No description available.

Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: CHAPTER 1 provides literature based background information to emphasize the use of a sequential, multistep diagnostic schedule for couples in an assisted reproductive program as well as the clinical importance of sperm morphology as recorded by strict criteria during the diagnostic approach of the infertile couple. Furthermore, the chapter includes evidence underlining the growing need for the implementation of the physiologically induced acrosome reaction as an important contribution to the assisted reproductive program. The zona pellucida-induced acrosome reaction (ZIAR), sperm-zona interaction as well as computer-assisted semen analyses were investigated. CHAPTER 2 provides detailed experimental protocols of the materials and methods used in the study. CHAPTERS 3-6 each represent a separate study that was prepared as a scientific paper and encompass the experimental research undertaken in the reproductive biology research laboratory at Tygerberg Hospital to address important aspects of human acrosome processes. In the first study, we aimed to evaluate the regulatory role of the Gi-like protein during the AR of normal sperm donors and the role of intact acrosomes during sperm-zona binding. It seems that pertussis toxin-sensitive Gi-like protein in human spermatozoa plays an important regulatory role in the ZIAR and this underlines the importance of intact acrosomes during sperm-zona binding. In the second study, we aimed to evaluate the relationship between the ZIAR and the percentage normal spermatozoa as well as the sperm-ZP binding potential among men referred for a routine semen analysis. ZIAR testing should become part of the second level of male fertility investigations, i.e., sperm functional testing, since 15% of the andrology referrals revealed an impaired AR response to solubilized ZP. In the third study, we aimed to evaluate the possible relationships between the sperm morphology, the acrosome responsiveness to solubulized human ZP and the sperm-zona binding potential among consecutive andrology referrals and randomly selected IVF cases. ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test since the results predicted fertilization failure during IVF treatment. In the fourth study, we aimed to evaluate changes in the sperm motion characteristics and the occurrence of hyperactivated motility after exposure to ZP among andrology referrals. Solubilized human ZP induces hyperactivated motility among sperm populations that have been capacitated under laboratory conditions. Capacitated spermatozoa have an elevated percentage hyperactivated cells that correlate with the percentage normal spermatozoa in the ejaculate. CHAPTER 7, the general discussion, is brief and concise to avoid unnecessary repetition, underlines the validity of a sequential, multistep diagnostic approach and concludes with the recommendation that the ZIAR should form part of the diagnostic tools in the assisted reproductive program. / AFRIKAANSE OPSOMMING: HOOFSTUK 1 bestaan uit ’n omvattende agtergrondstudie wat bestaan uit die ontwikkeling van diagnostiese toetse, die hantering van die egpaar in die reproduktiewe ondersteunings-program asook op die kliniese belang van spermmorfologie. Die toenemende behoefte aan die implementering van die fisiologies-geinduseerde akrosoomreaksie, as ’n belangrike bydrae tot die reproduktiewe ondersteuningsprogram, word ook beklemtoon. Die zona pellucida geinduseerde akrosoomreaksie (ZIAR), sperm-zonabinding asook rekenaar-bemiddelde semenanalises is ondersoek. HOOFSTUK 2 dek gedetailleerde eksperimentele protokolle van die materiale en metodes wat in die studie gebruik is. HOOFSTUKKE 3-6 behandel die eksperimentele navorsing wat in die laboratorium van die reproduktiewe biologie-eenheid te Tygerberg hospitaal uitgevoer is en wat as ses afsonderlike wetenskaplike publikasies aangebied word. Die doel van die eerste studie was om die regulerende rol van Gi-proteiene tydens die AR van normale spermdonors asook die rol