View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector HISTOCHEMICAL STUDIES ON THE SKIN II. THE ACTIVITY OF THE SUccINIC, MALIC AND LACTIC DEHYDROGENASE SYSTEMS DURING THE EMBRYONIC DEVELOPMENT OF THE SKIN IN THE RAT* KEN HASHIMOTO, M.D., KAZUO OGAWA, M.D., Ph.D. AND WALTER F. LEVER, M.D. As a continuation of our histochemical studies After an incubation for 3 to 12 hours at 37° C. on the skin (1), the changes in the succinic, maliethe sections were removed from the incubation medium, rinsed briefly in 0.1 M Sorensen's phos- and lactic dehydrogenase systems during thephate buffer, pH 7.6, and fixed in neutral formalin embryonic development of the skin have beenfor 2 to 3 hours at room temperature. investigated. Practically no work has been done In some instances, quinone compounds, such as as yet on the activity of any of these dehydroge-menadione (8, 9), phenanthraquinone (9) or Co- enzyme Q7 (8, 10), were added to act as a mediator nase systems during the embryonic developmentin the electron transfer between the succinic of the skin; and only the succinie dehydrogenasedehydrogenase and the tetrazolium salts. The activity has been investigated in adult skin byfinal concentration of menadione as well as of several authors (2—6). phenanthraquinone was 0.1 mg. per ml. of in- cubation medium. MATERIALS AND METHODS For controls were used a substrate-free medium and also the incubation medium containing, in Animal Material. Twenty-five rats of theaddition to sodium succinate, sodium malonate as Wistar strain were used. Twelve of these werecompetitive inhibitor in the same final molar embryos (3 each on the 12th, 14th, 16th and 18thconcentration as sodium succinate. days of gestation), 2 were new-born (the period Histochemical Demonstration of Malic (MDS) of gestation varied from 19 to 21 days), 3 wereand Lactic (LDS) Dehydrogenase Systems. The ten-day-old, 3 were fifteen-day-old and 5 weremethod of Nachlas et at. (11) was used to demon- adults. The adult rats weighed approximatelystrate the MDS and LDS. Diphosphopyridine 100 g. New-born and older rats were killed by anucleotide (DPN) was used as coenzyme. After 2 blow on the neck without anesthesia. Hair washours of incubation at 37° C. the sections were shaved off the back, and skin specimens weretaken out of the incubation medium, rinsed taken from there and cut into small square pieces.briefly in distilled water and fixed in neutral These pieces were immediately immersed for about formalin for 2 to 3 hours at room temperature. five minutes into ice-cold 0.1 M Sorensen's phos- In some case, quinones, such as menadione, phate buffer and then used for frozen sections.phenanthraquinone or Coenzyme Q, were added (The buffer had a pH of 7.6 for the study of theto the incubation media, just as had been done in succinic dehydrogenase system and a pH of 7.4the case of demonstrating the activity of the for the study of the malic and lactic dehydro-SDS. A substrate-free incubation medium was genase systems). In embryos, skin was removedused as a control. Also, incubation media con- from the back while they were still alive. Frozentaining iodoacetic acid as sulfhydryl inhibitor in sections, approximately 20in thickness, werea final concentration of 0.01 M, 0.05 M or 0.1 M made from the fresh skin specimens. were used. Histochem feat Demonstration of the Succinic With the methods described above, pink repre- Dehydrogenase System (SDS). Ogawa and Zim-sents a weak reaction of the enzymes, purple to merman's method (7) was employed to demon-blue a moderate reaction, and dark blue a strong strate the succinic dehydrogenase system. As areaction, although there have been objections to rule, nitroneotetrazolium chloride (nitro-NT)such a simple interpretation (12). A pink color was used as an electron acceptor. Occasionally,also occurs as the result of a non-enzymatic dis- however, nitro-blue tetrazolium (nitro-BT) wassolution of formazan granules (that have formed used, instead. in true reaction sites) in lipids, for example in sebaceous cells and subcutaneous fat cells (12, 13). * From the Department of Anatomy, Kyoto University School of Medicine, Kyoto, Japan, RESULTS and the Department of Dermatology, Tufts Uni- versity School of Medicine, Boston, Massachu- Succinic Dehydrogenase System (SDS). In 12-day setts. old embryos (Figs. 1 and 2) the epidermis con- This investigation was supported by grants from the Rockefeller Foundation (GA-MNS-60236), the sisted of an inner layer, the stratum germinati- Dome Chemicals Inc., New York, N. Y., and thevum (StGer), and an outer layer, the periderm. Duke Laboratories, Inc., South Norwalk, Con- necticut. The cytoplasm of the large vesicular cells in Received for publication August 3, 1961. these two layers stained diffusely pink, while 21 22 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY SOS LOS & MOS were sparsely distributed through the dermis and p.rtd 12-DAY contained small positive