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.

L’infertilité est définie par l’OMS comme l’incapacité à concevoir un enfant dans un couple, après au moins douze mois de rapports sexuels réguliers sans protection. Elle concerne 15 % des couples qui désirent avoir un enfant, un facteur masculin est retrouvé dans 50 % des cas. Dans le cadre de cette thèse, les principaux objectifs étaient de déterminer des causes génétiques d’infertilité masculine chez des patients atteints d'oligozoospermie sévère (OS) ou d'asthénozoospermie en lien avec des anomalies morphologiques des flagelles, afin de mieux comprendre la spermatogénèse, d’améliorer la prise en charge des couple infertiles et d'évaluer les risques de transmission à leur descendance. Nous avons abordé ces problématiques par le séquençage d’exome entier d'hommes ayant un de ces phénotypes au sein de deux familles consanguines. L'ensemble des résultats de cette thèse apportent des éléments de réponses sur de nouvelles causes génétiques d’infertilité masculine liée à une anomalie quantitative ou qualitative de la spermatogénèse. De plus, nos résultats concernant EXD1 nous interrogent sur la possibilité de transmission de mutations génétiques ou modifications de marques épigénétiques à la descendance en Assistance Médicale à la Procréation pour certains patients avec une oligozoospermie. / Infertility is defined by the WHO as the inability of a couple to conceive a child after twelve months of unprotected regular sexual intercourse. It concerns 15% of couples who wish to have a child, and a male factor is found in 50% of cases. In this thesis, the main objectives were to determine genetic causes of male infertility in patients with severe oligozoospermia (SO) or asthenozoospermia related to morphological abnormalities of the flagella, in order to better understand spermatogenesis, improve the care of infertile couples and inform risk evaluation for their offspring. We addressed these objectives by sequencing the whole exome of men with one of these phenotypes within two consanguineous families.The results presented in this thesis reveal new genetic causes of male infertility related to a quantitative or a qualitative abnormality of spermatogenesis. Moreover our findings concerning EXD1 raise questions about the risk that, for some patients with oligozoospermia, medically assisted reproduction could transmit de novo genetic or epigenetic modifications to future generations.

>Magister Scientiae - MSc / Male factor disorders affect more than 30% of infertile couples. Thus, it has become important to perform a andrological consultation and a basic semen evaluation in all male partners of couples consulting for infertility. The advent and development of assisted reproductive technologies has not only improved clinical results but also enhanced our basic understanding of the physiology of sperm and sperm preparation methods. Assisted reproduction has become among the more successful therapeutic modalities for a wide variety of sperm function disorders e.g. artificial insemination and in vitro fertilization (IVF) (Acosta et al. 1989). It is clear from recent experience that patients with male infertility showing oligozoospermia, asthenozoospermia, teratozoospermia (sometimes in combination), male immunological factor (antisperm antibodies) or ejaculatory problems as well as congenital abnormalities, can be successfully treated with IVF and embryo transfer. Prerequisite pre-fertilization changes by sperm, termed "capacitation" (Austin 1952) provides sperm with the capacity to fertilize eggs. These processes are generally regarded as encompassing all pre-fertilization changes occurring in sperm up 'to, but not including, loss of the acrosome (Bedford 1970). The endpoints of capacitation are often described as the acrosomal loss as well as changes in the motion characteristics. Capacitation alters the pattern of motility exhibited by freely swimming sperm, changing from a fairly rigid flagellar beat pattern to one of extreme flexure, often associated with increased thrust (Johnson et al. 1981) , which is referred to as hyperactivated motility (Yanagimachi 1981). without the transition to hyperactivated motility, sperm are unable to penetrate the zona pellucida (Fraser 1981), and possibly unable to fertilize eggs. Hyperactivation per se is marked by increased curvature in swimming trajectories and/or increased lateral displacement of the sperm head along their path (Burkman 1984). However, the physiological role of this change in motility is not clearly understood because almost all relevant data have been obtained under in vitro conditions. The association of specific seminal characteristics (sperm concentration, percentage motile cells and percentage normal sperm morphology) with the success rate of assisted particular has been The analysis of 1984; the reproductive techniques and IVF in under great scrutiny (Mahadevan and Trounson relationships between conventional semen parameters and fertilization rates in vitro has shown that sperm motility, concentration and morphology must be considered in estimating opportunities for successful intervention, as in the case of IVF and gamete intrafallopian transfer (GIFT) (Oehninger and Hodgen 1991). A reduction in the percentage of progressive motility alone does not seem to have a significant impact on IVF results unless it is below a threshold value of 10%. The semen sample should have an acceptable sperm concentration and morphology and/or that at least 1.5 X 106 motile spermatozoa can be recovered after swim-up separation (Acosta et al. 1989).

