Ultrastructure and immunohistochemistry of the embryonic type of fat identified in the human infant breast.код для вставкиСкачать
THE ANATOMICAL RECORD 241:129-135 (1995) Ultrastructure and lmmunohistochemistry of the Embryonic Type of Fat Identified in the Human Infant Breast RAMASWAMY ANBAZHAGAN AND BARRY A. GUSTERSON Section of Cell Biology and Experimental Pathology, Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, United Kingdom ABSTRACT In this paper we describe the light and electron microscopic appearance of the embryonic type of fat in human infant breast, together with immunocytochemical findings. This fat tissue was composed of numerous capillaries surrounded by a mixed population of undifferentiated mesenchymal cells and preadipocytes at various stages of differentiation. The preadipocytes were characterised by a number of cytoplasmic processes, varying numbers of lipid droplets, and an envelope of electrondense material outside the cell membrane. Immunocytochemistry showed a characteristic distributionof collagen type IV adjacent to and vimentin and SfOO @ein within the preadipveytes. This is the first report of the ukmstructure of the human mammary embryonic type of fat. The possible role of the embryonic type of fat in the development and growth of the human breast is discussed. o 1995 Wiley-Liss, Inc. Key words: Embryonic fat, Infant breast, Immunocytochemistry, Mesenchymal cells, Preadipocytes We have recently described the microanatomical and four were females. Tissue fixed in a 50:50 mixture of histological appearance of the human infant breast 2% glutaraldehyde and 2% formaldehyde was available (Anbazhagan et al., 1991). During the course of this in three cases. Slices of tissue from dissected areas adstudy we noticed the presence of characteristic fat tis- jacent to the ducts were carefully examined under a sue, which a t the light microscope level appeared to be stereomicroscope. The approximate location of the emembryonic in type, and its close association with the bryonic type of fat was identified in each case, aided by developing ducts. This embryonic type of fat could be the knowledge of light microscopic observation. These considered as the anatomical counterpart of fat pad areas were dissected out, cut into 1-2 mm pieces, and mesenchyme described in the developing mouse mam- processed for electron microscopy. Paraffin sections mary glands. The structure of mouse mammary fat pad from samples fixed in modified methacarn (Mitchell et mesenchyme and its possible inductive role in the de- al., 1985) were available from five cases and used for velopment of mammary glands have been described immunocytochemical study. The time of fixation after (Sakakura et al., 1982). There is only one brief report death was not known. describing the ultrastructure of human embryonic type of fat (Kats et al., 1984), and this was in inguinal tisProcessing for Electron Microscopy sue. It is important to have a proper knowledge of the The microdissected samples were postfixed in 1%osstructure of this tissue and its functional potential in mium tetroxide in phosphate buffer (pH 7.2-7.4) for a order to understand its possible role in human breast minimum of 1h, dehydrated through a graded series of development. We therefore carried out a n ultrastruc- alcohols, and embedded in Epon-Araldite via propylene tural and immunocytochemical study of this tissue to oxide (Mollenhauer, 1964). Semithin sections (1 Fm confirm the presence of embryonic type of fat, to com- thick) were stained with 1%toluidine blue and exampare the ultrastructure of this embryonic type of fat ined using a light microscope in order to select areas for tissue with the embryonic type of fat reported in the electron microscopy. Ultrathin sections (0.05-0.07 Fm) rodents, and to study the distribution of vimentin, col- were cut from selected areas of the block using a Reilagen type IV, and SlOO protein which are known to be chert Ultracut Microtome OMU4, collected on 200present in the rodent counterpart. mesh copper grids, and double stained (Stempack and Ward, 1964) with uranyl acetate and lead citrate in a n MATERIALS AND METHODS LKB Ultrastainer. The sections were viewed and photographed using a Philips CMlO electron microscope. Tissue Used The material used in this study is the same a s that used previously (Anbazhagan et al., 1991). The embryReceived March 23, 1994; accepted June 16, 1994. onic type of fat was observed in 7 out of 72 infant Address reprint requests to B.A. Gusterson, Section of Cell Biology breasts in the previous study. The age of these infants and Experimental Pathology, Institute of Cancer Research, Haddow ranged from 3 days to 2 months; three were males and Laboratories, Sutton, Surrey SM2 5NG, United Kingdom. 