| ObjectivesCaenorhabditis elegans(C.elegans)was used to establish the chronic damage model of Cadmium(Cd)exposure,to search for the sensitive index for evaluating the Cd exposure,and to explore the effect of Cd exposure on oogenesis.Transcriptome sequencing and bioinformatics analysis were used to screen genes and the underlying mechanisms of Cd reproductive toxicity were explored,which provided a theoretical basis for precise control and targeted treatment of Cd exposure.MethodsFirst,the effect of Cd exposure on the longevity of C.elegans was detected.The effects of Cd on growth and development,nervous system and reproductive capacity of C.elegans were examined using body length and width,frequency of head swing and body movement,number of offspring,instantaneous oviposition rate and number and state of fertilized eggs in uterus as observation indexes.Then the effects of Cd exposure on the specific process of oogenesis were investigated.The number of mitotic cells was used to evaluate the effects of Cd exposure on mitotic germ cells.For meiosis region,the number of cells and the morphology of nuclear were used to determine whether Cd affected the leptotene and zygotene stage.The effects of Cd exposure on pachytene were evaluated by the number of pachytene cells and the apoptosis level.The effects of Cd on diakinesis were evaluated by the number of diakinesis cells and the transverse diameter of-2 stage oocyte.Judging whether chromosomes were separated correctly in meiosis region after Cd exposure by the male rate of offspring.Using transcriptome sequencing of C.elegans,differential m RNA enrichment analysis and RT-q PCR,the target genes which may be related to Cd-induced reproductive damage were obtained,and its potential regulatory network was predicted by combining the competitive endogenous RNA(ce RNA)theory.The gene knockout mutant nematodes were also used to verify whether the target genes participated in the regulation of Cd-induced oogenesis injury.SPSS 21.0 software was used to analyze the statistical results,and the test level was α=0.05.Transcriptional sequencing results were analyzed by R 3.6.3 software.Results1.General toxicity of Cd to nematodes: The lifespan of nematodes decreased obviously after Cd exposure(10,30 and 60 μM).The locomotor ability of nematodes decreased obviously after 8d long-term exposure(P<0.05),suggesting that the nervous system of nematodes may be insensitive to Cd.Cd can obviously affect the growth and development of nematodes,and the obvious difference of nematodes’ bodyshape can be observed after short-term exposure for 2d(P<0.05).After Cd exposure,the number of offspring and fertilized eggs in uterus decreased,the reproductive peak and reproductive cycle prolonged and the number of late-stage fertilized eggs increased(P<0.05),indicating that Cd seriously damaged the reproductive capacity of nematodes.2.Cd had certain damages to C.elegans’ oogenesis: Cd reduced the number of total germ cells and the number of germ cells in mitosis and meiosis region(P<0.05),but the cell morphology did not change obviously.For meiosis cells,there was no significant difference in cell morphology and number of leptotene and zygotene after Cd exposure,suggesting that Cd may not affect the process of chromosome concentration and synapsis in leptotene and zygotene stage.The number of pachytene cells decreased,the level of apoptosis cells increased,and the number of diakinesis cells and the transverse diameter of-2 stage oocyte decreased significantly(P<0.05)after exposure,which indicated that Cd might affect the oogenesis of nematodes except for the leptotene and zygotene stage.3.Transcriptome sequencing and functional enrichment analysis to obtain reproductive genes: Transcriptome sequencing revealed the change of expression level of the whole transcriptome of nematodes,among which 1628 significantly up-regulated mRNA and 1539 significantly down-regulated m RNA were obtained(Fold Change > 1.5,P<0.05).The enrichment analysis of differentially expressed m RNA showed that the functions and pathways of down-regulating m RNA were mostly related to the genesis and development of oocytes.According to the results of enrichment analysis and the description of genes in wormbase database,the target genes mom-2,dsh-2,vang-1 and cul-6,which may be related to Cd-induced reproductive damage,were obtained.RT-q PCR results showed that the expression trend of the target genes after exposure was basically consistent with the sequencing results.ce RNA networks of target genes were finally constructed,which consisting of3 m RNA(except cul-6),4 mi RNA(mi R-1824-5p,mi R-2221,mi R-238-5p and mi R-243-5p),5 circ RNA and 26 lnc RNA.4.Study on the signal network related to Cd-induced reproductive impairment:Using mom-2,dsh-2,vang-1 and cul-6 gene mutant nematodes to observe the changes of mitosis,pachytene and diakinesis of meiosis after Cd exposure.There was no difference in the degree of cell reduction in mitotic region between the control and experimental groups of mom-2,dsh-2,vang-1 and cul-6 mutants after Cd exposure(P>0.05),which indicated that they probably did not participate in the regulation of cell proliferation damage in mitotic region caused by Cd.However,the reproductive apoptosis level of Wnt pathway related genes mom-2,dsh-2 and vang-1 mutated nematodes increased,and the number of cells in diakinesis phase decreased significantly(P<0.05).It is suggested that Cd may induce the germcell apoptosis by influencing Wnt signaling pathway,and then lead to reproductive toxicity.Conclusion1.The developmental and reproductive indexes of nematodes are sensitive indexes to evaluate Cd toxicity.2.Cd exposure may affect the oogenesis except leptotene and zygotene stage.3.The differentially expressed Wnt pathway key factors mom-2,dsh-2 and vang-1 obtained by transcriptome sequencing and RT-q PCR may be involved in negatively regulating Cd-induced germ cell apoptosis,and the ceRNA regulatory network has been preliminarily constructed. |
PDF Full Text Request