceptable distribution ranges(BPb< 10μg/d L)suggested by health regulators for people with non-occupational exposure.RNA sequencing technology(RNA-Seq),trace element analysis,ultrastructural observation,oxidative stress and glycolysis metabolism analysis were used to explore the dose-effect and time-effect relationship,and possible molecular mechanism of spermatogenesis impairment caused by low-dose Pb exposure at the onset of early puberty.At the same time,we established a mice model of joint intervention of Zinc(Zn)and Pb,to explore the protective effect of Zn on spermatogenesis dysfunction induced by low-dose Pb.This study provides evidence to cognize the testicular toxicity caused by long-term low-dose Pb exposure beginning from early puberty,and to make health policy on controlling the environmental limit concentration of Pb and preventing male infertility caused by long-term low-dose Pb exposure by the health department.Part Ⅰ The effect of low-dose Pb exposure on gene expression profiling of testis in miceObjective To understand the gene expression profiling of spermatogenesis of testis in mice exposed to low-dose Pb at the onset of early puberty,and provide theoretical basis for the subsequent functional study.Methods After 7 days of acclimatization,ten ICR mice(21 days old)were randomly allocated into control group and Pb-treated group.Each group comprised five individuals.The mice were given distilled water ad libitum with 0 or 200 mg/L Pb2+ for 90 consecutive days.Fresh testis was quickly freezed with liquid nitrogen as soon as possible.Total RNA was extracted from the testis and was fragmented to synthesize c DNA library.The libraries were sequenced on an Illumina Hiseq 4000 platform.The differential m RNA expression profiling of testis between control and Pb-treated groups were analyzed by Ballgown suite.Gene Ontology(GO)and KEGG pathways enrichment of differentially expressed genes were analyzed by GOSeq-top GO-hmmscan(Release2.12)and KOBAS(v2.0),respectively.The m RNA expression of sixteen spermatogenesis-related genes were determinated by RT-q PCR,which were used to verify the RNA-Seq data using Pearson correlation analysis.Results(1)A total of 57825 m RNAs were identified.There were 355 m RNAs differentially expressed between the control and Pb-treated groups.Compared to the control group,199 m RNAs were up-regulated and 156 m RNAs were down-regulated in the Pb-treated group.(2)GO enrichment results showed that GO terms of up-regulated m RNAs involved cellular component(such as organelle and cytoskeleton),biological process(such as organelle assembly and morphogenesis)and molecular function(such as protein binding).GO terms of down-regulated m RNAs involved catabolic process of protein or triglyceride,morphogenesis and organelle assembly.(3)KEGG pathways enrichment results showed that pathways of upregulated m RNAs involved amino acid degradation,focal adhesion and estrogen signaling pathway,et al.Pathways of down-regulated m RNAs involved AMPK signaling pathway,glycolysis metabolism and fatty acid biosynthesis et al.(4)RT-q PCR results showed that m RNA expression of GLUT1,MCT4 and ZAG up-regulated significantly(P < 0.05),AMPK-α1,GLUT3,PFK1,Ldh-a,Ldh-c,CD147,Spata6 and Fabp9 down-regulated significantly(P < 0.05),MCT1,Meig1,Spatc1 l,Tnp2 and Septin12 did not change significantly(P > 0.05)in testis of Pb-treated group,compared to the control group.Pearson correlation analysis results showed that there was a reasonably high correlation between RT-q PCR and RNA-Seq data,with the determination coefficient R2=0.9526.Conclusion There had different m RNA expression profilings between Pb-treated testis and normal testis.Spermatogenesis dysfunction in the Pb-treated male mice was associated with the abnormal expression of key genes on the sperm morphogenesis,glycolysis metabolism and AMPK signaling pathway.Spermatogenesis dysfunction induced by low-dose Pb exposure at the onset of early puberty was a complex regulation process,which involved catabolic process,morphogenesis and organelle assembly.These results provided theoretical basis for the subsequent functional study.Part Ⅱ The dose-effect relationship between the low-dose Pb exposure and spermatogenesis impairment of testis in miceObjective To explore the dose-effect relationship and possible molecular mechanism of spermatogenesis