0 1995 WILEY-LISS. INC 130 R. ANBAZHAGAN AND B.A. GUSTERSON Antibodies Used Anti-type IV collagen antibody This is a mouse monoclonal antibody (clone CIV 22) directed against collagen IV (Dakopatts, Glostrup, Denmark). The antibody recognizes a conformational epitope on a helical part of native collagen IV and shows a distinct reactivity with collagen IV in formalin-fixed and paraffin-embedded tissue. This was supplied a s tissue culture supernatant and used a t a dilution of 1:25. Anti-vimentin antibody This is a mouse monoclonal antibody (clone v9) raised against purified vimentin from porcine eye lens and supplied as tissue culture supernatant (Dakopatts). This antibody reacts with vimentin, the 57 kD intermediate filament protein. This antibody was used a t a dilution of 1:lO. Anti-S1 00 protein antibody This is a rabbit polyclonal antibody raised against S-100 protein isolated and purified from cow brain (Dakopatts). This antibody reacts with cow and human S-100 A and B. It was used at a dilution of 1:lO. lmmunocytochemistry Pretreatment of sections with 50 pgiml pronase (Calbiochem, La Jolla, CA) in phosphate buffered saline for 15 min at 37°C was found to be necessary for adequate staining with anti-type IV collagen antibody. Staining with this antibody was performed using a Vectastain ABC kit from Vector Laboratories (Peterborough, UK). Staining with anti-vimentin and anti-S100 protein antibodies was performed by a n indirect immunoperoxidase method as previously described (Anbazhagan et al., 1991). RESULTS The embryonic type of fat was successfully localised in two of the three cases processed for electron microscopy. One of these (male) showed poorly differentiated embryonic type of fat (PDEF), and the other (female) had a moderately differentiated embryonic type of fat (MDEF). This distinction is based on their difference in the light and ultrastructural appearance, which is described below. Light Microscopy The embryonic type of fat tissue was composed of developing adipocytes and capillaries which were organised in a number of distinct round to oval islands surrounded by fibrocollagenous tissue. There was considerable variation in the morphology of the developing adipocyte between cases. Within the same sample, however, the morphology of these cells showed only slight variation. These differences were mainly attributable to the different stages of maturation of these cells. Islands of PDEF were composed of sheets of preadipocytes with oval to polyhedral nuclei and dark eosinophilic cytoplasm. Similar cells were also seen in MDEF, but they were very few in number. These cells were so closely packed together that their cytoplasmic margins could not be distinguished clearly. Islands of MDEF were larger in size. They were composed of sheets of oval cells having centrally placed round nuclei and large amounts of pale eosinophilic cytoplasm. Some of these cells showed multiple clear vacuoles of varying sizes in the cytoplasm indicating the presence of fat. A varying number of mature fat cells with eccentric nuclei and large cytoplasmic vacuoles were seen a t the center of some of these islands. Numerous small capillaries were distributed throughout this embryonic type of fat in all cases. The histological appearance of the embryonic type of fat in semithin sections stained with toluidine blue was similar to that of the paraffin sections, but finer details were better appreciated. For example, in the case of PDEF the cell membrane of the immature adipocytes with a number of cytoplasmic processes could be clearly seen. Ultrastructure As this material was from postmortems and was not optimially fixed for electron microscopy, the ultrastructural detail was not ideal, but a number of characteristics could be clearly defined. Ultrastructurally, PDEF showed numerous capillaries lined by endothelial cells, each with a single, slightly irregular elongated nucleus and a thin cytoplasm forming the capillary wall. Surrounding these capillaries there were various cell types, including immature mesenchymal cells and preadipocytes a t different stages of maturation. The cytological features of various cell types found in the PDEF are a s follows. Mesenchymal Cells The mesenchymal cells were often seen in pairs, located near the capillaries and intermingled with preadipocytes and fibroblasts. These cells appeared stellate or elongated in shape and had a high nuclear cytoplasmic ratio (Fig. 1).The nucleus was often deeply indented, and the nuclear chromatin was condensed into course clusters near the nuclear membrane. The cytoplasm in each cell