Homogenates of hatching gland explants were fractionated by means of different electrophoretic techniques, and the fractions analyzed for proteolytic activity and for their capacity to affect the envelope of the eggs. Agar gel electrophoresis resulted in two fractions that were able to digest the zona radiata interna, and a third fraction that caused a significant swelling of the egg envelope. All three fractions were proteolytically active. Agarose gel and polyacrylamide gel electrophoresis gave only two fractions that showed proteolytic activity and were capable of digesting the zona radiata interna. The presence of these two fractions may imply that hatching enzyme is stored as proenzyme in the hatching gland granules. Swelling of egg envelopes was also observed during envelope digestion by thermolysin, pronase, and 0.5-1.0 N NaOH. Moreover, breakdown by hatching enzyme and pepsin under suboptimal conditions showed a slight swelling of the envelope. These results demonstrate that substances capable of solubilizing the zona radiata interna may cause envelope swelling. The swelling of the envelopes probably represents an intermediate phase in the proteolysis of the zona radiata interna. The agar gel electrophoresis fraction of hatching gland homogenates that causes swelling may contain a physicochemically different form of hatching enzyme.

Hatching gland cells (HGCs) of the teleost, Oryzias latipes, have been isolated. The optimum condition is mechanical dissociation of the buccal tissues in Ca-free saline containing 15% bovine serum albumin (BSA). The cell suspension is fractionated by centrifugation through 30-33.3% Percoll TM containing 5% BSA. The isolated HGCs are washed in 5% BSA. BSA in the latter two solutions can be omitted when a swing-out rotor is used. The recovery of total and viable HGCs was about 74% and 45%, respectively. The purity was 75-85%. In a typical experiment, 12 embryos were dechorionated manually with watchmaker's forceps in Ca-free Yamamoto's saline (7.5 gm NaC1,200 mg KCl, and 20 mg NaHC03 in one liter distilled water). The buccal tissues including hatching gland, gill cartilage, buccal epithelium, and adhering mesenchyme were explanted with forceps and tungsten needle. Xanthophores must be removed as thoroughly as possible because they cannot be separated from the HGCs in the system used.Explants were once washed in Ca-free saline for 15 min and the cells dissociated mechanically in 0.5 ml of the same saline containing various concentrations of bovine serum albumin (BSA, fraction V, Serva, Heidelberg) by repeated passage through a blunt syringe needle (0.7 mm in width). The cell suspension was then layered on 0.8 ml Percoll TM (Pharmacia, Uppsala) in Ca-free saline with or without BSA in a 2-ml siliconized tube and centrifuged at about 70 g for 15 min at 4°C. HGCs were collected as a pellet at the bottom, whereas the other cells remained near the surface of the Percoll. The pellet was washed once in 1 ml Ca-free saline with or without BSA and centrifuged under the conditions mentioned above. Cell counting was performed with a hemocytometer and the percentage of recovery was determined by assuming that each embryo has 930 HGCs (average of 10 embryos; SD = 80).Cells were considered viable when the plasma membrane was not wrinkled (Fig. 1). RESULTS AND DISCUSSIONA sheet of HGCs sandwiched by two layers of epithelial cells forms the wall of the buccal cavity (Yamamoto, '63; Yamamoto e t al., '79).Nono Yoshizaki is now w~t h the