(Eccrine and Apocrine Glands) During Childhood in Japanese, Especially on the PAS Positive Substance and Iron

Histochemical Investigation on the Axillary Sweat Glands (Eccrine and Apocrine Glands) during Childhood in Japanese, especially on the PAS Positive Substance and Iron

Iwao Yasui and Hiroshi Kagemoto

Department of Anatomy, School of Medicine, Keio University Shinjuku, Tokyo, Japan (Director : Prof. Dr. T. Taniguchi)

Introduction

The application of periodic acid to histochemical study on polysaccharide was made for the first time by Hot c h k i s s, M c Man u s, Lillie et G r e co and Marches e. This histochemical method was called for short PAS method " (namely, Periodic Acid S c h i f f). 'Recently , a good deal of the histochemical studies regarding glycogen, mucopolysaccharide and other substances in the sweat glands have been made by such various scholars as Bunt in g, Wislocki and Dempsey (1948), Montagna, Chase and Lobitz (1952), Montagna, Chase and M e l a r a g n o (1951) and others, apply- ing the PAS method. On the other hand, studies on the iron reaction in the sweat glands have been numerously reported since H o m m a (1925)'s research on the axillary sweat glands by Turnbull reaction, that is, Klaar (1926), Herzenberg (1927), Moriyama (1927), Oono and Kinoshita (1927), Wosllard (1930), Richter (1933), Manca (1934), Way and Memmesheimer (1938), Yoshi- mura (1942), Nagamitsu (1941), Yoshihiro (1942a), Cavaz- zana (1947), Bunting, Wislocki and Dempsey (1948),Iwa- shige (1951),Montagna,Chase and Lobitz (1953) and others. Nevertheless, there have been no reports which give histochemical observation on the axillary sweat glands during childhood by age group. The authors have tried a histochemical investigation on the 215 216 Iwao Yasui and Hiroshi Kagemoto axillary sweat glands (eccrine and apocrine glands) during childhood in Japanese by age group, especially on the PAS positive substance and iron.

Materials and methods

Ten Japanese children of ages varying from immediately after birth to 15 years, one embryo of 8 months and one adult of 25 years were used as experimental materials (Table 1). The skin which was taken from the central portion of the axillae of each body was fixed with pure alcohol and serially cut in paraffin sections of 5 ii in thickness. The sections were stained by PAS staining method and by Perls St i e d a's Berline blue reactive method for the detec- tion of iron. For the demonstration of glycogen the diastase digest test was used.

Table 1. Experimental materials.

Original findings and discussion

In regard to glycogen in the sweat glands many reports have been made since Bosse] l i n i (1902) for the first time touched upon this subject. Nowadays, it is certainly provable that glycogen is encountered in the glandular body of the eccrine gland, but there are opposite opinions in regard to the presence of glyco- Histochemical Investigation on the Axillary sweat glands 217 gen in the apocrine gland. I t o and others have asserted, that glycogen is found in the basal cells of the eccrine sweat gland and therefore two kinds of the cells, basal and surface ones are distinguishable. Recently, the PAS reactive method initiated by McManus has extensively been applied to the histochemical survey of polysaccharide, and at the same time, this method is also used for the observation of mucopolysaccharide, besides glycogen. B u n- t i n g, W i s l o c k i and Dempsey (1948) have proved the existence of glycogen in the eccrine sweat gland but not in the apocrine gland, while they have found mucopolysaccharide in both kinds of sweat glands. Montagna, Chase and Hamilton (1952) and others have reported that a good deal of glycogen and diastase resistant PAS positive substances are recognizable in both eccrine and apocrine sweat glands. Moreover, Montagn a, Chase and Lobitz (1952) and Montagna, Chase and Melaragno (1951) have asserted that glycogen is recognizable in the eccrine sweat gland but hardly in the apocrine, and they also have refered to the diastase resistant PAS positive substances, especially their significance. In the present investigation, the diastase resistant PAS positive substances were recognized in both, the eccrine and apocrine sweat glands, while on the other hand, glycogen was not recognized in either gland. On the PAS positive substances in the eccrine sweat gland. Extremely intensive PAS positive, coarse granular or small lumped substances and very fine granular PAS positive ones are recognizable in the cytoplasm of the eccrine sweat gland. Coarse granular, or small lumped substances which show very intensive PAS reaction are recognizable only in two cases of namely a 3 months old infant, and a 10 months old, that is, these substances are found in the cytoplasm of the basal cells (Figs. 5. 6 and 7). Though these findings are morphologically similar to glycogen, that is illustrated in Mon t a g n a's book (1956, The structure and function of skin ; Fig. 14, glycogen in eccrine sweat gland from the axilla. Observe the aisParity from cell to cell, (technique)), glycogen in the authors' material does not show any decrease or disappea- rance with the diastase digestive test. Notwithstanding it is widely known, that glycogen is detectable in the glandular cells of the eccrine sweat gland, no glycogen was demonstrated in the authors' materials. 218 Iwao Yasui and Hiroshi Kagemoto