was thin surrounding the nucleus and sometimes extending into slender processes. Many of these cytoplasmic processes were short, but some were long, extending between the capillaries and preadipocytes or in between the individual preadiopocytes. Preadipocytes The earliest identifiable preadipocytes were larger than the mesenchymal cells. The nuclear and cytoplasmic features were very similar to the mesenchymal cell. The cytoplasm showed one or two fat droplets, which identified them as preadipocytes (Fig. 2). The typical preadipocytes showed numerous cytoplasmic processes and moderate amounts of perinuclear cytoplasm (Fig. 3 ) . They had a number of cytoplasmic organelles and a n electron-lucent cytoplasmic matrix. Most of the organelles were located in the paranuclear area and appeared to be mitochondria. A few pinocytotic vesicles could be seen along the plasma membrane, and there were small lipid droplets in the cytoplasm. The nucleus mainly had a dispersed chromatin, with dense condensed clusters near the nuclear membrane. Some preadipocytes were larger and showed a more condensed nuclear chromatin and more electron-dense ULTRASTRUCTURE OF EMBRYONIC TYPE OF FAT 131 Fig. I . Electron micrograph of two mesenchymal cells showing a high nuc1ear:cytoplasmic ratio. Bar indicates 1 pm. Fig. 2. Electron micrograph of a preadipocyte with mesenchymal-like features and containing occasional fat droplets. Bar indicates 1 pm. cytoplasmic matrix. The organelles appeared to be few in number, but this is difficult to assess in large cells where only one plane of section is examined. The lipid droplets were larger and more numerous. Occasional cells with single large lipid droplets with similar cyto- plasmic features were seen. The nucleus was peripherally located and still showed a n indented outline (Fig. 4). Each preadipocyte was surrounded by a n envelope of electron-dense material, which ultrastructurally had 132 R. ANBAZHAGAN AND B.A. GUSTERSON Fig. 3. Electron micrograph of a preadipocyte with a number of fat droplets, multiple cytoplasmic processes, and electron-dense material adjacent to the plasma membrane. Bar indicates 2 pm. Fig. 4. Electron micrograph of a preadipocyte with a large fat droplet. Bar indicates 2 pm features of basal lamina-like material. This envelope was of variable thickness and filled the area between the cytoplasmic processes. This envelope was invariably lacking around the mesenchymal cells. In addition to the adipocytes, occasional mast cells, macrophages, and fibroblasts were also seen. The preadiopocytes of MDEF were much larger than the preadipocytes of PDEF. The cells were round, lacking cytoplasmic processes or electron-dense envelopes. They showed two to three large round lipid droplets filling the entire cytoplasm. Their cytoplasm was thin surrounding the lipid droplets. The nucleus was located 133 ULTRASTRUCTURE OF EMBRYONIC TYPE OF FAT Fig. 5. The embryonic type of fat immunostained for collagen type IV. Positive staining is seen outside the cell membrane. Fig. 6. The embryonic type of fat immunostained for vimentin. Positive staining is seen around lipid droplets. peripherally and was smoother with fewer nuclear indentations, but not compressed. Cytoplasmic organelles were rare. lmmunocytochernistry Collagen type IV Staining with anti-type IV collagen antibody revealed positive immunoreactivity associated with preadipocyte, in the PDEF cases, and in the basement membrane of the capillaries. In the preadipocytes, the staining was seen outside the cell membrane (Fig. 5) which corresponded to the electron-dense envelope observed outside the cell membrane with electron microsCOPY. Virnentin Staining with anti-vimentin antibody showed positive cytoplasmic staining in the preadipocytes. In the preadipocytes of PDEF, the staining was prominent in the perinuclear area. The cytoplasm of some of the preadipocytes showed numerous thin strands of positive staining which in some areas showed a mesh-like pattern. In the more differentiated preadipocytes of MDEF, the positive staining strands appeared to encase the cytoplasmic lipid droplets (Fig. 6). S-100 protein Staining with anti-S100 protein antibody was mainly localised in the cytoplasm of the preadipocytes. The endothelial cells were negative. The preadipocytes of PDEF showed diffuse intense staining in the cyto- plasm. The differentiated preadipocytes of MDEF showed weak to moderate staining. DISCUSSION Most studies on the ultrastructural appearances of the embryonic type of fat have been undertaken in rats and mice. Available literature regarding the adipocyte differentiation in rodent samples elaborately describes the morphological appearance at