impairment caused by low-dose Pb exposure at the onset of early puberty.Methods After 7 days of acclimatization,forty-five mice(21 days old)were randomly allocated into 0,50 and 200 mg/L Pb2+ groups.Each group comprised fifteen individuals.The mice were given distilled water ad libitum with 0,50 or 200 mg/L Pb2+ for 90 consecutive days.Concentrations of Pb and Zn in blood and testis were determined by atomic absorption spectrometry.Sperm quality(density,viability,morphology,and DNA fragmentation index [DFI])were analyzed by Neubauer hemocytometer,eosin–nigrosin smears,eosin smears and sperm chromatin structure assay,respectively.Ultramicroscopic morphology of sperms were observed by scanning electron microscopy(SEM).Ultrastructure of testicular cells were observed by transmission electron microscopy(TEM).Hematoxylin and eosin(HE)staining observed histopathological changes of testis and epididymis.The levels of oxidative stress and indexes of glucose metabolism in plasma and testis were detected by microplate reader.RTq PCR and Western blotting detected the expressions of target genes and proteins in testis and epididymis.Immunofluorescence detected the co-localization of MCT1-CD147 and MCT4-CD147 in testis.Results(1)There were no significant difference in body weight,water intake and food intake among the groups at the same exposure time point.Blood Pb(BPb)of 0,50 and 200 mg/L Pb groups were 0.70 ± 0.03,6.14 ± 0.34 and 11.92 ± 2.92 μg/d L,respectively,and those of Pbtreated groups were significantly higher than the control group(P < 0.05).Blood Zn(BZn)of 50 and 200 mg/L Pb groups were significantly lower than the control group(P < 0.05).(2)Compared to the control group,sperm density and viability were decreased while DFI was increased in Pb-treated groups with a dose-dependent manner.In 200 mg/L Pb group,the sperm abnormality rate was significantly higher than the control group(P < 0.05);the heads and headneck conjunctions of sperm were severly disrupted;the seminiferous tubules and interstitial structures of testis exhibited obviously degenerated and atrophy;the number of sperms were decreased markedly in the epididymis;TEM results showed that there had typical changes in testicular Sertoli cells and germ cells.(3)Compared to the control group,GSH,T-SOD and Cu/Zn-SOD of testis in 200 mg/L Pb group were decreased significantly(P < 0.05),while MDA were increased significantly(P < 0.05).(4)Blood glucose were increased significantly while testicular glucose were decreased significantly in Pb-treated groups with a dosedependent manner(P < 0.05).Testicular protein levels of GLUT1 and GLUT3 in Pb-treated groups were decreased in a dose-dependent manner.Testicular glucose,protein levels of GLUT1 and GLUT3 in 200 mg/L Pb group were decreased significantly compared to the control group(P < 0.05).(5)Testicular PFK1 and LDH activities,pyruvate and lactic acid content were decreased in a dose-dependent manner,and those of 200 mg/L Pb group were decreased significantly compared to the control group(P < 0.05).Also,m RNA expressions and protein levels of PFK1 and LDH of testis in 200 mg/L Pb group were decreased significantly compared to the control group(P < 0.05).(6)Testicular ATP concentrations,AMPKα1 m RNA expressions,P-AMPKα protein levels and P-AMPKα/T-AMPKα ratio were decreased in a dose-dependent manner and those of all the Pb-treated groups were significantly lower than the control group(P < 0.05).(7)The m RNA expressions and protein levels of MCT1 and MCT4 in testis of Pb-treated groups were increased significantly in a dose-dependent manner(P < 0.05).The m RNA expressions and protein levels of CD147 in testis were decreased in a dose-dependent manner,and those of 200 mg/L Pb groups were decreased significantly compared to control group(P < 0.05).The co-localization of MCT1-CD147 and MCT4-CD147 of testis in Pb-treated groups were inconsistent with those in the control group.