An Ultrastructural Study of Normal Human Mammary Epithelial Cells in Culture ' JOSE RUSSO, PHILIP FURMANSKI AND M A R V I N A. RICH Division of Biological Sciences, Michigan Cancer Foundation, 110 East W a r r e n A v e n u e , Detroit, Michigan 48201
ABSTRACT The ultrastructure o f normal human mammary cells cultured from post-weaning breast fluids is described. Cells from confluent monolayers in two week old cultures were studied. The epithelial nature of these cells was established by the demonstration of a well developed system of cell-to-cell interdigitation and numerous desmosomes. These cells also share with breast epithelial cells in vivo, polarity, with blunt short microvilli on the apical surface and an oriented arrangement of organelles in the basal and apical portions of the cells. The Golgi apparatus, which is the most highly developed organelle, is localized in the apical pole and contains substantial quantities of secretory material in the cisternae and vesicles. A variegated palisade of finely granular material mixed with tonofilaments is seen in the basal portion of the cells; many of these tonofilaments end in the terminal web of the desmosomes. The regular occurrence of these cells in breast fluids during the terminal phases of lactation suggests that th9ir separation is a part of normal breast involution.
Many methods have been used i n attempts to isolate and cultivate epithelial cells from human mammary glands and mammary tumors (Buehring, '72; Feller, Stewart and Kantor, '72; Lasfargues et al., '72). Recently, we reported that milk obtained from human donors contains few cells (generally less than lo4 cells per sample of 30-160 ml); these cells did not grow in culture. The concentration and total number of cells increase dramatically at weaning (Furmanski et al., '74a,b). These cells can be placed in culture and, in the presence of autologous serum, grow with a generation time of 60-72 hours. Such cultures of normal human mammary epithelial cells, free from contamination by fibroblasts, provide a n important source of cells for both comparison with mammary tumor cells in a variety of experimental studies and as a substrate for infection with candidate human tumor viruses (Keydar et al., '73; Moore et al., '69; Schlom et al., '72). We therefore initiated detailed studies on the morphological characterization of these cells, the establishment of their epiAM. J. ANAT., 142: 221-232.
thelial nature and a comparison of their morphological characteristics with those described for normal human mammary epithelium i n vivo. M A T E R I A L S A N D METHODS
Breast fluid obtained from human donors after weaning was centrifuged at low speed (200 X g). The cells collected were diluted in culture medium and counted in a Hemacytometer. The culture medium consisted of Dulbecco's modified Eagle's medium containing autologous serum (15% v/v), insulin (10 pg/ml) and antibiotics, a s described previously (Furmanski et al.,'74a,b). The cells rapidly attached and grew, with a doubling time of 60-72 hours. The cultures were used for morphological studies when they had formed confluent monolayers, approximately two weeks after plating. Cells from five different donors were used in the studies described here. 1 This work was supported i n part by Contract NIHNCI-E-71-2421 and grant CA 14100 from the National Cancer Institute and an Institutional Grant to the Michigan Cance; Foundation from the United Foundation of Detroit, Michigan.
