Enzymatic Activity in the Chorion for Hatching in the California Grunion

Leuresthes tenuis is a small, silverside fish that spawns on the beaches during some of the highest tides of the summer months. Of the many unique traits to the species including that the eggs develop fully out of water to the point of hatching competence but will not hatch until presented with an environmental cue, which causes them to hatch in less than a minute. The purpose of this study is to better understand the role of enzymes called chorionases, which act to break down the chorion (egg membrane). I hypothesize that the chorion begins to weaken in this species when it is hatching competent but before it receives the stimulus to hatch. Unlike most organisms, the grunion embryo reaches hatching competence when it is fully developed, once it reaches competence, it stalls development and waits for an environmental cue. Before this, the egg is not hatching competent and will not hatch even if the trigger is there. The fact that it hatches so quickly could be evidence that an enzyme is acting to break down the chorion and this is what I hypothesize causes hatching competence. Some related fish have two chorionases while some only have one. To see whether one or two enzymes act in hatching in the grunion, I made a solution of the hatching enzyme, concentrated it, ran both SDS-PAGE and native-PAGE gels to separate out the protein by size, and cut out and sent those bands away for sequencing. To test whether the egg weakens upon hatching competence, I measured change in diameter of eggs that were pressed under a weight. We found no significant difference in pliability between the two groups of eggs using this methodology. If there are two enzymes, one may be acting before the other. If there is only one enzyme, another mechanism is at play