different stages of differentiation. There is considerable controversy regarding the histogenesis and the cell of origin of preadipocytes (reviewed in detail by Nnodim, 1987). Napolitano (1963) first outlined the ultrastructural appearances of developing rat inguinal and epididymal fat pads. He described fibroblast-like preadipocytes with long tenuous cytoplasmic extensions and profuse endoplasmic reticulum. He observed a gradual change in the shape of the cell, transitory appearance of glycogen, accumulation of cytoplasmic non-membranebound lipid droplets, and changes in other organelles during differentiation. Simon (1965) coined the name “adipogenic reticular cell” to describe the precursors of rat interscapular brown adipocytes. He described the ultrastructural appearance of this adipogenic reticular cell and emphasized that this was quite distinct from a “fibrocyte,” the latter being characterised by cytoplasmic processes and small, inconspicuous mitochondria. Usuku e t al. (1978) studied the differentiation of epididymal fat pads of rats and showed that in early preadipocytes a small number of microvesicles and smooth or occasionally rough endoplasmic reticulum was in 134 R.ANBAZHAGAN AND R.A. GUSTERSON close contact with the surface of the lipid droplets. At later stages lipid droplets were surrounded by welldeveloped smooth endoplasmic reticulum which also connected with rough endoplasmic reticulum in some places. They considered that smooth endoplasmic reticulum was closely related to active formation of lipid droplets. Lyama et al. (1979) found some similarity between the cytoplasmic processes of preadipocytes and the cytoplasmic process of pericytes in transplanted epididymal fat pads. From this they concluded that immature pericytes of capillaries could differentiate into preadipocytes. Slavin (1979) described ultrastructural features of differentiating adipocytes from different regions in mice. His important observations include peculiar intercellular contacts between the cellular processes of preadipocytes and the presence of a n external lamina. The electron-dense envelope seen around the preadipocytes contains type IV collagen, supporting the view that this is basal lamina material synthesized by these immature fat cells. Later, Bani-Sacchi e t al. (1987) described adipocyte differentiation in the mouse mammary gland following stimulation with 17P-estradiol and relaxin. They suggested four stages of adipocyte differentiation. The earliest “pale preadipocytes” were characterised by their irregular shape due to cytoplasmic processes, well-developed organelles, few small lipid droplets, and numerous pinocytotic vesicles. The next stage “dark preadipocytes” showed a denser cytoplasmic matrix, reduced organelles, and more abundant lipid accumulations. Subsequent stages were a globular multivacuolated adipocyte and univacuolated, signet-ring adipocyte. Light microscopic appearance of the embryonic type of fat at different stages of maturation has been described previously (Anbazhagan e t al., 1991). To our knowledge there is only one earlier report of the ultrastructure of the human embryonic type of fat (Kats et al., 1984). These authors mainly described the ultrastructural features of embryonal liposarcoma and gave a brief account of the similarities and differences between the human embryonic type of fat and the embryonal liposarcoma. The embryonic type of fat was obtained from the inguinal fat pad of a 27 cm length human embryo. No electron micrographs of normal immature adipocytes were included in this paper. The ultrastructural details of the human breast embryonic type of fat described by us showed many features similar to that seen in rodents. Since the material was obtained from necropsy samples, the finer features of the organelles could not be resolved. There are a number of theories regarding the cell of origin of preadipocytes. Mesenchymal cells, fibroblasts, endothelial cells, and pericytes have all been put forward a s possible progenitor cells. These considerations were based on the morphological similarities between the preadipocytes and the proposed progenitor cells. In the present study, the presence of fat droplets was identified in some cells which otherwise resembled mesenchymal cells. This would suggest mesenchymal cells as the favourable candidate for the progenitor cell, but definite confirmation would require a detailed study of a large number of samples and the use of immunocyt,ochemical markers. ...