22 1
222
JOSE RUSSO, PHILIP FURMANSKI AND MARVIN A. RICH
Cultures were fixed in place using a glutaraldehyde-osmium-uranyl acetate sequence (Hirsch and Fedorko, '68). After dehydration, the cells were removed from the flask with propylene oxide and rinsed three times in order to remove plastic residue from the layer of cells. The material was embedded in Epon (Luft, '61) and sectioned with a diamond knife in an LKBultratome. The sections were stained with lead citrate (Reynolds, ' 6 3 ) and examined in a Siemens Elmiskop IA. RESULTS
Cells in culture, from each of the five donors studied, exhibited a homogenous pattern consisting of a single cell type (figs. 1, 2). Monolayers formed by these cells showed some degree of overlapping. The shape of the cells, while variable, could generally be described as cuboidal. The cells were joined by a well developed system of cytoplasmic interdigitations and numerous desmosomes (fig. 5). More than one desmosome was frequently observed to connect two cells. In some instances, chains of three to four desmosomes were observed. The other characteristics which these cells shared with normal mammary epithelium are a distinct cellular polarity, surface specialization in the form of projecting short, blunt microvilli (fig. 2 ) , and the arrangement of organelles within the cell. The latter is especially evident for Golgi apparatus, secretory material and centrioles in the apical pole of the cell (figs. 2, 7, 9 ) . The basal portions of the cells were filled with condensed, finely granular material, distributed as a variegated palisade adjacent to the cell membrane where it was attached to the culture vessel (figs. 2, 3 , 5). This granular material was mixed with longitudinal and cross sections of tonofilaments (fig. 5). The condensed granular material had a diameter of 80 to 90 A. Similar material was observed inside of vesicular structures in the basal portions of the cells (figs. 3 , 4). These vesicles were surrounded by a unit membrane, and some opened into the cytoplasm. The presence of this granular material
i n the basal portion of the cell may be considered abortive hemidesmosome formation, and may be related to the strong adhesion of these cells to the culture substrate. Tonofilaments were also distributed throughout the remainder of the cytoplasm, some of which were in the form of bundles of fibrils connected to the desmosomes, forming a terminal web (fig. 5). A polymorphic population of mitochondria, differing in size and shape, was found throughout the cytoplasm of human mammary epithelial cells i n culture. In some cells they were more concentrated in the apical and lateral areas (figs. 2, 8). All the mitochondria possessed numerous cristae and a dense matrix with one or two small dense granules (fig. 8). Rough endoplasmic reticulum was distributed randomly in the cytoplasm. This organelle was not well developed in these cells, but was more evident than smooth endoplasmic reticulum, which was very scarce (fig. 8). Free ribosomes and polyribosomes were observed more frequently in the basal portion of the cells (fig. 5). The Golgi apparatus was the most highly developed organelle in these cells. I t consisted of numerous flat cisternae and vesicles filled with granular material of differing electron density (fig. 9 ) , in various stages of packaging as secretory products, with a n approximate diameter of 25-50 A. It is of interest to note, that this is the diameter reported for casein (Bargmann and Welsh, '69). Occasional dense lysosomes were seen in the cytoplasm. The centriolar apparatus (fig. 7 ) was generally localized in the apical portion of the cell among the Golgi vesicles and lipid inclusions. The concentration of lipid droplets varied greatly from one cell to another. Some cells were extremely rich in lipid droplets while others possessed few droplets (figs. 7, 8). The lipids did not have the characteristics of secretory material and we have not observed secretion of this material from the cells. The nucleus of these cells was generally ovoid (figs. 1, 2 ) , but cells with spherical or elongated nuclei were also observed. In some cells the spherical nucleus was indented. Heterochromatin was distributed
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
around the periphery of the nucleus in contact with the inner layer of the nuclear envelope, Usually single, rounded nucleoli were observed, in contact with the nuclear envelope. DISCUSSION
