The Mam mary F at Pad

Journal of Mammary Gland Biology and Neoplasia,Vol.3,No.2,1998Editorial CommentaryThe Mammary Fat PadMargaret C.Neville,1,4Daniel Medina,2Jenifer Monks,1and Russel C.Hovey3The mammary fat pad is essential of the mammary epithelium,providingsignals that mediate ductal alveolar Theªclearedºfat pad is often used as a transplantation the crucial of fat pad,itsproperties have received relatively little attention from researchers in the field.Some of thequestions whose investigation is pertinent to understanding both normal mammary developmentand carcinogenesis are outlined in this commentary in the spirit of stimulating enquiry intothis important subject.It is clear from a brief perusal of the available literature that untilstudies are specifically designed to clearly differentiate between functional effects of thefibrous and the adipose stroma,more substantive information about their differential effectson mammary development and tumorigenesis will not be forthcoming.Development of the mammary epithelium is reproductive states has received relatively little unique in that mammary ducts must grow into a pad of attention.adipose tissue if morphogenesis is to proceed normally.Most work on the mammary fat pad has been The result is that the mammary epithelium is associated conducted in mice,a species with a relatively low with an adipocyte-containing stroma throughout all proportion of fibrous connective tissue investing the phases of development.In many women the ducts and ducts and alveoli.In the virgin female the mammary lobules(equivalent to the alveoli in mice)are invested gland consists of fine ducts bearing sparse or frequent with a dense connective tissue matrix(1)outside of alveolar buds,depending on the strain,coursing islands of adipocytes comprise a fair proportion through a copious adipose stroma(Fig.1A).During the stroma.According to Bartow there is a greater pregnancy the alveoli multiply and expand into the preponderance of adipose stroma in some populations adipose stroma in such a way that islands of adipocytes of women with a lower incidence of breast cancer are interspersed around the alveolar structures(Fig.(2).However,the relative distribution of fibrous and1B).In lactation,at least in rodents,the adipocytes adipose stroma in the human breast across various lose most of their fat content;however,the cells arestill present as very long projections in the interstitialspace between alveoli(3,4),(Figs.1C and2).In themouse the layer of fibrous connective tissue around 1Department of Physiology,University of Colorado School of Med-both small ducts and secretory alveolar cells is much icine,Box C240Denver,Colorado80262.thinner in the pregnant and lactating gland,and mam-2Department of Cell Biology,Baylor College of Medicine,Onemary epithelial cells in these developmental stages Baylor Plaza,Houston,Texas77030.3Laboratory of Tumor Immunology and Biology,National Institutes are associated quite intimately with the surrounding of Health,Building10,Room5B56,Bethesda,Maryland20892.adipocytes as illustrated in Figs.2D and2E.Within a 4To whom correspondence should be addressed at Departmentday or two of the cessation of milk removal the stromal of Physiology,Box C240,Room3802,University of Coloradoadipocytes refill with fat and can be clearly seen inter-Health Sciences Center,Denver,Colorado80262.e-mail:peggy.neville@ spersed among the milk-filled alveoli.1091083-3021/98/0400-0109$15.00/0q1998Plenum Publishing Corporation110Neville,Medina,Monks,and HoveyA that has been extensively utilized as a transplantationsite for both normal mammary epithelium and tumors(9±13).In one experiment the fat pad itself was trans-planted and shown to have age-dependent effects onthe acquisition of progesterone receptors by the epithe-lium(14).It is important to recognize that the clearedmammary fat pad comprises more than adipose tissueand includes several other cell types such as fibro-blasts,endothelial cells and cells of the immunesystem,any or all of which could influence mammo-B genesis.However,the specific effects of the mammaryfat pad relative to other mesenchymes in the recombi-nation experiments of Sakakura and others,describedlater,suggest that differential morphogenetic responsesmay be directly or indirectly due to the presence ofthe adipocytes within the mammary fat pad.Fat cells have also been shown to enhance thegrowth of mammary cells in vitro.For example,pri-mary mammary epithelial cells from a pregnant mouseC cultured with an adipocyte cell line(3T3-L1)grewmore rapidly than cells plated on plastic(15)and alsosynthesized milk components in response to lactogenichormones(16).The growth response could be mim-icked by plating the mammary cells on the extracellularmatrix laid down by the adipocytes;however,the adi-pocytes themselves had to be present for casein synthe-sis to occur.Fibroblast lines did not duplicate theseeffects.Fig.1.Cross sections of mouse mammary gland showing fatty Considering the crucial role of the fat pad in stroma in(A)virgin;(B)pregnant;and(C)lactating gland.Figuremammary development as well as its frequent utiliza-used by permission from Ref.