RUMBLE, J.*; MARTIN, K.; VANDERGON, T.: Choriolytic enzyme in the California grunion fish, Leuresthes tenuis

The grunion Leurethes tenuis, a southern California fish, has the only known vertebrate eggs that do not hatch unless presented with a mechanical trigger. Grunion fish spawn terrestrially on the spring tide, leaving their eggs to incubate in the sand. Although the embryos are ready to hatch by the next spring tide, they can delay hatching until waves of a succeeding tide are high enough to wash them out to sea. Embryos can remain in the eggs, ready to hatch, for several additional weeks. Agitation in seawater by waves is the mechanical trigger that stimulates the eggs to hatch, and upon stimulation, hatching is completed rapidly, usually within a few minutes. The hatching process of grunion eggs is unique, and so it is uncertain whether the mechanisms involved are different from those of other fish. In most aquatic eggs, a choriolytic enzyme aids the hatching process. This enzyme, called chorionase because it digests the chorionic membrane surrounding the egg, is a protein that is highly conserved through many phyla. Using biochemical and molecular techniques, we found evidence for the presence of a hatching enzyme in the grunion eggs. A crude enzyme extract was obtained for use in assays on the effects of different salinities on hatching success, and for colorimetric assays of protein hydrolysis over time. Using redundant DNA primers based on highly conserved chorionase sequences, a fragment of the grunion chorionase mRNA was amplified by reverse transcription PCR, isolated, and cloned. We conclude that not only do the grunion have a hatching enzyme, but it is very similar to the zinc endopeptidases of the astacin family. This work was supported in part by NSF DBI-99-87543.