The observations presented here clearly show that these cultured cells isolated from post-weaning human breast fluids are epithelial. An extensive system of cell junctions, cellular polarity, surface differentiation and secretory activity, all features of mammary epithelium, are prominent in these cells. Finely granular material found in the Golgi vesicles, very similar to that described in the Golgi vesicles of the lactating mammary gland in mice (Bargmann and Welsh, '69; Wellings, '69), may represent the product of synthesis and secretion of milk protein. While it is difficult to establish the exact size of this granular material, our estimate of particles with diameters of 25-50 A is in accord with previous reports on milk casein (Bargmann and Welsh, '69; Waugh, '67; Wellings, '69). Biochemical analysis for the synthesis of casein and a-lactalbumin, currently in progress, will clarify this point. The finely granular material in the basal portion of the cells may be synthesized in the endoplasmic reticulum and released into the cytoplasm from vesicles in which it was also seen. The relationship between this granular material and tonofilaments is not yet clear; both have a diameter of 8090 A and are seen in close proximity along the cell membrane attached to the culture vessel. The topographic origin of these cells in the mammary gland is of great interest. The mammary gland is a secretory organ covered by epithelium along the ducts and ductules (Sykes et al., '68; Tannenbaum et al., '69; Toker, '67; Waugh and van der Haeven, '62). Two types of cells are found in the glandular epithelium. One is the epithelial cell that surrounds the lumen into which milk is secreted. The other is the myoepithelial cell, which is located in the basal portion of ducts and ductules, between the luminal epithelial layer and the basal membrane. The ultrastructural
223
characteristics of cells cultured from postweaning fluids are consistent with those of luminal epithelial cells. This conclusion is in agreement with previous observations that epithelial cells in lactating milk arise from the ductal epithelium (Papanicolaou et al., '58). Histochemical and immunological studies in progress are aimed at establishing the specific topographical 10calization of these cells. The tremendous increase in the concentration and total number of cells secreted at or following weaning may be due to random detachment of breast epithelium, or may be a consequence of a specific physiological mechanism involved in the involution of the gland at the end of lactation.' In milk, epithelial cells, fibroblasts, macrophages and a large quantity of cellular debris are observed (Papanicolaou et al., '58). Attempts to culture cells from this fluid were consistently unsuccessful (Furmanski et al., '74a). Postweaning fluids, on the other hand, contain a very much higher concentration of epithelial cells, which retain a capacity to grow in short term cultures (Furmanski et al., '74a,b). These cells, therefore, may represent breast secretory epithelium which is released during the process of breast involution after weaning, a time in which the levels of hypophyseal hormones that maintain secretion and the tissues of the breast during lactation, decrease (Bargmann and Welsh, '69; Cross and Findlay, '69; Meites et al., '69). This detachment of epithelial cells from the ductal lining may represent a mechanism for the involution of the breast at the end of lactation, alternative or in addition to the mechanisms proposed by other authors (Helminen and Ericsson, '68; Mumford, '63; Richards and Benson, 771). ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Mr. Ronald H. Bradley, Robert Kaspark, Clifford Longley and the assistance of Ms. Ruth Rich in the acquisition of biological resources. LITERATURE CITED
Bargmann, W., and U . Welsh 1969 On the ultrastructure of the mammary gland. In: 2
Furmanski et al., manuscript in progress.
224
JOSE RUSSO, PHILIP FURMANSKI AND MARVIN A. RICH
“Lactogenesis, The Initiation of Milk Secretion and Parturition.” M. Reynolds and S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 43-52. Buehring, G. C. 1972 Culture of human mammary epithelial cell: Keeping abreast with a new method. J. Natl. Cancer Inst., 49: 14331434. Cross, B. A., and A. L. R. Findlay 1969 Comparative and sensory aspects of milk ejection. In: “Lactogenesis, The Initiation of Milk Secretion and Parturition.” M. Reynolds and S . J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 245-252. Feller, W. F., S. E. Stewart and J. Kantor 1972 Primary tissue culture explants of human breast cancer. J. Natl. Cancer Inst., 48: 11171120. Furmanski, P., C. Longley, D. Fouchey, R. Rich and M. A. Rich 1974a Normal human mammary cells in culture: Evidence for 0ncornav:rus-like particles. J. Natl. Cancer Inst., 52: 975-977. - 