(58).Magnification50.tion as a transplantation site,the properties of the fatpad itself have received relatively little attention fromresearchers in the field.Some of the questions whose The mammary fat pad arises in the mouse embryoas a large,separate condensation of mesenchyme that investigation is pertinent to understanding both normal appears posterior to the mammary rudiment on daymammary development and carcinogenesis are out-14of development and occupies more than half the lined here in the spirit of stimulating enquiry into this subcutaneous space of the trunk(5).A similar structureimportant subject.appears at mid-pregnancy in the human fetus(Fig.Does the mammary fat pad differ from other adi-3),(6).Preadipocytes in the mammary fat pad of thepose depots with respect to its ability to support the embryonic mouse begin to accumulate fat droplets on growth of mammary epithelium?In early experiments day16of gestation;differentiation into monolocularHoshino(9,17)and Faulkin and DeOme(8)showed adipocytes is complete two to three days after birth(7).that epithelial elements would grow and respond to Around day16the rudimentary ducts of the mammarylactogenic hormones in any of four fat depots,mam-epithelium begin to expand into the fat pad;however mary,perirenal,mesometrial and subcutaneous.How-much of the fat pad is devoid of parenchyma untilever,the extent of growth was less in the perirenal after puberty.At puberty ducts elongate and mesometrial fat layers than in the cleared fourthmammary fat pad(17).Later work also showed that to fill the entire fat pad,their growth asthey approach its margins.brown fat would support mammary growth(18).Pieces The epithelial rudiment can be removed in itsof mammary gland transplanted to subcutaneous sites entirety from the fourth mammary gland of the three devoid of adipose tissue could be maintained but did week old mouse(8)resulting in aªclearedºfat padnot grow outside the confines of the original transplantEditorial Commentary:The Mammary Fat Pad111F i g .2.A d i p o c y t e s i n t h e l a c t a t i n g m o u s e m a m m a r y g l a n d .A ±C .I m m u n o c y t o c h e m i c a l s t a i n i n g f o r p e r i l i p i n i n s e c t i o n s f r o m (A )a t h r e e -d a y l a c t a t i n g m o u s e ;(B )a s e v e n -d a y l a c t a t i n g m o u s e ;a n d (C )a 16-d a y l a c t a t i n g m o u s e .T h e a l v e o l i a r e f a i n t l y v i s i b l e d u e t o a u t o f l u o r e s c e n c e i n t h e s e c t i o n .F a t d r o p l e t s w i t h i n a d i p o c y t e s a r e o u t l i n e d b y t h e i r p r o t e i n c o a t s t a i n e d w i t h a n a n t i b o d y t o p e r i l i p i n .I n s e c t i o n s f r o m t h e 7-a n d 16-d a y l a c t a t i n g g l a n d s t h e s t r i n g s o f s m a l l f a t d r o p l e t s i n c l o s e p r o x i m i t y t o e a c h o t h e r p r o b a b l y r e p r e s e n t t h e m u l t i l o c u l a r d r o p l e t s o f a s i n g l e f a t -d e p l e t e d a d i p o c y t e ;(D )E l e c t r o n m i c r o g r a p h o f a s e c t i o n f r o m a t h r e e -d a y l a c t a t i n g m o u s e i l l u s t r a t i n g t h e i n t i m a t e a s s o c i a t i o n b e t w e e n a n a d i p o c y t e (A d i p )a n d t h e b a s a l s u r f a c e o f a m a m m a r y e p i t h e l i a l c e l l (M E C ).N o t e t h e t w o b a s e m e n t m e m b r a n e s s e p a r a t i n g t h e c e l l s a n d t h e l a r g e n u m b e r o f r e g u l a r l y -s p a c e d c a v e o l a e a t t h e s u r f a c e o f t h e a d i p o c y t e (a r r o w h e a d s );(E )A l o n g p r o j e c t i o n o f p a r t o f a f a t -d e p l e t e d a d i p o c y t e i s s h o w n (A d i p )b e l o w a c a p i l l a r y (C a p ).N o t e t h e v e r y s m a l l l i p i d d r o p l e t s a n d t h e c a v e o l a e s t i l l p r e s e n t i n t h e a d i p o c y t e .112Neville,Medina,Monks,and Hoveythe adipose stroma to accept tumor tissues.Othersfound that normal rat mammary parenchyma,but