Ultrastructure of the human mammary gland. II. Postpartum lactogenesis.

Ultrastructure of the Human Mammary Gland. II. Postpartum Lactogenesis HECTOR TOBON AND HERNANDO SALAZAR Department of Pathology, Magee-Womens Hospital, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania devoid of limiting membrane. Abundant fat droplets were present in the basal and apical regions and "pinched off' into the lumen, apparently surrounded by a limiting membrane. The lactogenic cells were richly endowed with slender microvilli in their luminal pole and related to one another by cytoplasmic prolongations, desmosomes and tight junctions. The myoepithelium was stretched and thinned out toward the periphery of the acini between the lactogenic epithelium and the basement lamina. Abundant myofilaments were present in the myoepithelial cells. Pinocytotic vesicles were also present, although not very numerous, in both the lactogenic and the myoepithelial cells. In general the human mammary gland during lactopoiesis follows similar structural changes as those previously described in other mammals. (/ Clin Endocrinol Metab 40: 834, 1975)

ABSTRACT. The histology and fine structure of 7 human mammary glands were studied in the postpartum. Widespread hypertrophy and hyperplasia of the mammary acini were observed accompanied by dilatation and engorgement of the lumen by milk. Loose strands of connective tissue surrounding the mammary lobules and ductules contained dilated, engorged vascular channels. The lactogenic epithelial cells displayed rich cytoplasm containing prominent layering stacks of rough endoplasmic reticulum closely related to the enlarged oval mitochondria, on occasion surrounding their entire circumference. Also noted were numerous ribosomes and polyribosomes. The markedly hypertrophied Golgi apparatus revealed cisternae containing particulate electron dense material and vesicles with dense granules. The latter were frequently seen being discharged in the lumen

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XTENSIVE light and electron microscopic studies of the mammary gland during lactogenic activity have been done in numerous mammals (1-4). Surprisingly, however, no studies have been published of the fine structural changes of the human mammary gland during the postpartum period and lactation, except for our own observations (5). This has constituted an obvious gap in the general knowledge of the human mammary gland. Undoubtedly, the understanding of the morphology of cellular changes of this gland during its various phases of growth, development and maturation with detailed knowledge of the fine structural modifications under different normal and abnormal conditions, are basic for the analysis of the subcellular functional phenomena occurring in the mammary gland as an endresponse organ to the various hormonal influences. As part of our continuing studies on the human mammary gland under various Received November 15, 1974.

physiological and pathological conditions, it is the purpose of this communication to present a correlative account of the histological and ultrastructural modifications observed during lactogenesis in the postpartum. Our findings are correlated with the various endocrinologic events normally occurring during this period. Materials and Methods These studies were carried out in mammary gland tissue, normal in appearance, obtained at surgery from seven lactating women biopsied from 1 day to 5Y2 months postpartum. Although several of these patients had lactogenic adenomas or galactoceles, for which they were operated upon, the tissues studied were adjacent to but not involved in these processes. Small fragments of mammary tissue were fixed in cold 2.5% gluteraldehyde in Millonig's buffer, pH 7.35, post-fixed in buffered osmium tetroxide, and dehydrated in graded alcohols and propylene oxide. Embedding was carried out in Epon-Araldite. Areas of mammary acini were screened in 1 fim thick sections with toluidine blue and adequate tissue phases selected for thin sectioning with diamond

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knives. Thin sections mounted on uncoated copper grids and stained with uranyl acetate and lead citrate were studied and photographed in a Philips 300 electron microscope.

