Molecular cloning and expression of ovarian cathepsin D in seabream, Sparus aurata

In marine fish producing pelagic eggs, the acquirement of buoyancy by the e ggs through the hydration process is a key event of reproduction; moreover, the yolk proteolysis, which leads to buoyancy, seems to affect the fertili ty and survival of the spawned eggs. Recently we demonstrated that cathepsi n D is the aspartic protease responsible for this intraoocytic processing o f vitellogenin into yolk proteins. In the present study, we isolated, clone d, and sequenced the cDNA encoding cathepsin D and studied expression of th e message by Northern blotting and whole-mount in situ hybridization. The full-length seabream cathepsin D cDNA is 1837 base pairs long, encoding a protein of 400 amino acids (aa) consisting of a signal peptide of 19 aa, a prosequence of 44 aa, and a mature peptide of 336 aa. An absolute sequen ce conservation at the aspartyl residues (+33 and +221) was found, and ther e are three potential N-glycosylation sites at +70, aa +189, and aa +274. T he aa sequence of seabream cathepsin D reveals a high degree of sequence si milarity with cathepsin D mRNAs from other organisms (73% sequence homology to mouse and rat, 72% to human and trout, 69% to chicken, 66% to pig, and 65% to Xenopus). The cathepsin D mRNA in floating eggs was present as a single band that was approximately 1.9 kilobases (kb) in size, while in the sinking eggs there were several fast-migrating bands (size range 1.3-0.2 kb). Whole-mount hybr idization was used to investigate transcription of cathepsin D in the devel oping embryo; during the hatching period, cathepsin D mRNA-positive cells w ere distributed in a wide region between the trunk and the tail, and in the ventral region over the yolk ball. The highest levels of cathepsin D enzym atic activity were found in the sinking eggs and during the hatching period of embryonic development. These data suggest that cathepsin D can be considered a possible marker for egg quality.