A Preliminary Study On The Zona Pellucida Function Of Dissosticus Mawsoni

Dissostichus mawsoni,the Antarctic toothfish,the species of cold icefish that inhabit the Southern Ocean in subzero degree water of-1.9°C,also the eggs are produced in seawater with low temperature.The involvement in antifreeze glycoproteins（AFGPs）is an important part of mechanism to adapt to low temperature environment.Without protection for AFGPs,then how can it was able to survive in freezing temperature? The zona pellucida protein（ZP）is a glycoproteins that recognize sperm-egg bindings.Studies have shown that the Antarctic fish,subfamily Notothenioids,ZP has been extensively expanded and may be associated with Antarctic fish in extreme cold waters.Previous studies have shown that ZPC5（zona pellucida c5,ZPC5）is expressed extensively in the Antarctic fish ovaries,but its specific function is unclear.The previous study also found that the ovary of the Dissostichus mawsoni contains three kinds of ZPC5 and the length of protein encoding gradually shortened.These three kinds of ZPC5 are DmZPC5₁（zona pellucida protein ZPC5 isoform 1）,DmZPC5₂（zona pellucida protein ZPC5 isoform 2）,DmZPC5₃（zona pellucida protein ZPC5 isoform 3）.In order to clarify whether ZPC5 is involved in mechanism of cold resistance to the low temperature,here,we attempted the overexpression of ZPC5 in Chinese Hamster Ovary（CHO）cells,and then treated CHO cells in the low temperature,by monitoring the survival rate of CHO cells,thus we revealed the function of ZPC5 in fish.Our data suggested that ZPC5 may be involved in the cold resistance process of the Antarctic notothenioid fish towards cold environment.The detailed results are summarized as follows:1)The sequencing results in recombinant plasmid showed that the coding regions of Dm ZPC5₁,DmZPC5₂,DmZPC5₃ genes were 1629 bp,1482bp and 1389 bp,respectively.The number of amino acids encoded were 543,494,and 463,respectively.2.The results of double digestion of recombinant plasmid showed that DmZPC5₁,DmZPC5₂ and DmZPC5₃ had two corresponding electrophoresis bands,which indicated that the target genes were inserted into the p IRES2-EGFP vector correctly.3)The Western Blot analysis showed that the Dm ZPC5₁,DmZPC5₂ and DmZPC5₃ were highly expressed in CHO cells and the expression of DmZPC5 was significantly higher than that of 37 ℃,which proved that the stability of DmZPC5 at low temperature was correlated with temperature,exogenous protein DmZPC5 was more stable at low temperature.4)The survival rate of CHO cells treated with 0℃ for 16 h or-2℃ for 4h and 8h was significantly higher than the mock（P<0.01）,which proved that ZPC5 improved the cold resistance of the cells.Moreover,DmZPC5₁,Dm ZPC5₂ and DmZPC5₃ had different effects on the survival rate of CHO cells.The survival rate of CHO cells transfected with Dm ZPC5₁ was the lowest,and the survival rate of CHO cells transfected with DmZPC5₃ was the highest.5)Native PAGE of the treated CHO cells in 0℃ showed that DmZPC5₁ protein polymers increased significantly,but no protein polymers DmZPC5₂,DmZPC5₃,the only protein monomer was significantly increased,this may be associated with the difference in survival of CHO cells at low temperatures..In conclusion,this study investigated the expression of DmZPC5₁,DmZPC5₂,DmZPC5₃ in CHO cells and the survival rate of CHO cells in low temperature stress.First,under low temperature,the expression of DmZPC5 protein in CHO cells was higher than 37 ℃,and it is possible that DmZPC5 proteins which was exogenous were more stable at low temperatures.up-regulated in CHO cells,the expression of DmZPC5 protein may have low temperature regulation;second,low temperature（-2℃）after the treatment,the survival rate of CHO cells increased significantly than the mock.The temperature of this low-temperature treatment is similar to condition of the Antarctic waters,which is a good basis of the study of the molecular mechanism of Antarctic fish’s low temperature adaptation.Third,DmZPC5₁,DmZPC5₂ and DmZPC5₃ had different protein characteristics at low temperature,which is a foundation for future studies of low temperature adaptation of Antarctic fish.