Results

Grossly, the specimens obtained from the mammary glands were multilobulated, rubbery and tannish-yellow. The acini and ducts were distended and a thick whitishyellow, milky material was easily expressed. The adipose portions of the breast were not as abundant as in the normal resting mammary gland. These macroscopic observations were similar, although at different levels of magnitude, in all the glands studied at variable times in the postpartum. The histological and ultrastructural features described and discussed below represent a cross section of the subcellular morphological events occurring during lactogenesis, generally observed at the different times studied. The only significant exception is that of involutionary changes secondary to waning of lactation in one case. Histology. Sections stained with hematoxylin and eosin and toluidine blue revealed hyperplastic mammary lobules occupying most of the microscopic field (Fig. 1). They were surrounded by strands of collogenous fibers and numerous enlarged and engorged vascular channels. There seemed to be a decreased amount of adipose tissue, relative to the breast parenchyma that had become greatly hypertrophied and secretory. Throughout the breast, however, the mammary acini varied in size, resulting from variations in the height of the epithelial cells and the caliber of the lumina, which contained different amounts of secretory material. On occasion, foci of resting mammary lobules, devoid of secretory activity, were seen. The distended terminal mammary ductules were filled with finely granular eosinophilic secretory material, sometimes partially basophilic, containing fat

FIG. 1. Histologic appearance of a human mammary gland during active lactogenesis. The mammary acini are greatly enlarged and surrounded by thick strands of fibrocollagenous tissue. Notice the varying amounts of secretory material within the lumens of alveoli and terminal ductules. H & E. (x53)

globules, fragments of cells and, at times, entire desquamated epithelial cells. The dilated ductules displayed an inner coat of large, vacuolated cuboidal cells with abundant eosinophilic cytoplasm containing various sized lipid globules at different levels within the cytoplasm (Fig. 2). The nuclei were large, round to ovoid, had a finely granulated chromatin and eosinophilic nucleoli of various sizes which, in general, were quite prominent. The myoepithelial cells were inconspicuous and the few observed had a clear halo around the nuclei. Ultrastructure. The distended mammary ductules contained electron dense, granular material admixed with fat globules of various sizes. Also present were large fragments of lactogenic epithelial cells and naked nuclei in various stages of karyorrhexis.


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FIG. 2. Close-up view of mammary gland from Fig. 1. The alveoli are greatly distended with granular secretion and fat globules. There are fat droplets in the lactogenic cells, some already discharged into the lumen (short arrows). Note narrow ductules with little or no secretion. Myoepithelial cells are inconspicuous. H & E. (x210)

The most striking changes were noted in the lactogenic epithelial cells (Fig. 3). They occupied the entire thickness of the alveolar and ductular walls between the lumen and the peripheral basement lamina, except in places where narrow segments of myoepithelial cytoplasm were interposed. Cells richly endowed with a large number of organelles were interpreted as undergoing high metabolic activity. The most prominent features were abundant free ribosomes and polyribosomes, as well as layering stacks of rough endoplasmic reticulum (RER) which gave a dark appearance to the cytoplasm. The enlarged cisternae of the RER contained finely granular

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electron dense material and occupied very extensive portions of the cell body. The Golgi apparatus was mostly located towards the luminal pole of the lactogenic cells, although it was frequently present all around the nuclei reaching into the basal regions of the cytoplasm (Figs. 3 and 4). Its dilated cisternae and vesicles contained osmiophilic granular material organized as tortuous, chain-like structures which seemed to coalesce into larger irregular granules. This material became more clumped, coarser and seemed to form larger and denser granules as the cisternae approached the secretory pole of the cell. The largest and most compact of these granules were adherent to the vesicular walls (Fig. 4). Numerous elongated or oval shaped mitochondria, present throughout the cytoplasm, had prominent transverse cristae embedded in a finely granular matrix. Quite frequently, the outer contour of mitochondria was silhouetted by RER lamella, even surrounding their entire circumference (Fig. 5). Lipid inclusions of various sizes were observed in several regions of the cytoplasm, but particularly in a supranuclear location (Figs. 3 and 4). They appeared as homogeneous material of medium electron density, occasionally observed in the process of being discharged into the lumen by a "pinching off" mechanism, apparently coated by a close limiting membrane. On occasion, a few lysosomes could be observed as well as a few myelin bodies and discreet clusters of fine cytoplasmic filaments. The cells contained scanty pools of glycogen. The general shape of nuclei was irregularly round to ovoid but frequently displayed a tortuous contour, sometimes with deep indentations. The chromatin was finely granular forming irregular heterochromatinic clumps which appeared floating free in the nuclear sap but most of the time attached to the inner coat of the nuclear envelope.