notrat mammary tumors,would grow in the cleared mousefat pad(13)suggesting species-specificity in some ofthese effects.In more recent studies Anderson has found thatnormal tissue from human breast will survive and showhormone responsiveness in nude mice(19).However,it was necessary that stroma be transplanted with theepithelium if the tissue was to be maintained subcutis.Neither human nor bovine mammary epithelium growswell in the mouse mammary fat pad.In early studiesJensen and Wellings found that human mammary lob-ules were maintained in mouse mammary fat pads asrounded structures encapsulated by a sheath of connec-tive tissue(24).Similar results have been reportedfrom a number of laboratories(25,26).In these investi-gations it was clear that the encapsulated transplantsmaintained hormone responsiveness but did not showbranching growth into the fat pad.Both human andbovine mammary glands have a larger amount offibrous stroma than the interstitium of the mouse mam-mary gland.The possibility that this difference isresponsible for the lack of growth of human and bovineepithelium in the nude mouse requires additionalinvestigation.Are there metabolic differences between the mam-Fig.3.Mammary bud and developing fat pad in a19-week-old mary fat pad and adipose tissue in other sites?Are human fetus[Used by permission from Ref.(6).]differences between the mammary fat pad and adiposetissue in other locations due to inherent characteristics (17,19).Faulkin and DeOme also showed that theof the mammary adipocytes or are they an effect of presence of epithelial elements inhibited the growth the adjacent mammary parenchyma?These questionshave been addressed in a few laboratories.The fatty of both normal and hyperplastic alveolar nodule trans-plants.They interpreted this finding,correctly as it acid composition of intact mouse mammary glands, turns out(20),as resulting from the epithelial secretioncleared mammary fat pads and abdominal adipose tis-of an inhibitory substance whose action is to appropri-sue was compared as a function of reproductive state ately space the ducts within the stroma.(27).The levels of the essential fatty acids-linolenicUnder many circumstances mammary tumors acid and linoleic were higher in abdominal adiposetissue at midpregnancy;this adipose pad also accumu-grow better in a fat pad than when transplanted tosubcutaneous sites.For example,Elliot and colleagues lated more lipid during pregnancy than the cleared fat (21)found a much higher proportion of tumorªtakesºpad.The results from the intact gland in this study are when a mouse mammary tumor cell line,SP1,was more difficult to interpret.In a study of breast and transplanted into one of several fat pads rather thaniliac fat from postmenopausal women undergoing sur-into the subcutis or the peritoneal cavity.Interestingly,gery for breast cancer(28),the mammary adipose tis-sue was enriched in saturated fatty acids and depleted some authors found that mammary tumors from bothmouse(22)and rat(23)grew better in the intact fat of mono-and poly-unsaturated fatty acids.In terms of pad than in the cleared fat pad while hyperplasticresponsiveness to lipolytic agents like norepinephrine, alveolar nodules grew better in the cleared fat pad(22).the mammary fat pad resembled the abdominal fat These observations suggest that mammary tumors arepad and,in premenopausal women,was much more unresponsive to the normal signals that regulate ductal responsive than the femoral fat pad(29).Although spacing andor that the native epithelium may conditionthis increased responsiveness to lipolytic agents wasEditorial Commentary:The Mammary Fat Pad113 hypothesized to result from a difference in estrogen branched structure similar to that induced by salivary sensitivity,there is no evidence to support this con-mesenchyme(5).In contrast,recombination with fat tention.The presence of mammary parenchyma was pad precursor cells led to formation of normal end buds unlikely responsible for the enhanced lipolysis since and ductal outgrowths given the appropriate hormonal the effect was seen in both the mammary and abdomi-stimuli(5).Using transplantation into the cleared fat nal fat pads.However,the liberation of fatty acids pad,Daniel and colleagues(37)showed that neither from mammary adipocytes in vitro was altered by the co-injected collagen nor fibrous connective tissue septa presence of the epithelium(30).The increased vascu-allowed normal outgrowth of transplanted terminal end larization of the stroma associated with hormone buds.However,when the transplanted end buds