Presence of Active Hatching Enzyme in the Secretory Granule of Prehatching Medaka Embryos

Secretory granules of hatching gland were isolated from a 0.3 M sucrose homogenate of whole medaka embryos at prehatching stage by differential centrifugation, followed by a Percoll density gradient centrifugation. The obtained preparation was almost free of melanosomes and composed exclusively of the secretory granules of hatching gland (hatching enzyme granules), as judged by morphological as well as enzymological criteria.
The aqueous extracts of the purified secretory granules showed a specific choriolytic activity as high as about 40 times that of a partially purified secretory granule preparation, P_1,000, and represented a single protein band with molecular weight of about 21,000 on SDS-polyacrylamide gel electrophoresis. It was also revealed that a major component of the hatching enzyme preparation (P II–0.3 enzyme, 13) purified from the hatching liquid was identical with the 21,000 molecular weight band.
These results suggest that the hatching enzyme is present in the secretory granules of prehatching embryos in an active molecular form.