Preliminary Study On Screening Genes Related To Sperm DNA Damage Based On RNA-seq Data

Sperm quality is one of the determinants in male fertility.Adverse external factors and internal environment have been reported to cause sperm DNA damage,resulting in sperm quality decline,eventually leads to male infertility.Semen analysis is a critical means to evaluate sperm quality and male fertility.But due to the lack of sperm DNA integrity evaluation,the traditional semen analysis has been limited.Sperm DNA fragmentation index(DFI)detection plays an increasingly important role in reproductive medicine clinic and breeding stock production.However,the molecular mechanism of sperm DNA damage and its exact role in male fertility remains to be elucidated between sperm DFI and sperm-related parameters.To explore the effect of sperm DNA damage on male fertility,we performed human sperm and Landrace boar sperm as experimental materials to investigate the correlation between sperm DFI and sperm-related parameters.The sperm of human male and Landrace boar with different DFI were sequenced by transcriptome sequencing(RNA-seq)technology,and the genes related to sperm DNA damage were screened by bioinformatics analysis.The detailed research results are as follows:1.Correlation between sperm DNA damage and sperm related parametersIn order to explore the correlations between male sperm DNA damage and sperm related parameters,we divided sperm samples into high,medium and low DFI groups according to DFI.Next,we performed routine semen analysis,sperm morphology and ultrastructure evaluation and other methods,revealed that sperm DNA damage related to decreased sperm motility,increased deformity rate,ultrastructural abnormalities,the decline of mitochondrial membrane potential(MMP)level and rising of ROS level and apoptosis level.2.Screening differential genes related to DNA damage in human sperm by RNA-seqTo further explore the molecular mechanism of sperm DNA damage,we conducted RNA-seq on three groups of sperm samples respectively.Our results suggested that DNA damage could cause sperm gene expression profile changes in male sperm.We identified 10288 differentially expressed gene(DEGs)in high DFI group and low DFI group,including 4737 up-regulated and 5551down-regulated DEGs.Go analysis showed that the abnormal expression DEGs in biological processes such as spermatogenesis,sperm motility,reaction of ROS.KEGG analysis showed that the differential genes related to sperm DNA damage were mainly enriched in the signal pathways related to oxidative stress and spermatogenesis,including MAPK signal pathway,Rap1 signal pathway and PI3K-Akt signal pathway.3.Correlation among boar sperm DNA damage,sperm related parameters and litter sizeIn order to explore the relationship among boar sperm DNA damage,sperm related parameters and litter size,Landrace boars were divided into high and low reproductive performance groups according to litter size.Next,we performed routine semen analysis,sperm ultrastructure evaluation,DFI and ROS level detection on the samples.The results showed that DNA damage of Landrace boar sperm related to the decline of semen quality and sperm motility,as well as abnormal sperm and sperm ultrastructure damage,which led to the decline of litter size of mated sows.4.Screening differential genes related to DNA damage in boar sperm by RNA-seqIn order to explore the molecular mechanism of DNA damage in boar sperm with different reproductive performance,we use the transcriptome technology to sequence and analyze two groups of sperm.Sequencing results showed that boar sperm DNA damage would cause changes in sperm gene expression profile.1349 DEGs were identified between high reproductive performance(HRP)group and low reproductive performance(LRP)group,of which 163 genes were up-regulated and 1186 genes were down-regulated in LRP group compared with HRP group.The DEGs were mainly enriched in GO biological processes such as apoptosis and transcriptional regulation,and pathways related to oxidative stress and spermatogenesis,including PI3K-Akt signaling pathway,MAPK signaling pathway,and so on.In conclusion,our results demonstrated that sperm DNA damage related to decreased sperm motility,increased deformity rate,ultrastructural abnormalities,the decline of mitochondrial membrane potential(MMP)level and rising of ROS level and apoptosis level,and ultimately leads low male fertility.Among,sperm gene expression varies with DFI.In this study,we screened genes related to sperm DNA damage as biomarkers to predict male fertility,providing a reference for further investigation on sperm DNA damage mechanism.