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Ultrastructure and immunohistochemistry of the embryonic type of fat identified in the human infant breast.

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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.
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