Study On Differential Expression Of Transcriptome And MiRNA Before And After Boar Sperm Capacitation

Sperm capacitation is one of the important processes of sperm maturation in mammals.Sperm must be incubated in the female reproductive tract or in vitro for a certain period of time,then sperm can be able to recognize and bind to zona pellucida of the oocyte.Then,the sperm is fused with the oocyte to form the zygote.Studies showed that many factors,such as heparin,serum albumin,calcium and bicarbonate,induce the loss of sperm plasma membrane cholesterol,increase of Ca²⁺ and cAMP concentration,and result in protein tyrosine phosphorylation,which are thought to be involved in a series of physiological and biochemical changes of sperm capacitation.In addition,miRNA and mRNA are also demonstrated to be involved in sperm capacitation.However,the molecular mechanism of sperm capacitation and its regulation are still unclear.Therefore,in this study,transcriptome and small RNA sequencing methods were used to compare the expression differences of miRNA and mRNA in fresh and capacitated boar sperm.The functions of miRNA and mRNA genes in fresh and capacitated sperm were analyzed by GO and KEGG.The results will provide important theoretical basis to understand the process of fertilization of sperm in vivo and in vitro.The main results are as follows:（1）Transcriptome sequencing was performed on the fresh and capacitated sperm using Illumina Hi Seq 2500 platform,we obtained 26,843,452 and 29,925,873 clean data respectively,and Q30 value was higher than 86%.5,342 mRNAs were found to be differentially expressed between the fresh and capacitated sperm.Among them,503 mRNAs were up-regulated and 4,839 mRNAs were down-regulated in capacitated sperm.GO and KEGG showed that 5,342 differentially expressed mRNAs were mainly involved in sperm capacitation-related pathways,such as Wnt,MAPK,PI3K-AKt,energy metabolism etc.（2）Small RNA sequencing was performed on the fresh and capacitated sperm using Illumina Hi Seq 2500 platform.We totally obtained 12,561,033 and 11,222,990 clean data respectively,and Q₃₀ value was higher than 86%.The sequencing results obtained204 miRNAs that were differentially expressed.Among them,there were 86 miRNAs and 118 miRNAs that were significantly up-regulated and significantly down-regulated,and 66 miRNAs were known,152 miRNAs were newly discovered.9 miRNAs were specifically expressed in the capacitated sperm,and 16 miRNAs were specifically expressed in fresh sperm.All differentially expressed miRNAs were analyzed with target genes prediction,GO and KEGG software package,and results indicate that these differentially expressed miRNAs were mainly involved in the process of cell process,protein binding and metabolic.（3）15 miRNAs（miR-34c、miR-1、miR-214、miR-374b、miR-1343、miR-127、miR-151-3p、miR-1285、miR-133a-3p、miR-101、miR-378、conservation＿3＿15893、conservation＿7＿221178、unconservation＿7＿234335、unconservation＿11＿42222）andtheir corresponding differentially expressed 20 mRNAs（ADPGK、PFKP、PGK2、PHKG2、CD36、PSPI、PDE4A、AKAP3、VDAC1、Cat Sper4、CABYB、ACRBP、COL11A1、HSPA2、CYP19A1、MYC、PRDX5、DNM1、ABCA1、CLU）are involved in capacitation-related pathways,such as PI3K-Akt,MAPK,cAMP-PKA and Calcium signaling pathways.