Delineation of downstream signalling components during acrosome reaction mediated by heat solubilized human zona pellucida

Abstract

Background: Human egg is enveloped by a glycoproteinaceous matrix, zona pellucida (ZP), responsible for binding of the human spermatozoa to the egg and induction of acrosomal exocytosis in the spermatozoon bound to ZP. In the present manuscript, attempts have been made to delineate the downstream signalling components employed by human ZP to induce acrosome reaction.

Methods: Heat-solubilized human ZP (SIZP) was used to study the induction of acrosome reaction in capacitated human spermatozoa using tetramethylrhodamine isothiocyanate conjugated Pisum sativum agglutinin (TRITC-PSA) in absence or presence of various pharmacological inhibitors. In addition, intracellular calcium ([Ca2+]i) levels in sperm using Fluo-3 acetoxymethyl ester as fluorescent probe were also estimated in response to SIZP.

Results: SIZP induces acrosomal exocytosis in capacitated human sperm in a dose dependent manner accompanied by an increase in [Ca2+]i. Human SIZP mediated induction of acrosome reaction depends on extracellular Ca2+ and involves activation of Gi protein-coupled receptor, tyrosine kinase, protein kinases A & C and phosphoinositide 3 (PI3)- kinase. In addition, T-type voltage operated calcium channels and GABA-A receptor associated chloride (Cl-) channels play an important role in SIZP mediated induction of acrosome reaction.

Conclusions: Results described in the present study provide a comprehensive account of the various downstream signalling components associated with human ZP mediated acrosome reaction.

Figure 1

SIZP mediated intracellular calcium release profile in capacitated human sperm in presence or absence of pharmacological inhibitors of L- and T-type VOCCs. Fluo-3/AM labelled capacitated human sperm (1 × 10⁶) were pre-incubated with either L- type VOCC inhibitor, Verapamil (10 μM) or T-type VOCC inhibitor, pimozide (20 μM), for 10 minutes and then exposed to SIZP (~5 zonae) at 0 seconds. Changes in intracellular calcium levels (nM; y-axis) have been plotted as a function of time (seconds; x-axis) for ~10 min. Blue line represents SIZP mediated calcium increase profile whereas pink and green lines represent SIZP mediated calcium release profile after prior treatment of labelled sperm with Pimozide and Verapamil respectively. Values are presented as Δ [Ca²⁺]_i obtained by subtracting the respective mean resting [Ca²⁺]_i values preceding SIZP addition from the peak [Ca²⁺]_i.

Figure 2

SIZP mediated induction of acrosome reaction in capacitated human sperm in presence or absence of pharmacological inhibitors of L- and T-type VOCCs. Capacitated human sperm (1 × 10⁶) pre-incubated for 10 min at 37°C in presence of 5% CO₂, with L-type VOCC inhibitors (Nifedipine, Verapamil), T-type VOCC inhibitors (Pimozide, Mibefradil) or respective vehicle controls (Panels 2a and 2b respectively) were subsequently incubated with SIZP (~5 zonae). After 60 min incubation at 37°C in presence of 5% CO₂, sperm samples were analyzed for their acrosomal status by TRITC-PSA staining as described in Methods. Y-axis represents percent induction of acrosomal exocytosis calculated by dividing the number of acrosome-reacted sperm by total number of sperm counted and multiplied by 100. Values are Mean ± SEM of six different experiments using semen samples from at least six different male donors. Calcium ionophore A23187 (10 μM) was used as a positive control in the experiment. Asterisk (*) represents p < 0.05 with respect to the solvent control and hash (#), p < 0.05 with respect to SIZP mediated induction of acrosome reaction.