RADIOAUTOGRAPHIC STUDIES OF KERATIN FORMATION

Abstract

Young mice of strains C57 or A were used in studies of the distribution of S35.labeled 1-cystine, p32-labeled phosphate, C14-labeled carbonate and protein-hydrolyzate in various tissues. Radioautographs of skin made 1, 2, 4, 6, and 8 hours after radiocystine injection (approximately 3 [mu]c/g body weight) showed an appreciable uptake of isotope by the keratogenous zone 1 hour after administration, with an increase in intensity of the radioautograph and an increase in the radioactive region of the hair follicle up to 6 hours after injection. In skin and tongue sections made 1 or more days after injection, the radioactive zone followed the growth of the hair shaft or conical papilla. Treatment of skin sections with various agents to determine mode of binding of the cystine in the keratogenous zone indicated that the cystine was not bound to side groups by disulfide linkages but was assumed to be incorporated into the main chains of the keratin molecule by peptide linkages. Eight hours after injection of approximately 40 [mu]c of C14-labeled algal protein-hydrolyzate into a young mouse in anagen, skin sections produced lightly positive radioautographs after exposure to the film for 8 or more weeks. Activity was seen in the hair bulb, extending up the hair follicle into the keratogenous zone. Similar results were obtained in another mouse 6 weeks after injecting 300 [mu]c of radiocarbonate (after 9 weeks exposure to film). Eight hours after injecting 200-500 [mu]c P32/mouse, the amount of radioactivity was high in the hair bulb and decreased towards the keratogenous zone, and 18 - 24 hours after injection there was a generalized uptake of P32 by the lower part of the follicle including the keratogenous zone. In order to study the distribution of radiocystine in the female genital tract under various hormonal stimuli, 6 5-months-old virgin female mice, castrated for 90 days, were injected intramuscularly daily for 3 days with 0.01 mg estradiol in sesame oil. Eight hours before sacrifice 3 [mu]c of radiocystine per g body weight were injected subcutaneously. Segments of vagina and uterus were imbedded in paraffin and exposed to film for 3 weeks. Statistical analyses of the mean grain counts for paired regions showed that the radiocystine concentration in the uterine lining or glandular epithelium was not significantly different from that in the vaginal stratum basale, but the vaginal stratum spinosum had significantly more cystine than other regions. Radioactivity was least in the stratum basale and greatest just below the keratin layer of the vagina. Autographs of the vagina 4 hours after injection of radiocystine showed a distribution of cystine similar to that seen 8 hours after injection. Twenty-four hours after injection the basal region of the keratin layer became radioactive, and vaginal smears at 48 hours showed considerable activity. The mechanism of keratin formation is discussed in the light of these results.