Studies On The Mechanism Of Male Fertility And Sterility Of Different Ploidy Cyprinids

In nature,polyploidization exists in various organisms,which is significant in the formation of new species and evolution.Researches on polyploids and polyploidization are very valuable,it is not only beneficial to study the important role of polyploidization in biological evolution,but also important for guiding the polyploid breeding in animals and plants.In the past years,we got F₃-F₂₄ allotetraploid fish?4n=200?by crossing the female red carp?Carassius auratus red var.??2 n=100?with male common carp?Cyprinus carpio??2 n=100?.Males and females tetraploid individuals are fertile.Then,we got sterile triploid fish by crossing the male tetraploid with female diploid red carp.The testis of tetraploid individuals?AT?develop normally and produce normal diploid sperm steadily,while the testis of triploid fish develop normally,but can not produce normal sperm.Germ cells of male triploid fish can develop to the stage of round spermatid,then degenerate.Sterile triploid and fertile tetraploid fish group are rare precious materials for the study of the impact which the change of polidy has on fish fertility mechanism.In addition,the artificial bisexual fertile tetraploid strain is obtained for the first time in fish,and may be the first in vertebrate,it is also very meaningful for study of polyploidization.In this thesis,we mainly use the RNA-seq and proteomics technology to perform whole transcriptome analysis of mature testis in three different polidy fish,respectively,and proteome analysis of common carp sperm,tetraploid fish sperm.Using different polidy fish mature testis transcriptome and the basis of selected differentially expressed genes in testis of different ploidy fish,we discussed molecular mechanism of the sterility in triploid male fish and molecular mechanism of fertility in tetraploid male fish.Using 2-DE maps,we analyzed differentially expressed protein spots,in order to reveal the change of breeding characteristics in male tetraploid fish and the meaning of diploid sperm production.1.Transcriptome analysis of testes from three different ploidy CyprinidsWe obtained the testis transcriptomes for diploid,triploid,and tetraploid cyprinid fish and tested for the presence of differentially expressed genes in the testis.After filtering,we obtained 53,51,and 53million clean reads in the testis of diploid,triploid,and tetraploid fish,respectively.And a total of 71,006,94,968 and 95,276 transcripts were identified in the testis of diploid,triploid,and tetraploid fish.The annotation revealed that 60,562?85.29%?,78,165?82.31%?,and 77,402?81.24%?of the assembled transcripts in the diploid,triploid,and tetraploid transcriptomes,respectively,had at least one positive hit.The annotation and enrichment of the GO,COG,and KEGG pathways was similar among these three transcriptomes.Thus,although there was phenotypic variation of the testes of among these three groups,but,the range of expressed transcripts was limited,and the most important factor may be the differences in the expression level of partial genes.2.Analysis of differently expressed transcripts in testis from triploid fishAfter bioinformation analysis,a total of 2,779 and 1,591 transcripts were differentially expressed between triploid fish and diploid and tetraploid fish,respectively.Furthermore,450 transcripts were simultaneously downregulated and 179 transcripts were simultaneously upregulated in triploid compared with diploid and tetraploid fish,respectively.The functional analysis revealed that genes related to reproductive,developmental,locomotion processes and axoneme part were differently expressed in triploid compared with those in diploid and tetraploid fish.Pathway analysis indicated that variation of gene expression levels in protein synthesis-related pathways,oxidative phosphorylation,ubiquitin mediated proteolysis played a central role in affecting the sterility of male triploid fish.A series of genes?DNAHs,DNALs,IFTs,and DNAAFs?associated with sperm flagellar assembly and motility,and testis-specific candidate markers?Tcte1,Tekt1,Tsga10,and Spef1?had lower expression levels in the testis of triploid fish.We validate the veracity of these differently expressed genes in triploid fish by the qPCR of five representative genes?DNAH1,DNAH3,DNAH7,DNAH9l,DNAH10?.Furthermore,19 putative transcription factors;one chromatin remodeling factor;and eight transcription co-factors exhibited differential expression in triploid compared with diploid and tetraploid fish.This study provides insight into the regulatory mechanism regarding the sterility in male triploid fish.3.Analysis of differently expressed transcripts in testes between diploid and tetraploid fishSimilarly,bioinformation analysis shows that 663 transcripts were upregulated and 1449 transcripts were downregulated in tetraploid compared with that diploid fish.Among these differentially expressed transcripts,partial genes are enriched in axoneme?GO:0005930?and axoneme part?GO:0044447?,which are indicated to be associated with the formation of sperm flagellum.Other partial genes are enriched in reproduction?GO:0000003?,sexual reproduction?GO:0019953?,reproductive process?GO:0022414?,cell motion?GO:000692?,microtubule-basedprocess?GO:0007017?,cellproliferation?GO:0008283?,actin filament-based process?GO:0030029?,microtubule organizing center attachment site organization?GO:0034994?,gamete generation?GO:0007276?,cell motility?GO:0048870?,which are indicated to be associated with the reproduction and spermatogenesis.In addition,partial differentially expressed genes are enriched in“Oxidative phosphorylation”,“Cell cycle”,“Pyrimidine metabolism”,“mRNA surveillance pathway”,“Oocyte meiosis”,and“Progesterone-mediated oocyte maturation”.Either we analyse focusing on single genes,or systematic pathways,we all could conclude that the expressed level of some pivotal genes which take part in spermatogenesis changed during the spermatogenesis in tetraploid fish.We should do further researches to verify the founction of these differentially expressed genes during the spermatogenesis in tetraploid fish.4.The detection of DNA content and sperm motility of diploid spermAfter the measurement by flow cytometry,the mean DNA content of diploid spermatozoa was double of the control haploid spermatozoa from common carp.And whether actived in distilled water or in activation solution,the rapid motility time and overall duration of motility of diploid spermatozoa was significantly longer than that of haploid spermatozoa.And these verified the variation of the ability of diploid sperm motility.5.The composition variations between diploid and haploid spermatozoaproteinUsing two-dimensional electrophoresis and LC-MS/MS,21 protein spots which in abundance were analyzed.As common carp or tetraploid fish was not a fully sequenced model organism,we firstly idenified proteins by MASCOT search against protein database of danio rerio in NCBI,and then verified the veracity referring to the predicted homologous proteins derived from transcriptome of testis in common carp and tetraploid fish.Of these,20 spots were successfully identified.And these identified proteins primarily belonged to four categories:cytoskeletal,energy metabolism,ubiquitin-proteasome system and other functions,indicating these three processes may be crucial in causing variation of diploid sperm.Overall,it is the first time to provide us new perspective to investigate the reproductive characteristics of male polyploidy.But future work will be required to explore the influencing mechanism of each protein in diploid sperm.In the same time,our attempt to use predicted amino acid sequences for protein identification indicates that transcriptome data is useful for proteomic analyses in non-model organisms.In conclusion,functional analyses of differentially expressed transcripts in testis from triploid fish reveals that the molecular mechanism of male sterility in triploid fish was strongly related to the disruption of gene expression during spermiogenesis.Specifically,genes which are associated with flagellar assembly and motility and some testis-specific genes were disrupted and directly caused the abnormal formation of sperm flagellar.In this paper,we also investigated the variation of protein composition between diploid sperm and haploid sperm.We found that the higher level of cytoskeletal proteins,energy metabolism proteins and lower level of ubiquitin-proteasome proteins in diploid sperm may be the indicators for longer motility of diploid sperm.