con-(estrogen and progesterone)administration is not seen tacted the surrounding adipose stroma they grew into in the cleared fat pad(31),indicating that the vasculari-normal ductal structures.These elegant studies provide zation of the stroma is dependent on stimuli from the incontrovertible evidence that only the adipose stroma parenchyma,as is DNA synthesis(32).This rather supports ductal elongation via end buds.However, heterogeneous assortment of data suggests that the the signals responsible for this phenomenon are not mammary fat pad has some unique characteristics that understood although some information regarding are currently ill-defined but are likely,in some or all stroma-derived growth factors is now available. cases,to be profoundly influenced by the presence of In the mouse,the layer of fibrous connective tis-the epithelium.sue around both small ducts and secretory alveolar How does the adipose stroma of the mammary cells is much thinner in the pregnant and lactating gland gland differ from the fibrous stroma of the gland in terms and mammary epithelial cells in these developmental of the signals passed to the epithelium?The mammary stages are associated more intimately with the sur-rudiment in the12day mouse embryo is surrounded by rounding adipocytes(3,4).The intimate contact is illus-a layer of fibrous connective tissue derived from the sub-trated by the electron micrographs shown in Fig.4. dermis(33).In the gland of the mature virgin a layer of What are the signals that pass between the adi-fibrous connective tissue is intimately associated with pose stroma and the mammary epithelium?Mammary mammary ducts,providing an intervening layer fat pads cultured in serum-free medium released a between the epithelium and the adipose stroma(1).This substance or substances that stimulated growth of a connective tissue layer arises from the fat pad as shown mammary cell line derived from a pre-neoplastic out-by transplantation of rat mammary epithelium intogrowth(38).Similar diffusible factors also modulated cleared mouse mammary fat pads(34).As the rat ductalthe ability of a normal epithelial cell line to respond epithelium grew out it associated with mouse stromalto specific growth factors in vitro(39).Hosick and cells,distinguishable from rat stromal cells by theircolleagues(38)identified a heat and trypsin stable nuclear staining with Hoechst dye.In another set ofsubstance with a molecular weight greater than10,000 experiments in mice,undifferentiated mesenchymalkD as well as certain free fatty acids as enhancers of cells were attracted to the portion of the duct subtendingcell growth in their studies.Rudland et al.(40)found the end bud as the myoepithelial cells matured(35).that mammary stromal derivatives capable of forming These mesenchymal cellsmatured into fibrocytes whichfat cells in culture secreted PGE2and that this PGE2 began to lay down collagen fibrils.An interesting ques-stimulated epithelial cell proliferation.Another candi-tion is whether undifferentiated precursors of both fibro-date stroma-derived growth factor for epithelia is hepa-blasts and adipocytes are present in the mammarytocyte growth factor/scatter factor(HGF/SF).HGF/SF stroma and are involved in the extensive remodeling ofmRNA is present at high concentrations in the virgin the gland that occurs both during reproductive develop-mammary gland and during the post-involutional ment and tumorigenesis.reconstitution of the mammary ductal system(41,42).In early embryonic life the subdermis-derivedIt stimulates branching growth of mammary epithelial fibrous connective tissue may be the source of signalscells in culture(41,43).Because it is secreted by both that direct the epidermis to form a mammary bud(5).adipocyte and fibroblastic cell lines,it is not clear It is certainly the source of signals that mediate thewhether it derives from the adipose or the fibrous androgen-stimulated ablation of the nipple in malestroma of the mammary gland.Niranjan et al.