Study Of The Critical Genes In Male Germ Cell Differentiation From Chicken Embryonic Stem Cell

In the sexual reproduction of organisms,germ cells undertake the genetic information passed in generations and generate new individuals by sperm-egg binding.The characteristic of the germ cells make it become one of the research objects of animal embryology,developmental biology and cell biology.Male germ cells could be used to replace embryonic stem cells for disease therapy and genetic modification since it has the merits of pluripotency and do not involve in ethical and immune rejection problem,therefore,it caused great concern in the biological community.Until now that the molecular mechanisms during embryonic stem cells differentiation into germ cells is not fully confined,only found some signal gene or pawthway can regulate this process,do not have a whole understanding.Therefore,in this study,we choose three kinds of cells in this differentiation process:embryonic stem cells?ESCs?,primordial germ cells?PGCs?and spermatogonial stem cells?SSCs?,by two high-throughput methods Microarray and RNA-seq to detect the genes changes in three cells,in order to have a fully understanding of the gene trend s,to find the critical genes.Through this study will help us have a in-depth knowledge and understanding of the process of germ cells generation,also providing a theoretical basis and reference for future experiments.Meanwhile,the study of male germ cell differentiation will help us to establish the gene regulatory networks and provide the theoretical foundation for future studies.The results were as follows:1.The Microarray experiments of the three avian stem cells showed that there are 20,087 differentially expressed genes between ESCs and PGCs,of which 9,393 up-regulated and 10,694 down regulated while between ESCs and SSCs,there are total 20,020 differentially expressed genes?9209 raised and 10,811 decreased?,also our results revealed 17,090 differentially expressed genes between PGCs and SSCs?8611 raised and 8479 downward genes?.Microarray exists between the three types of cells to detect differences in these results as the number of different genes is reduced with increasing of the fold differences.The fold difference of approximately 57%of multiple genes is less than two;only about 0.1%is more than 10-fold difference.The GO results showed the gene difference in a biological process,mainly to the transcriptional regulation;differences in the composition of the cell nuclei of most genes;the combined molecules to the functional categories,including nucleic acid binding,metal ion binding,etc.2,Using of Illumina Solexa transcriptome sequencing of three avian stem cells transcriptome sequencing results showed a difference of 7,697 genes in male ESCs vs PGCs group,of which 4,187 genes were up-regulated in male PGCs,the 3510 genes were down-regulated in the male PGCs.In ESC svs SSCs group,a total of 7,968 differentially expressed genes,of which 4,944 genes up-regulated in male PGCs,the 2924 genes were down-regulated in the male PGCs.In male PGCs vs SSCs group,there are a total of 6,226 differentially expressed genes,of which 3,876 genes upregulated in male PGCs and 2350 genes were down-regulated in the male PGCs.GO analysis showed that after comparison of ESCs and male PGCs,a total of 7697 differentially expressed genes,of which there are 5,825 differentially expressed genes GO annotations to 40 entries.In the 40 entries,21 with biological processes,8 related to cellular components and the remaining 11 associated with molecular function.After ESCs and SSCs comparison,there were a total of 7968 differentially expressed genes,of which there are 5,968 differentially expressed genes GO annotations to 40 entries.In the 40 entries,21 with biological processes,eight were related to cellular components and the remaining 11 associated with molecular function.After male PGCs and SSCs comparison,a total of 6226 differentially expressed genes,of which there are 4,482 differentially expressed genes GO annotations to 37 entries.In the 37 entries,21 with biological processes,7 were related to cellular components and the remaining 9 were associated with molecular function.3.Preliminary screening of 173 candidate key genes such as GAL9,AMH,PLK1,PSMD7 etc.,revealed that they include continued 25 up-regulated genes,14 sustained down-regulated genes,18 ESCs specific genes,58 PGCs specific genes,16 SSCs specific genes,other 46 genes;later classification by function,it was found that there were 98 genes involved in cell differentiation,19 genes involved in the metabolic process,56 genes involved in the differentiation and common metabolic processes.The experimental data results found 115 unknown new genes?fold difference between cells more than 7 times?where 52unknown genes were specifically expressed in ESCs,19 were specifically expression in PGCs and 46 were specifically expressed in SSCs.4.Four unknown new genes which specifically expressed in PGCs?XM₀01233327,XM₄24100,NM₀01030786,XM₄16629?were selected for depth analysis.While the candidate key genes bioinformatics analysis showed that the selected four genes have a major high homology with birds,but also are unknown homologous proteins,genes are not named;candidate key genes domain analysis showed that,in addition to the predicted transmembrane transporter protein of XM₄24100,it was found 52 superfamily?transmembrane protein 52,TMEM52?extra-territorial structure,but the remaining three genes are not found in specific domains of gene function and so require further study.In summary,this study of transcriptome expression profiling and data analysis of chicken ESCs,PGCs and SSCs revealed 173candidate key genes were screened to regulate ESCs cell differentiation into male germ cells,115 new genes of unknown function and initial features of the four new genes were studied.The results will reveal the mystery of germ cell development provides an important reference for access to reproductive cells in vitro and to provide strong theoretical support.