Proteomic analysis of human sperm proteins in relation to sperm motility, morphology and energy metabolism

[摘要] ENGLISH ABSTRACT: Male infertility is often associated with impaired sperm motility and morphology(asthenoteratozoospermia) for which there is no specific therapeutic treatment. It hascome to light that the modification and expression of human sperm proteins play acrucial role in sperm function. In the present study, we present proteomic data of humanspermatozoa in the context of sperm dysfunction. Novel techniques have been used tosuccessfully isolate and identify differences in protein expression on a cellular levelassociated with asthenoteratozoospermia.In the first part of the study, differences in protein expression within the total spermproteome were investigated between immature and mature sperm populations. Semenwas collected from healthy donors (n=23) and separated into mature and immaturesperm populations by 3-layer Percoll gradient centrifugation. Cells were washed andmotility and morphology were measured by computer assisted sperm analysis (CASA).For the proteomic investigation cells were lysed and proteins separated by means oftwo-dimensional gel electrophoresis (2D electrophoresis). PD-Quest was used toidentify the differentially expressed proteins. The protein spots of interest were excisedand subjected to in-gel digestion. Peptides were separated by High Pressure LiquidChromatography (HPLC) analysis and amino acid sequences determined by massspectrophotometry. Proteins were identified by Mascot, using the Swiss Prot database.The results show that the motility (immature; 26.1±1.75% total motile cells vs. mature;60.93±3.24% total motile cells; p<0.001) and morphology parameters (immature;64.1±2.75% normal head morphology vs. mature; 87.63±3.24% normal headmorphology; p<0.001) of the two populations differed significantly. After 2Delectrophoresis, 16 differentially expressed protein spots were identified within the totalsperm proteome between the immature and mature sperm populations. 56% of thedifferentially expressed proteins were more abundant in the immature sperm populationcompared to the mature sperm population. Functions have been ascribed to theseproteins of which only four proteins, namely Tubulin-3C/D chain, Tubulin-2C chain,Outer dense fibre protein 2 and A-Kinase anchoring protein 4 precursor, were directlyrelated to sperm motility and morphology.In the second part of the study the expression of nuclear proteins in humanspermatozoa was investigated between immature and mature sperm populations.Semen was collected from healthy donors (n=156) and further separated from theseminal plasma by PureSperm® gradient centrifugation. The immature and maturesperm populations were retrieved and used during further analysis. For the proteomicanalysis of nuclear proteins, cells were fractionated into four different subcellular proteinfractions, instead of analyzing the whole sperm proteome. The results show that themotility (immature; 32.33±0.51% total motile cells vs. mature; 88.67±0.85% total motilecells; p<0.0001) and morphology parameters (immature; 13.51±0.87% normal headmorphology vs. mature; 20.89±1.20% normal head morphology; p<0.0001) of the twopopulations differ significantly. After 2D electrophoresis, 21 differentially expressednuclear proteins were identified between the immature and mature sperm populations.95% of the differentially expressed nuclear proteins were less abundant in the immaturepopulation compared to the mature population. Only one nuclear protein namely 78kDaGlucose regulated protein was more abundant in the immature population compared tothe mature population. Functions ascribed to these individual proteins were directlyrelated to sperm motility, morphology and energy metabolism.In conclusion,In conclusion, in the current study novel techniques have been employed to investigateprotein differences between immature and mature sperm populations. From theseresults it is evident that protein expression in the total sperm proteome and nuclearprotein fraction is significantly different and incomplete in the immature population,compared to mature population. Based on these findings, it is recommended that furtherstudies should be done on human spermatozoa to validate the role of the individualproteins in sperm function. Proteomics is an ideal tool to identify idiopathic causes ofmale infertility, as it can help to identify novel receptors (and signal transductionpathways) that can be used in the screening of drugs to alleviate sperm dysfunction.

[发布机构] Stellenbosch University