On the extremely fine granular PAS positive substance. M o n t a g n a (1956) stated, that the glandular cells of the eccrine sweat gland contained PAS positive substances which did not fade away with the diastase digest test, besides glycogen and these substances might be mucopolysaccharide. In the authors' observation, an extremely fine granular PAS positive substance is encountered in the cytoplasm of the glandular cells in the whole cases examined. The results of the observation by age group are as follows. In the cases from the 8th fetal month to one month after birth, the above mentioned reaction is intensive in the cytoplasm of the glandular cells near the glandular lumen at these periods the difference between the so-called superficial and basal cells is not yet distinct (Figs. 1, 2 and 3). In the infants of 3 months and of 10 months, the intensity of the PAS reaction is about the same in both surface and basal cells, and this is weaker than in the adult (Figs. 4, 5, 6 and 7). In the cases of over 4 years of age, the PAS positive substance in both types of the cells is similar in its intensity or somewhat more intensive in the basal cells (Figs. 8, 9, 10, 11 and 12). The PAS positive substance in the cytoplasm of the superficial cells is intensive in the cytoplasm on the luminal side in the cases of ages varying from the 8th fetal month to one month after birth and the intensity is gradually decreased towards the basal side (Fig. 1). In the cases of over 3 months old, the PAS positive substance is uniformly and diffusely recognizable throughout the cytoplasm (Fig. 4). In the children of ages ranging from 6 to 12 years, a somewhat larger granular PAS positive substance is uniformly and diffusely observable in addition to the extremely fine granular PAS positive substance. A small quantity of the PAS positive substance in the cytoplasm of the basal cells is found on the basal side of the glandular cells in the 8th fetal month embryo and in the new born infant (Fig. 1). In the cytoplasm of the basal cells in the 1 month old infant, two types of the cytoplasm are re.cognizable, that is, the one of which contains a small quantity of the PAS positive substance and the other that contains a somewhat large quantity, and with the advance of age two types of the cytoplasm are distinctly distinguishable from each other (Figs. 9 and 12). Only in the infants of 3 months and 10 months after birth, coarse granular or small lumped PAS- reaction is intensively observable as mentioned before. Histochemical Investigation on the Axillary Sweat Glands 219

About the PAS-reaction in the boundary portion between the glandular cells. In the present investigation, the boundary portion between the cells of the basement is chiefly observed because that of the superficial area is unclearly recognizable so that in the superficial area the spaces between the cells are observed. In the embryo of the 8th fetal month and the new born infant, the boundary portion between the glandular cells is recognizable as a feebly filamentous PAS positive substance (Fig. 1). In the children under 4 years, the PAS positive substances are narrowly recognizable (Fig. 8). In the child of 6 years PAS positive substances, at last, get to be somewhat distinctly recognizable, thereafter in the children of 12 and 15 years old, they are clearly recognizable (Figs. 9, 10 and 12). In the adult, the PAS positive reaction exhibits a thick and filamentous picture, though its intensity is somewhat weak as compared with that in the 15 years old adolescent. Changes of the PAS positive substances in the boundary portion between the glandular cells in children are thought to follow the increase of the boundary portion between the cells with the advance of age. The portion where the superficial cells face the glandular lumen is recognizable as a filamentous PAS positive substance, but it is similar in thickness to that in the boundary portion between the cells. In several cases (the new born, 15 years and adult cases), the intensive PAS positive substance is observable in this portion (Figs. 10, 11 and 12). In the 15 years old adolescent, a filamentous or small granular PAS positive substance with a strong intensity is sporadically recognizable (Fig. 10). In the adult, small secretory process-like substances are recognizable. Montagna, Chase and Hamilton (1951) have reported,; that there are cells which contain a good deal of S c h i f f reactive granules at the apex of the small and somewhat darkish glandular cells, and moreoverMontagna, Chase and Melaragno (1952) have asserted, that these granules may contain mucopolysaccharide because they can be stained metachromatically with toluidin blue. According to Mon t a g n a (1956) these granules were found in the respective sections of the head skin obtained from Alopetia areata, and he writes that though it is hard to explain what they are, the quantity of these granules is not altered in the course of perspi- ration, caused by administration of acetylcholine. Generally, the eccrine sweat gland is an organ which performs the secretory 220 Iwao Yasui and Hiroshi Kagemoto

