Cloning of a quail homologue of hatching enzyme: its conserved function and additional function in egg envelope digestion

The aim of the present study was to reveal molecular entities participating in the digestion of the egg envelope in the Japanese quail, Coturnix japonica. We isolated a 1,510-bp cDNA from extraembryonic tissues of developing embryos and designated it quail hatching enzyme (QHE) cDNA. The QHE cDNA was found to code a protein molecule comprising an astacin protease domain in the N-terminal half and a complement subcomponents C1r/C1s, Uegf, Bmp1 (CUB) domain in the C-terminal half. A phylogenetic analysis showed that QHE belonged to the hatching enzyme group and was distinct from other proteases in the astacin family. Northern blotting and in situ hybridization demonstrated that expression of the QHE mRNA occurred twice during the development: first in ectodermal cells of the yolk sac on days 0–5, then in those of the albumen sac on days 8–13. Zymography revealed that proteolytic activity in extracts of days 3–4 and 9–12 embryos appeared at the position of 40 kDa. Immunoblotting tests showed that anti-QHE antiserum stained a 40-kDa molecule in extracts of day 3 area vitellina. Anti-QHE antibody stained the ectodermal cells of the area opaca on days 0–1, those of the area vitellina of the yolk sac on days 2–5, and those of the albumen sac on days 9–12. The temporal and spatial expression pattern of QHE mRNA was closely associated with digestion of the vitelline membrane occurring on days 1–4, and with that of the egg white on days 9–12.