van intakte akrosome tydens sperm-zonabinding te evalueer. Dit kom voor asof Gj-protei'ene in spermatozoa ’n belangrike regulerende rol in die ZIAR speel. Dit beklemtoon die belangrikheid van intakte akrosome tydens sperm-zonabinding. Die doel van die tweede studie was om die verhouding tussen die ZIAR en die persentasie normale spermatozoa asook die sperm-zonabindingspotensiaal tussen mans wat vir ’n roetine semenanalise verwys is te evalueer. ZIAR-toetsing moet deel uitmaak van die tweede vlak van manlike fertiliteitsondersoeke, d.w.s. funksionele toetsing, aangesien 15% van die andrologie pasiente ’n verswakte AR respons tot opgeloste ZP openbaar. In die derde studie was die doel om die moontlike verhoudinge tussen sperm-morfologie, die ZIAR en die sperm-zonabindingspotensiaal onder opeenvolgende andrologie-pasiente asook lukraak geselekteerde IVB-pasiente te evalueer. Die ZIAR-resultate bied verdere informasie aangaande disfunksionele spermatozoa en kan gebruik word as ’n addisionele diagnostiese toets aangesien hierdie resultate mislukte bevrugting tydens IVB behandeling voorspel. Die vierde studie het ten doel gehad om veranderinge in spermmotiliteitseienskappe asook hiperaktiwiteit na die blootstelling aan opgeloste zona onder andrologiepasiente te evalueer. Daar is afgelei dat opgeloste menslike zona hiperaktiwiteit induseer in spermpopulasies wat onder gunstige laboratoriumkondisies gekapasiteer is. Die gekapasiteerde spermatozoa het ’n verhoogde persentasie gehiperaktiveerde spermatozoa getoon wat met die persentasie normale spermatozoa in die ejakulaat korreleer. In HOOFSTUK 7 word aangetoon dat dit noodsaaklik is om die diagnostiese skedule by die hantering van die onvrugbare egpaar te gebruik asook dat die ontwikkeling van die funksionele toestand belangrik is vir die bepaling van ZIAR.

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

No description available.

No description available.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2007. / For spermatozoa to gain access to the oocyte for fertilization, lytic enzymes need to be released during the acrosome reaction. These enzymes, which are stored and transported within an organelle termed the acrosome, make it possible for spermatozoa to collectively penetrate the layers of cells and glycoproteins that surround and protect an oocyte. Acrosomes may thus be viewed as essential for fertilization and their shape, size and volume were examined morphometrically by utilizing automated morphometric analysis equipment. In addition to the acrosome being necessary for normal unassisted fertilization, spermatozoa also need the ability to migrate to the oocyte. Following zona pellucida binding, sperm tail thrust movement initiates zona penetration into the space created by the digestive action of the acrosomal enzymes. Therefore the motion characteristics of spermatozoa were also quantified in terms of kinematic properties. In the treatment of male sub fertility, assisted reproductive techniques are applied. In the application of such techniques, a motile sub-population of spermatozoa was obtained by employing a procedure (swim-up selection) that selects cells on the basis of their kinematic ability. This study presents an analysis of the morphometric and kinematic qualities of spermatozoa populations that are subjected to swim-up selection and investigates the relationship of these morphometrical and kinematic qualities. Computer-assisted semen analysis, swim-up selection and automated sperm morphology analysis tests were all used to evaluate spermatozoa populations. Results indicated that, irrespective of acrosome size, higher kinematic parameter measurements were observed post-swim-up. A significant inverse relationship between the population’s average acrosome size and a number of kinematic parameters was observed. Our results indicated that for a post-swim-up population of spermatozoa an increase in the average acrosome size was significantly related to a decrease in the kinematic parameters VAP, VCL and the VSL within the same population.