197413 Oncornavirus-like particles in human mammary cells in culture. Fed. Proc., 33: 753 (abstract). Helminen, H . J., and J. L. E. Ericsson 1968 Studies on mammary gland involution. I. On the ultrastructure of the lactating mammary gland. J. Ultrast. Res., 25: 193-213. Hirsch, J. G., and M. E. Fedorko 1968 Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and postfixation in uranyl acetate. J. Cell Biol., 38: 615-627. Keydar, J., Z. Gilead, S. Karby and E. Hare1 1973 Production of virus by embryonic cultures co-cultivated with breast tumor cells or infected with milk from breast cancer patients. Nature New Biol., 242 : 49-52. Lasfargues, E. Y., W. G. Coutinho and D. H. Moore 1972 Pitfalls in the isolation of a human breast carcinoma virus in tissue culture. J. Natl. Cancer Inst., 48: 1101-1105. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9 : 409-414. Meites, J., M. Sar and J. L. Voogt 1969 Effect of suckling and pituitary release of prolactin, ACTH, GH and TSH in the rat. In: ‘Lactogenesis, The Initiation of Milk Secretion and
Parturition,” M. Reynolds and S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 171-179. Moore, D. H., J. Charney, B. Kramarsky, E. Y. Lasfargues. N. H. Sarkas, M. J. Brennan, J. H . Burrows, S . M. Sirsat, J. C. Paymaster and A. B. Vaidya 1969 Search for a human breast oncornavirus. Nature (London), 229: 611-614. Munford, R. E. 1963 Changes in the mammary glsnds of rats and mice during pregnancy, 1actat:on and involution: I. Histological structure. J. Endocrinol., 28: 37-45. Papanicolaou, G. N., D. G. Holmquist, G. H. Bader and E. A. Falk 1958 Exfoliative cytology of the human mammary gland and its value in the diagnosis of cancer and other diseases of the breast. Cancer, 11: 3 7 7 4 0 9 . Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Richards, K. C., and G. K. Benson 1971 Ultrastructural changes accompanying involution of the mammary gland in the albino rat. J. Endocrinol., 51: 127-132. Schlom, J., S. Spiegelman and D. H. Moore 1972 Reverse transcriptase and high molecular weight RNA in particles from mouse and human milk. J. Natl. Cancer Inst., 48: 1197-1203. Sykes, J. A., L. Recher, P. H. Jernstrom and J. Whitescarver 1968 Morphological investigation of human breast cancer. J. Natl. Cancer Inst., 40: 195-223. Tannenbaum, M., M. Weiss and A. J. Marx 1969 Ultrastructure of the human mammary ductule. Cancer, 23: 958-978. Taker, C. 1967 Observations of the ultrastructure of a mammary ductule. J. Ultrastruct. Res., 21: 9-25. Waugh, D. F. 1967 The interaction of a’, p and k-caseins in micelle formation. Disc. Faraday SOC.,25: 186-192. Waugh, D. F., and E. van der Haevea 1962 Fine structure of the human adult female breast. Lab. Invest., 11: 220-228. Wellings, S. R. 1969 Ultrastructural basis of lactogenesis. In: “Lactogenesis, The Initiation of Milk Secretion and Parturition,” M. Reynolds and S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 5-25.
PLATES
PLATE 1 EXPLANATION OF FIGURES
226
1
One micron section of human epithelial cells from post-weaning fluid in culture. x 850.
2
Human mammary epithelial cells from post-weaning fluid after two weeks in culture. Microvilli M, desmosomes D, mitoch-ndria m, filaments F, Golgi apparatus G, lysosome L, granular material in the basal portion of the cell GM. x 7,500.
3
Basal portion of the epithelial cells, with the granular material GM attached to the membrane, forming a variegated palisade; vesicles V with granular material of the same characteristics. x 28,000.
4
High magnification of a vesicle V apparently releasing granular material GM into the basal portion of the cell. x 44,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 1
227
PLATE 2 EXPLANATION OF FIGURES
228
5
TWOcells joined by desmosomes in the apical and basal poles of the cells. Numerous tonofilaments T are present, some confluent, forming tonofibrils ending i n the terminal web of the desmosomes. Free ribosomes R and polyribosomes PR are i n the cytoplasm. x 34,500. The upper inset shows a desmosome. x 60,000.
6
Cytoplasmic interdigitation (arrows) between the epithelial cells from the post-weaning fluid. The granular material GM is apparently attached to the cell membrane. >; 18,200.
7
Golgi apparatus G and vesicles with secretory material S surrounding the centrioles C. Upper left corner and lower right corner, lipid droplets Lip. x 18,200.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 2
229
PLATE 3 EXPLANATION OF FIGURES
230
8
Portion of the cytoplasm showing mitochondria m with different size and shape, granular endoplasmic reticulum ger, lysosomes L, lipid droplets Lip, tonofilaments T. x 15,700.
9
Putative secretory material S and its relation to Golgi apparatus G , lipid droplets Lip and mitochondria rn. x 15,700. Inset: High magnification of the finely granular material in a secretory vesicle. x 72,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 3
231
ABSTRACT The ultrastructure o f normal human mammary cells cultured from post-weaning breast fluids is described. Cells from confluent monolayers in two week old cultures were studied. The epithelial nature of these cells was established by the demonstration of a well developed system of cell-to-cell interdigitation and numerous desmosomes. These cells also share with breast epithelial cells in vivo, polarity, with blunt short microvilli on the apical surface and an oriented arrangement of organelles in the basal and apical portions of the cells. The Golgi apparatus, which is the most highly developed organelle, is localized in the apical pole and contains substantial quantities of secretory material in the cisternae and vesicles. A variegated palisade of finely granular material mixed with tonofilaments is seen in the basal portion of the cells; many of these tonofilaments end in the terminal web of the desmosomes. The regular occurrence of these cells in breast fluids during the terminal phases of lactation suggests that th9ir separation is a part of normal breast involution.
Many methods have been used i n attempts to isolate and cultivate epithelial cells from human mammary glands and mammary tumors (Buehring, '72; Feller, Stewart and Kantor, '72; Lasfargues et al., '72). Recently, we reported that milk obtained from human donors contains few cells (generally less than lo4 cells per sample of 30-160 ml); these cells did not grow in culture. The concentration and total number of cells increase dramatically at weaning (Furmanski et al., '74a,b). These cells can be placed in culture and, in the presence of autologous serum, grow with a generation time of 60-72 hours. Such cultures of normal human mammary epithelial cells, free from contamination by fibroblasts, provide a n important source of cells for both comparison with mammary tumor cells in a variety of experimental studies and as a substrate for infection with candidate human tumor viruses (Keydar et al., '73; Moore et al., '69; Schlom et al., '72). We therefore initiated detailed studies on the morphological characterization of these cells, the establishment of their epiAM. J. ANAT., 142: 221-232.
thelial nature and a comparison of their morphological characteristics with those described for normal human mammary epithelium i n vivo. M A T E R I A L S A N D METHODS
Breast fluid obtained from human donors after weaning was centrifuged at low speed (200 X g). The cells collected were diluted in culture medium and counted in a Hemacytometer. The culture medium consisted of Dulbecco's modified Eagle's medium containing autologous serum (15% v/v), insulin (10 pg/ml) and antibiotics, a s described previously (Furmanski et al.,'74a,b). The cells rapidly attached and grew, with a doubling time of 60-72 hours. The cultures were used for morphological studies when they had formed confluent monolayers, approximately two weeks after plating. Cells from five different donors were used in the studies described here. 1 This work was supported i n part by Contract NIHNCI-E-71-2421 and grant CA 14100 from the National Cancer Institute and an Institutional Grant to the Michigan Cance; Foundation from the United Foundation of Detroit, Michigan.
