| Animals are inevitably suffered the starvation stress upon the seasonal changes and environmental fluctuations in nature or rearing condition.Especially in aquatics,starvation stress is common to fish due to the natural activities or the temporal and spatial fluctuations of food supplment in the environment.To meet this challenge,organisms mobilize a series of modulations with physiological and biochemical activities to maintain homeostasis and survival,and develop some adaptive phenotypic changes and intergenerational effects.Besides,the research of environmental epigenetics reveal that starvation stress could induce epigenetic changes,which exhibit modulations to phenotypic variations.DNA methylation as the best studied epigenetic and its role in fish physiological activities needs further investigation.In the present study,zebrafish(Danio rerio)were used to detect the responsive mechanism of starvation stress on fish reproduction,cell growth,autophagy,and DNA methylation with the physiological and biochemical examination,histological observation,locomotor activity analysis,transmission electron microscope observation,transcriptome and whole genome methylation analyses.The main results were as following:(1)After 3 weeks of starvation stress,the adult female zebrafish showed decreased condition factor,hepatosomatic index,and gonadosomatic index.The blood glucose level,blood triglyceride content,blood total cholesterol content,and the RNA/DNA ratios in tissues were reduced.The hepatic lipid storage was reduced with elevated gluconeogenesis activity.Besides,the follicle development was retarded and the ovarian fecundity was reduced,accompanied by upregulated autophagic genes expression,which is account for maintaining ovarian function and reproductive output.The global DNA methylation level was increased and the methylation levels of specific Cp G loci in the 5' flanking region of amh and cyp19a1 a were changed,which could affect the binding of transcription factors to their respective sites and the gene expression.(2)The starved female fish were mated with normal male fish to investigate the impairment of maternal starvation on offspring fitness.Results showed that maternal starvation decreased the embryonic spontaneous coiling rate,increased the heart rate,delayed the hatching time,and decreased the hatching rate.The larval malformation rate was enhanced,the body length and the survival rate was decreased.And the yolk sac ratio to body lateral area in the larvae were higher than control group,which is consistent with the increased egg size that might related to the reproductive strategy of female fish.Besides,the decreased locomotor activity was observed in larvae after maternal starvation with differential decreased total distance,velocity,acceleration,motility,and highly mobile time.The autophagic genes expression were downregulated in larvae that might contribute to the poor health and reduced the tolerance to environmental stress.(3)The zebrafish embryonic fibroblast(ZF4)cells were processed serum starvation for 48 h to detect the cellular changes under starvation stress.Results showed that starvation stress changed cell shape,decreased cell viability,induced cell death,suppressed cell proliferation and cell cycle.Autophagic activity was enhanced with increased autophagosome/autolysosome number,lysosomal activity,and autophagic genes and proteins expression at early-stage,while decreased at later-stage.The apoptosis was gradually increased with the prolonged starvation time.Besides,the cellular methyl metabolism was disrupted and the elevated global DNA methylation level,which were consistent with the autophagic activity that suggests the recyclable activity induced by autophagy increased the transmethylation activity could accelerate the methyl group metabolism and subsequently change the 5m C level in the zebrafish cells.(4)The ZF4 cells upon starvation stress of 12 h was used to find out the transcript and whole genome DNA methylation changes.Results showed that the differential expressed genes and differential methylated genes were overrepresented in the pathways of DNA replication,cell cycle,cell growth and death were linked to phenotypic variation.The methyl cytosine types and sequence preferences with DNA methylation trends and levels showed no significant changes between starved group and control group.The differential methylation regions exhibited dense in some chromosomes and enriched in the non-coding regions of intron and intergenic region.In an integrated analyses of transcriptional level and promoter methylation level,the negatively correlated hub genes of gins2,cdca5,fbxo5,slc29a2,suv39h1 b,and zgc:174160 were determined by the interaction networks and were predominant responsive to the nutrient condition changes.Besides,nutrient recovery and DNA methyltransferases inhibitor(5-Azacytosine,5-Aza-2'-deoxycytidine,RG-108)supplements partly rescued cell proliferation state with decrease of DNA methylation and reactivation of several depressed genes involved in cell proliferation pathways.In summary,present study systemically elaborated the impairments of starvation stress on zebrafish reproduction and intergenerational effects by multiple experimental technologies both in vivo and in vitro,and indicated the crucial role of autophagic activity in maintaining homeostasis,reproductive output,and environmental resistance.Besides,current study revealed the cellular changes of cell proliferation,autophagy,apoptosis,and methyl metabolism with their intrinsic correlations.The analyses of transcriptome and methylome showed the responsive effects of starvation stress on DNA methylation,which exhibited crucial role in the modulation of cellular stress responses. |
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