fnuction and sometimes the cells are metachromatically stained or contain some substances showing the PAS-reaction. Thereupon he concluded, that these substances might be mucopolysaccharide. In the authors' investigation, such granules as described by Mont a g- n a are fewly recognized. Moreover in the sections which were fixed with Z e n k e r-formalin, so numerous granules were found that some part of the granules were probably diminished away on the way of fixative routine. There are many cases in which the intercellular secretory canaliculi do not exhibit a distinct reaction, nevertheless the cases exhibit reaction so that intercellular secretory canaliculi show more intensive PAS reaction than the boundary portion between the cells of the basement. In the embryo of the 8th fetal month, the boundary portion between the glandular cells and the intercellular secretory canaliculi are not yet distinguishable. Though in the infant of 1 month old, some of the canaliculi show a somewhat intensive PAS- reaction, most of them do not show the reaction. In the infant of 3 months, the PAS-reaction of the intercellular secretory canaliculi is somewhat intensive as compared with that of the boundary portion between the glandular cells (Fig. 4). In the 15 years old adolescent, there are secretory canaliculi, showing an intensive PAS positive reaction at a certain region of the intercellular secretory canaliculi (Figs. 9 and 10). In the adult as well as in the 15 years old adolescent, secretory canaliculi, which demonstrate a striking. PAS-reaction are encountered (Figs. 11 and 12). Montagna, Chase and Lobitz (1952), in view of the studies on the distribution of glycogen in the skin, made an observ- ance on the localization of glycogen. They have insisted, that some of the cells in the eccrine sweat gland contain a good deal of glycogen and the others do not. In the cells which are abundant in glycogen, the intra- and intercellular secretory canaliculi are also abundant in glycogen. Moreover along the walls of the secretory canaliculi, the granules of glycogen are acumulated and give a clear outline, and most of them are diminished by the digestive test of saliva or diastase. In the authors' materials, the PAS positive substances are recognizable as clear two lines which indicate the walls of the intercellular canaliculi. In the 15 years old adolescent, partly intensive PAS positive' substances are sporadically recognizable on the intercellular secretory canaliculi, and these substances , are not decreased or diminished by the diastase digest test though Histochemical Investigation on the Axillary Sweat Glands 221 this finding is similar to Mon t a g n a and others' findings on glycogen. On the myoepithelial cell. Any of the myoepithelial cells exhibit a weak PAS-reaction with feebly purplish-red colour. In some cases under 6 years of age, the myoepithelial cells do not show the reaction. The reaction in the new born infant is less intensive than that in the adolescent of 15 years. On the proper layer of mucosa. On the proper layer of mucosa the PAS positive substances are observable throughout all the cases and they are devided into two layers, that is, the inner layer shows intensively filamentous reaction and the outer one shows clear and feeble purplish-red color. In the embryo of the 8th fetal month and the new born infant, the inner layer only is recognizable and the outer one is unclearly recognizable but in the children over 1 month after birth, the outer layer as well as inner one is clearly observable (Figs. 1, 2 and 4). With the advance of age, the outer layer increases in its thickness, that is, the width in the transversal sections of the gland increases, nevertheless the PAS reaction is not so increased in its intensity as the outer layer. The inner layer becomes thick in the older children but the increase in the thickness is small (Figs. 9 and 10). According to Montagna (1956), the proper layer of the mucosa is composed of two layers, the inner layer of which shows the PAS positive reaction and the outer one is negative. With the advance of age, the proper layer of the mucosa is increased in its thickness, but this increase is limited to the outer layer which does not show a hyaline- PAS-reaction. In the present investigation, the findings on the proper layer of the mucosa are about the same as those described by Mon t a g n a, but if remotely compared with both of them, the outer layer shows a feeble PAS-reaction and besides, the inner one also increases a little to thickness.

About the PAS positive substance in the apocrine sweat gland. Contents in the glandular lumen. According to Mon t a g n a (1956), protein, carbohydrate, ferric iron and ammonia are obtained from the secretory substances of the apocrine sweat gland. Besides he writes, that the secretion from the glandular cells is slow and small in its quantity. Sc h e l 1ey (1951) has described, that the glandular cell does not 222 Iwao Yasui and Hiroshi Kagemoto , always perform the secretory activity, but between the active cycle of the secretory activity a latent period extending over 24 hours is put. Therefore, if the secretion is so slow as mentioned -above, it may be possible to observe the contents in the glandular lumen and the PAS positive substance in the glandular cells by way of comparative survey. The results obtained from the authors' investigation on the PAS positive substance in the luminal 4contents. by age group are as follows. In the whole case of ages varying from the 8th fetal month to 6 years, the PAS positive substances with purplish red color that is almost the same red color of the iris are recognizable (Fig. 15). In the child of 8 years, reddish purple or much purplish red PAS positive substances are recognizable, that is in this case, such bright coloration as in the case under 6 years of age is not observable. In the children of 12 years, of 15 years old, and the adult, the coloration of the contents of the glandular lumen is in accord with that of the PAS positive substance in the cytoplast" of the glandular cells, namely they present two tone coloration much reddish purple and much purplish red (Figs. 27 and 28). In the luminal contents which are surrounded by the high columnat glandular cells, substances that do not often give the PAS-reaction are observable. From the description mentioned above, varieties in the luminal contents are suggested in an about 8 years old child as a divided point, from the charactor of the PAS positive substances only. The hyaline cuticular border The hyaline cuticular layer is not observable in the embryo ol the 8th fetal month and in the new born (Fig. 13). In the infan1 of 1 month after the birth, though a hyaline cuticular layer of somewhat intensive PAS positive reaction is recognizable, it is no yet so clear as in the adult (Fig. 15). In the 4 years old child, th( hyaline cuticular layer, which exhibits a somewhat intensive PM positive reaction is clearly observable (Fig. 16). In the child of years of age, the PAS positive substances in the hyaline cuticula] layer are very intensive. In each case of the 12 years, 15 year: old, and the adult, the PAS positive substances which are very in tensive are encountered (Fig. 26). Namely, the hyaline cuticula] layer appeared in the 4 years old child. The portion where the glandular cells face the glandular lumei and the brush border. Ito, Takahashi and Kitagawa (1956), on the study 0] Histochemical Investigation on the A1,cillarySweat Glands 223 the axillary apocrine sweat glands of healthy adult men using the PAS technique, have reported, that a thin border which shows PAS positive reaction is observable on the surface of the cuticular layer in the glandular cells throughout all sections in which nuclei . are stained. On this border as well as on the cuticular border and brush border, numerous and fine small rods which present reddish purple PAS reaction are arranged vertically on the free surface. I t o and others (1956) approve of the opinion, that this border is called " brush border " because of it being finer than the cuticular border of the epithelium of the intestine in its structure. The small rods in our observation are somewhat different from those described by I t o and others. The authors have observed them as the brush border which includes the PAS positive substances look- ing at the luminal end of the small rod. Accordingly, the portion that corresponds to the small rod is a bright and unstainable thin filament in the authors' observation. At this portion, the 15 years old adolescent only shows the small rod (Figs. 17, 23, 24, 25 and 26). Viewed from the point of the age group, the findings on the PAS positive substances in the portion that faces the glandular lumen and at the brush border are as follows. In the cases of ages from the 8th fetal month to 3 months after birth, these portions are very rarely recognizable as an extremely thin filamentous PAS positive substance. In the 10 months old infant, the presence of the extremely fine, bright and unstainable portion is noticeable between the filamentous PAS positive substances and the free surface of the cytoplasm of the glandular cells. However, in the children under 2 years old, the free surface of the glandular cells is recognizable still as a fine line with feeble purplish red color being slightly leaning toward purple, so that it is hard to designate it as the brush border. In the 4 years old child, linear substances with an intensive PAS positive reaction are intermittently and clearly observable for the first time in the hyaline cuticular border and in the free surface and moreover, an extremely narrow and bright unstainable portion is encountered between the hyaline cuticular border and the free surface. This portion is presumably to corre- spond to the brush border (Fig. 16). In the 8 years old child, the brush border exhibits its variety. That is, in some cases intensive PAS positive substances forming a thin filament are intermittently recognizable attached on the free surface of the hyaline cuticular layer, through an extremely narrow and bright, unstainable portion 224 Iwao Yasui and Hiroshi Kagemoto

