The core challenge of the fertilization process is one of pathfinding: how do motile sperm cells locate the immotile oocyte? In some species, this process is well studied, e.g. sea urchin and human. Both species employ chemotaxis—the orientation along a three-dimensional gradient of a chemoattractant—to facilitate pathfinding of sperm towards the oocyte. But the molecular implementation differs considerably between the two species. Overall, great variation is found in fertilization processes even between closely related species. Zebrafish is a widely used vertebrate model system, yet very little is known about how their sperm function. In my thesis, I approached the question of pathfinding in zebrafish sperm from several angles. First, I performed extensive bioinformatic studies in jawless, cartilaginous, and bony fishes to elucidate the presence or absence of several components of the signaling cascades involved in pathfinding. I showed a fine- grained pattern of many independent gene losses for the CatSper channel complex and soluble adenylate cyclase (sAC), both of which are considered essential for sperm motility in species as distantly related as sea urchin and human. In particular, both CatSper and sAC are clearly absent in zebrafish. Synteny analysis between the genome of zebrafish and the closely related goldfish, which possesses CatSper, showed chromosomal breakpoints as likely origin of the loss for all four subunits of the CatSper channel. By comparison with related species I could deduce that CatSper was lost at the origin of the family Danionidae, whereas sAC was already lost earlier, at the origin of the order Cypriniformes. The absence of these proteins in zebrafish means that signaling in zebrafish sperm must function in a manner yet unknown. Second, I investigated the proteome of the egg envelope (chorion) using mass spectroscopy to identify potential chemoattractant candidates for haptotaxis (orientation along a two-dimensional gradient of a tethered chemoattractant). Zebrafish chorion possess a micropyle, an opening that allows the sperm access to the oocyte. I found no difference in the proteome between chorion samples that contained the micropyle and those that did not. The main components of the zebrafish chorion are zona pellucida (ZP) proteins. I identified 20 ZP proteins in the proteome and assigned their respective subfamilies. Interestingly, ZPB and ZPC proteins are present, which are required for the acrosome reaction in mammals, although zebrafish possess a micropyle and therefore do not use the acrosome reaction. Third, I established a baseline of motility behavior and internal ion changes of activated but unstimulated zebrafish sperm through dark-field and fluorescence microscopy. Swimming paths vary greatly in curvature, although the majority displays lower curvature. Cytosolic calcium increases during initiation of motility, via release from internal stores. The sperm also acidify, in contrast to sea urchin, human, and mouse, where the sperm alkalizes. Next, I tested oocyte-derived stimulus on the sperm, but found no indication that zebrafish sperm employ chemotaxis. Last, I discovered regularly spaced, dye-accumulating structures in the sperm flagellum. They may correspond to vesicular bodies of unknown function described so far only morphologically. My results form a basis for future studies to fully elucidate signaling in zebrafish sperm
