Bicarbonate, calcium ions, hydrogen peroxide and trypsin modulate activation of Anopheles gambiae sperm motility and protein tyrosine phosphorylation

With the increasing concern of potential loss of transgenic mosquitoes which are candidates as new tools for mosquito-borne disease control, methods for cryopreservation are actively under investigation. Methods to cryopreserve Anopheles gambiae sperm have recently been developed, but there are no artificial insemination or in vitro fertilization tools available. As a step to achieve this, we sought to identify a suitable medium for in vitro incubation of An. gambiae sperm and to tease out critical components that are involved in the sperm motility activation process. Using two cell viability assays, we identified the Biggers-Whitten-Whittingham (BWW) medium as suitable for in vitro incubation of An. gambiae sperm isolated from testes. We then modified the medium for motility assays by testing different HCO₃^- and Ca²⁺ concentrations. Our results show that there is an HCO₃^- and Ca²⁺ concentration-dependent activation of An. gambiae sperm motility. We further demonstrated that H₂O₂ can be produced by the testes in vitro and that the addition of 5.3 μM of H₂O₂ to the medium improves sperm motility and increases protein tyrosine phosphorylation in An. gambiae. Finally, we show a dose-dependent activation of sperm motility by the addition of trypsin to the medium and more than a 2-fold increase in sperm motility when modified BWW (mBWW) medium is supplemented with H₂O₂ and trypsin. Our in vitro results suggest that protein tyrosine phosphorylation, intracellular ionic influx, intrinsic production of H₂O₂ and trypsin-like proteases play a vital role in signal transduction that leads to the activation of An. gambiae sperm motility.