ABSTRACT
Cleavage-stage embryos of the neotenic urodele Ambystoma mexicanum are surrounded by a fertilization envelope and four macroscopic jelly coats termed J1 (innermost) through J4 (outermost). In sections prepared for light microscopy, each of the jelly layers stained with protein stains and the periodic acid-Schiff's reagent, but only J1 stained with alcian blue at pH 2.5. These results suggest that each layer consists of proteins and glycoproteins and that J1 uniquely contains some sulfate esters. Only J4 was solubilized with alkaline mercaptan treatment in situ, however, the isolated inner jelly complex (J1 , J2 and J3 ) was easily dissolved in this reagent suggesting that solvent access is impaired in situ. A single alcian blue-staining component plus one protein-staining component were detected on reducing polyacrylamide gel electrophoresis of outer jelly (J4 ). In the inner jelly complex (J1 , J2 , J3 ), two protein-staining components were detected and no alcian blue-staining components were observed. A predominant polypeptide of 110,000 molecular weight was detected and purified to homogeneity on reducing and denaturing gels of the inner jelly complex. Amino acid analysis of the polypeptide demonstrated a slightly higher fraction of acidic over basic amino acids (Glx+Asx=18.1 mole%vs. Arg + Lys = 11.7 mole%). The N-terminal amino acid was Glu and the sequence of the first eleven amino acids was determined.
ABSTRACT
Larval stomach development was studied in the obligate carnivorous larva of the frog Lepidobatrachus laevis. Pepsin producing cells of the larval stomach were identified using rabbit anti-porcine pepsin and immunohistochemical techniques. Pepsin production was detected at a very early stage of development (stage 24: during opercular development) when the larvae were first competent for feeding. Peptic activity in isolated larval stomachs was demonstrated in a microassay using acid denatured hemoglobin at pH 1.7. The total activity per stomach increased 5,400 fold through the beginning of metamorphosis and the specific activity increased 345 fold through the same period. Electrophoretic analysis of the larval pepsinogens, using a caseinolytic assay revealed the presence of one major pepsinogen at stage 24; two additional isozymes were observed during later larval development. The molecular weight of the isopepsinogens was 34,800.
ABSTRACT
Eggs and cleavage-stage embryos of the frog Lepidobatrachus laevis are encased by 3 µm thick vitelline/fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). Based on light and transmission electron microscopy, J1 had a dense reticular appearance whereas J2 had a laminar structure. Direct dissolution of the jelly coats was accomplished by reduction of disulfide bonds with 0.08 M 2-mercaptoethanol at pH 10. Soluble jelly preparations were uncontaminated with nucleic acid (A280 /A260 =1.44) and yielded an average of 150 µg protein/egg or embryo (n=5). The biochemical composition of the jelly coats in unfertilized eggs was different from that in embryos. When examined via gel permeation chromatography, soluble jelly from unfertilized eggs contained macromolecules which were markedly larger and more heterogeneous (earlier eluting and broader peaks) than jelly from embryos. Differences in the components of jelly from unfertilized eggs and embryos were also observed by electrophoresis, however, a 29,700 molecular weight glycoprotein chain was common to both jelly preparations. The electrophoretic pattern of jelly obtained from parthenogenetically activated eggs was identical to that of unfertilized eggs, therefore the fertilization-associated changes are not due to the exclusive action of cortical granule products.
