DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Ulrich Severin :
The role of pertussis-toxin-sensitive G-proteins in the induction of the acrosome reaction in mammalian spermatozoa
Die Rolle Pertussistoxin-sensitiver G-Proteine bei der Auslösung der Akrosomreaktion in Säugetierspermien

FU Logo


|Abstract| |Table of Contents| |More Information|

Abstract

The participation of G-proteins belonging to the Gi-family in the induction of the acrosome reaction has been discussed for a long time using pertussis toxin (PTX) as a cell biological tool for the investigations carried out for this purpose. The efficiency of a PTX-treatment on spermatozoa of different species has never been verified. The aim of this work was to examine systematically the influence of PTX on G-proteins involved in the acrosome reaction (AR), employing functional and biochemical assays to differentiate the toxin?s catalytic properties from a non-specific action on mammalian spermatozoa. An influx of Ca2+ following the attachment of sperm to the ZP is supposed to be a major event in fertilization, triggering the acrosome reaction. Fluorimetric determination of [Ca2+]I in boar sperm showed no PTX-sensitivity for the influx of Ca2+ induced by the Gi-protein activator mastoparan. Boar spermatozoa that had undergone PTX-treatment prior to determination of [Ca2+]I as well as those of the solvent control reacted with a quick, long lasting influx of Ca2+ up to nearly identical plateaus. PTX is not inactivated during the six-hour capacitation as shown by fluorescence microscopic assessment of chemokine-stimulated Ca2+-transients in co-incubated HL-60 cells. A non-specific effect of PTX on spermatozoa affecting the cell?s functional integrity was excluded as shown by the results of the zona pellucida-binding assay. Bull and boar spermatozoa capacitated in the presence of PTX attached to homologous zonae with comparable efficiency as did solvent-pretreated sperm. Because only fully capacitated spermatozoa can bind to the zona pellucida, this assay allows to assess capacitation as a functional parameter. In connexion with the PTX-insensitivity of the mastoparan-induced Ca2+-influx, the effect of PTX pre-treatment on the mastoparan-induced acrosome reaction of boar sperm was investigated. Mastoparan induced the acrosome reaction to comparable extent in PTX pre-treated and non pre-treated boar spermatozoa. In order to investigate the influence of PTX on the zona pellucida-stimulated AR, the acrosomal status of zona-bound boar sperm was determined using a FITC-PNA/Ethidium-homodimer-1 double stain. It could be evidenced that the zona pellucida-induced AR was not abolished by PTX pre-treatment. These findings implicated PTX might not be able to cross the plasma membrane of intact mammalian spermatozoa during capacitation and thus is unable to carry out ADP-ribosylation of Gi-proteins. For this reason, expression rates of Gi-proteins in membranes of human, porcine, murine and bovine sperm were examined by metabolic marking with 32P-NAD+ as a substrate for PTX. Expression of Gi-proteins was clearly confirmed in any of the species tested except for bovine spermatozoa. Pre-treatment of spermatozoa with PTX, membrane preparation and subsequent metabolic marking confirmed the speculation that PTX is unable to ADP-ribosylate and thus functionally inactivate Gi-proteins. It could be established that PTX-treatment of spermatozoa from different species does not result in a functional uncoupling. The toxin probably cannot cross the intact plasma membrane and thus is not suitable to function as a cell biological tool to investigate the role of Gi-proteins in the zona pellucida-induced acrosome reaction. Within a group of several ZP3 receptor candidates, b-1,4-galactosyltransferase is supposed to be involved in the activation of Gi-proteins and the induction of the acrosome reaction. Given the results of the present study, it is doubtful whether the ZP3-mediated AR via G-proteins represents the only acceptable signal transduction pathway. The data presented in this study give new significance to other G-protein-independent concepts such as a p95 tyrosinkinase or Ca2+-permeable ion channels as candidates for signal transduction mechanisms induced by zona pellucida glycoproteins.

Table of Contents

Download the whole PhDthesis as a zip-tar file or as zip-File

For download in PDF format click the chapter title

Titelblatt, Inhaltsverzeichnis, Lebenslauf
Kapitel 1 und 2
Kapitel 3 bis 8
Literaturverzeichnis

More Information:

Online available: http://www.diss.fu-berlin.de/2002/138/indexe.html
Language of PhDThesis: german
Keywords: Spermatozoa; Acrosome-reaction; G-Proteins; PTX-sensitive
DNB-Sachgruppe: 34 Veterinärmedizin
Date of disputation: 28-Jun-2002
PhDThesis from: Fachbereich Veterinärmedizin, Freie Universität Berlin
First Referee: Prof. Dr. Dr. habil. Peter Siegfried Glatzel
Second Referee: Prof. Dr. Günter Schultz
Third Referee: Prof. Dr. Michael F. G. Schmidt
Contact (Author): ulle.severin@web.de
Contact (Advisor): glatzel@vetmed.fu-berlin.de
Date created:02-Aug-2002
Date available:05-Aug-2002

 


|| DARWIN|| Digitale Dissertationen || Dissertation|| German Version|| FU Berlin|| Seitenanfang ||


Mail-Icon Fragen und Kommentare an:
darwin@inf.fu-berlin.de

© Freie Universität Berlin 1999