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FIG. 3. This electronmicrograph illustrates a portion of a human mammary gland 1 day postpartum. This hypertrophic lactogenic cell whose luminal pole (L) shows short microvilli and relates to other lactogenic cells (La) by interlocking microvilli, tight junctions and desmosomes (short arrows). Parallel layering stacks of RER are noted throughout as well as fat globules (F). The mitochondria are enlarged and abundant. The Golgi apparatus (G) shows dilated cisternae, some containing secretory material (long arrows). Note prominent nucleolus and folded nucleus, (x 10,100)


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a constant feature at the luminal pole. The secretory surface of the lactogenic cells was studded with short, thin microvilli between which secretory and pinocytotic vesicles were present. Large, conical cytoplasmic prolongations containing fat vacuoles and profiles of RER were also observed at the luminal pole in the process of being detached and released into the lumen in a "microapocrine" fashion. The basal pole of the epithelial cells was either supported by a thin, single, well formed basement lamina, or adjacent and attached to myoepithelial elements. Epithelial cells in a "resting" phase (Fig. 6) were observed chiefly in groups within inactive alveoli and also isolated among

FIG. 4. This tall columnar lactogenic cell is apposing directly the basement lamina at the bottom and shows short, stubby microvilli at the luminal surface. Note large elongated mitochondria towards the center and hypertrophic, dilated vesicles of Golgi apparatus (G) containing varying sized granules adherent to their inner surface (short arrows). Abundant free ribosomes are noted throughout as well as profiles of RER (long arrows). A portion of a "resting" lactogenic cell is seen on the right side with narrow Golgi cisternae. A portion of its nucleus and nucleolus is also seen, (x 5,320)

The epithelial cells related to each other and to the stretched myoepithelial cells by long, slender cytoplasmic prolongations protruding and interlocking in the intercellular spaces. Desmosomes were not very numerous. Tight epithelial junctions were

FIG. 5. Cluster of large, ovoid, and elongated mitochondria whose outer contour is partially or totally surrounded by RER (arrows). Tiny empty vesicles of the Golgi (G) system are also observed, (x 13,000).



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FIG. 6. "Resting" epithelial cells (Ep) showing dark cytoplasm containing abundant free ribosomes. The Golgi apparatus (G) shows dilated and narrow empty cisternae. Note the slender profiles of RER (long arrows), the tiny fat globules (F) and the clusters of tonofilaments (short arrows). The cells appose directly the basement lamina (arrowheads), have scanty pinocytotic activity and present short stubby microvilli towards the lumen (L). Mitochondria are abundant, (x9,340)

actively secreting cells. These resting cells showed abundant cytoplasm with clusters of oval or slightly elongated mitochondria whose finely granular matrix embedded cristae of varying lengths. They had abundant free ribosomes and slender, parallel profiles of RER cisternae containing scant,

fine granular material. The Golgi system varied in appearance being either inconspicuous or slightly dilated with empty sacs and channels. Fat bodies were seen in small numbers and sizes. The luminal pole of these cells showed short, stubby microvilli and no evidence of release of secre-


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FIG. 7. Myoepithelial cells (My) at the periphery of a ductule depicting characteristic folded nuclei with dense chromatin clumps adherent to the nuclear membrane. Their richly fibrillar cytoplasm contains tiny clusters of small mitochondria (long arrow), scanty ribosomes and inconspicuous slender RER profiles. Note abundant branching prolongations surrounded by tortuous multiple layers of basement lamina (short arrows) and hemidesmosomes (arrowheads) probably representing a state of contraction. The inset shows a thin cytoplasmic prolongation of myoepithelium rich in myofilaments and studded with dense bodies, (x8,900)

tory products. The nucleus in general bore little differences with that of the fully active secretory cells. Complete myoepithelial cells were rarely seen in the distended acini (Fig. 7). More frequently, small elongated portions of cytoplasmic extensions containing myofibrils were observed, always located peripherally between the plump epithelial cells and the basement lamina. Their relatively small and elongated nuclei had irregular outlines, deep indentations and large heterochromatin clumps under the

nuclear membrane. The cytoplasm was narrow and richly endowed with myofilaments and dense bodies. Small oval mitochondria and pools of glycogen were scanty. The Golgi system and the RER were inconspicuous and never secretory. Portions of the myoepithelium, adjacent to the basement lamina, presented hemidesmosomes and pinocytotic vesicles. In some places, finely slender prolongations of the myoepithelial cells reached out into the adjacent connective tissue in an arborescent fashion, usually coated by a thin and