(41) mice(36).If this fibrous stroma was recombined withfound the mRNA to be present in interlobular fibro-the mammary epithelial rudiment and transplantedunder the kidney capsule,it induced a compactly blasts closely associated with organoids from the114Neville,Medina,Monks,and Hovey human breast,but not with intralobular fibroblasts that estrogen receptors in virgin mice.Since these receptors possibly arise from the less intimate fatty stroma.have not been colocalized with markers of cell type, There is evidence that many other growth factors the question of whether they are expressed in fibrous are also derived from the stroma.For example,in theor adipose stroma has not been answered.However, adult virgin mouse,heregulin transcripts were localized estrogen receptors have not been localized to the by in situ hybridization to the layer of fibroblastic stromastroma in the human breast,but appear to be present tightly associated with the ducts,but were distributed in a non-proliferative population of epithelial cells more widely in the mammary stroma of the pregnant(19).To what extent these systemic hormones affect mouse(42).There is definitive evidence that IGF-I and characteristics of the fat pad other than the possible IGF-II originate in the stroma rather than the epithelialsecretion of growth factors is not known. compartment(44±46).Evidence exists for the stromal Does the fat pad play a role in the normal function origin of the fibroblast growth factors FGF-1,FGF-2of the mammary gland,e.g.,milk synthesis and secre-(47)and keratinocyte growth factor(FGF-7)(48)as well tion?Teleologically,the necessity for adipose stromain the mammary gland is not immediately obvious;no as for transforming growth factor-(TGF-),(49).TheWnt’s represent a family of genes thought to encode other epithelial gland has a similar requirement to grow secreted proteins involved in cell-cell communication.into a fat pad.The advantage could be anatomical: They are developmentally regulated in the mammary The distribution of ducts in the fat pad may simply gland and there is evidence that Wnt-2,Wnt5a,and Wntprovide the space necessary for alveolar expansion 6are expressed in the stroma whereas Wnt4,Wnt5b,during pregnancy and lactation.The possibility of and Wnt7b are associated with the epithelium(50).another,more functional,advantage is raised by evi-Finally mammary adipose tissue,like other adipose tis-dence that the enzyme lipoprotein lipase derives from sue depots,is capable of transforming androgens intomammary adipocytes rather than the epithelium(54). estrogen through aromatase activity.This wealth of This enzyme is responsible for hydrolyzing plasmatriglycerides into fatty acids for uptake and synthesis growth factor interactions suggests that the stroma maymediate and integrate signals from systemic hormones into milk lipid during lactation.to regulate an orderly pattern of development.However,In lactation the mammary epithelium exerts a lipid in general the adipose and fibrous stroma have yet to be depleting effect on proximal adipocytes(3),(Fig.2) distinguished in growth factor studies.suggesting that the fat pad could also serve as a source The possibility that extracellular matrix mole-of lipid substrate for milk synthesis.However,theproportion of lipid that could be provided from this cules themselves may be responsible for parenchymalgrowth induction by the mammary fat pad has been source is limited.For example,a mouse suckling eight raised by Sakakura and colleagues(33)who suggestedor more pups secretes its entire body weight in lipid that different types of basement membrane compo-during a single three-week lactation.Since the mam-nents were elaborated by the fibrous and adiposemary fat pad cannot comprise more than5%of the stroma.Santoro and colleagues observed that expres-total body weight of the animal the contribution of sion of the21integrin and its three major ligands,lipid substrate from this compartment must be small. collagen I,collagen IV and laminin were all develop-The mechanistic question of how fat-filled adipocytes mentally regulated in the mammary gland in a mannerrapidly reappear when the pups are removed from consistent with a role in the regulation of mammary the lactating dam has not been adequately addressed.Clearly those adipocytes that are still differentiated morphogenesi s(51).Finally,the role of the epithelial component in simply reaccumulate their fat stores once the lipid stimulating the production of stromal growth factorssubstrates arriving in the plasma are no longer needed has not been well-defined,in part because the proper-for milk lipid synthesis.Whether others have dediffer-ties of the cleared fat pad with respect to growth factorentiated into preadipocytes or have actually undergone production have only begun to be investigated(46).apoptosis and must be replaced is not known.Does the fat pad play a role,either positive or nega-Do hormones involved in mammary developmentaffect the fat pad as well as the epithelium?There is tive,in mammary tumorigenesis?Recent work in the substantial evidence that both growth hormone andlaboratory of Barcellos-Hoff shows that