Thesis (MSCMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / In this study, we presented data on the role of free radicals in human spermatozoa, particularly in the context of centrifugation and the potential development of defective sperm function. In order to achieve this, methods were developed to directly measure intracellular free radicals in human sperm and the effects of exogenously applied free radicals on sperm function were established. The role of brief and prolonged centrifugation and the associated generation of free radicals was also investigated.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The application of assisted reproductive biotechnologies in sheep flocks is hampered by the susceptability of ovine sperm to cryodamage. There is still considerable scope in the improvement of cryopreservation protocols for ovine sperm to minimize the degree of damage to sperm during the cryopreservation process. Pre-cryopreservation processing has a definite effect on the survivability, motility, and fertilizing ability of sperm. Little information is however available on the potential contribution of the genetic make-up of rams, divergently selected for fecundity, on the ability of sperm to offer resistance to the damage caused by cooling, cryopreservation and thawing. The study aimed to investigate the influence of genetic selection for prolificacy (i.e. High Merino Line and Low Merino Line in terms of fecundity) on the ability of ovine sperm to offer resistance to cryodamage. The study investigated the effect of pre-cryopreservation processing by comparing motility and morphometry traits recorded for fresh- and post-thaw Merino ejaculated and epididymal sperm samples obtained form the High and Low lines, respectively. The effect of different sperm concentrations, equilibration periods and the addition or omission of seminal plasma from cryopreserved samples on the viability and morphometrical traits were also investigated. Ejaculate samples were collected by means of the artificial vagina (AV) method from 8 High Line rams and 7 Low Line rams. Epididymal samples were collected from 6 rams of each of the High and Low lines respectively, by recovering the epididymal sperm via aspiration from the cauda epididymides post mortem. Ejaculate samples were subjected to macroscopic and microscopic evaluation, and epididymal samples only to microscopic evaluation, for which the Sperm Class Analyzer® program was used for the evaluation of motility and morphometric measurements. Sperm motility recordings were captured at 100 frames per second. From findings of the study, it was concluded that genotype had no positive influence on the conception rate of the ewes mated to the High or Low Line rams, even though the rams from the two lines differed significantly in terms of their serving capacity. When sperm morphometry was evaluated for fresh ejaculate samples, the two lines differed significantly in terms of the morphometric traits elongation and ellipticity. Epididymal and ejaculated sperm obtained from Low Line rams had broader and rounder heads, compared to sperm obtained from High Line rams. When morphometry was assessed for sperm samples between the two methods of sperm recovery (collected with an AV or recovery via aspiration from the cauda epididymides of sacrificed rams), no morphometrical differences were observed. Significant differences were reported for the majority of the sperm motility traits (i.e. percentage motile, rapid-, medium-, slow swimming, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and amplitude of the lateral head displacement (ALH)) recorded for ejaculated and epididymal sperm. The motility traits ALH and beat-cross frequency (BCF) analysed for epididymal sperm differed significantly between the two lines. When epididymal sperm were evaluated post-thaw, it became evident that the sperm obtained from the High Line rams had a larger acrosome surface cover when compared to that of the Low Line ram sperm. The addition of seminal plasma to epididymal samples did not result in an improvement of the preservation of sperm motility. It is known from the literature that cryopreservation causes a decrease in sperm head size. Head width was unaffected by cryopreservation with the addition of seminal plasma in this study, indicating a potential benefit with the use of seminal plasma in the cryopreservation protocol of epididymal ram sperm. The study compared two pre-processing techniques, i.e. the more time consuming swim-up technique (SUT) with a more time-efficient ‘flush technique’ (FT) to optimize the pre-processing protocol for motility assessment of sperm samples before cryopreservation of ram sperm. Comparison of the SUT and FT indicated that almost all of the motility parameters measured using the FT compared favourably with those obtained using the SUT. The results indicated that the FT can be used a more time-efficient technique to use for determining the motility of a sperm sample prior to cryopreservation. In conclusion, line differences associated with reproduction were observed in terms of the serving capacity of the rams, with selection for fecundity influencing the morphometric traits elongation and ellipticity