(Fig. 20) and in the other case, the brush border is recognizable as an extremely thick layer (Fig. 19). In the 12 years old child, high columnar cells and cubical cells are observable and the brush border is clearly recognizable as a thin filamentous substance. The brush border in the adolescent of 15 years is various in its appearance. Thin filamentous PAS positive substances are observable on the free surface of the hyaline cuticular layer through the bright and unstainable portion in two types of the glandular cells, one is of high columnar cells which are abundant in the PAS positive substance with feeble purplish red color leaning towards purple, and the other is of about cubical cells which numerously contain reddish purple PAS positive substances. In the bright portion fine small rod- like PAS positive substances are observable as if they were the teeth of large comb (Figs. 23 and 24). Chiefly in high columnar glandular cells which are abundant in the reddish purple PAS positive substances being tinged with red, the brush border is attached to the same colored PAS positive substances. These are also of the same coloration as the PAS positive substance in the cytoplasm (Fig. 27). In the adult, the PAS positive substance is observable as a thin filament. I t o and others (1956) have reported, that the significance of the brush border in the epithelial cells of the sweat gland is un- known. In the authors' experiment, such phenomena are observed as in some glandular cells, which contain an intensive PAS positive substance in the cytoplasm are gathered in the cytoplasm of the cells near the glandular lumen, and moreover after they are attached to the brush border, probably they finally isolate into the luminal contents ; namely an eccrine-like secretion is suggested. Besides, in the other glandular cells, substances which are unreactive to the PAS method are accumulated between the brush border and the surface of the glandular cell, and then after the brush border gradually swells, these substances move to the outline of the secretory process ; namely apocrine-like secretion is observable. Furthermore, in the authors' observation the secretory substances which are presumable to form the secretory type, where the secretory process is not formed, are mostly PAS positive substances. On the other hand, it is thought, that secretory substances which are of the secretory type with the formation of a secretory process, are chiefly composed of substances that do not exhibit PAS- Histochemical Investigation on the Axillary Sweat Glands 225 reaction. There are two types of coloration in the PAS positive substance in the cytoplasm of the glandular cell, that is, feeble purplish red tinged much with purple, and reddish purple tinged much with red. The PAS positive substances with the former coloration are very fine granular and these substances are diffusely and uniformly found in the cytoplasm of the whole cells. In children over 8 years of age, some glandular cells are hard to find, because they are covered with the PAS positive substances with a reddish purple color that is leaning toward red. In the adolescent of 15 years and the adult, the formation of the secretory process is recognizable in the glandular cells which contains fine garnules with feeble purplish red color that exceeds in purple (Figs. 25 and 30). The PAS positive substances which present a reddish purple color that exceeds in red are recognizable as a bright red lump in the cytoplasm of the glandular cell in the 4 years old child. In the 8 years old child, there are two kinds of the cells, that is, one is abundant in the small granules with a reddish purple color that exceeds in red and the other contains the lumped PAS positive substances. In the children of 12 years and of 15 years of age, a good deal of the intensive and coarse PAS positive granules are recognizable (Figs. 22 and 27). In the adult, some cells are diffusely abundant in the fine granules (Fig. 29).

