| Buffalo(Bubalus bubalis)is one of most important livestock breed in China.Buffalo show the better characters,such as crude feed tolerance,higher feed conversion rate and dairy nutritional value.Buffalo industry will contribute to great economics benefits.However,buffalo have lower reproductive rate.One reason why the low reproduction trait is that the poor quality of germinal cells development.Germinal cells maturation are complex biological processes.These complexity manifests not only genomic transcription and translation,but also post-translation modification and gene regulatory networks.Proteins are the direct executant of life functions,high-throughput proteomics study can yield the information of protein expression and post-translation modification in a large scale.Which contribute to uncover the protein-protein networks,and discover the key genes or biomarkers.So far,"omics" have been comprehensively researched in model mammalian.Nevertheless,proteomics seldom has been reported on buffalo species.In this thesis,proteomic study was carried out to explore the influencing factor of motility in buffalo sperm.The protein changes and mRNA variation during oocytes maturation were obtained and subsequent analyzed using multi-omics correlation.This study provided a new approach for buffalo reproductive physiology,which is the theoretical base for developmental mechanism of sperm and oocytes.The results were shown as follows:(i)proteome profiling of buffalo germinal cells(sperm and oocytes)and its microenvironment(seminal plasma and follicular fluid)were constructed and subsequently analyzed by bioinformatics tools.Using multi-dimensional protein identification technology(MudPIT),2,147,864,1,710 and 363 proteins were identified in sperm,seminal plasma,oocytes and follicular fluid,respectively.An open-access buffalo germinal cells proteome databases were established including the parameters of accession ID,protein name,molecular weight,isoelectric point and MS spectrums.The database providing value information for developmental biology.Secondly,bioinformatics analysis and data mining were carried out by Gene Ontology(GO)and KEGG.Base on cellular component,biological process and molecular function,the protein enrichment with specific terms were presented in sperm,oocytes,seminal plasma and follicular fluid.Considerable protein candidates involving in fertilization,meiosis,et al.,were defined.Quite a number of metabolism pathway in KEGG were discovered.In sperm and oocytes,more proteins participated in energy metabolism and anabolism.In seminal plasma and follicular fluid,complement cascade and proteosome pathways play a crucial role in microenvironment maintenance.Base on the protein function and relationship,global protein-protein interaction(PPI)networks were created.Some PPI events associated with germinal cells development were discovered.These results enriched the knowledge of buffalo proteome and functional gene,and provided the research foundation for illustrating the maturation mechanism of sperm and oocytes.(ii)Sperm motility is one of the key factors relate to fertilizing capacity.In order to explore the mechanism of sperm motility maintenance,we compared and validated the sperm protein variation and phosphorylation modification.The high-and low-motility sperm were separated by gradient density percoll centrifuge,after proteins extraction,TMT labeling combined with high resolution mass spectrometry were utilized to analysis the differential experssional proteins(DEPs).The results indicated that a total of 145 DEPs were found,52 proteins were up-regulated in low-motility sperm,while 93 proteins were down-regulated.The DEPs variation trend was declining.The up-regulated proteins were involved in morphogenesis and regulation of cell differentiation.The down-regulated proteins were involved in transport,oxidation-reduction,microtubule-based,sperm motility,regulation of cAMP metabolism and regulation of DNA methylation.The phosphoproteomics study revealed that 197 phosphoproteins,384 phosphopeptides and 441 phosphorylation sites were identified in high-motility sperm group.In the low-motility sperm group,178 phosphoproteins,361 phosphopeptides and 424 phosphorylation sites were confirmed.Phosphoproteins are universal in sperm,and which are associated with many biological events and signaling pathways,suggested that phosphorylation plays a key role in sperm function.The comparison of phosphopeptides indicated that 22 phosphorylation sites were different between high-and low-motility sperm.The modification sites derived from 9 proteins,including 4 structural protein,AKAP3,AKAP4,ODF1 and FSIP2.All the protein variation in quantitative and phosphorylation revealed that lower energy metabolism,insufficient protamine and dynein,tail structural proteins modification variation in low-motility sperm.Six proteins,PRAM1,SDC2,TEKT3,AKAP3,CCDC40 and IDH1,were validated by western blot assay and RT-PCR.The results of western blot consistent with the MS quantification.RT-PCR results confirmed that SDC2,TEKT3 and IDH1 proteins have barely correlation between mRNA level and protein level.These DEPs were mediated by translational regulation.(iii)The molecular mechanisms of in vitro oocyte maturation are not fully known.In order to acquire the mature oocytes changes in nuclear and cytoplasm,comparative proteomics was applied to immature and mature oocytes.Combined with the gene transcriptional data,a system biological correlation was analyzed.In comparative proteomic study,TMT labeling combined with mass spectrometry was utilized to screen the oocyte DEPs,compared with immature oocytes,a total of 62 proteins were defined as DEPs in mature oocytes,including 38 proteins up-regulation and 24 proteins down-regulation.The DEPs were involved in several biological processes,energy synthesis,RNA splicing and transport,and so on.The expression of HSP60 and GEMIN8 proteins were validated by western blot assay.In order to verify the related mRNA expression in transcriptional level,immature and mature oocytes were applied to RNA sequencing and were compared subsequently.The comparison rates on reference genome were 86.4%and 86.8%,respectively.Based on transcriptome,the comparison rates were 22.4%and 23.7%,respectively.The differential expressional genes(DEGs)were defined according to comparison of FPKM value.A total of 2,667 DEGs were found in mature oocytes,1659 DEGs were down-regulated while 972 DEGs were up-regulated in mature oocytes.COG function classification indicated most DEGs involved in transcription,replication and recombination,signal transduction mechanism,ribosomal structure,and posttranslational modification.The DEGs associated with some signaling pathways.The functional classification was different from of DEGs and DEPs.The transcriptome and proteome were compared by BLASTx,co-expressed unigenes were selected for correlation analysis.The r value is 0.49.This results indicated the low correlation in transcript level and translation level during oocytes maturation.A total of 27 proteins were correlated among 62 DEPs,the r value is 0.57.14 proteins were both significant differently expressed in mRNA and protein.In summary,high throughput proteomics and phosphoproteomics were utilized in the study of buffalo sperm motility,which will help to clarify the relationship of protein changes and sperm motility maintenance.The"multi-omics" approach were applied to oocytes development,to clarify the dynamic changes of mRNA and proteins during oocyte maturation.This conclusions are significant for discovering the mechanism of oocytes maturation and developing the IVF efficiency. |
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