22 1
222
JOSE RUSSO, PHILIP FURMANSKI AND MARVIN A. RICH
Cultures were fixed in place using a glutaraldehyde-osmium-uranyl acetate sequence (Hirsch and Fedorko, '68). After dehydration, the cells were removed from the flask with propylene oxide and rinsed three times in order to remove plastic residue from the layer of cells. The material was embedded in Epon (Luft, '61) and sectioned with a diamond knife in an LKBultratome. The sections were stained with lead citrate (Reynolds, ' 6 3 ) and examined in a Siemens Elmiskop IA. RESULTS
Cells in culture, from each of the five donors studied, exhibited a homogenous pattern consisting of a single cell type (figs. 1, 2). Monolayers formed by these cells showed some degree of overlapping. The shape of the cells, while variable, could generally be described as cuboidal. The cells were joined by a well developed system of cytoplasmic interdigitations and numerous desmosomes (fig. 5). More than one desmosome was frequently observed to connect two cells. In some instances, chains of three to four desmosomes were observed. The other characteristics which these cells shared with normal mammary epithelium are a distinct cellular polarity, surface specialization in the form of projecting short, blunt microvilli (fig. 2 ) , and the arrangement of organelles within the cell. The latter is especially evident for Golgi apparatus, secretory material and centrioles in the apical pole of the cell (figs. 2, 7, 9 ) . The basal portions of the cells were filled with condensed, finely granular material, distributed as a variegated palisade adjacent to the cell membrane where it was attached to the culture vessel (figs. 2, 3 , 5). This granular material was mixed with longitudinal and cross sections of tonofilaments (fig. 5). The condensed granular material had a diameter of 80 to 90 A. Similar material was observed inside of vesicular structures in the basal portions of the cells (figs. 3 , 4). These vesicles were surrounded by a unit membrane, and some opened into the cytoplasm. The presence of this granular material
i n the basal portion of the cell may be considered abortive hemidesmosome formation, and may be related to the strong adhesion of these cells to the culture substrate. Tonofilaments were also distributed throughout the remainder of the cytoplasm, some of which were in the form of bundles of fibrils connected to the desmosomes, forming a terminal web (fig. 5). A polymorphic population of mitochondria, differing in size and shape, was found throughout the cytoplasm of human mammary epithelial cells i n culture. In some cells they were more concentrated in the apical and lateral areas (figs. 2, 8). All the mitochondria possessed numerous cristae and a dense matrix with one or two small dense granules (fig. 8). Rough endoplasmic reticulum was distributed randomly in the cytoplasm. This organelle was not well developed in these cells, but was more evident than smooth endoplasmic reticulum, which was very scarce (fig. 8). Free ribosomes and polyribosomes were observed more frequently in the basal portion of the cells (fig. 5). The Golgi apparatus was the most highly developed organelle in these cells. I t consisted of numerous flat cisternae and vesicles filled with granular material of differing electron density (fig. 9 ) , in various stages of packaging as secretory products, with a n approximate diameter of 25-50 A. It is of interest to note, that this is the diameter reported for casein (Bargmann and Welsh, '69). Occasional dense lysosomes were seen in the cytoplasm. The centriolar apparatus (fig. 7 ) was generally localized in the apical portion of the cell among the Golgi vesicles and lipid inclusions. The concentration of lipid droplets varied greatly from one cell to another. Some cells were extremely rich in lipid droplets while others possessed few droplets (figs. 7, 8). The lipids did not have the characteristics of secretory material and we have not observed secretion of this material from the cells. The nucleus of these cells was generally ovoid (figs. 1, 2 ) , but cells with spherical or elongated nuclei were also observed. In some cells the spherical nucleus was indented. Heterochromatin was distributed
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
around the periphery of the nucleus in contact with the inner layer of the nuclear envelope, Usually single, rounded nucleoli were observed, in contact with the nuclear envelope. DISCUSSION