for sperm obtained from the two lines. Future studies should be aimed at investigating morphometric traits of ovine sperm, to correlate it with fertilizing ability of sperm post-thaw and ensure optimal cryopreservation processing. / AFRIKAANSE OPSOMMING: Die toepassing van ondersteunende reproduksie tegnieke in skaaptroppe word bemoeilik deur die onvermoë van ram sperme om weerstand teen bevriesingskade te bied. Daar is nog baie ruimte vir die verbetering van die bevriesingsprotokolle vir skaap sperm om die omvang van bevriesingskade te verminder. Voor-bevriesing verwerking het dan 'n besliste uitwerking op die oorlewing, beweeglikheid en bevrugtingsvermoë, van skaap sperme. Min inligting is beskikbaar oor die potensiële bydrae van die genetiese samestelling van ramme wat uiteenlopend op grond van vrugbaarheid geselekteer is, op die vermoë van skaap sperme om weerstand te bied teen die skade wat deur verkoeling, diepbevriesing en ontdooiing, veroorsaak word. Die doelwit van die studie was om die invloed van genetiese seleksie vir fekunditeit (d.i. Hoë Merino Lyn en Lae Merino Lyn in terme van fekunditeit) op die vermoë van skaap sperme om weerstand teen bevriesingskade te bied, te ondersoek. Die studie het getoets wat die bevriesing proses se effek op epididimale sperme is, deur sperm motiliteit en -morfometrie te vergelyk tussen vars gekollekteerde sperme en sperm monsters na ontdooiing. Die effek van verskillende sperm konsentrasies, ekwilibrasie tydperke en die byvoeging of uitsluiting van seminale plasma op die lewensvatbaarheid en morfometriese eienskappe van Merino ramsperme is ondersoek in die studie. Geëjakuleerde monsters is versamel met behulp van 'n kunsmatige vagina (AV) van 8 Hoë Lyn en 7 Lae Lyn ramme. Epididimale monsters is verkry van 6 ramme van elk van die Hoë en Lae Lyne, deur middel van aspirasie van die sperme uit die cauda epididimii nadoods. Geëjakuleerde sperm monsters is met behulp van makroskopiese en mikroskopiese metodes geëvalueer, en epididimale sperm monsters slegs mikroskopies geëvalueer, met behulp van die Sperm Class Analyzer® program wat vir die evaluasie van beweeglikheid en morfometriese afmetings gebruik is. Sperm beweeglikheids opnames is opgeneem teen 100 raampies per sekonde. Die resultate van die studie het aangedui dat genotipe geen effek het op besetting van die ooie gepaar met die Hoë of Lae Lyn ramme gehad het nie, terwyl die dekvermoë aansienlik tussen ramme van die twee lyne verskil het. Wanneer die morfometriese eienskappe van vars geëjakuleerde sperme vergelyk was, het die lyne beduidend in terme van die morfometriese eienskappe van verlenging (elongation) en elliptisiteit verskil het. Die epididimale en geëjakuleerde sperme verkry vanaf die Lae Lyn ramme het ʼn breër en ronder kopvorm getoon as sperme wat verkry is van die Hoë Lyn ramme. Wanneer die morfometriese eienskappe van sperme versamel met die twee verskillende metodes (d.i. kunsmatige vagina of aspirasie vanuit die cauda epididimides) vergelyk was, is geen morfometriese verskille waargeneem nie. Die meeste sperm beweeglikheidseienskappe (d.i. persentasie beweeglike, vinnig-, medium- en stadig-swemmende sperme, VCL, VSL, VAP en ALH) van geëjakuleerde en epididimale sperme het verskil. Die beweeglikheidseienskappe amplitude van die laterale verplasing van die spermkop (ALH) en frekwensie waarmee sperm sy eie pad kruis (BCF), soos bepaal vir epididimale sperme, het beduidend tussen die twee lyne verskil. Met die evaluering van epididimale sperme na ontdooiing was dit duidelik dat sperme verkry van die Hoë Lyn ramme 'n groter mate van akrosoom-oppervlak gehad het, in vergelyking met sperme van die Lae Lyn ramme. Die byvoeging van seminale plasma by epididimale monsters het nie bygedra tot 'n verbetering van spermbeweeglikheid nie. Bestaande literatuur dui aan dat diepbevriesing 'n afname in die kopgrootte van sperme veroorsaak. In hierdie studie het die byvoeging van seminale plasma ʼn verandering in kopgrootte voorkom, wat dui op ʼn potensiële voordeel om seminale plasma in die bevriesingsprotokol van epididimale ramsperme in te sluit. Die studie het twee beweeglikheid bepalingstegnieke vergelyk om te bepaal of die tydrowende “opswem” tegniek (SUT) vervang kan word met 'n meer tyd-doeltreffende "spoel tegniek” (FT) in die voorbevriesing verwerking protokolle van ram sperme. Vergelyking van die twee tegnieke het aangedui dat die meeste van die kinematiese eienskappe van die FT gunstig met die waardes soos verkry met die SUT, vergelyk het. Resultate het getoon dat die FT parameters goed vergelyk met die beweeglikheid parameters van die SUT, dus kan dit aangeneem word dat die FT ʼn meer tyd-doeltreffende tegniek is wat vergelykbare sperm beweeglikheidsinligting oor skaap sperm monsters voor bevriesing sal verskaf. In samevatting is verskille in terme van die dekvermoë en op morfometriese vlak, meer spesifiek die eienskappe van verlenging (elongation) en elliptisiteit, tussen die twee lyne waargeneem. In toekomstige studies moet die morfometriese eienskappe van skaapsperme verder bestudeer word, asook die korrelasie daarvan met die bevrugtingsvermoë na ontdooiing bepaal om sodoende die diepbevriesing protokolle van skaapsperme te optimaliseer.

