RESUMO
Fish embryonic hatching glands (HGs) secrete choriolysin-zymogens, which when activated degrade the chorion, allowing larval exit. Thus, hatching encompasses two dissimilar choriolysin-processes: pre-choriolysis including activated choriolysins accessing the perivitelline space (PVS), followed by choriolysis. Discovery of serine-proteolytic zonase in Atlantic salmon hatching fluid (HF) raises questions concerning its participation in hatching. This work aims to identify salmon choriolysins, and evaluate their role and that of zonase during hatching. Precocious salmon hatching occurs under alkaline conditions, producing an HF containing similar metallo- and serine- proteolytic activities. Purified zonase is selectively inhibited by peFabloc, whose MW (580 Da) allows diffusion through the chorion into the PVS. Without apparent toxicity, brief peFabloc-pretreatment (2 mM) of salmon eggs reduced precocious hatching substantially, compatible with a zonase-relevance for hatching. Atlantic salmon differed from other fishes since their HGs were not immuno-stained by polyclonal antibodies against pike choriolysins. However, cloning and sequencing experiments revealed a single low choriolytic enzyme (LCE) of 69% identity to pike LCE. Similar experiments with high choriolytic enzymes (HCEs) revealed two types (HCE-1 and HCE-2) with respectively 71% and 91% identity to pike HCE-1 & HCE-2. In situ hybridization revealed typical HGs. However, zebrafish-choriolysis is achieved by HCE-class choriolysins alone. Another zebrafish choriolysin (HE2) was not expressed. Peptide-bond hydrolysis by non-choriolytic zonase mimicks HCE-action generating hydrophilic sites for LCE-choriolytic catalysis. Ultimately, precise definitions of choriolytic and pre-choriolytic catalysis requires developmental genetics. Our data are compatible with a complex pre-choriolytic hatching-stage in Atlantic salmon, before LCE-choriolysis degrades the chorion.