Male infertility may be due to oligozoospermia, asthenozoospermia and teratozoospermia. Intracytoplasmic sperm injection is used to address male infertility. However, the percentage of viable embryos obtained by this technique is very low. Pronucleus formation has been identified as one of the key events in fertilisation and gamete decondensation is vital for this process to take place. Decondensation can be initiated by chemicals such as DTT that reduce the disulphide groups between the protamine proteins that keep the DNA of the gamete condensed. An increase in decondensation should translate into a higher fertilization rate and a higher yield of embryos. The research from this thesis has compared the decondensation ability via DTT in human spermatozoa and bovine spermatozoa, to study pronucleus formation in bovine zygotes and bovine embryo formation in the presence of DTT; and lastly the cytotoxic effect of DTT using somatic cells in culture has been investigated. In this study 12 semen samples for either fertile or subfertile subjects were collected, isolated and exposed to 25 mM DTT for 0, 5, 7, and 10 minutes, washed and the morphological changes associated with decondensation was evaluated by phase contrast microscopy. After 5 and 7 minutes 11 of the 12 samples underwent decondensation while after 10 minutes several samples showed a lower rate of decondensation and this was associated with and unusual hypercondensed state, CMA3 staining revealed all spermatozoa samples evaluated were mature. However, after treatment with DTT for 5, 7 and 10 minutes an increase in fluorescence was observed indicating increased protamine thiol group reduction and subsequently increased CMA3 accessibility. For some samples reduced fluorescence was observed possible due to the supercoiling of the DNA. DTT successfully induces decondensation of human spermatozoa, however does this lead to the formation of viable embryos? Due to ethical issues associated with working with human embryos all further studies were done using bovine embryos. Spermatozoa used were derived from Friesian bulls and the samples were pooled to prevent sample bias and interindividual variation. Spermatozoa were exposed to 25 mM of DTT at 5, 7, and 10 minutes as used for human spermatozoa. No decondensation was observed using the same conditions as for human spermatozoa, therefore the ‘swim up’ medium containing heparin and regularly used in IVF procedures for bovines was used, and this resulted in successful decondensation of bovine spermatozoa after 30 minutes. The effects of DTT on pronucleus formation and embryo development were evaluated in three bovine specimens. In the first group, DTT had no significant effect on the parameters measured, namely the number of oocytes that were in metaphase II, with one pronucleus, with two pronuclei, with degeneration of the nucleus and polyspermia. In the second group the percentage cleavage and embryo formation was determined on Day 1 (group 2) and 7 (group 3) respectively and statistical differences were obtained between the control and the DTT group. DTT had no significant effect on all the early parameters measured however later in development DTT had a significant adverse effect on cleavage and eventual embryo development. <p)Cleavage and embryo formation is a process of multiple mitotic divisions resulting in an increase in the number of cells that become smaller with each cell division, while somatic cells also undergo mitotic division although the cell size remains constant. Therefore the L929 cell line, a standardized system used to test toxicity, can be used to investigate the toxic effects of DTT on a dividing cell population. In this study L929 cells were expose to 25mM DTT for 30 minutes, and lysosomal membrane integrity, cell viability and number was determined immediately following exposure and after 48 hours growth. In another experiment the L929 cell line was exposed to all concentrations used in this and other studies for 5, 10 and 20 minutes. At all concentrations and exposure times DTT was found to be cytotoxic to the L929 cell line. How exactly DTT mediates this toxic effect is unknown, however due to its high solubility DTT can cross the cell membranes. The tertiary structure of proteins, enzymes and DNA is vulnerable to the reducing effects of DTT. In conclusion, although DTT induces decondensation in human and bovine spermatozoa, in the bovine model it does not lead to viable embryo formation and this has been confirmed in cell culture where DTT at all concentrations used was found to be cytotoxic. / Dissertation (MSc (Anatomy))--University of Pretoria, 2006. / Anatomy / unrestricted

La protéine TAT1 (Testis Anion Transporter 1 ; SLC26A8) appartient à la famille des SLC26, une famille de transporteurs d’anions qui contribuent dans différents épithelia à l’homéostasie cellulaire. La protéine TAT1 s’exprime exclusivement dans les cellules germinales mâles, chez l’homme et chez la souris. Sur le spermatozoïde mature, la protéine TAT1 est localisée à la jonction des pièces intermédiaire (PI) et principale (PP) du flagelle, au niveau de l’annulus, une structure en forme d’anneau composée de différents polymères de Septines (1, 4, 6, 7 et 12).Le modèle murin d’invalidation du gène Tat1 présente une infertilité mâle par asthénozoospermie totale (absence de mobilité des spermatozoïdes) et des défauts de capacitation associés à des anomalies structurales du flagelle (plicature du flagelle, disjonction entre la PI et la PP, atrophie de l’annulus). Ce modèle indique que la protéine TAT1 pourrait avoir un rôle structural dans le maintien de l’annulus et dans la mise en place du flagelle. Par ailleurs, la protéine TAT1 possédant