Regulatory Role Of MEI1 With Alternative Splicing Event On Spermatogenesis In Mongolian Horse

Mongolian horse is one of the most famous local breeds in China and even in the world.It has strong adaptability,cold resistance and crude feeding tolerance,and can adapt to harsh climate and extensive feeding conditions.In mammals,MEI1 is the first meiosis specific mutant gene identified by forward genetics,and it is highly expressed in human testis,and is related to the formation of homologous recombination in meiosis.Our previous study found that in the transcriptome sequencing study of Mongolian horse testicle tissue after sexual maturity,there are two kinds of variable splicing patterns of MEI1(Exon skip-ES and Mutually exclusive exons-MEX),indicating that the variable splicing pattern of MEI1 may be related to spermatogenesis.By exploring the regulation effect of MEI1 with ES event on spermatogenesis of Mongolian horses,the molecular regulation technology can be utilized to improve the sperm number and semen quality of Mongolian horses,which can not only effectively improve reproductive efficiency and reduce production costs,but also effectively accelerate genetic progress and promote population reproduction.In this study,testicular sertoli cells of Mongolian horses were used as research objects,and the recombinant plasmid MEI1-1-pIRES2-EGFP without ES event and the recombinant plasmid MEI1-2-pIRES2-dsred with ES event were constructed and transfected into testicular sertoli cells.The proliferation and activity of the transfected cells were detected by CCK-8,the transfected cells were screened by G418 antibiotics,the differential expression of meiosis related genes in cells was detected by q RT-PCR,and the regulatory effect of the variable splinter event of MEI1 gene on the spermatogenesis of Mongolian horse was investigated by RNA-seq and other experiments.The results are as follows:1.The vector of MEI1-1-pIRES2-EGFP and the vector of MEI1-2-pIRES2-dsred were successfully constructed,and the vector was successfully constructed by agarose gel electrophoresis and sequencing experiments,and the recombinant plasmid mass spectrum was plotted.2.Two recombinant plasmids,MEI1-1-pIRES2-EGFP and MEI1-2-pIRES2-dsred,were transfected into sertoli cells of Mongolian horses.The proliferation and activity of the transfected cells were detected by CCK-8 kit at 0 h,24 h,48 h and 72 h,respectively.The results showed that the cells transfected with the two plasmids had the highest cell activity and good proliferation at 48 h after transfection,and the cells transfected with MEI1-2-pIRES2-dsred had the highest cell activity.3.The differential expression of meiosis related genes in the two plasmid transfected cells was detected by q RT-PCR.The results showed that The results showed that the meiosis pathway gene(BUB1、 CUL1、 CCNB1、 PPP2R5C、CCNB2,et al)and part of the supporting cell signaling pathway regulating sperm meiosis(ARID4A、 FSH、 GDNF、 Stra8、 NRG1,et al)were highly expressed in the experimental group,and the differences were significant,indicating that MEI1 gene with ES event was more conducive to the regulation of germ cell meiosis and spermatogenesis.4.Cells with and without variable splicing were sequenced using RNA-Seq.The cells without variable splicing were used as the control group to screen the differentially expressed genes of the two types of cells and annotate their functions.A total of 191 differentially expressed genes were screened,including 25 up-regulated genes including MEI1 and 166 down-regulated genes.GO annotation differentially expressed genes were mostly concentrated in cellular components and molecular functional regions.KEGG annotation analysis screened 44 signal regulatory pathways,most of which were related to spermatogenesis.These results indicate that the variable splicing event of MEI1 can promote cell proliferation and up-regulate the expression of spermatogenesis related genes,and ultimately affect the spermatogenesis process.The results of this study laid a theoretical foundation for exploring the regulatory mechanism of variable shear events affecting spermatogenesis of Mongolian horses.