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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52709 |
| Date | 12 1900 |
| Creators | Du Plessis, S. S.(Simon Stephanus) |
| Contributors | Franken, D. R., Page, C., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 193 p. : ill. |
| Rights | Stellenbosch University |
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