| The global climate became warming and increased temperature was concerned in recent years.The warming climate leads to the changed habitat,the recession of animal's physiological function,the small of individual size and the short of life times.For the environment-dependent sex determination,temperature is the key factor for the sex ratio shifts of fish,amphibian and reptiles.In studies,the rised temperature could affects on the sex ratio of poikilotherm absolutely.And the temperature-sensitive period is crucial for the sex ratio shifts.The temperature-sensitive period in zebrafish is unknown,and the mechanism of temperature-dependent sex shifts is unclear.Related researches indicated that the sex-related genes expression and the produce of gonadal hormone lead to the sex ratio shifts.Few studies focused on the epigenetic mechanism involved in the sex reversal and changed sex ratio.Most of these studies are refer to DNA methylation and several genes modification by epigenetic or the up regulation of DNA methyltransferase.However,the specific sites of DNA methylation are confused.To investigate the questions described above,we selected zebrafish(Danio rerio)as experiment objective and exposed them with different temperature(28~oC,23~oC and 33~oC)for a 50days period(10-60 days post fertilization).After then,we detected the sex ratio.For further investigation of the temperature-sensitive period,zebrafish were exposed with different period(10-60,10-20,20-30,30-40 and 40-50 days)with different temperature.With this period,we investigated the expression of several sex related genes at different temperature.Moreover,we treated zebrafish with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine(5-AZA)at different temperature and then measured the expression of DNA methyltransferase and sex related genes.Finally,we detected promoter methylation status used methylation-specific PCR(MSP),and studied the genes expression that promoter methylation changed including esr1 and sox9b.In addition,we detected methylation status of esr1 and sox9b promoter with bisulfite sequencing PCR(BSP).At the same time,5-AZA and high temperature combined treatment group was set up to investigate the function of DNA methylation in temperature induced masculinization.The result showed that,the normal temperature(28~oC)lead to the sex ratio with 1:1 in zebrafish,while the feminization was produced with the sex ratio 7:3 in the lower temperature(23~oC)and masculinize with sex ratio 3:7 in the higher temperature(33~oC).Further,we found the20-30 days of post fertilization is the thermal sensitive period.With this thermal sensitive period,the expression of female related genes including cyp19a1a(Cytochrome P450 family 19 subfamily A member 1a),foxl2(Forkhead box L2),esr1(Estrogen receptor 1)and sox9b(Sex determining region Y-box 9b)were increased and the expression of male related genes such as dmrt1(Doublesex and mab-3 related transcription factor 1),amh(Anti-Müllerian Hormone),ar(Androgen receptor)and sox9a(Sex determining region Y-box 9a)were decreased in the lower temperature(23~oC)group,while in the higher temperature(33~oC)group,male related genes(dmrt1,amh,ar and sox9a)were up regulated and female related genes(cyp19a1a,foxl2,esr1and sox9b)were down regulated.Besides,the expression of heat shock protein hsp90 and hspb1were also arised by real-time qualification PCR.Combined with the DNA methylation inhibitor5-AZA,we found that the expression of dnmt3a2,dnmt3b4,esr1 and sox9b were down regulated compare with the only higher temperature(33~oC)treatment.The 5-AZA had significant negative effect on the expression of temperature-induced sex related genes and methyltransferase,which indicates DNA methylation plays a role in temperature-dependent sex ratio shifts.Moreover,we also found that higher temperature enhanced the promoter methylation of esr1 gene(chr20:26459670-26460005)and sox9b gene(chr3:62343318-62343367)to inhibit its transcription and masculinize the zebrafish group in higher temperature(33~oC)with MSP.The results from BSP showed that higher temperature(33~oC)enhanced promoter hypermethylation of esr1 and sox9b genes and 5-AZA recovery the methylation status.Besides,we predicted the combined transcription factor in the islands of esr1 and sox9b promoter and we found that the key transcription factors are CREB(c AMP-responsive element binding proteins),GATA(GATA binding factors),SP1(GC-Box factors SP1/GC),FOX(Forkhead box),NF?B(Nuclear factor kappa B/c-rel)and SORY(SOX/SRY-sex/testis determine and related HMG box factors).And the key methylation sits of esr1 are Cp G5(chr20:26459832),Cp G73(chr20:26459910).The sox9b'methylation sites that play the important role are Cp G8(chr3:62343326),Cp G34(chr3:62343352)and Cp G67(chr3:62343385).All in all,lower temperature(23~oC)has feminization in the zebrafish group and masculinization in the higher temperature(33~oC)treatment group.This occurred in the thermal sensitive period of 20-30 post days of fertilization.With this period,the temperature enhanced the promoter methylation to inhibit the transcription of sex genes and shift the sex ratio. |
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