(8)The m RNA expressions and protein levels of ZAG in testis and epididymis of Pb groups were increased significantly in a dose-dependent manner(P < 0.05).The m RNA expressions and protein levels of Spatc1 l,Meig1,Fabp9,Tnp2 and Spata6 in epididymis of Pb groups were decreased significantly while those of Sept12 were increased significantly in a dose-dependent manner(P < 0.05).The variation trends of Spatc1 l,Meig1,Fabp9,Tnp2,Spata6 and Sept12 in testis were similar to those in epididymis,but not as remarkable.Conclusion Exposure to low-dose Pb at the onset of early puberty reduced sperm quality,and damaged the structure and function of testicular cells,with a dose-dependent manner.Pb might inhibit AMPK activity of testicular cells and then inhibited the downstream glucose uptake,glycolysis metabolism and lactate transport,and resulted in energy metabolism disorder of germ cells.Furthermore,Pb could disorganized the expressions of spermatogenesis-related genes both in testis and in epididymis.All of above might be the mechanisms that caused testicular cell death and lowered sperm quality.Part Ⅲ The time-effect relationship between the low-dose Pb exposure and spermatogenesis impairment of testis in miceObjective To explore the time-effect relationship and possible molecular mechanism of spermatogenesis impairment caused by low-dose Pb exposure at different growth and development period of mice.Methods After 7 days of acclimatization,ninety mice(21 days old)were randomly allocated into two groups(0 and 200 mg/L Pb).Each group comprised forty-five individuals and they were averagely divided into three different subgroups in which mice were exposed to Pb for 30,60 or 90 days.The mice were given distilled water ad libitum with 0 or 200 mg/L Pb2+ for 30,60 or 90 consecutive days.Research indicators and detection methods were the same as described in Part Ⅱ.Results(1)BPb of 30,60 and 90 days Pb-treated groups were 7.78 ± 2.46,9.20 ± 1.55,and 11.92 ± 2.92 μg/d L,respectively.Pb concentration of testis in Pb-treated groups were increased significantly in a time-dependent manner(P < 0.05).Zn concentration in blood and testis of Pb-treated groups were decreased significantly in a time-dependent manner(P < 0.05).(2)Compared to the control groups at the same period,sperm density of 30 days Pb-treated groups were decreased significantly while sperm abnormality rate and DFI were increased significantly,and those of 60 and 90 days Pb-treated groups were more remarkable(P < 0.05).Slight abnormality on the sperm head of 30 days Pb-treated groups and moderate abnormality on the sperm head with shrinkable acrosomal cap of 60 days Pb-treated groups were observed while severe abnormality on the sperm head and head-neck conjunctions of 90 days Pb-treated groups were observed.Under optical microscopy,a slight damage was observed in the seminiferous tubules and epididymis of 30 days Pb-treated group,parts of cells with irregular arrangement and parts of sperms loss were observed in 60 days Pb-treated group,while plenty of cells with irregular arrangement and plenty of sperms loss were observed in 90 days Pbtreated group.TEM results showed that typical ultrastructure changes of testicular Sertoli cells and germ cells were observed in 60 and 90 days Pb-treated groups.(3)Compared to the control groups at the same period,testicular MDA was increased while GSH,T-SOD and Cu/Zn-SOD were decreased in 30 days Pb-treated group,and the changes were more remarkably in 60 and 90 days Pb-treated groups(P < 0.05).(4)Blood glucose were increased in Pb-treated groups with a time-dependent manner.Pb exposure for 30 days significantly increased testicular glucose,GLUT1 protein levels while Pb exposure for 60 and 90 days significantly decreased testicular glucose,GLUT3 protein levels(P < 0.05).(5)Pb exposure for 30 days significantly increased testicular pyruvate and PFK1 m RNA expression while Pb exposure for 60 and 90 days significantly decreased testicular PFK1 and LDH activity,pyruvate and lactic acid content(P < 0.05).Testicular m RNA expression and protein levels of PFK1 and LDH were decreased in Pb-treated groups with a time-dependent manner.(6)Testicular ATP concentrations,AMPKα1 m RNA expressions,P-AMPKα protein levels and P-AMPKα/T-AMPKα ratio were decreased in Pb-treated groups with a time-dependent manner,and those of 30 days Pb-treated groups were increased significantly while 60 and 90 days Pb-treated groups were decreased significantly compared to the controls(P < 0.05).(7)Testicular MCT1 and MCT4 protein levels in Pb-treated groups were increased significantly compared to the controls(P < 0.05).Testicular protein levels of CD147 in Pb-treated groups were decreased in a time-dependent manner,and that of 30 days Pb-treated groups were increased significantly while 60 and 90 days Pb-treated groups were decreased significantly compared to the controls(P < 0.05).The co-localization of MCT1-CD147 and MCT4-CD147 in testicular cells among 30,60 and 90 days Pb-treated groups were inconsistent.