Studies of Sparus aurata sperm motility by computer-assisted sperm analysis (CASA).

Aim: Sperm of Sparus aurata, like those of other teleosts, become activated when spawned into the external medium. Several extracellular factors (ions, osmotic pressure, O2/CO2, sperm activating peptides) have been reported to control sperm motility activation. These factors act on the flagellar motile apparatus, the axoneme, through signal transduction across the plasma membrane and determining the dynein-mediated sliding of the axonemal outer-doublet microtubules through protein phosphorylation. In the present study we have investigated the role of different proteins involved in this signal transduction cascade using cryopreserved sperm of Sparus aurata. Methods: Studies have been performed by using cryopreserved sperm (Fabbrocini et al., Cryobiology 40:46-53, 2000). To investigate the role played by different proteins involved in the signal transduction cascade we evaluated: (1) the effect of specific protein inhibitors on sperm motility (assessed by CASA), (2) quantitative and qualitative evaluation of phosphorylated proteins (tyrosine-phosphorylated, serine-phosphorylated and threonine-phosphorylated) by western blot analysis in activated and non-activated sperm. Results: Results obtained indicated that: (1) calcium and potassium were not involved in sperm motility activation; (2) osmolality values <1,000 mOsm/Kg inhibited the sperm motility activation; (3) inhibition of Adenylyl Cyclase by 500 μM MDL-12330A hydrochloride and Protein-Kinase A (PKA) by 50 μM U73122 prevented the sperm activation; (4) inhibition of Tyrosine-kinase (by 100 μM AG18) and CAM-Kinase (by 50 μM KN93) were ineffective on sperm activation, but determined changes in sperm motility parameters. Particularly, AG18 significantly decreased the Curvilinear Velocity (VCL), while KN93 significantly decreased both Curvilinear Velocity (VCL) and Straight Line Velocity (VSL). Conclusion: In Sparus aurata sperm, an important factor controlling sperm motility activation was the osmolality of the external medium. The osmotic shock induces sperm activation by cAMP/PKA signaling pathway. In addition also Tyrosine-kinases and CAMkinases seems to be involved in the control of sperm motility.