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In adult female rainbow trout (Oncorhynchus mykiss), an immunocytochemical study of the oocyte has shown that a proteolytic enzyme, cathepsin D, is localized in multivesicular bodies (MVB) which begin to differentiate before the phase of vitellogenesis. Estrogens cause the liver to synthesize the protein vitellogenin (VTG), which then enters the systemic circulation. During vitellogenesis, endocytosed VTG is co-localized with cathepsin D in the MVB.Assays of oocyte cathepsin activities have shown that the only proteolytic activity of note is that of cathepsin D. Along with the yolk proteins derived from VTG, this enzyme will be included in the central coalescent yolk mass.With the installation of the yolk syncytial layer and the surrounding yolk vascular system, embryonic development is characterized by high lysosomal activity, especially in the syncytial layer. At this level, proteolytic activity concerns a cathepsin L, secreted as a proenzyme. We propose the hypothesis that cathepsin D, along with the yolk proteins in the yolk globules that break away from the yolk mass in the vitellolysis zone, activates the proenzyme and leads to protein degradation, which then becomes very rapid. 0 1994 Wiley-Liss, Inc.Embryonic development in oviparous vertebrates is related to the size of egg reserves, namely vitellus or yolk. In fish as in amphibians, protein accumulation is the essential process involved in oocyte growth. The sequence of events contributing to protein "vitellogenesis" has been thoroughly described and illustrated in several reviews (Wallace, '85; Wallace and Selman, '90). In the rainbow trout, a salmonid teleost, large quantities of vitellogenin (VTG), a phosphoglycolipoprotein, are synthesized by the liver cells of mature females under estrogenic control. The VTG transported by the blood flow is incorporated in oocytes by receptor-mediated endocytosis (Tyler et al., '88, '90, '91). Cytoplasmic translocation in the oocyte is accompanied by the proteolytic processing of VTG, which undergoes specific cleavage into yolk proteins: lipovitellin (LV), phosvitin (PV), and possibly others.Multivesicular bodies (MVB) are differentiated during the initial phase of oocyte growth. Their role in the processing of endocytosed VTG in amphibians has been proposed by Wall and Patel ('87a).MVB are considered as potential contributors to hydrolase activity. Passage via MVB is required to cleave VTG to yolk proteins, as well as for their subsequent distribution to mature "heavy yolk platelets" (Wall and Patel, '87b).The first step of the present study was a n at-tempt to delineate specific yolk processing pathways in the trout oocyte by means of a n immunocytochemical study and by measuring lysosomal activities. It was found that cathepsin D is co-localized with VTG in the MVl3, which have become pre-yolk bodies. Cathepsin D is subsequently found in the yolk bodies and in the central yolk mass resulting from their coalescence, along with yolk proteins arising from VTG hydrolysis. Cathepsin D is the only protease act...

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Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC(50) values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.