(8)The m RNA expressions of ZAG in testis and epididymis of Pb-treated groups were increased significantly in a time-dependent manner and protein levels of ZAG in 30,60 and 90 days Pb-treated groups were higher than their severally control groups(P < 0.05).Conclusion Exposure to low-dose Pb at early puberty reduced sperm quality,and damaged the structure and function of testicular cells,with a time-dependent manner.Pb induced slight oxidative stress,positively regulated glucose uptake,glycolysis metabolism and lactate transport,and caused unconspicuous spermatogenesis impairment at the early exposure period.Over time,Pb induced higher levels of oxidative stress,inhibited glucose uptake,glycolysis metabolism and lactate transport,and caused conspicuous spermatogenesis impairment.Part Ⅳ The intervention effect of Zn on the spermatogenesis impairment caused by lowdose Pb exposure in miceObjective To explore the protective effect of Zn on spermatogenesis impairment caused by low dose of Pb exposure at the onset of early puberty,and understand the possible mechanism that Zn antagonize the testicular toxicity caused by Pb.Methods After 7 days of acclimatization,forty-five mice(21 days old)were randomly allocated into control,Pb-treated and Zn-intervention groups.Each group comprised fifteen individuals.The mice were given distilled water ad libitum with 0 or 200 mg/L Pb2+,or 15 mg/L Zn2+ mixed with 200 mg/L Pb2+ for 90 consecutive days.Research indicators and detection methods were the same as described in Part Ⅱ.Results(1)Pb concentrations of blood and testis in Zn-intervention group were significantly lower than Pb-treated group(P < 0.05).Zn concentrations of blood and testis in Zn-intervention group were significantly higher than Pb-treated group(P < 0.05).(2)Sperm density and viability were increased significantly while sperm abnormality rate and DFI were decreased significantly in Zn-intervention group compared to Pb-treated group(P < 0.05).Znintervention alleviated the severe abnormality caused by Pb on the sperm heads and head-neck conjunctions.Zn-intervention alleviated the histopathological changes caused by Pb on seminiferous tubules and epididymis.Zn antagonized the structural damage on testicular Sertoli cells and germ cells caused by Pb.The sperm quality of Zn-intervention group did not return to the levels of the control group,though.(3)Testicular GSH level,T-SOD and Cu/ZnSOD activity of Zn-intervention group were increased significantly while MDA content was decreased significantly compared to Pb-treated group(P < 0.05).The same phenomenon was observed in in blood.(4)Blood glucose of Zn-intervention group were decreased significantly while testicular glucose and protein level of GLUT1 were increased significantly compared to Pb-treated group(P < 0.05).Zn intervention significantly increased testicular LDH activity,pyruvate and lactic acid content,m RNA expressions and protein levels of PFK1 and LDH(Ldh-a)compared to Pb-treated group(P < 0.05).Zn intervention significantly increased testicular ATP concentrations,AMPKα1 m RNA expressions,P-AMPKα protein levels and PAMPKα/T-AMPKα ratio compared to Pb-treated group(P < 0.05).Testicular m RNA expressions and protein levels of MCT1 and MCT4 in Zn-intervention group were decreased significantly,and those of CD147 were increased significantly compared to Pb-treated group,but still were lower than the control group(P < 0.05).The co-localization of MCT1-CD147 and MCT4-CD147 in testicular cells in Zn-intervention group were consistent with those in the control group.(5)The m RNA expressions and protein levels of ZAG in testis and epididymis in Zn-intervention group were significantly lower than Pb-treated group,but still were higher than the control group(P < 0.05).Conclusion Certain concentration of Zn alleviated injury on sperm quality caused by low-dose Pb,alleviated the inhibition on glucose uptake,glycolysis metabolism and lactate transport caused by low-dose Pb,and motivated AMPK and ZAG to regulate glucose transport and glycolysis metabolism,and then conduced to spermatogenesis.