Validation Of Differentially Expressed Genes During Spermiogenesis In Mice

Spermatagonial stem cells eventually formed mature sperm cells through a series of complex and regular cell differentiation process. Many related studies have showed that RNA did existed in sperm and the expression of genes in sperm were also affected by the regulation of relevant transcription factors. A growing number of spermatogenesis related genes have been found, but the study about gene expression in whole stage of spermatogenesis have not been seen. Our lab previously extracted total RNA of round spermatid, elongating spermatid and mature sperm, constructed cDNA labrary and performed ultra-high-throughput RNA-sequencing. We got 12105 differentially expressed genes through the analysis of DEG-seq software(P﹤0.5).Due to the differential expression of genes are closely related to biological characters and function of tissue and cell, with the extensive study on the differentiation of tissue and organs and the growth and development of cells and individuals, the validation of differentially expressed genes are getting more and more important. However, the validation for the samples of difficult sampling like germ cell and less amount of RNA containing like sperm is still a great challenge. This experiment combined frozen section production and Laser Capture Microdissection, obtained about 5×103 round spermatid and elongating spermatid respectively. It solved the problem that the other methods(such as the density gradient centrifugation, etc.) can’t get high purity aim cells and the problem of difficult sampling. We successfully extracted good integrity and high purity RNA of aim cells.In order to verify the accuracy of high-throughput sequencing results, we use GO(Gene Ontology) terms and KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway to do the gene-enrichment analysis. We selected 10 differentially expressed genes which played an important role in spermatogenesis related pathways, the 10 Genes were Gabarap, Gkap1, Spaca4, Ndufb8, Atp5g1, Atp5 o, Hagh, Tfam, Mbd2, Tbpl1, respectively. We used fluorescence quantitative polymerase chain reaction(qPCR), beta actin as house keeping gene, to verify the RNA-seq results. The qPCR results showed that, the expression level of eight genes in round spermatid and elongating spermatid were higher than that in mature sperm(P<0.05), but expression level of the other two in mature sperm were higher than that in elongating spermid, but the difference was not significant(P≥0.05). The result indicated that the high-throughput sequencing result is reliable.