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

Rapuling, Llewelen (Stellenbosch : University of Stellenbosch, 2010-12)

Thesis

ENGLISH ABSTRACT: Male infertility is often associated with impaired sperm motility and morphology (asthenoteratozoospermia) for which there is no specific therapeutic treatment. It has come to light that the modification and expression of human sperm proteins play a crucial role in sperm function. In the present study, we present proteomic data of human spermatozoa in the context of sperm dysfunction. Novel techniques have been used to successfully isolate and identify differences in protein expression on a cellular level associated with asthenoteratozoospermia. In the first part of the study, differences in protein expression within the total sperm proteome were investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=23) and separated into mature and immature sperm populations by 3-layer Percoll gradient centrifugation. Cells were washed and motility and morphology were measured by computer assisted sperm analysis (CASA). For the proteomic investigation cells were lysed and proteins separated by means of two-dimensional gel electrophoresis (2D electrophoresis). PD-Quest was used to identify the differentially expressed proteins. The protein spots of interest were excised and subjected to in-gel digestion. Peptides were separated by High Pressure Liquid Chromatography (HPLC) analysis and amino acid sequences determined by mass spectrophotometry. 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 head morphology; p<0.001) of the two populations differed significantly. After 2D electrophoresis, 16 differentially expressed protein spots were identified within the total sperm proteome between the immature and mature sperm populations. 56% of the differentially expressed proteins were more abundant in the immature sperm population compared to the mature sperm population. Functions have been ascribed to these proteins 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 directly related to sperm motility and morphology. In the second part of the study the expression of nuclear proteins in human spermatozoa was investigated between immature and mature sperm populations. Semen was collected from healthy donors (n=156) and further separated from the seminal plasma by PureSperm® gradient centrifugation. The immature and mature sperm populations were retrieved and used during further analysis. For the proteomic analysis of nuclear proteins, cells were fractionated into four different subcellular protein fractions, instead of analyzing the whole sperm proteome. The results show that the motility (immature; 32.33±0.51% total motile cells vs. mature; 88.67±0.85% total motile cells; p<0.0001) and morphology parameters (immature; 13.51±0.87% normal head morphology vs. mature; 20.89±1.20% normal head morphology; p<0.0001) of the two populations differ significantly. After 2D electrophoresis, 21 differentially expressed nuclear proteins were identified between the immature and mature sperm populations. 95% of the differentially expressed nuclear proteins were less abundant in the immature population compared to the mature population. Only one nuclear protein namely 78kDa Glucose regulated protein was more abundant in the immature population compared to the mature population. Functions ascribed to these individual proteins were directly related to sperm motility, morphology and energy metabolism. In conclusion,In conclusion, in the current study novel techniques have been employed to investigate protein differences between immature and mature sperm populations. From these results it is evident that protein expression in the total sperm proteome and nuclear protein fraction is significantly different and incomplete in the immature population, compared to mature population. Based on these findings, it is recommended that further studies should be done on human spermatozoa to validate the role of the individual proteins in sperm function. Proteomics is an ideal tool to identify idiopathic causes of male infertility, as it can help to identify novel receptors (and signal transduction pathways) that can be used in the screening of drugs to alleviate sperm dysfunction.