Myoepithelial cell In the children under 6 years of age, feeble purplish red and weak PAS positive substances are recognizable only in the 10 months old infant. In all cases over 8 years of age, feeble purplish and weak reactive PAS positive substances are observable and then the PAS positive substances are gradually increased with the advance of age. In the embryo of the 8th fetal month and in the new born infant, the proper layer of the mucosa is recognizable as extremely thin, purplish red and intensively reactive PAS positive substances. The layer is not distinguishable in two layers, inner and outer ones. In the children over 1 month after birth, the inner layer is recognizable as thin, purplish red and extremely intensive PAS positive substances. On the other hand, the outer layers are observable as feeble purplish red and they have weak reactive PAS positive substances. The PAS positive substances of the outer layer are slightly 226 Iwao Yasui and Hiroshi Kagemoto increased with the advance of age. About the starting time of the apocrine-like secretion. K a n (1941) has insisted, that in the embryonal period and childhood even the apocrine sweat gland takes the same secretory process as in the eccrine gland, and in 8 years of age the glandular. cells which show a proper morphologic change in the apocrine gland is for the first time, present. In the authors' observation, the contents of the glandular lumen in the children under 6 years are composed of almost structureless, uniform and intensively reactive PAS positive substances. In every case the PAS positive substances in the cytoplasm of the glandular cell are few, but only in the case of the 4 years old, small granular and lumped intensive PAS positive substances are encountered. In the child of 8 years, the PAS positive substances in the luminal contents, brush border and cytoplasm of the glandular cell become much colorful and numerous, and the formation af the secretory process is for the first time, recognizable. From these facts in addition to K a n's findings, the apocrine-like secretion begins at 8 years of age, and under 6 years of age the eccrine-like secretion may be carried out. Furthermore, in children over 8 years, not only the apocrine-like secretion but also eccrine-like one may be carried out, from the consideration on the relationship of PAS positive substance with the cytoplasm of the glandular cell, brush border and the contents of the glandular lumen. In both, the apocrine and eccrine sweat glands, any distinct decrease or diminishment of the PAS positive substances by means of the diastase digestion test is not recognizable. About the iron positive substance in the apocrine sweat gland. With respect to the iron reaction in the sweat glands it is widely known, that the iron reaction in the apocrine sweat gland is positive, on the other hand, it is negative in the eccrine sweat gland. However, on the iron reaction in the apocrine sweat gland of the child and of the women before menarche, K la a r (1926), Richter, (1933) and Way and Memmesheimer (1938) have reported respectively, but in all these reports the iron reaction is negative. The proportion of the positive appearance of the iron reaction in the apocrine sweat gland obtained from several scholars is shown in Table 2. Namely, according to European and American scholars the negative appearance is ranging from 5% to 97.5%. The reports made by Japanese are divided in osmidrotic and non-osmidrotic cases. According to M o r i y am a (1927) N a g a- Histochemical Investigation on the Axillary Sweat Glands 227

Table 2. Frequency in positive appearance of the iron reaction in the apocrine sweat gland.

m i t s u (1941) and I w a s h i g e (1951), 100% of the osmidrotic cases are positive and none of the non-osmidrotic cases show a positive iron reaction. Only Yoshi h i r o (1942a) has reported that 82.86% of the osmidrotic cases are positive by Turnbull reaction , and 82.27% are positive by Berlin blue method, that is, the positive iron reaction does not apper in all the cases. The reports described by Europeans and Americans are not divided into osmidrotic and non-osmidrotic cases but gather both. N a g a m i t s u (1941) has proved the result obtained by M o r i y a m a (1927) that in the axillary apocrine sweat gland of Japanese, the iron reaêtion is positive only in an osmidrotic case, and he, too, has confirmed such findings that in the axillae 228 Iwao Yasui and Hiroshi Kagemoto of every osmidrotic person the iron reaction in the apocrine sweat gland is positive and in every non-osmodrotic person the reaction is negative, therefore the presence of the iron granules in the apocrine sweat gland is an important key to discriminate the osmidrosis. Yoshi h i r o (1942a) has almost the same opinion as N a g a- m i t s u (1941), and I was h i g e (1951) who also has reported the same findings to N a g a m i t s u. About the area which shows the iron reaction in the glandular cells, Homma (1952), Herzenberg(1927),Richter (1933) and Yo s hi h i r o (1942a) have similarly reported, that it appears in that area, which is accordant with the pigment granules in the cells near the luminal surface. On the other hand, Sc ha f f e r (1927) and Mel z e r (1935) have described, that the area where the iron positive substance described by Ho m ma appears, is accordant with the Golgi apparatus. I w a s h i g e (1951) has reported in detail on the iron positive area, that is, the iron reaction is the greatest in the cytoplasm between the infra-hyaline cuticular border and Golgi area, and a few inside the Golgi area and area between the Golgi area and nucleus. However, in the hyaline cuticular border and secretory process the iron positive granules are not observable. Besides, according to I w as h i g e, the iron positive granules are the most numerous in the columnar cells, less in the cubical cells and the least in the flat cells. In the authors' investigation, the iron positive substances on the luminal side of the cytoplasm of the glandular cell, are recognizable at the bright portion just below the hyaline cuticular layer, that is, this portion is accordant with the portion where the socalled mature secretory granules that were found by Min a m i t a n i (1941b), are recognizable, at the same time the iron positive substances are found in the luminal contents. However they are not observable in such portions as the hyaline cuticular layer, brush border and secretory process. Portions where the iron positive substances are observable are in detail shown in Table 3 (Figs. 31-38). Similarly, in the authors' observation, the iron positive substances are observable in every such shaped glandular cells as the columnar cells, cubical cells and the flat cells without any discre- pancy with I w a s h i g e's finding, but the iron positive substances are so small in quantity, that the difference of the positive intensity between the various typed cells is not clearly observable. Concerning the iron positive substances in the contents of the Histochemical Investigation on the Axillary Sweat Glands 229

Table 3. P e r I s S t i e d a method. Iron positive substance.