une activité de transport d’anions, il est vraisemblable qu’elle puisse influer directement sur la régulation de la mobilité et de la capacitation puisqu’il est bien établi que les échanges ioniques sont essentiels au contrôle de ces deux processus.En effet, les ions chlorure, bicarbonate et calcium participent à l’activation de la voie de signalisation AMPc/PKA, au cours des processus de mobilité et de capacitation (i.e. processus de maturation ayant lieu dans le tractus génital féminin et conférant au spermatozoïde un mouvement hyperactivé et la capacité à interagir avec l’ovocyte).Plusieurs travaux ont montré une interaction physique et fonctionnelle des membres de la famille SLC26 avec le canal chlorure/bicarbonate CFTR (Cystic Fibrosis Transmembrane conductance Regulator) dont les mutations sont responsables de la mucoviscidose. De manière intéressante des données récentes ont montré l’expression de CFTR dans le spermatozoïde et son rôle dans la régulation des flux de chlorure au cours de la capacitation. Au cours de ma thèse, nous avons testé la coopération entre les protéines TAT1 et CFTR ; nous avons pu montrer que la protéine TAT1 est capable d’interagir physiquement avec CFTR et de stimuler son activité de transport d’anions, suggérant qu’in vivo les deux protéines forment un complexe moléculaire impliqué dans la régulation des flux de chlorure et de bicarbonate dans le spermatozoïde.Tout comme TAT1, plusieurs membres de la famille SLC26 ont une expression tissulaire spécifique. Par ailleurs, les mutations génétiques de certains SLC26 sont associées à des pathologies humaines (surdité, diarrhée chlorurée congénitale et chondrodysplasie). De par le phénotype du modèle murin Tat1 et l’importance des SLC26 en pathologie humaine, TAT1 constitue un bon candidat dans la recherche des causes génétiques des asthénozoospermies humaines.Le laboratoire a mis en place au cours de ma thèse, un projet de recherche de mutations du gène TAT1 dans les asthénozoospermies humaines. Le séquençage des régions codantes du gène TAT1 dans une cohorte de 147 hommes infertiles par asthénozoospermie a ainsi permis d’identifier des variations de séquence inédites du gène chez 7 sujets. L’étude in vitro de certains variants indique pour trois d’entre eux une instabilité des formes mutantes associée à un défaut de stimulation du canal CFTR, in vitro. Par ailleurs, les spermatozoïdes de ces patients présentent d’importantes anomalies flagellaires dans la mise en place de la pièce intermédiaire, compatible avec un rôle de la protéine TAT1 et de ses partenaires (les septines) dans la genèse du flagelle / TAT1 (Testis Anion Transporter 1 ; SLC26A8) belongs to the SLC26 family of anion transporters, which is implicated in cellular homeostasis of different epithelia. TAT1 is exclusively expressed in male germ cells, in human and mouse. On mature spermatozoa, TAT1 is located at the annulus, a ring-shaped structure composed of different septins polymers (1, 4, 6, 7 and 12), at the junction of the midpiece (MP) and principal piece (PP) of the flagellum.The knock-out mouse model of Tat1 gene shows a male infertility by complete asthenozoospermia (lack of sperm motility) and capacitation defects combined with flagellar structural abnormalities (flagella bending, MP and PP disjunction and atrophy of the annulus). This model suggests that the TAT1 protein could fulfill structural roles in the annulus and during flagellum biogenesis. Moreover TAT1 displayind an anion transport activity, it could also be implicated in the control of sperm motility and capacitation by regulating anions exchannges, which are well known to be essential for both processes.Indeed, chloride, bicarbonate and calcium ions are involved in the activation of the cAMP/PKA pathway, controlling sperm motility and capacitation processes (i.e. maturation events occuring in the female genital tract and providing the spermatozoa an hyperactivation movement and the ability to interact with oocyte).Several publications have reported a physical and functionnal interaction between SLC26 family members and the chloride/bicarbonate CFTR channel (Cystic Fibrosis Transmembrane conductance Regulator), which mutations are responsible of cystic fibrosis. Interestingly, recent data showed CFTR expression in spermatozoa and its role in the regulation of chloride fluxes during capacitation. During my thesis, we tested TAT1 and CFTR cooperation; we showed that TAT1 can interact physically with CFTR and stimulate its anion transport activity, suggesting that in vivo they form a molecular complex involved in the regulation of chloride and bicarbonate fluxes during sperm capacitation.Like TAT1, several SLC26 family members have a tissue specific expression. Furthermore genetic mutations in several SLC26 members result in human pathology such as deafness, congenital chloride diarrhea and chondrodysplasia. According to the phenotype of the KO Tat1 mouse model and the role of SLC26 members in human pathology, TAT1 constitutes a good candidate for the search of genetic causes of human asthenozoospermia.During my thesis, the laboratory has set up, a research project aiming at identifying mutations in the TAT1 gene that are responsible for human asthenozoospermia.Sequencing of the TAT1 gene coding regions in a cohort of 147 infertile men presenting with asthenozoospermia allowed us to identify several new sequence variations in in the TAT1 gene. In vitro study of these variants shows that 3 of them are associated with protein instability and abrogate CFTR stimulation. Besides, patients sperm show important flagellar abnormalities in the midpiece, consistent with a role of TAT1 and its partners (septins) in flagellum biogenesis.