AFRIKAANSE OPSOMMING: Manlike infertiliteit word dikwels geassosieer met verlaagde sperm motiliteit en morfologie (asthenoteratozoospermia) waarvoor daar tot dusver nog geen spesifieke terapeutiese behandeling is nie. Dit het aan die lig gekom dat die modifisering en uitdrukking van menslike sperm proteïene ‘n belangrike rol speel in spermfunksie. In die huidige studie stel ons data voor van proteiene in menslike sperme in die konteks van abnormale spermfunksie. Unieke tegnieke was gebruik om verskille in proteïen uitdrukking op sellulêre vlak suksesvol te isoleer en identifiseer wat verband hou met asthenoteratozoospermia. Tydens die eerste deel van die studie was verskille in proteïen uitdrukking binne die totale spermproteoom tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme van gesonde skenkers (n=23) is geskei in twee spermpopulasies (onvolwasse en volwasse sperme) deur middel van ‘n 3-laag Percoll gradiënt sentrifugasie tegniek. Selle is gewas en sperm motiliteit en morfologie is gemeet deur rekenaar geassisteerde sperm analise (CASA). Vir proteomiese analise is selle geliseer en proteïene geskei deur twee dimensionele gel elektroforese (2D-elektroforese). PD-Quest sagteware is gebruik om statisties beduidende proteïen verskille aan te dui. Die proteïene van belang is uitgesny en onderwerp aan in-gel vertering. Peptiede is geskei met behulp van hoë druk vloeistof chromatografie (HPLC) analise en aminosuurvolgordes is bepaal deur massa spektrofotometrie. Proteïene is geïdentifiseer met behulp van Mascot deur van die Swiss Prot databasis gebruik te maak. Die resultate toon dat die sperm motiliteit (onvolwasse; 26.1±1.75% totale motiele selle vs. volwasse; 60.93±3.24% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 64.1±2.75% normale kop morfologie vs. volwasse; 87.63±3.24% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2Delektroforese is 16 proteïen kolle geïdentifiseer wat beduidend verskil het, tussen die totale sperm proteoom van onvolwasse spermpopulasies en volwasse spermpopulasies. 56% van die proteïene wat beduidend verskil het, was meer uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse sperm populasie. Funksies is toegeskryf aan hierdie proteïene waarvan net vier proteïene naamlik Tubulin -3C/D ketting, Tubulin -2C ketting, Buite digte vesel proteïen 2 en A-Kinase anker proteïen 4 voorloper direk verband hou met sperm motiliteit en morfologie. In die tweede deel van die studie is die uitdrukking van nukluêre proteïene in menslike spermatozoa tussen onvolwasse en volwasse spermpopulasies ondersoek. Sperme was van gesonde skenkers (n=156) versamel en verder geskei van seminale plasma deur middel van ‘n PureSperm® gradiënt sentrifugasie tegniek. Vir die proteomiese analise van nukluêre proteïene is selle gefraksioneer in vier verskillende sub-sellulêre proteïen fraksies, in plaas van analise van die totale spermproteoom. Die resultate toon aan dat die sperm motiliteit (onvolwasse; 32.33±0.51% totale motiele selle vs. volwasse; 88.67±0.85% totale motiele selle; p <0,001) en morfologiese parameters (onvolwasse; 13.51±0.87% normale kop morfologie vs. volwasse; 20.89±1.20% normale kop morfologie; p <0,001) tussen die twee populasies beduidend verskil. Na 2D-elektroforese is 21 kern proteïen kolle geïdentifiseer wat betekenisvol uitgedruk was tussen onvolwasse en volwasse spermpopulasies. 95% van die nukluêre proteïene wat beduidend verskil het, was minder uitgedruk in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Slegs een kern proteïen naamlik 78kDa Glukose gereguleerde proteïen was meer uitgedruk in die onvolwasse spermpopulasie in vergelyking met die volwasse spermpopulasie. Funksies is toegeskryf aan hierdie proteïene wat direk verband hou met sperm motiliteit, morfologie en energie metabolisme. Ten slotte, in die huidige studie is unieke tegnieke geïmplementeer om proteïen verskille tussen onvolwasse en volwasse spermpopulasies te ondersoek. Uit hierdie resultate is dit duidelik dat proteïen uitdrukking in die totale sperm proteoom en in die kern proteïen fraksie beduidend verskil en onvolledig is in die onvolwasse spermpopulasie ten opsigte van die volwasse spermpopulasie. Op grond van hierdie bevindinge word aanbeveel dat verdere studies op menslike sperme gedoen moet word ten einde die rol van individuele proteïene in sperm funksie te kan bepaal. Proteomika is ‘n ideale tegniek om die iodiopatiese oorsake van manlike infertiliteit te identifiseer, aangesien dit kan help in die identifisering van unieke reseptore (en seintransduksie paaie) wat gebruik kan word om sperm disfunksie te verbeter deur farmaseutiese behandeling.

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