± scarce, + a little, * moderate, *numerous glandular lumen, Richter (1933) has not found any iron positive granules, on the other hand, M or i y a m a (1927), N a g a m i t s u (1941) and Bunting, Wislocki and Dempsey (1948) have respectively observed them in the luminal contents. According to I was hi g e (1951), iron positive substances were recognized in the glandular cells which were desquamated into the lumen though it was reported that the iron positive substances were not observable in the luminal lumen. Z or z o l i (1952), Mon t a g n a, Chase and L o b i t z (1953) have asserted, that in the lumen the iron positive substances are not observable. In the authors' investigation, though some iron positive granules and substances, which do not form granules are observable, glandular cells which are thought to be desquamated in the lumen are not found (Figs. 34 and 38). The authors have not yet received any report, that iron positive substances are observed in the karyoplasm of the glandular cells. However, in the authors' investigation, the granular iron positive substances are recognizable in the karyoplasm on the trans- 230 Iwao Yasui and Hiroshi Kagemoto versal section of the glandular duct, which is composed of somewhat higher glandular cells than the cubical ones in the 15 years old adolescent. In the transversal sections of the glandular duct, the iron positive granules are more or less visible in the other shaped cells besides the high cubical ones. The quantity of the iron positive substances in the cytoplasm of the glandular cells is rather less than that in the karyoplasm. The authors observed the cells in which the cytoplasm arround the nucleous does not show any iron reaction, lest they should not mistake the observation, because the nucleus sometimes falls on the iron positive substance in the cytoplasm and its figure is cast on the karyoplasm. Under such careful observation as mentioned above, it was confirmed, that the iron positive substances are recognizable in the karyoplasm. L i s o n (1953), in his book described as follows. Though it is classical theory under the influence of Schneider and Mac allu m, that the nucleus of the cell contains iron, this is certainly an error by the tainted staining. Already Gilson in 1892 has insisted, that the nucleus has a re- markable affinity with iron, and that iron which is demonstrated in the nucleus by means of a chemical method is, in in fact, carried by a reagent. After the precise investigation Wiener has actually affirmed the finding obtained by G i l s o n. As mentioned above, such character as to absorb iron from a solution containing a very small amount of iron, and to accumulate, is found in various elements inside the tissue, besides it is very interesting that these elements are basophilic in every case. Also our finding, that the iron reaction is positive in the nucleus may depend on such cause as described by Lis° n. In the authors' findings, some iron positive substances present clear blue granules and the others present blue amorphous substances. The latter is probably recognizable to be out of focus of the microscope, though on the occasion of the granular iron positive substances being a little out of focus of the microscope these substances are visible as an uniformly structureless structure because of very intensive blue coloration of the iron positive granules. In the adolescent of 15 years, the uniform substances of iron positive reaction is recognizable in the hyaline cuticular layer and the glandular cells with the iron positive granules are observable at a bright area of the cytoplasm under the hyaline cuticular layer (Fig. 35). In these glandular cells, the iron reaction is actually Histochemical Investigation on the Axillary Sweat Glands 231 positive in the hyaline cuticular layers or it is seemingly uniformly positive in the layer ; because the transversal surface of the sections is out of the top of the glandular cells so that both, hyaline cuticular layer and cytoplasm of the glandular cell overlap each other at the same area and the coloration of the iron positive substances in the cytoplasm reflects on the hyaline cuticular layer. In the authors' findings on the other larger parts of the glandular cells, they do not show iron positive reaction in the hyaline cuticular layer, and also these findings are common to the opinions of many other scholars' so that it is probably appropriate to conclude, that actually in the authors' observation the hyaline cuticular layer does not give any iron positive reaction. Bunting, Wislocki and Dempsey (1948) have described,, that in the axillary hairs sometimes a trifle of Berlin blue recog- nizably adheres to the hair shaft. In the authors' experiment, however, no iron positive substances are observable, not only in the hair shaft but also in the hair root and hair follicle. Only in the 12 years old child, irregularly formed and coarse granular iron positive substances adhere to the surface of the corneus layer of the epithelium, and these substances are rather numerously observable and accumulated on the surface of the corneus layer in the canalicules. Furthermore, these iron positive substances are recognizable at the area, where the hair follicle opens its orifice, and each of them still adheres to the surface of the corneus layer but does nOt penetrate into the hair follicle, that is, such findings, that are obtained by Bunting, Wislocki and Dempsey (1948) as the iron positive substances are adherent to the hair shaft and to the hair root, are not observable in the present investigation. Though the iron positive substances which are found in the cytoplasm of the glandular cells, present a beautiful blue color, those on the surface of the corneus layer are dirty blue. Namely, the iron positive substances on the surface of the corneus layer perhaps are thought that some kinds of substance which are adherent on the surface of the epithelium while alive, are attached to a pigment during the staining procedure. Iron positive substance in the apocrine sweat gland viewed , from the age group. In children under 8 years of age, not any iron positive substances are found. On the other hand, in all of the children over 12 years of age, the iron positive substances are recognizable in the 232 Iwao Yasui and Hiroshi Kagemoto

apocrine sweat gland. The 12 years old case has less quantity of the iron positive substances than the 15 years old one, but in the adult these substances are clearly more scanty as compared with the above mentioned two cases. Generally, in the present study the cases where the iron positive substances are found are scarce and the intensity of the iron reaction is also very feeble.

