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. |