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: In this study the author presents new data that will shed light on the fairly nebulous knowledge of intracellular pathways involved in the physiologically induced acrosome reaction and the subsequent events leading to fertilization. The zona pellucidainduced acrosome reaction, sperm-zona interaction as well as various sperm motion characteristics were investigated. The first part of the study focussed on the role of extracellular signal regulated kinase (ERK), a member of the family of mitogen activated protein kinases, during the zona pellucida-induced acrosome reaction and sperm-oocyte interaction. It was shown that the inhibition of ERK significantly reduced the zona pellucida-induced acrosome reaction as measured by fluorescent microscopy. This suggests that ERKs are directly or indirectly involved in the signal transduction pathway through which the human sperm acrosome reaction is induced by the zona pellucida. In an attempt to provide further proof that ERK was involved in human acrosome signalling hemizona assays were employed to test sperm-oocyte binding. From these sperm-oocytebinding experiments it was clear that the inhibition of ERK leads to increased binding. These results support the idea that the zona pellucida-induced acrosome reaction, and therefore the physiologically relevant exocytotic event, is regulated by an ERKmediated signal transduction process. In the second part of the study the significance of phosphatidylinositol 3-kinase (PI3K) in the process of human sperm motility, acrosome reaction and sperm-oocyte binding, was investigated by employing the specific PI3K, LY294002. PI3K inhibition increased the percentage motility and percentage progressive motility in asthenozoospermia patients. Under the present laboratory conditions PI3K inhibition furthermore did not influence the acrosome reaction, whilst it enhanced sperm-oocyte binding. These results therefore imply that PI3K negatively affect sperm motility and zona-binding. In the last part of the study the possible effects of intracellular cGMP accumulation via acute in vivo sildenafil citrate (ViagraTM) administration was investigated on seminal parameters, induction of the acrosome reaction, sperm-oocyte binding and sperm motility. As was expected no changes in the macro- and microscopically seminal parameters were caused by sildenafil citrate when compared to placebo. Furthermore the acrosome reaction was also not initiated or potentiated by sildenafil citrate at concentrations of 50mg orally. Sperm-oocyte binding, smooth path velocity, straight line velocity and the percentage rapid cells all increased after sildenafil citrate treatment. From these results it appear that there are various role players in the zona pellucidainduced acrosome reaction intracellular signalling system. A thorough understanding of such signal transduction systems and the crosstalk in-between will ultimately yield information regarding the nature of receptors to which these signal transduction pathways are coupled in human spermatozoa as well as the intracellular effectors that ultimately regulate sperm function. Moreover, an understanding of these regulatory pathways will be essential for the future development of clinical approaches designed to enhance or preclude fertilization. / AFRIKAANSE OPSOMMING: Die outeur lê in hierdie studie nuwe data voor ten einde meer lig te werp op die relatief vae veld van intrasellulêre seintransduksie paaie betrokke by die fisiologiesgeïnduseerde akrosoomreaksie en die daaropvolgende gebeure wat aanleiding gee tot bevrugting. Die zona pellucida-geïnduseerde akrosoomreaksie, sperm-zona