Bioinformatic Analyses Of Zona Pellucida Genes In Vertebrates And Their Expression In Nile Tilapia

The zona pellucida（ZP）protein is a type of glycoprotein encoded by the ZP gene family,which constitutes the envelope of animal eggs and early embryos.This extracellular membrane plays an important role in structural and physiological function of living organisms,especially in the formation of the egg,fertilization,and the development of the embryo.More and more studies on the composition,formation process and physiological functions of ZP proteins have been carried out based on the rapid development of molecular biology.However,with more and more ZP genes identified and cloned from different species,there is no unified nomenclature system of ZP gene until now.Genome-wide identification of ZP genes in vertebrates needs to be carried out.The expression of ZP genes in tetrapods is clear,being consistent in different lineages.Nevertheless,the number of ZP genes in fishes is quite large.Subsequently the expression profile of ZP genes in fishes is quite complicated and distinct expression profiles across different lineages have been reported.In this study,a genome-wide identification of vertebrate ZP genes was carried out,and a nomenclature system with better prevalence,generality,and parsimony was proposed for vertebrate ZP genes.Besides,expression patterns of ZP genes were analyzed based on the transcriptomic data of seven different tissues and gonads at different developmental stages.Moreover,RT-PCR was carried out to validate the expression of ZP genes in different adult tissues.The cellular location of ZP genes was observed using in situ hybridization（ISH）.Transcriptomic analysis of the dax1 and 42Sp50 homozygous mutant ovary was also conducted.To ascertain the impacts of estrogen on expression of ZP genes,transcriptomic analysis of sex reversed XY testis induced by 17β-estradiol（E₂）and XX ovary induced by fadrozole treatment was carried out.Simultaneously,we studied the expression of ZP genes in E₂ incubated XY adult liver and testis,as well as methyltestosterone（MT）and letrozole incubated XX adult liver and ovary,respectively.In conclusion,our study laid a solid foundation for further study of ZP gene function.The main results are as follows:1.Identification of ZP genes in vertebratesTotally 19 ZP genes were isolated from the genome of the Nile tilapia.16,9,17,27,21,20,26,14,11,24,17,9,18,8,11,9,8,5,and 4 ZP genes were identified fromthe sea lamprey,elephant shark,coelacanth,spotted gar,zebrafish,medaka,stickleback,Amazon molly,platyfish,seahorse,Northern snakehead,cavefish,tetraodon,western clawed frog,Chinese softshell turtle,chicken,platypus,kangaroo,and human,respectively.The number of ZP genes is related to the environment where the eggs are fertilized and the embryos develop.2.Phylogenetic analysis of vertebrate ZP genesThe amino acid sequences of ZP genes from coelacanth,zebrafish,Nile tilapia,western clawed frog,turtle,chicken,and human were used to construct phylogenetic tree of ZP proteins by NJ method.Based on phylogenetic analysis,the ZP genes of vertebrates can be divided into five subfamilies:ZPA,ZPB,ZPC,ZPD and ZPAX.ZPA exists in vertebrates except chondrichthyans and euteleosteans;ZPB and ZPC exist in all vertebrates;ZPD exists in vertebrates except cyclostomes,cavefish,marsupial and eutherians while ZPAX exists in vertebrates except eutherians.The ZP gene name were suggested follow the hierarchy of the phylogeny:subfamily first（uppercase letter）,then a first paralogue label（Arabian number）,then a second paralogue label（lowercase letter,when applicable）and finally a tandem label（dash plus Arabian number）.Tandem duplicates were distinguished by dash plus Arabian number,for example,ZPB2a-2.3.Expression of ZP genes in 7 different tissues of adult tilapiaIllumina RNA-seq data from brain,head kidney,heart,liver,ovary,testis,and kidney of adult tilapia indicated that 2 ZP genes（ZPB1a and ZPB2c）were expressed at background level,and 2（ZPB2b and ZPC2）were expressed highly and exclusively in the liver.The remaining 15 ZP genes were expressed dominantly in the ovary,with background expression in other 7 tissues.RT-PCR also revealed that ZPB2b and ZPC2were expressed highly in the liver of XX adult fish,while ZPAX1,ZPAX4,ZPC5-1,and ZPC5-2 were expressed highly in the ovary.4.Expression of ZP genes in tilapia gonads at different developmental stagesZPB1a,ZPB2b,ZPB2c,ZPC2,and ZPAX2 were not found to be expressed at any examined developmental stages.The other 14 ZP genes were expressed extremely low in both ovaries and testis at 5 dah,and began to express in ovaries at 30 dah.They were highly expressed in ovaries at 90 and 180 dah,with FPKM of each gene higher than 100.5.Validation of the ZP gene expression profile by ISHTo ascertain which population of cells in the ovary and liver expresses ZP genes,in situ hybridization（ISH）of ZPC2,ZPC5-2,and ZPAX1 was performed using tilapia livers,ovaries,and testes.ZPC2 was found to be expressed in the hepatocytes cells of the liver,while it was not observed in the ovary.ZPC5-2 was expressed in phase I and II oocytes,and ZPAX1 was expressed in phase I,II,and III oocytes.Consistent with transcriptomic data,ZPC5-2 and ZPAX1 were not expressed in the liver and testis.6.Expression of ZP genes in dax1 and 42Sp50 homozygous mutant ovaryTranscriptomic analysis of the dax1 and 42Sp50 mutant ovary revealed that at 30dah,the expression level of all ZP genes was lower in dax1^-/-ovary than that of the control.In contrast,at 180 dah,the expression level of all ZP genes was higher in the42Sp50^-/-ovary than that of the control.This is consistent with the number of phase I and II oocytes in the mutant and control ovary.7.Impacts of estrogen treatment on expression of ZP genesTranscriptomic analysis of the sex reversed XY ovary induced by E₂ revealed that the expression level of all ZP genes was higher in E₂ induced ovary than that of the control XY testis.Similarly,E₂ treatment significantly increased the expression level of ZP genes in the in vitro cultured adult male liver and testis compared with the control.On the other hand,the expression of all ZP genes were down-regulated in the fadrozole induced XX testes when compared with the control XX ovaries.Similarly,MT and letrozole treatment significantly decreased the expression level of ZP genes in the in vitro cultured female adult liver and ovary compared with the control,respectively.These results indicated that the expression of ZP genes can be induced by E₂.In summary,the ZP genes of vertebrates can be divided into five subfamilies,namely ZPA,ZPB,ZPC,ZPD,and ZPAX.The number of ZP genes in vertebrates generally decreased from lower to higher,from aquatic to terrestrial and from oviparous to viviparous.Totally 19 ZP genes were isolated from the genome of the Nile tilapia.Among them,no expression of ZPB1a and ZPB2c were detected in different tissues and gonads at different developmental stages,while two（ZPB2b and ZPC2）were expressed exclusively and highly in the liver.Other ZP genes were mainly expressed in the phase I and II oocytes of the ovaries.The expression of ZP genes,either in the liver or ovary,could be induced by estrogen.Taken together,these results present a thorough overview of ZP gene family and their expression profile,providing a new perspective on the evolution and function of this family in vertebrates.