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tortuous basement lamina which frequently tules delimiting the epithelial-stromal space appeared multilaminated (Fig. 7). described by Ozzello (6). The endotheThe fibroconnective tissue surrounding lium of the dilated capillaries was richly the mammary acini was composed of large endowed with pinocytotic vesicles, had prominent fibroblasts with large and di- long intraluminal slender prolongations and lated RER cisternae filled with fine particu- was constantly surrounded by a well delate material. Sometimes elongated fibro- fined basement lamina. blasts were observed containing groups of well formed intracytoplasmic fibrils of Discussion tropocollagen (Fig. 8). Slender prolongations of these fibroblasts constantly coated It is currently accepted that, as a target the outer portion of the acini and duc- organ, the mammary gland undergoes a

FIG. 8. This micrograph illustrates the epithelial-mesenchymal region including a segment of a lactogenic cell (La) and a portion of underlying connective tissue containing sections of three capillaries. The large fibroblast (Fi) in the center shows a markedly folded nucleus that has trapped large RER cisternae filled with finely granular material. Numerous fibrils traverse the irregular cytoplasm. The capillaries have dilated lumens and the endothelium is richly endowed with pinocytotic vesicles and slender superficial processes (arrows). The epithelial-stromal junction space (double head arrow) is obvious. A basement lamina is seen surrounding the three capillaries and the epithelial cells, (x 7,800)


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series of hormonal effects during gestation resulting in the "priming" of the epithelial cell machinery for lactogenesis to occur in the postpartum. These processes seem to be similar in all mammals and characterized by stages of mammary growth with enlargement of the acinary system, epithelial hyperplasia and lobular hypertrophy, developed prior to the actual secretion of milk. This gestational maturation includes, at the subcellular level, the formation of a rich ribosomal system, both free and clustered, hyperplasia of the RER, enlargement of the Golgi system and increase in the number and volume of mitochondria (8). Using the method originally developed by Lyons (9) and Meites and Turner (10), we have demonstrated in virgin rabbits treated with human chorionic gonadotrophin (hCG) the remarkable ultrastructural changes occurring in the epithelial cells during the pseudo-pregnant state and the cellular events of lactogenesis induced in the same animals by the intraductal injection of ovine pituitary prolactin or human placental lactogen (hPL) (11). In short, during pseudo-pregnancy the epithelial cells become hypertrophic and hyperplastic with enlargement of mammary lobules. Their cytoplasm shows marked development of the RER, the Golgi apparatus, the ribosomal system and the mitochondria. Cells thus prepared are rendered lactogenic by a single intraductal injection of ovine pituitary prolactin or hPL. The most remarkable changes are observed in the RER and Golgi systems whose cisternae become full of proteinaceous secretory material discharged into the alveolar lumen as dense granules. Abundant lipid globules are "pinched off" into the lumen. This process of lactogenesis can be inhibited in situ by ovine prolactin antiserum as shown by Saji and Crighton (12). These varied morphologic changes in the mammary gland, as shown by Fiddler et al. (13), are accompanied by a fast increase of RNA and DNA, followed by

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an increase in the amounts of lactose and casein-like proteins. Our observations reported in this paper show that during human lactogenesis in the postpartum probably a comparatively active protein and carbohydrate synthesis also occurs, judging from the dilatation of the RER and its high content of participate material and from the enlargement of the Golgi system. Large lipid droplets appear also in the cytoplasm and are secreted during this lactopoietic phase. The large number of mitochondria, which are sometimes gigantic during this active secretory period, may be related to the high demand for energy necessary for synthesis and transport. Although fine structural characteristics of the milk-forming products differ from one species to another, generally speaking, the ultrastructural features observed in the human lactogenic process resemble those of the glands of lactating cows (1), rabbits (2), mice (3) and rats (4). Numerous advances have been gained in the understanding of some of the actions of the pituitary prolactin upon the mammary gland at various stages of growth and differentiation. As recently summarized by Friesen et al. (14), maternal serum prolactin levels begin to rise and then increase progressively to term, reaching approximately 200 ng/ml. Tyson et al. (15) have shown that, during the first postpartum

week, suckling induces a further but smaller increase in maternal prolactin levels within 30 mins; however, between the 10th and 40th day postpartum, the same suckling stimulus elicits about a 6-fold increase in circulating prolactin. Morphologically, this increase apparently results in a larger number of acini being engaged in active secretion at a given time, but not in subcellular differences related to hormonal levels at various stages of the post-partum. According to the same investigators (15), basal levels of prolactin are reached again at about 80 days postpartum and no further


ULTRASTRUCTURE OF HUMAN LACTOGENIC BREAST increase is produced after suckling. Basal concentrations are also observed 2 to 3 weeks postpartum in mothers who do not breast-feed their babies (14). From the ultrastructural data here presented, we can infer that an advanced stage of metabolic function has been attained by the epithelial lactogenic cells by the first day of the postpartum. It is in general characterized by the peak hypertrophy and hyperplasia of the cell machinery developed during gestation. The extreme richness of the cytoplasm denotes a remarkable development for the synthesis of enzymes and milk proteins as well as other milk components. This activity is depicted by the dilated cisternae of the RER which is arranged in large layering stacks, very closely related to the fat droplets and abundant mitochondria. At the same time aremarkable hypertrophy of the Golgi system is observed. These two systems denote, therefore, a rich production of proteins and/or glycoproteins being carried on. The electron-dense granular proteinaceous material of the endoplasmic reticulum seems to go through a process of concentration in the Golgi system, with dense granules being carried within vesicles across the cytoplasm and discharged into the lumen intermixed with lipids and cellular fragments. Accompanying these changes there are large, elongated and pleomorphic mitochondria forming a great part of the cell volume, perhaps providing the necessary chemical energy for the active metabolic process involved in the secretion of milk. The large nuclei of lactogenic cells with their abundant chromatin and prominent nucleoli are probably indicative also of the great synthetic activity demanding abundance of nucleic acid precursors. All of the morphologic parameters observed during the first day postpartum are maintained more or less throughout the lactogenic period. There is, however, a cell-to-cell variation in the time sequences of the secretory activity. We have shown at

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the light microscopy level a variation in the secretory activity of adjacent lobules as well as of acini within a given lobule. In addition, we have observed by electron microscopy a similar variation between individual cells within the same alveolus or ductule. Cells seem to be at different stages of secretory activity judging from the development of cytoplasmic organelles and the varying amount of secretory materials observed. Thus, it is possible that in the human gland there is a hormonedependent secretory cycle in each cell or group of cells, independent from each other and operating asynchronously in its various stages of synthesis, storage, transport and release; this active cycle is followed, perhaps, by a period of inactivity or recovery. The asynchrony of this cycle probably guarantees the continuity of the lactogenic function. As the process of secretion continues, the acini becomes dilated, storing milk; the stellate myoepithelial cells are stretched out and compressed against the basement lamina by the flattening epithelial cells and the intraluminar accumulation of milk. It seems that the stretching of myoepithelial cells advances with the formation of large gaps between the elongated cytoplasmic processes, through which the epithelial cells appose directly the basement lamina. The modification of myoepithelial cells during lactogenesis and their functional implications in milk ejection were elegantly demonstrated histologically by the unsurpassed work of K.C. Richardson in lactating goats (16). In conclusion, this fine structural study of the cellular elements of the human breast during the postpartum, shows that the harmonious functions of the various organelles of the mammary epithelial cells follow a sequence of cell growth and differentiation conducive to the production of milk. These processes, which are regulated by complex endocrinologic conditions during pregnancy, "prime" or prepare


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the lactogenic cells for a rich secretory activity and the myoepithelium for a supportive and contractile function in the postpartum, in a similar fashion to that observed in other mammals. These functions of lactogenesis, lactopoiesis and milk ejection are, then, accomplished under a purely maternal hormonal milieu, stimulated by the suckling of the offspring. Acknowledgments The authors gratefully acknowledge the expert technical assistance of Robert Florida and Alice Olah.