interaksie sowel as spermmotiliteitseienskappe is ondersoek. Die eerste gedeelte van die studie fokus op die rol van ekstrasellulêreseingereguleerde- kinase (ERK), 'n lid van die familie van mitogeen-geaktiveerde proteïenkinases, tydens die zona pellucida-geïnduseerde akrosoomreaksie en sperm-oosiet interaksie. Daar word aangetoon dat die inhibisie van ERK die zona pellucida geïnduseerde akrosoomreaksie, soos gemeet met behulp van fluorosensie mikroskopie, betekenisvol verminder. Dit suggereer dat ERK direk of indirek betrokke is by die seintransduksie paaie waardeur die akrosoomreaksie van die menslike sperm deur die zona pellucida geïnduseer word. In 'n poging om onomwonde te bewys dat ERK betrokke is by menslike akrosoom-seintransduksie, is hemizona essais gebruik om sperm-oesiet binding te bepaal. Van hierdie sperm-oosiet bindingeksperimente is dit duidelik dat die inhibisie van ERK aanleiding gee tot verhoogde binding. Hierdie resultate ondersteun dus die idee dat die zona pellucidageïnduseerde akrosoomreaksie en dus die fisiologies relevante eksositotiese gebeurtenis gereguleer word deur 'n ERK-gemediëerde seintransduksie proses. In die tweede gedeelte van die studie is die belang van fosfatidielinositol 3-kinase (PI3K) in die proses van menslike spermmotiliteit, akrosoomreaksie en sperm-oesiet binding ondersoek deur van die spesifieke PI3K inhibitor LY294002, gebruik te maak. Pl3K-inhibisie het die persentasie motiliteit en progressiewe motiliteit by astenozoospermiese pasiënte verhoog. Onder hierdie laboratoriumtoestande het Pl3K-inhibisie nie die akrosoomreaksie beïnvloed nie, terwyl sperm-oosiet binding verhoog is. Hierdie resultate beteken dus dat PI3K spermmotiliteit en zona-binding negatief beïnvloed. In die laaste gedeelte van die studie is die effekte van intrasllulêre cGMP akkumulasie deur akute in vivo sildenafil sitraat (ViagraTM) toediening op seminale parameters, induksie van die akrosoomreaksie, sperm-oesiet binding en spermmotiliteit ondersoek. Soos verwag is geen veranderinge in die makro- en mikroskopiese seminale parameters deur sildenafil sitraat in vergelyking met plasebo veroorsaak nie. Verder is die akrosoomreaksies ook nie deur 50mg orale sildenafil sitraat geïnisieer of potensieer nie. Sperm-oosiet binding, geplaneerde snelheid, reguitlyn snelheid en persentasie vinnigbewegende selle was almal vehoog na sildenafil sitraat behandeling. Uit hierdie resultate blyk dit dat daar verskeie rolspelers in die zona pellucidageïnduseerde akrosoomreaksie is. 'n Deeglike insig van al hierdie seintransduksiepaaie en die onderlinge kruiskontak tussen mekaar sal uiteindelik die nodige inligting rakende die reseptore waaraan hierdie seintransduksie paaie gekoppel is, verskaf sowel as die intrasellulêre effektore wat uiteindelik spermfunksie beheer. Nog te meer sal die begrip van hierdie regulatoriese paaie verder noodsaaklik wees vir die toekomstige ontwikkeling van kliniese benaderings om bevrugting te bevorder of te voorkom.

Current sperm processing methods used in assisted reproductive technologies can cause damage to the sperm cell. New ways that mimic the natural guidance mechanisms present in the female genital tract may offer ways to sort sperm with better fertility parameters. Sperm that respond to these cues may have better over sperm health. Human sperm exhibit positive rheotaxis by orienting and swimming against the fluid released by the female genital tract. At certain flow rates sperm can actively orient and swim against the flow. Sperm retrieved that exhibit positive rheotaxis have higher motility and better morphology than the original semen sample. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection