On the Origin and Morphology of Myoepithelial Cells of Apocrine Sweat Glands* D J

Home , Apocrine, Basement membrane, Duct (anatomy), Myoepithelial cell

ON THE ORIGIN AND MORPHOLOGY OF MYOEPITHELIAL CELLS OF APOCRINE SWEAT GLANDS* D J. GOLDSTEIN, MB., B.Cn., B. Sc.Horcs.

In view of the importance of myoepithelial MATERJAL AND METHODS cells in the functioning of sweat glands (Shelley The glands studied were apoerine sweat glands and Hurley (1); IHiurley and Shelley (2)), it is("ceruminous glands"—Shelley and Perry (17)), surprising that more attention has not been paidof the external auditory meatus of man aud of the to their origin and development (Hdggqvist (3)).Vervet monkey (Cercopithecus acthiops). First described by Koelliker in 1547, myo- Paraffin sections of material freshly fixed in epithelial cells were claimed to be ectodermal inHelly's or Bouin's fluids were stained with hema- origin by Ranvier in 1875 (both cited bytoxylin and eosin, the periodic acid-Schiff (P.A.S.) Haggqvist, (3)). Supporting this view is the situa-method, a modified Wilder's method for reticulin, tion of the myoepithelial cells superficial to theReidenhain's iron-hematoxylin or Altmann's aniline-acid fuchsine. Free-floating frozen sections basement membrane (Schaffer, (4); Leeson, (5)).of monkey material fixed in chilled formalin were Further, it has been claimed that in the embryostained with Scharlach H for fat, or were incubated the basal layer of the originally two-layered sweatfor varying times in the Gomori glycero -phosphate gland tube differentiates into myoepithelial cellssubstrate for the demonstration of alkaline phos- in the secretory portion of the gland, while thephatase (Pearse, (18)). epithelium retains its stratification in the duct In addition, mitotic figures were sought in (Schaffer, (4)). Myoepithelial cells of embryonicserial sections of the external auditory meatuses salivary glands are similarly related to the basalof a female Vervet monkey treated witb eolchicine. cells of the duct (Heidenhain, (6)). This animal, weighing approximately 2.7 Kg, was given 6mg colchicine in 3 ml saline subcutaneously In the adult, cells intermediate in structureat 10.30 p.m., and was killed five hours later, at between myoepithelial cells and the secretory3.30 a.m. An ampoule of hyaluronidase cells of the sweat gland have been described by("Hyalase") was added to the injection to facili- Hibbs (7) in electron micrographs, but were nottate absorption. found in salivary or lacrimal glands by Scott and Pease (8), using the same technique. In oBsEnvATroNs general, workers with the light microscope have Occurrence of Transitional Forms failed to find such transitional forms in sweat, In large apocrine sweat glands of the human salivary or laerimal glands. external auditory meatus, stained with P.A.S. It has often been noted (Brunn, (9); Stohr,and hematoxylin, or with hematnxylin and (10); Hoepke, (ii)) that the myoepithelial celleosin, typical myoepithelial cells are visible layer of adult sweat glands is a continuation ofbetween the basement membrane and the secre- the basal cell layer of the duct. Cells interme- tory cells of the gland. They are pale-staining diate in structure between the two types do notelongated cells with their long axes approxi- however seem to have been described in themately parallel to the long axis of the tubule. adult, and a number of authors have stated orNuclei of myoepithelial cells are elongated or implied that such transitional forms do not flattened, are usually darkly stained, and contain occur (Alverdes, (12); Way and Memmesheimer,rather coarsely granular ehromatin. (13); Bunting et at., (14); Hurley and Shelley, Basal cells of the two- or three-layered duet (2); Horstmann, (15); Hibbs, (7); Lee, (16)). epithelium characteristically have round or The present paper discusses the relationshipslightly oval, pale-staining nuclei, containing of myoepithelial cells to the basal cells of thefine granules of ehromatin. duets of adult, functioning apocrine sweat At the junction between the secretory portion glands, and presents evidence relating to this. of the gland and the duct, nuclei are frequently *Fromthe Department of Anatomy, Univer-found which are intermediate in appearance sity of the Witwatersrand Medical School, Hos-between the nuclei of the myoepithelial cells pital St., Johannesburg, South Africa. Received for publication January 26, 1961. and those of the duct cells (figs. 1, 2, 3 and 4). 301 302 THEJOURNAL OF JNVESTIGATIVE DERMATOLOGY

On following a series of basal nuclei from the The basal cells of the duct arc devoid of hire- duct towards the secretory portion of the gland,fringcncc, whether examined in transverse or in typical duct nuclei as described above arclongitudinal section (figs. 17—20). The more gradually replaced by nuclei which progressivelysuperficial cells of the duct frequently contain become flatter and more darkly stained, untildiscrete bircfringcnt fibrils. These do not re- finally the typical appearance of a myocpithelialsemble the more diffusely bircfringent myofibrils ccli nucleus, as described above, is found. Similarof myocpithclial cells, and arc probably tono- transitional forms arc sometimes found in thefibrils (figs. 17—20). apocrine glands of the external auditory meatus of the Vervct monkey. Basement Membrane A small number of cccrinc sweat glands from The basement membrane underlying the various parts of the human body has beensecretory portion of the apocrinc sweat gland examined, and well marked transitional formsbears a characteristic relation to the myoepithclial are not present in most glands. Careful study ofcells. Not only does it underlie the cells, but pro- serial sections has however revealed them in ajections from the basement membrane penetrate few glands, and transitional cells possibly occurfor some distance into the cpithclium, thus in most or all types of sweat gland, but happencnsheathing the myocpithclial cells in a sort of to be particularly easily demonstrated in thetrough. Since the long axes of the elongated apocrine glands of the ear. myocpithclial cells lie roughly parallel to the In the present material no cells have beenlong axis of the gland tubule, these projections found intermediate in appearance betweenof the basement membrane arc well seen in trans- myoepithclial cells and the secretory cells of theverse or tangential sections (figs. 7, 15), but not gland. in sections parallel to the long axis of the gland The occurrence of cells intermediate in char-(fig. 11). The intra-epithelial projections of acter between myoepithelial cells and the basalbasement membrane stain with P.A.S., but not cells of the duct, suggests that the two cell typeswith Wilder's reticulin method. arc related in some way. To investigate this Similar invaginations of the basement mem- relationship, several properties of the cells havebrane were not found related to the duct. been investigated and compared. Several differences which have been found are reported here. Alkaline Phosphatase Myocpithclial cells are strongly positive for Myofibrils Stainable with Iron-Hematoxylin alkaline phosphatasc. After short (30 minutes) Myofibrils of myocpithclial cells, althoughincubation, myocpithclial cells are blackened usually not individually resolvable with thealmost selectively, with little or no staining of light micrpscope, are readily stainable with iron-the overlying secretory cells (figs. 6, 21). Studies hematoxylin. Under similar conditions of stain-with polarized light (figs. 21, 22) indicate that ing, the cytoplasm of the basal duct cells remainsthe initial deposition of precipitate is along the unstained (fig. 5). cell borders of the myocpithelial cell, rather than in the substance of the cytoplasm. This is shown Birefringence by the fact that bircfringencc (due to myofibrils The highly oriented molecules of the myo-in the cytoplasm) and deposition of precipitate fibrils impart positive bircfringcncc to the cyto-are not found in the same region of the cell. plasm of myoepithclial cells. This bircfringcncc, After longer (two hours) incubation, the entire which appears to fill the entire cytoplasm, is seensecretory portion of the sweat gland is intensely in longitudinally or tangentially, but not inblackened, but the duct is still completely free transversely sectioned cells (figs. 11—16). from precipitate (fig. 23). Birefringent material is occasionally seen in the cytoplasm of cells morphologically inter- Incidence of Mitosis mediate between myocpithelial cells and duct After preliminary studies on material fixed at cells (figs. 15, 16). The material studied is9 a.m. had indicated that mitoscs were rare in insufficient to generalize about the stage of theVervet apocrine sweat glands, tissues were taken transition sequence in which cytoplasmic hire-in the early hours of the morning from an animal fringence can first be detected. treated with colchicine (see above for details). ORIGIN AND MORPHOLOGY OF MYOEPITHELTAL CELLS 303

This was done in ease a diurnal cycle of mitosisFirstly, myocpithclial cells and duct cells may occurs in this animal (Bullough. (19, 20)). have certain common functions, and secondly, Due to the spindle destruction caused by theone cell type may become transformed into the coichicine, typical mctaphasc plates were notother even in adult life. seen, and it was sometimes difficult to distinguish Since the only known function of myocpi- mitotic figures from pyknotic nuclei. Excludingthelial cells is contraction, particular attention doubtful mitoses, approximately 30 cell divisionswas paid to morphological or cytochcmical were found in 36 glands completely traced infeatures of the duct cells which might indicate serial sections from one external auditory mcatusthat these cells, also, are contractile. (designated "Ear A"). All these cell divisions were found in ducts (figs. 8, 9 and 10), and none Myofibrils in the secretory portions of the glands. The presence in myoepithclial cells of fibrils Mitotic figures were found all along the duct,demonstrable with iron-hematoxylin or polarized from the junction with the secretory portion tolight (Haggqvist, (3); Bunting et al. (14); Hurley the entrance into the associated hair follicle. Itand Shelley, (2)) has been confirmed in this has not been established in this material thatstudy. Contraction in biological systems depends, mitosis occurs preferentially at any particularin general, on the presence of oriented, elongated site along the duct, but the impression has beenmolecules, and the absence of fibrils from the gained that mitotic figures may be somewhatbasal cells of the ducts (indicated by their lack more frequent near the junction of the duct withof birefringence and their failure to stain with the secretory portion. iron-hematoxylin) is therefore strongly against A study of serial sections from the opposite earthese cells being contractile. ("Ear B") of the same animal revealed no mitotic figures in 18 complete sweat glands Basement Membrane traced. The tissue from car B had been taken Although it is not possible with the light from a more superficial level of the meatus thanmicroscope to distinguish basement membrane the tissue from ear A, but it was felt that thisfrom material just inside the cell membrane was insufficient to explain the difference in the(Goldstein, (22)), the basement membrane incidence of mitoses in glands from the two sides.underlying the secretory portion of the sweat In view of the known correlation between mitoscsgland appears to form P.A .S.-positivc intra- in epidermis and in the associated hair folliclescpithclial projections, which partially enclose (Montagna, (21)), it was thought that a relationeach myoepithclial cell. These projections do not should be sought between mitotic incidence inappear to have been reported previously in light epidermis and in the underlying sweat glands. Amicroscopic studies, but probably correspond to mitotic count was accordingly made in the epi-an electron-dense, homogeneous material which dermis from the two sides, 30 sections beingseparates myoepithelial cells of human axillary counted in each case. The epidermis from ear Aapocrine glands from each other and from the showed a mean of 5.3 mitoses per section, with aunderlying dermis (Charles, (23)). The signifi- standard deviation of while the com-cance of these projections of basement membrane parable figures from car B were 1.73 and is obscure, but in any event their absence from The difference between the two means was 3.57,the sweat duct is another way in which the basal more than five times the standard error of thecells of the duct differ from myocpithelial cells. difference (±0.61). There was thus a highly significant difference between the incidence of Alkaline Phosphatase cpidermal mitoses in the two ears, the higher The presence of large amounts of alkaline incidence occurring on the side where mitoscs were found in the ducts of the sweat glands. phosphatase in apocrine gland myocpithelial cells (Bunting, (24); Bunting et at. (14); Shelley and Mescon, (25)) has been confirmed in this study. DISCUSSION The initial appearance of precipitate on or near The existence of cells intermediate in mor-the cell membrane (see above) may be due to phology and situation between myocpithelial cellslocalization of the enzyme in this site, or may be and the basal cells of the ducts of adult apocrinedue to an adsorption artifact. sweat glands, may be interpreted in two ways. At various times it has been suggested that 304 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY alkaline phosphatase functions in active trans-the basal cells of the duet, are described in port, synthesis of fibrillar proteins, cell differen-apoerine sweat glands of the adult ear. tiation and cell migration (Novikoff, (26); Myoepithelial cells differ from the basal duet Chiquoine and Rothenburg (27)). It is temptingcells in being birefringent, showing a positive to speculate about the possible role of the enzymereaction for alkaline phosphatase, staining readily in myoepithelial cells, but real understanding ofwith iron-hematoxylin and being partially its function or functions is lacking (Danielli,enveloped by intra-epithelial projections of the (28)), and one may simply conclude that thebasement membrane. From this it is concluded presence of the enzyme in myoepithelial cells,that the basal cells of the duet and the myo- and its absence from duct cells, indicate yetepithelial cells are dissimilar in structure, and another functional difference between the twohence probably also in function. cell types. Preliminary results indicate that cell divisions In view of this and the other differencesare common among duet cells, but that myo- between myoepithelial cells and the basal cellsepithelial cells may be incapable of mitosis. It is of the duet of apoerine sweat glands, it seemssuggested that the occurrence of transitional extremely unlikely that the two cell types sharecells may indicate that myoepithelial cells any major specific function. differentiate from duet cells even in the adult.

Mitosis and Origin of Myoepithelial Cells AcKNOwLEDGMENTs The transitional forms intermediate in struc- I wish to thank Prof. P. V. Tobias and Dr. ture and situation between myoepithelial cellsA. G. Oettlé for helpful criticism of this manu- and the basal cells of the duets may simplyscript. Mr. D. S. Dry and Mr. R. Herman reflect the probable eetodermal origin of theassisted with the photography. myoepithelial cells in the embryo. The possibility nevertheless exists that myoepithelial cells may REFERENCES continue to differentiate from duet cells even in 1. SHELLEY, W. B. AND HUELEY, H. J.: The the adult, and that the observed transitional physiology of the human axillary apoerine sweat gland. J. Invest. Derm., 20: 285—95, forms are stages in this transformation. This is 1953. supported to some extent by the presently 2. HUELEY, H.J. ANDSHELLEY, W. B.: The role of myoepithelium of the human apoerine reported incidence of mitosis in the cells of the sweat gland. Ibid., 22: 143—55, 1954. duet, and the apparent absence of cell division 3. HAooovis'r, C.: In"Handb. der mikr. Anat. from cells of the secretory portion of the gland. des Menseh." Vol. 2 part 3. Ed. W. v. Mollendorff, Berlin Springer Verlag, 1931. The negative finding of the absence of mitosis 4. SeHAFFEE, J.: Das Epithelgewebe. In "Handb. in myoepithelial cells, is based on only one animal der mikr. Anat. des Menseh." Vol. 2 part 1. and of course requires confirmation in more Ed. W. v. Mollendorff, Berlin Springer Verlag, 1927. extensive material. Smallness of the sample 5. LEESON, C. R.: The histoehemieal identifica- also perhaps explains the fact that cell divisions tion of myoepithelium, with particular reference to the harderian and exorbital have been reported (although rarely) in secretory lacrimal glands. Aeta Anat., 40: 87—93, 1960. cells of apoerine sweat glands (Montagna, (21)), 6. HEIDENHAIN, H. (1921): Quoted by Zimmer- but have not been found in the present study. mann, K. W., in "Handb. der mikr. Anat. des Menseh." Vol. 5part 1. Ed. W. von The correlation between the incidence of Mollendorff, Berlin Springer Verlag, 1927. mitoses in sweat glands and in the overlying 7.Hinas, R. C.: The fine structure of human epidermis, and the apparent regional differences eecrinesweat glands. Amer. J. Anat., 103: 201—17, 1958. found in a single animal are also presented here S. Seorr, B. L. ANDPEASE, D.C.: Electron as interesting findings which need confirmation. microscopy of the salivary and lacrimal glands of the rat. Ibid., 104: 115—61, 1959. Among the possibilities which arise is the ques- 9. BRUNN,A.vms: Haut. In "Handb. der Ariat. tion of mitotie waves, known to occur in some des Mensch." Vol. 5 part 1. Ed. K von other species (Montagna, (21)). Bardeleben,Jena Fischer Verlag, 1897. 10. STonE, P.: Lehrbuch der Histologie, 12th Ed. JcnaFischer Verlag, 1906. SUMMARY 11. HOEPKE,H.:Die Haut. In "Handb. der mikr. Transitional cells, intermediate in appearance Anat.des Mensch." Vol. 3 part 1. Ed. W. v. MOllendorff, Berlin Springer Verlag, 1927. and position between myoepithelial cells and12. ALvEEDES, K.: Die apokrinen DrOsen im ORIGIN AND MORPHOLOGY OF MYOEPITHELIAL CELLS 305

Vestibulum nasi des Menschen. Z. Mikros-21. MONTAGNA, W.: The Structure and Function kopisch-anat. Forsch., 28:609—43, 1932. of Skin. New York, Academic Press, 1956. 13.WAY, S. C.ANDMEMMESHEIMEE, A.: The 22. GOLDSTEIN, D. J.: Some histochemical ob- sudoriparous glands. I. The eccrine glands. servations of human striated muscle. Anat. A.M.A. Arch. Derm., 34: 797—SOS, 1936. Eec., 134: 217—37, 1959. 14. BUNTING, H., WIsLocKI, 0. B. AND DEMPSEY, 23. CHAELES, A.: An electron microscopic study E. W.: The chemical histology of human of the human axillary apocrine gland. J. eccrine and apocrine sweat glands. Anat. Anat., 93:226—32,1959. Rec., 100: 61—77, 1948. 24. BUNTING, H.:Cytochemicalproperties of 15. HOE5TMANN E.: Die Haut. In "Handb. der apocrine sweat glands normally present in mikr. Anat. des Mensch." Suppl. to Vol. 3 the human mammary gland. Anat. Rec., 101: part 1. Ed. W. v. MOllendorff, Berlin 5—12, 1948. Springer Verlag, 1957. 25. SHELLEY, W. B. AND MEscoN, H.: Histochemi- 16. LEE, M. M. C.: Histology and histochemistry cal demonstration of secretory activity in of human eccrine sweat glands, with special human eccrine sweat glands. J. Invest. reference to their defence mechanisms. Anat. Derm., 18: 289—301, 1952. Eec., 136: 97—105, 1960. 26. NovIKoYP, A. B.: Histochemical and cyto- 17. SHELLEY, W. B. AND PEEEY, E. T.: The phys- chemical staining methods. In "Analytical iology of the apocrine (ceruminous) gland Cytology". Ed. R. C. Mellors, New York, of the human ear canal. J. Invest. Derm., 26: McGraw-Hill Book Co., 1955. 13—20, 1956. 27. CHIQUOINE, A. D. AND ROTHENBUEG, E. J.: 18. PEAR5E, A. 0. E.: Histochemistry (2nd Ed.) A note on alkaline phosphatase activity of London, Churchill, 1960. 19. BULLOUGH, W. S.: The relation between the germ cells in amblystoma and chick em- epidermal mitotic activity and the blood- bryos. Anat. Eec., 127: 31—5, 1957. sugar level in the adult male mouse, Mus28. DANIELLI, J. F.: The Calcium Phosphate musculus L. J. Exp. Biol., 26: 83—99, 1949. Precipitation Method for Alkaline Phos- 20.BULLOUGH, W. S.: The action of colchicine in phatase. General Cytochemical Methods, arresting epidermal mitosis. Ibid., 26: 287— 1:423—43,Ed. J.F.Danielli, New York, 91,1949. Academic Press, 1958. 306 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY

PLATE I All photomicrographs were taken on panehromatic film, using the following filters: yellow (figs. 8, 9 and 10), green (figs. 1—5, 7, 11, 13, 15, 17, 19 and 21) and crossed Polaroid filters (figs. 12, 14, 16, 18, 20 and 22). FIGs. 1—4. Junctions hetween secretory portions of human apocrine sweat glands, on the right, and ducts, on the left. A gradual transition is seen from the basal cells of the duct to the myoepithelial cells of the secretory portion. Hematoxylin and eosin. X 850. Fin. 5. Apocrine sweat gland from the ear of a Vervet monkey, stained with iron-hematoxylin. The cytoplasm of the myoepithelial cells stains darkly, while that of the basal cells of the duct is pale. The transition between duct and myoepithelial cells in this gland is abrupt. X 850. Fin. 6. Alkaline phosphatase in a monkey apocrine sweat gland (30 minutes incubation). Intense activity is present in the myoepithelial cells, but little activity is seen in the overlying secretory cells at this stage (see also figs. 21 and 23). )< 400. Fin. 7. Monkey sweat gland, stained with P.A.S. Note the intra-epithelial projections of the basement membrane. X 850. Fins. 8—10. Mitoses in ducts of monkey apoerine sweat glands, treated with colehicine. Hematoxylin and eosin. X 1000. ORIGIN AND MORPHOLOGY OF MYOEPITHELIAL CELLS 307

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PLATE II FIG. 11. Vervet myoepithelial cell in longitudinal section. P.A.S. and hematoxylin. X 850. FIG. 12. The same field as fig. 11, polarized light. Note the birefringence in the cytoplasm of the myoepithelial cell. X 850. FIG. 13. Tangential section through human apocrine sweat gland, showing myoepithelial cells and intra-epithelial projections of the basement membrane. P.A.S. and hematoxylin. X 850. FIG. 14. The same field as fig. 12, polarized light. Note the birefringence in the cytoplasm of the myoepithelial cells. X 850. FIG. 15. Transverse (above) and longitudinal (below) sections through parts of a human apocrine sweat gland. In the transverse section one may see intra-epithelial projections of the basement membrane. In the longitudinal section a myoepithelial cell nucleus may be seen, and on the right the nucleus of a cell intermediate in character between a myoepithelial cell and basal cells of the duct (not seen in this field). P.A.S. and hematoxylin. X 850. FIG. ifi. The same field as fig. 15, polarized light. Birefringence may be seen both in the cytoplasm of the myoepithelial cell and in that of the transitional cell. In the transverse section of a tubule (above) birefringence is present only in the basement membrane. )< 850. FIG. 17. Transverse section of the duct of a homan apocrine sweat gland. Heinatoxylin and eosin. x850. FIG. 18. The same field as fig. 17, polarized light. No birefringence is present in the basal cells of the duet, but some birefringent tonofibrils are to be seen in the more superficial cells. X 850. FIG. 19. Longitudinal section through duct of human apocrine sweat gland. Hematoxylin and eosin. x850. FIG. 20. The same field as fig. 19, polarized light. No birefringence is seen in the cytoplasm of the basal cells, but birefringenee is present in tonofibrils and in the basement membrane. >< 850. FIG. 21. Alkaline phosphatase in myoepithelial cell of Vervet apocrine sweat gland (30 minutes incubation). Activity is also present in a capillary on the right. x850. FIG. 22. The same field as fig. 21, polarized light. Birefringenee is seen in the cytoplasm of the myoepithelial cell, bot not in the same region as the precipitate seen in fig. 21. X 850. FIG. 23. Alkaline phosphatase in Vervet apoerine sweat gland (2 hours incubation). A sweat duct is seen running from the secretory portion (below) to a hair follicle (above). Despite the fact that the entire epithelium of the secretory portion is intensely blackened after two hours incubation, there is no precipitate in the duct (the nuclei are lightly stained with hematoxylin). X 200. ORIGIN AND MORPHOLOGY OF MYOEPITHELIAL CELLS 309