The portion where the iron positive -substance is found. In the 12 years old child, most of the iron positive substances are observable in the bright portion just below the hyaline cuticular layer of the cytoplasm of the glandular cell but both are not found in the hyaline cuticular layer and the contents of the glandular lumen. In the 15 years old adolescent, iron positive substances are observable in the portion just below the hyaline cuticular layer of the cytoplasm of the glandular cell and in the contents of the glandular lumen, but they are not recognizable in such portions as the hyaline cuticular layer, brush border and secretory process. In the adult, they are recognizable only in the contents of the glandular lumen. I t o (1949), who investigated the literatures on the iron reaction in the sweat glands, has insisted, that such findings obtained by Japanese scholars as the iron reaction is positive only in osmidrosis, and show, perhaps that the iron appears in the glandular cells when the function of the apocrine sweat gland is vigorous . However it is hard to conclude, that iron has a direct reaction with the odor of osmidrosis. In consideration of the PAS positive substance as well as the iron positive substance in the apocrine sweat gland , and meeting with I t o's opinion, the activity of the apocrine sweat gland in children under 6 years of age is perhaps extremely feeble within the materials that were examined by the authors. However in the child of 8 years old, the activity suddenly becomes vigorous, and with the advance of age in the 12th year and 15th year the activity is more vigorous. Nevertheless , in the adult the activity of the apocrine sweat gland is less intensive than in the children of 12 and 15 years and more than in the 8 years one. On the other hand, taking in the consideration N a g a- mi t s u and I w a s h i g e's opinions, the children over 12 years must be osmidrotic. Histochemical Investigation on the Axillary Sweat Glands 233

Summary The axillary sweat glands (eccrine and apocrine glands) in ten Japanese children of ages varying from immediately after birth to 15 years, one embryo of 8 fetal months and one adult of 25 years were histochemically investigated by means of PAS technique and Perls Stied a's Berline-blue reactive method and the following results were obtained. 1. In the eccrine sweat glandular duct, the PAS positive substances with an extreme intensity were recognized in the glandular cells of the basement in only two cases during the infant period but PAS positive substances in the other portions were gradually and slightly increased in their qnantity with the advance of age. On the other hand, in the glandular duct of the apocrine sweat gland, PAS positive substances exsisted of but a trifle in the children before the first half of the school period. 2. The PAS positive substances in the glandular cells of the eccrine sweat gland were numerous in the cytoplasm near the lumen during the new born period. After and at the infant period, the PAS positive substances were similarly abundant in the basal and surface cells or somewhat more numerously in the basal cells. In the children of school age, small granular PAS positive substances were recognizable in the surface cells. 3. The PAS positive substances in the intercellular canaliculi were recognized in children after the infant period and they were slightly increased with the advance of age. 4. The PAS positive substances that are intensively reactive in the brush border and cytoplasm of the glandular cells of the apocrine sweat gland, were recognizable during the later period of infant time, and they were various and numerous after the middle period of school age. Moreover, the contents of the glandular lumen were various and numerous after the middle period of school age. 5. In the myoepithelial cells, the PAS positive substances were recognized in the eccrine sweat gland of the whole cases examined and in the apocrine sweat gland of the children after the later period of school age. That is, the PAS positive substances in the children at the first half period of school age are observable in only one case in the apocrine sweat gland. 6. In the proper layer of the mucosa the PAS positive substances were recognizable in the inner layer of the whole examined 234 Iwao Yasui and Hiroshi Kagemoto cases in both kinds of the sweat gland, and they were also observable in the outer layer of the cases after the infant period. 7. The iron positive substances were found in the cytoplasm just below the hyaline cuticular layer of the glandular cells of the apocrine sweat gland and in the contents of the glandular lumen in the persons after puberty.

Literatures

1) Bunting, H., G.B. Wislocki and E.W. Dempsey, 1948. The chemical histology of human eccrine and apocrine sweat glands. Anat. Rec., 100 : 61-77. 2) H e r z en b e r g, H., 1927. Neue Beitrage zur Lehre von den apokrinen Schweiss- drilsen. Virchows Arch., 266 : 422. 3) H o m m a, H., 1925. Uber positive Eisenbefunde in den Epithelien der apokrinen Schweissdriisen menschlicher Axillarhaut. Arch. Dermat. u. Syphilis, 148: 463- , 469. 4) I t o, T., 1949. Histology and cytology of sweat glands. Igakunoshinpo, 6 : 106, (in Japanese). 5) , T. T a k ahash i and T. K itaga w a, 1956. Uber der Vorkommen des Briistensaums an den Epithelien der Schweissdriisen des Menschen. Arch. hist. jap., 11 : 317. 6) I w a s h i g e, K., 1951. Beitrage zur Kenntniss der Eisenreaktion bei den apokrinen Schweissdriisen der Achselhaut von Japanern. Arch. hist. jap., 2 : 367-374. 7) K a n, M., 1941. Histologische und histo-entwicklungsgeschichtliche Untersuchun- gen der Schweissdriisen der Achselhaut von Japanern. Nagoya Gakkai Z., 56 : 365, (Japanisch). 8) K 1 a a r, J., 1926. Zur Kenntnis des weiblichen Axillarorgans beim Menschen. Wien Klin. Wochschr., 39 : 127. (cited from I t o, 1949). 9) Lis() n, L., 1953. Histochemie et cytochernie animals. Principls et methodes, Gauthiervillars, Paris. 10) M a n c a, P. V., 1934. Ricerche sulla strutura delle ghiandole apocrine. Giorn. ital. dermat. e. sifilol., 75 : 187-193, (cit.d from Mont a g n a, 1956). 11) Mon t a g n a, W., H. B. C h a se and J. B. Ha mil to n, 1951. The distribution of glycogen and lipids in human skin. J. Invest. Dermat., 17 : 147-157. 12) M o n t a g n a, W., 1956. The structure and function of skin. Academic Press Inc., New York. 13) , H. B. Chase and W. C. Jr. L obi t z, 1952. Histology and cytochemistry of human skin. II. The distribution of glycogen in the epidermis, hair follicles, sebaceous glands and eccrine glands. Anat. Rec., 114: 231-248. 14) and , 1953. Histology and cytochemistry of human skin. V. Axillary apocrine sweat glands. Ame. J. Anat., 92 : 451-470. (cited from Montagna, 1956). 15) and H. P. Me la r a g n o, 1951. Histology and cytochemistry of human skin. I. Metachromasia in the mons pubis. J. Nat. Cancer Inst., 12: 591-597, (cited from Mon t a g n a, 1956). 16) M o r I y a m a, G., 1927. Mikro-chemische Untersuchung fiber die Achseldriise, inbesondere Beriicksichtigung der Osmidrosis axillae. Nagasaki Igakkai Zasshi,. Histochemical Investigation on the Axillary Sweat Glands 235