granules. EMBRYO In 14-day old embryos (Figs. 1 and 3) the large, Mc AnN columnar, palisade-like cells of the stratum germinativum showed a strong reaction; while 14-DAY the stratum intermedium and the vesicular cells EMBRYO / / - of the periderm stained less strongly. The primary epithelial germs, which at this stage of develop- IS-DAY ment appeared as bulbous pegs, had as an outer EMBRYO lining large columnar cells which resembled the P&Bb cells of the stratum germinativum and contained fine purplish formazan granules in their cyto- IB-DAY plasm above and below their nuclei. Thus, two EMBRYO purplish lines of granules were seen running parallel over the peg-shaped protrusions of the NEW - primary epithelial germs, while the cells located BORN in the center of the germs stained diffusely pink. The anlagen of the hair papillae (PA) were moderately reactive. In the dermis an interlacing 5-DAY OLD network of capillaries (C) stained faintly against a negative background. Weakly reactive mast cells (Mt) were concentrated in the uppermost dermis. Dermal muscles (M) stained uniformly ID-DAY pink in all fibers. OLD & In 16-day old embryos (Figs. 1 and 4) the basal ADULT layer of the epidermis reacted more intensely than the other layers. The intensity of the reac- tion gradually decreased toward the surface of Fxo. 1. Schematic outline of the succinie, malicthe epidermis. The gradual transformation of and lactic dehydrogenase systems (SDS, MDS and LDS) during the embryonic development of eachlarge squamous cells, rich in cytoplasm, to par- skin element. A: Arrector pili muscle. AnPG:tially keratinized, flattened cells caused in the Anlage of primary epithelial germ. B: Bulge. C:upper stratum malpighii a condensation of intra- Capillary. CH: Club hair. F: Fat cell. HC: Hair cone. HG: Hair germ. K: Keratogenous zone. M:cytoplasmic formazan granules. The granules Muscle fiber. m: Intermediary fiber. MC: Mesen-were arranged in several stratified, undulating ehymal cell. Mt: Mast cell. P: Hair papilla. Pp: Anlage of hair papilla. P and Bb: Hair papilla andlines running parallel to the alignment of the hair bulb. Perid: Periderm. PC: Primary epithelialpartially keratinized cells and to the undulating germ. r: Red muscle fiber. 5: Sebaceous gland.surface of the epidermis (See arrows in Fig. 4). SC: Secondary epithelial germ. St.Cer: StratumThe rather thin horny layer was composed of not germinativum. V: Vessel. w: White muscle fiber. yet completely keratinized cells stippled with fine formazan granules. However, the reaction along the cell walls fine, blue formazan granulesin the horny layer was in part non-enzymatic, were deposited. The large nuclei of these cellsbecause the cells of the horny layer stained not did not stain. The primordia of primary epi-only in substrate-containing media but also in thelial germs, which were observed occasionallysubstrate-free media, though to a lesser degree, when nitro-BT was used as electron acceptor, as slight bulges extending downward from the and, furthermore, this staining reaction could be stratum germinativum, showed a slightly moreinhibited by the addition of iodoaeetate in a final intense reaction than the stratum germinativum.concentration of 0.01 M or higher. When control There was an increase in the number of mesen-sections were incubated in substrate-free media chymal cells (Mc) in the dermis immediatelywith nitro-NT rather than with nitro-BT, no beneath each bulge of the stratum germinativum.formazan granules formed in the cells of the These cells, representing the anlage of the hairhorny layer. The primary epithelial germs (PC), papilla, stained weakly. Large mesenchymal cellswhich were in the late bulbous peg stage, showed DEHYDROGENASE SYSTEMS DURING HISTOGENESIS OF THE SKIN 23 FIG. 2. SDSstain.12-day old embryo. The epidermis consists of an inner layer, the stratum germi- nativum (StGer), and an outer layer, the periderm. There is shght downward bulging of the stratum germinativum, indicating the primordium of the primary cpithelial germ, with increased enzymatic activity. Fine formazan granules are located in the periphery of most of the large vesicular cells of the epidermis. Beneath the bulge of the stratum germinativum, there is a concentration of mcscnchymal cells (Mc) indicating the earliest stage in the formation of a hair papilla. (X 600.) Mt PA C tt/iM FiG. 3. SDS stain. 14-day old embryo. The large cuboidal cells of the stratum germinativum and of the outer layer of the primary cpithelial germs show a palisade-like arrangement and stain alike. Large nuclei occupy a considerable part of the cells resulting in the accumulation of reactive granules above and below the nuclei, which thus form two dark hnes, one above and one below the nuclei, parallel to the surface of the epidermis. Mast cells in the upper dermis (Mt), the anlagc of a hair papilla (PA), muscle fibers (M) and faintly stained capillaries (C) show enzymatic activity.