6. 7.

8.

9. 10. 11.

References 1. Feldman, J. D., Lab Invest 10: 238, 1961. 2. Hollmann, K. H., Z Zellfors Mikrosk Anat 69: 395, 1966. 3. , Ultrastruct Res 2: 423, 1959. 4. Bargmann, W., and U. Welsch, In Reynolds, M., and S. J. Folley (eds.), Lactogenesis: The Initiation of Milk Secretion at Parturition, ed. 1, University of Pennsylvania Press, Philadelphia, 1969, p. 43. 5. Salazar, H., and H. Tobon, In Josimovich, H. B., M. Reynolds, and E. Cobo (eds.), Lactogenic Hormones, Fetal Nutrition and Lactation, ed. 1,

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John Wiley and Sons, Inc., New York, 1974, p. 221. Ozzello, L., Cancer 25: 586, 1970. Rivera, E. M., In Josimovich, J. B., M. Reynolds, and E. Cobo (eds.), Lactogenic Hormones, Fetal Nutrition and Lactation, ed. 1, John Wiley and Sons, Inc., New York, 1974, p. 279. Hollman, K. H., In Reynolds, M., and S. J. Folley (eds.), Lactogenesis: The Initiation of Milk Secretion at Parturition, ed. 1, University of Pennsylvania Press, Philadelphia, 1969, p. 27. Lyons, W. R., Proc Soc Exp Biol NY 51: 308, 1942. Meites, J., and C. W. Turner, Am J Physiol 150: 394, 1947. Josimovich, J. B., R. J. Stock, and H. Tobon, In Josimovich, J. B., M. Reynolds, and E. Cobo (eds.), Lactogenic Hormones, Fetal Nutrition and Lactation, ed. 1, John Wiley and Sons, Inc., New York, 1974, p. 335. Saji, M. A., and D. B. Crighton, J Endocrinol 41: 555, 1968. Fiddler, T. J., M. Birkinshaw, and I. R. Falconer,/ Endocrinol 49: 459, 1971. Friesen, H. G., P. Fournier, and P. Desjardins, In Osofsky, H. J. (ed.), Clinical Obstetrics and Gynecology, ed. 1, Harper and Row, Hagerstown, New York, 1973, p. 25. Tyson, J. E., P. Hwang, H. Guyda, and H. G. Friesen, Am] Obstet Gynecol 113: 14, 1972. Richardson, K. C , Proc R Soc Med 136: 30, 1949.


Inhibition by low concentrations of ouabain of prolactin-induced lactogenesis in rabbit mammary-gland explants.

Activity of DNA-dependent RNA polymerases in rabbit mammary gland during lactogenesis.

Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution.

Gland ultrastructure in human gall bladder.

Ultrastructure of chromophobe adenoma of the human pituitary gland.

MHC class II expression in the bovine mammary gland.

Ultrastructure of a spindle cell carcinoma in the mammary gland of a dog.

Lactogenesis in explant cultures of mammary tissue from pregnant cows.

IRE1 phosphatase PP2Ce regulates adaptive ER stress response in the postpartum mammary gland.

Examination of Duct Physiology in the Human Mammary Gland.

Precursors of hexoneogenesis within the human mammary gland.

The mammary gland.

Ultrastructure of the mink parotid gland.

Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression.

Prolactin and the onset of mammary extraction of plasma triacylglycerols during lactogenesis in the goat.

Mammary gland involution as an immunotherapeutic target for postpartum breast cancer.

Effects of the presence of indium on the mammary gland ultrastructure, body weight, food intake and plasmatic prolactin concentration.

Alteration of human placental lactogen by mammary gland.

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure.

Postpartum psychosis and the pineal gland.

Ultrastructure of human organ-cultured cornea. II. Stroma and epithelium.

Ultrastructure of the parotid gland of the nine-banded armadillo.

Membranes of mammary gland. XI. Marker enzyme distribution profiles for membranous components from bovine mammary gland.

On the ultrastructure of the parotid gland in miniature pigs.