~~ Until recently there was no realiable marker to iden- tify early preadipocytes. Cinti et al. (1989) have suggested S-100 protein as a useful marker for the identification of early preadipocytes in vivo and in vitro. This is a calcium-binding protein that was first described in nervous tissue (Moore, 1965). Although earlier it was considered specific for glial cells, it was subsequently demonstrated in various epithelial (including mammary) and connective tissue cells (Haimoto et al., 1987). S-100 protein is present in mature adipocytes (Suzuki et al., 1982; Nakajima et al., 1982; Hidaka et al., 1983; Kato e t al., 1983; Michetti et al., 1983) and in early preadipocytes (Cinti et al., 1989) but is absent in fibroblasts (Cocchia et al., 1981,1983; Hashimoto et al., 1984; Haimoto et al., 1987). The biological role of this molecule is not well established, but it has been suggested that it acts as a carrier of fatty acids (Haimoto et al., 1985). Until more such markers are identified and employed in the identification of the progenitor cell, the cell of origin of preadipocytes will remain elusive. Vimentin intermediate filaments have been identified in the differentiating preadipocytes in vivo and in vitro and appear to be the only intermediate filament present in the preadipocytes (Frankey et al., 1987). In normal 3T3 cells and in early stages of adipose conversion, the vimentin filaments appear to be distributed over most of the cytoplasm and show association with the nascent lipid droplets. At the more advanced stages of adipose conversion they appear to be localized around fat globules which are in t u r n surrounded by endoplasmic reticulum, resulting in complex cage-like structures. This arrangement might play a role in favouring interaction between lipids and other diffusible components of the cytoplasm, including triacylglycerol and the enzymes involved in their biosynthesis. Adipocyte precursors are capable of synthesizing collagen (Cryer and Van, 19821,and 3T3-Ll preadipocytes also synthesize substantial levels of collagen (Green and Meuth, 1974). Changes in collagen mRNA expression during adipocyte differentiation have been reported (Weiner et al., 1989). The presence and distribution of S-100 protein, vimentin, and type IV collagen in the embryonic type of fat of the infant breast were similar to those reported in the rodent tissue. This would suggest that the preadipocytes of the human breast are very similar to the rodent counterpart in structure as well a s function and possibly play a similar role in mammary development. It is known that adipocytes in the bone marrow play a crucial role in providing the microenvironment for growth and proliferation of haematopoietic cells. In the developing mammary gland, embryonic mesenchyme is required for normal growth and development both in vivo and in vitro (Krotochwil, 1969; Sakakura et al., 1976). The 3T3-Ll cell line which undergoes adipocyte differentiation promotes the growth of mammary epithelium in vitro (Levine and Stockdale, 1984). While it is obvious that the preadipocytes support the growth of a variety of cells, the mechanism by which they influence mammary epithelial growth is largely unknown. Rudland et al. (1984) have tested media which have been exposed to different rat mammary fibroblasts and fibroblast cell lines which have the potential to yield adipocytes in vitro. They identified a common trophic substance, prostaglandin E,, that can stimulate the growth of Drimarv cultures and cell lines of normal and ULTRASTRUCTURE OF E MBRYONIC TYPE OF FAT neoplastic rat mammary epithelium. Further studies on the structural and functional aspects of adipocyte differentiation and its interaction with epithelial cells are essential for a more complete understanding of mammary epithelial growth and differentiation. The close association of the embryonic type of fat and the ductal elements is consistent with the rodent and in vitro data suggesting that this embryonic type of fat may play a n inductive role in glandular morphogenesis. ACKNOWLEDGMENTS The Institute of Cancer Research is supported by funds from the Cancer Research Campaign and the Medical Research Council. R. Anbazhagan is supported by a generous grant from the Lady Joseph Fund. LITERATURE CITED Anbazhagan, R., J . Bartek, P. Monaghan, and B.A. Gusterson 1991 Growth and development of the human infant breast. Am. J. Anat., 192:407-417: Bani-Sacchi. T.. S. Bianchi. G. Bani. and M. Bicrazzi 1987 Ultrastructural studies on white adipocyte differentiation in the mouse mammary gland following oestrogwt and relaxin. Acta Anat., 129:l-9. Cinti, S., M. Cigolini, M. Moorroni, and M.C. Zingaretti 1989 S-100 protein in white preadipocytes: An immunoelectronmicroscopic study. Anat. Rec., 224r466-472. Cocchia, D., L. Lauriola, V.M. Stolfi, G. 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