5 : 302, (Japanisch). 17) Naga in its u, G., 1941. Pathologisch- histologische Untersuchungen aber die Osmidrosis axillae. Keio-Igaku, 21 : 1011, (Japanisch). 1) 0 h t a k e, Y., 1959. Comparative histological studies of the axillary glands. Acta. Anthropologica, Anthropohereditologica et Somatologica, 31 : 1, (Japanese). 19) Ric h t e r, W., 1933. Beitrage zur normalen und pathologischen Anatomie der apokrinen Hautdriisen des Menschen mit besonderer Beriicksichtigung des Ach- selhählenorgans. Virchows Arch., 287 : 277. 20) SC he 1 1 e y, W. B., 1951. Apocrine sweat. J. Invest. Dermat., 17 : 255, (cited from from Montagna, 1956). 21) W a y, S. C. and A. Memmesheime r, 1938. The suboriporous glands. II. The apocrine glands. Arch. of Dermat., 38 : 373. 22) Wool 1 a r d, H. H., 1930. The cutaneous glands of man. Jour. Anat. 64 : 415. 23) Y 0 s h i h i r o, I., 1942a. Beitrage zu klimishen und pathologischen Untersuchungen der Osmidrosis axillae. II. Histologische Untersuchungen iiber die Osmidrosis axillae. Hifuka-Seibyoka Zasshi, 55 : 557, (Japanisch). 24) Zorzol i, G., 1952. Primi dati par una classificatione istochimica dei pegmenti cromolipoidi. Boll. soc. ital. sper., 28 : 1078-1080, (cited from Mon t a g n a, 1956).

Explanation of figures

A) PAS positive substances in the eccrine sweat gland. Fig. 1. Transversal surface of the glandular duct in the 8th fetal months. Fig. 2. Transversal surface of the glandular duct in the new born. Fig. 3. 1 month. Fig. 4. Transversal surface of the glandular duct in the 3 months old. Fig. 5. Transversal surface of the glandular duct in the 3 months old. Fig. 6, Transversal surface of the glandular duct in the 10 months old. Fig. 7. Transversal surface of the glandular duct in the 10 months: old. Fig. 8. Transversal surface of the glandular duct in the 4 years old. Fig. 9. Transversal surface of the glandular duct in the 15 years old. Fig. 10. Transversal surface of the glandular duct in the 15 years old. Fig. 11. Transversal surface of the glandular duct in the adult old. Fig. 12. Transversal surface of the glandular duct in the adult old.

B) PAS positive substances in the apocrine sweat gland. Fig. 13. 18 fetal month. Fig. 14. 1 month. Fig. 15. 10 months. Fig. 16. 4 years old child. Fig. 17. 8 years. Low cells. Fig. 18. 8 years. Low glandular cells. Fig. 19. 8 years old child. Fig. 20. 8 years. Brush border. Fig. 21. 8 years. Secretory process. Fig. 22. 12 years old child. Fig. 23. 15 years. Brush border. Fig. 24. 15 years old child. 236 Iwao Yasui and HiroshilKagernoto

Fig. 25 and 26. 15 years old child. Fig. 27 and 28. 15 years old child. Fig. 29. Adult. Fig. 30. Adult. C) Iron. (P e r 1 s-S tied a's method) Fig. 31. 12 years old child. Fig. 32. 12 years. Granules of iron positive reaction. Fig. 33. 12 years. Low glandular cells. Fgi. 34. 15 years old child. Fig. 35. 15 years old child. Fig. 36. 15 years old child. Fig. 37. 15 years. Flat cells. Fig. 38. Adult. 237

Plate I

I. Yasui and H. Kagemoto Z3U

Plate II

I. Yasui and H. Kagemoto 241

Plate III

I. Yasui and H. Kagemoto Plate IV

I. Yasui and H. Kagemoto 245

Plate V

I Yasui and H. Kagemoto