The observations presented here clearly show that these cultured cells isolated from post-weaning human breast fluids are epithelial. An extensive system of cell junctions, cellular polarity, surface differentiation and secretory activity, all features of mammary epithelium, are prominent in these cells. Finely granular material found in the Golgi vesicles, very similar to that described in the Golgi vesicles of the lactating mammary gland in mice (Bargmann and Welsh, '69; Wellings, '69), may represent the product of synthesis and secretion of milk protein. While it is difficult to establish the exact size of this granular material, our estimate of particles with diameters of 25-50 A is in accord with previous reports on milk casein (Bargmann and Welsh, '69; Waugh, '67; Wellings, '69). Biochemical analysis for the synthesis of casein and a-lactalbumin, currently in progress, will clarify this point. The finely granular material in the basal portion of the cells may be synthesized in the endoplasmic reticulum and released into the cytoplasm from vesicles in which it was also seen. The relationship between this granular material and tonofilaments is not yet clear; both have a diameter of 8090 A and are seen in close proximity along the cell membrane attached to the culture vessel. The topographic origin of these cells in the mammary gland is of great interest. The mammary gland is a secretory organ covered by epithelium along the ducts and ductules (Sykes et al., '68; Tannenbaum et al., '69; Toker, '67; Waugh and van der Haeven, '62). Two types of cells are found in the glandular epithelium. One is the epithelial cell that surrounds the lumen into which milk is secreted. The other is the myoepithelial cell, which is located in the basal portion of ducts and ductules, between the luminal epithelial layer and the basal membrane. The ultrastructural
223
characteristics of cells cultured from postweaning fluids are consistent with those of luminal epithelial cells. This conclusion is in agreement with previous observations that epithelial cells in lactating milk arise from the ductal epithelium (Papanicolaou et al., '58). Histochemical and immunological studies in progress are aimed at establishing the specific topographical 10calization of these cells. The tremendous increase in the concentration and total number of cells secreted at or following weaning may be due to random detachment of breast epithelium, or may be a consequence of a specific physiological mechanism involved in the involution of the gland at the end of lactation.' In milk, epithelial cells, fibroblasts, macrophages and a large quantity of cellular debris are observed (Papanicolaou et al., '58). Attempts to culture cells from this fluid were consistently unsuccessful (Furmanski et al., '74a). Postweaning fluids, on the other hand, contain a very much higher concentration of epithelial cells, which retain a capacity to grow in short term cultures (Furmanski et al., '74a,b). These cells, therefore, may represent breast secretory epithelium which is released during the process of breast involution after weaning, a time in which the levels of hypophyseal hormones that maintain secretion and the tissues of the breast during lactation, decrease (Bargmann and Welsh, '69; Cross and Findlay, '69; Meites et al., '69). This detachment of epithelial cells from the ductal lining may represent a mechanism for the involution of the breast at the end of lactation, alternative or in addition to the mechanisms proposed by other authors (Helminen and Ericsson, '68; Mumford, '63; Richards and Benson, 771). ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Mr. Ronald H. Bradley, Robert Kaspark, Clifford Longley and the assistance of Ms. Ruth Rich in the acquisition of biological resources. LITERATURE CITED
Bargmann, W., and U . Welsh 1969 On the ultrastructure of the mammary gland. In: 2
Furmanski et al., manuscript in progress.
224
JOSE RUSSO, PHILIP FURMANSKI AND MARVIN A. RICH
“Lactogenesis, The Initiation of Milk Secretion and Parturition.” M. Reynolds and S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 43-52. Buehring, G. C. 1972 Culture of human mammary epithelial cell: Keeping abreast with a new method. J. Natl. Cancer Inst., 49: 14331434. Cross, B. A., and A. L. R. Findlay 1969 Comparative and sensory aspects of milk ejection. In: “Lactogenesis, The Initiation of Milk Secretion and Parturition.” M. Reynolds and S . J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 245-252. Feller, W. F., S. E. Stewart and J. Kantor 1972 Primary tissue culture explants of human breast cancer. J. Natl. Cancer Inst., 48: 11171120. Furmanski, P., C. Longley, D. Fouchey, R. Rich and M. A. Rich 1974a Normal human mammary cells in culture: Evidence for 0ncornav:rus-like particles. J. Natl. Cancer Inst., 52: 975-977. - 197413 Oncornavirus-like particles in human mammary cells in culture. Fed. Proc., 33: 753 (abstract). Helminen, H . J., and J. L. E. Ericsson 1968 Studies on mammary gland involution. I. On the ultrastructure of the lactating mammary gland. J. Ultrast. Res., 25: 193-213. Hirsch, J. G., and M. E. Fedorko 1968 Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and postfixation in uranyl acetate. J. Cell Biol., 38: 615-627. Keydar, J., Z. Gilead, S. Karby and E. Hare1 1973 Production of virus by embryonic cultures co-cultivated with breast tumor cells or infected with milk from breast cancer patients. Nature New Biol., 242 : 49-52. Lasfargues, E. Y., W. G. Coutinho and D. H. Moore 1972 Pitfalls in the isolation of a human breast carcinoma virus in tissue culture. J. Natl. Cancer Inst., 48: 1101-1105. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9 : 409-414. Meites, J., M. Sar and J. L. Voogt 1969 Effect of suckling and pituitary release of prolactin, ACTH, GH and TSH in the rat. In: ‘Lactogenesis, The Initiation of Milk Secretion and
Parturition,” M. Reynolds and S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 171-179. Moore, D. H., J. Charney, B. Kramarsky, E. Y. Lasfargues. N. H. Sarkas, M. J. Brennan, J. H . Burrows, S . M. Sirsat, J. C. Paymaster and A. B. Vaidya 1969 Search for a human breast oncornavirus. Nature (London), 229: 611-614. Munford, R. E. 1963 Changes in the mammary glsnds of rats and mice during pregnancy, 1actat:on and involution: I. Histological structure. J. Endocrinol., 28: 37-45. Papanicolaou, G. N., D. G. Holmquist, G. H. Bader and E. A. Falk 1958 Exfoliative cytology of the human mammary gland and its value in the diagnosis of cancer and other diseases of the breast. Cancer, 11: 3 7 7 4 0 9 . Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212. Richards, K. C., and G. K. Benson 1971 Ultrastructural changes accompanying involution of the mammary gland in the albino rat. J. Endocrinol., 51: 127-132. Schlom, J., S. Spiegelman and D. H. Moore 1972 Reverse transcriptase and high molecular weight RNA in particles from mouse and human milk. J. Natl. Cancer Inst., 48: 1197-1203. Sykes, J. A., L. Recher, P. H. Jernstrom and J. Whitescarver 1968 Morphological investigation of human breast cancer. J. Natl. Cancer Inst., 40: 195-223. Tannenbaum, M., M. Weiss and A. J. Marx 1969 Ultrastructure of the human mammary ductule. Cancer, 23: 958-978. Taker, C. 1967 Observations of the ultrastructure of a mammary ductule. J. Ultrastruct. Res., 21: 9-25. Waugh, D. F. 1967 The interaction of a’, p and k-caseins in micelle formation. Disc. Faraday SOC.,25: 186-192. Waugh, D. F., and E. van der Haevea 1962 Fine structure of the human adult female breast. Lab. Invest., 11: 220-228. Wellings, S. R. 1969 Ultrastructural basis of lactogenesis. In: “Lactogenesis, The Initiation of Milk Secretion and Parturition,” M. Reynolds and S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 5-25.
PLATES
PLATE 1 EXPLANATION OF FIGURES
226
1
One micron section of human epithelial cells from post-weaning fluid in culture. x 850.
2
Human mammary epithelial cells from post-weaning fluid after two weeks in culture. Microvilli M, desmosomes D, mitoch-ndria m, filaments F, Golgi apparatus G, lysosome L, granular material in the basal portion of the cell GM. x 7,500.
3
Basal portion of the epithelial cells, with the granular material GM attached to the membrane, forming a variegated palisade; vesicles V with granular material of the same characteristics. x 28,000.
4
High magnification of a vesicle V apparently releasing granular material GM into the basal portion of the cell. x 44,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 1
227
PLATE 2 EXPLANATION OF FIGURES
228
5
TWOcells joined by desmosomes in the apical and basal poles of the cells. Numerous tonofilaments T are present, some confluent, forming tonofibrils ending i n the terminal web of the desmosomes. Free ribosomes R and polyribosomes PR are i n the cytoplasm. x 34,500. The upper inset shows a desmosome. x 60,000.
6
Cytoplasmic interdigitation (arrows) between the epithelial cells from the post-weaning fluid. The granular material GM is apparently attached to the cell membrane. >; 18,200.
7
Golgi apparatus G and vesicles with secretory material S surrounding the centrioles C. Upper left corner and lower right corner, lipid droplets Lip. x 18,200.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 2
229
PLATE 3 EXPLANATION OF FIGURES
230
8
Portion of the cytoplasm showing mitochondria m with different size and shape, granular endoplasmic reticulum ger, lysosomes L, lipid droplets Lip, tonofilaments T. x 15,700.
9
Putative secretory material S and its relation to Golgi apparatus G , lipid droplets Lip and mitochondria rn. x 15,700. Inset: High magnification of the finely granular material in a secretory vesicle. x 72,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Jose Russo, Philip Furmanski and Marvin A. Rich
PLATE 3
231
