Immunocytochemical localization of keratins, associated proteins and uptake of histidine in the epidermis of fish and amphibians.

Keratinization and the role of histidine in some species of fish and amphibians have been analyzed by immunocytochemistry and autoradiography. In cartilaginous and bony fishes, staining of acidic (AE1-positive) and basic (AE3-positive) keratins was strong and their distribution patterns were uniform in all epidermal layers. The AE2 antibody (for keratins K1 and K10 that are typical for keratinization) did not produce any positivity. This was also observed in lungfish epidermis but the AE2 antibody often produced some positivity in the more keratinized layers. In the axolotl (urodele), that is adapted to aquatic conditions, as well as in other species of urodele (newts) that are more adapted to terrestrial conditions, the same pattern was present as in fish. In the latter, the AE2 antibody non-specifically stained all epidermal layers. In more terrestrially-adapted anurans (frog and toad) AE1 immunopositivity was mainly found in basal layers, the AE3 antibody stained the entire epidermis, and AE2 immunopositivity was often localized in the external layers of the epidermis. This pattern resembled that in the epidermis of amniotes. Administration of tritiated histidine to goldfish epidermis showed that at 1, 4 and 24 h after injection, labelling was low and uniformely distributed in all epidermal layers. In newt and toad epidermis, histidine labelling increased from 1 to 4 h after injection but tended to remain evenly distributed throughout the epidermis. However, from 4 up to 24 h after injection, labelling became concentrated in the upper intermediate and replacement layers, suggesting that turnover proteins were produced. Histidine was probably converted into other metabolites at 4-24 h after injection. Whether the newly synthetized proteins were a form of keratin or a specific histidine-rich protein remains to be determined biochemically. Uptake of tritiated thymidine in newt epidermis indicated that keratinocytes move into the uppermost stratum intermedium within 4 days, and reach the replacement layer in approximately 6 days. Taken together, the data obtained with tritiated histidine and thymidine suggest that most histidine is taken up in the upper intermedium and replacement layer at 4-24 h after injection. Neither a granular layer nor crossreaction with filaggrin and loricrin were observed in fish and amphibian epidermis. Although the cell membrane of superficial corneous cells of amphibian epidermis became thicker, the absence of loricrine immunolabelling suggests that a cell corneous envelope containing this protein is not present or undetectable.