Differential Expression Of MRNA And LncRNA Between Fresh And Frozen-thawed Giant Panda Sperm

As an important part of ex-situ conservation strategy of wild species,sperm cryopreservation plays an important role in giant panda breeding and long-term preservation of genetic resources.Sperm cryoinjuries(structural and functional damages)lead to decreased sperm motility,viability and lower conception rate,which limits the use of artificial insemination with frozen-thawed giant panda sperm.However,the cryodamage or cryoinjury mechanism during cryopreservation is remain unknown.Numerous studies have revealed that mRNA and non-coding RNAs(miRNA and lncRNA)are usually involved in regulating spermatogenesis,apoptosis and regulation of sperm quality parameters.However,the roles of mRNA and lncRNA in the process of cryopreservation remain largely unexplored.Thus,the purpose of this study is to identify differentially expressed mRNAs and lncRNAs associated with cryodamage or freeze tolerance in the frozen-thawed sperm.The results are as follows:(1)The results of lncRNA sequencing of giant panda fresh and frozen-thawed sperm showed that 61.05 Gb of Clean reads were obtained;22,774 lncRNAs were detected,including 1086 antisense strand lncRNAs,4369 intron lncRNA,1209 sense strand lncRNAs and 16,110 intergenic lncRNAs.A total of 32,322 mRNAs were detected by mRNA sequencing.(2)2873 differentially expressed lncRNAs were detected in frozen-thawed sperm compared with fresh sperm.Of which 1477 are up-regulated,and 1396 are down-regulated.A total of 5,226 significantly differentially expressed mRNAs were detected,of which 3,581 mRNAs were significantly up-regulated and 1,645 significantly down-regulated in frozen-thawed sperm.(3)GO analysis of the cis target genes of differentially expressed lncRNAs revealed 202 significantly enriched GO terms and 10 KEGG pathways;GO analysis of trans target genes of lncRNAs revealed 233 significantly enriched GO terms and 6 significantly enriched KEGG pathways.GO and KEGG analysis of 5226 differentially expressed mRNAs revealed 365 significantly enriched GO terms and 14 significantly enriched pathways.Differentially expressed mRNAs and lncRNAs are mainly associated with cell membrane-related terms,and are distributed mostly on the olfactory factor transduction pathway.(4)There were 432 differentially expressed lncRNAs and their differentially expressed target mRNAs.13 lncRNAs and 11 target mRNAs were found to be involved in sperm fertilization,spermatogenesis and sperm capacitation;22 differentially expressed mRNAs were involved in sperm cell apoptosis(8 anti-apoptosis and 14 pro-apoptosis).In conclusions,our work is the first,systematic dissection of lncRNA and mRNA profiling in fresh and frozen-thawed giant panda sperm.Numerous differentially expressed mRNAs and lncRNAs were identified and focused on membrane,olfactory transduction signaling pathway and thought to be associated with membrane cryodamages,metabolism and apoptosis.Our results will provide significant insights into the mechanism of sperm cryodamage and freeze tolerance.