Influence of the ionic and protein environment on sperm motility activation in the European eel

Zusammenfassung

In general, fish spermatozoa are immotile in the testis. Movement is activated due to the osmotic shock (hypo- or hyperosmotic, depending on fish origin: freshwater or sea water species) experienced when they are released into the external medium. However, there is no consensus regarding the mechanisms that occur after activation. The aim of this project was to study the importance of the environmental ions and proteins on the activation of European eel sperm, and to apply this knowledge to the improvement of sperm quality preservation. Our results demonstrated that there was a notable reduction in sperm motility when either Na+ or K+ was removed from the seminal plasma, but not when they were removed from the activation media. Therefore, our results demonstrated that the presence of Na+ or K+ in the seminal plasma is necessary for the preservationof sperm motility in European eel. However, the presence of Na+ or K+ in the activation media is not essential for the initiation of sperm activation. In contrast, the presence of the ion Ca2+ in the seminal plasma (or the activation media) was not essential for sperm motility activation in this fish species. Moreover, several authors have hypothesised that the hyperosmotic aquatic environment causes an efflux of water through the spermatozoa membrane, and this efflux causes an increase in the intracellular ion concentration (due to the decrease in cellular volume). However, this hypothesis has never been proven. In this study, sperm size (sperm head area) was studied pre- and post-activation in sea water, and in different conditions. For the first time in a marine fish, a significant decrease in sperm head area post activation in sea water was demonstrated. Also the results of this thesis show a notable reduction in sperm head area when either Na+ or K+ was removed from the seminal plasma, as well as a marked reduction in motilityed. Thus, our results demonstrate that the presence of K+ and Na+ in the seminal plasma is important for the preservation of sperm motility in the European eel, at least in part by maintaining the right sperm cell volume in the quiescent stage. In the this study, a method for the quantitative analysis of the intracellular [Na+ ] ([Na+ ]i) and pH (pHi) levels of eel sperm was created, and used to study the variations in [Na+ ]i (for first time in a marine fish) and pHi during motility activation. The pHi in the quiescent stage was 1.3 units lower than the pH of the seminal plasma, indicating that a H+ gradient exists in the quiescent stage, with higher [H+ ]i levels than those found in the seminal plasma; an important difference for sperm motility. In contrast, the results show that Na+ levels are the same both outside and inside the spermatozoa in the quiescent stage. Our results demonstrate that for sperm motility activation to be successful, the pHi should be lower than the seminal plasma pH and lower or equal to the activation media pH. The absolute [Na+ ]i concentration of marine fish spermatozoa was 1.5 times higher after motility activation than in the quiescent stage, and this increase in intracellular Na+ after activation could be caused both by a cell volume decrease and by the influx of external Na+ . Regarding the ion Ca2+, our results strongly suggest that an increase in intracellular [Ca2+ ] SUMMARY 2 ([Ca2+]i) is not a pre-requisite for the initiation of sperm motility in European eel sperm. Nevertheless several sperm velocity parameters (VCL, VSL, VAP) decreased in the absence of Ca2+ , thus supporting the theory that Ca2+ has a modulatory effect on sperm motility. In addition, it appears that th