Bibliography / Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2010. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5205 |
| Date | 12 1900 |
| Creators | Rapuling, Llewelen |
| Contributors | Du Plessis, Stefan, Hattingh, Suzel, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xxiii, 180 p. : ill. |
| Rights | University of Stellenbosch |
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