ionizing radia-estrogen act on the stromal compartment of the virgin tion is associated with an immediate(within one hour) mammary gland to promote parenchymal growth(52,activation of TGF-in the adipose stroma of the mam-53),and the mammary stroma appears to be a site of mary gland with subsequent changes in the distributionEditorial Commentary:The Mammary Fat Pad1153.J.J.Elias,D.R.Pitelka,and R.C.Armstrong(1973).Changes of collagen III in both the adipose and peri-epithelialin fat cell morphology during lactation in the mouse.Anat. stroma.Barcellos-Hoff hypothesizes that these changes Rec.177:533±547.may contribute both to radiation induced fibrosis of the4. E.J.Blanchette-Mackie,N.K.Dwyer,T.Barber,R.A.Coxey,T.Takeda, C.M.Rondinone,J.L.Theodorakis, A.S. mammary gland and carcinogenesis(55).When Saka-Greenberg,and C.Londos(1995).Perilipin is located on the kura transplanted salivary mesenchyme into the mam-surface layer of intracellular lipid droplets in adipocytes.J.mary gland a more condensed stroma developed thatLipid Res.36:1211±1226.5.T.Sakakura,Y.Sakagami,and Y.Nishizuka(1982).Dual origin fostered greater tumor development in certain strains ofof mesenchymal tissues participating in mouse mammary gland mice bearing MMTV or treated with DMBA(56)than embryogenesis.Devel.Biol.91:202±207.the normal adipocyte containing stroma.This experi-6. C.Pechoux,P.Clezardin,R.Dante,C.M.Serre,M.Clergot,N.Bertin,wler,P.D.Delmas,J.L.Vauzelle,and L. ment,plus the evidence cited earlier that mammaryFrappart(1994).Localization of thrombospondin,CD36and tumors tend not to grow well in the cleared fat pad,pro-CD51during prenatal development of the human mammarymotes the speculation that the fibrous connective tissuegland.Differentiation57:133±141.7.T.Sakakura,I.Kusano,M.Kusakabe,Y.Inaguma,and Y. supports tumorigenesis whereas the adipose pad may beNishizuka(1987).Biology of mammary fat pad in fetal mouse: relatively inhibitory.The stromal reaction prominent in capacity to support development of various fetal epithelia insome types of mammary tumors where large amounts ofvivo.Development100:421±430.8.L.J.Faulkin and K.B.DeOme(1960).Regulation of growth dense connective tissue separate the mammary elementsand spacing of gland elements in the mammary fat pad of the from the adipose stroma(57)leads to the question of C3H mouse.J.Natl.Cancer Inst.24:953±969.whether the signals arising from the fibrous stroma are9.K.Hoshino(1962).Morphogenesis and growth potentiality ofmammary gland in mice.Transplantability of growth potential-different in intensity or type from the signals arisingity of mammary tissue of virgin mice.J.Natl.Cancer Inst. from the more distant adipose stroma.Although the gen-29:835±849.eral role of the stroma in directing development and10.K.B.DeOme,L.J.Faulkin,H.A.Bern,and P.B.Blair(1959).Development of mammary tumors from hyperplastic alveolar tumorigenesis is receiving increased attention,as evi-nodules transplanted into gland-free mammary fat pads of denced by several articles in this issue,the particular female C3H mice.Cancer Res.19:515±525.idea that the adipose stroma has differential effects from11.P.A.W.Edwards, C.Abram,and J.M.Bradbury(1996).Genetic manipulation of mammary epithelium by transplanta-the fibrous stroma has rarely been followed up in anytion.J.Mam.Gland Biol.Neoplasia1:75±89. systematic way.12. D.Medina(1996).The mammary gland:A unique organ forWe have raised a number of questions about thethe study of development and tumorigenesis.J.Mam.GlandDevel.Neoplasia1:5±19.function of the mammary fat pad and the mechanisms by13. C.W.Welsch,D.H.O’Connor,C.F.Aylsworth,and L.G. which it exerts its influence on mammary development.Sheffield(1987).Normal but not carcinomatous primary ratWhat is clear from this brief perusal of the availablemammary epithelium:readily transplanted to and maintainedin the athymic nude mouse.J.Natl.Cancer.Inst.78:557±565. literature is that until studies are designed specifically14.S.Z.Haslam and L.J.Counterman(1991).Mammary fibroblast to differentiate between functional effects of the fibrous influence on normal mouse mammary epithelial cell responsesand the adipose stroma,more substantive informationto estrogen in vitro.Endocrinology129:2017±2023.15.J.F.Levine and F.E.Stockdale(1984).3T3-L1adipocytes about their differential effects on mammary developmentpromote the growth of mammary epithelium.Exp.Cell Res. and tumorigenesis will not be forthcoming.151:112±122.16.J.F.Levine and F.E.Stockdale(1985).Cell-cell interactionspromote mammary epithelial cell differentiation.J.CellBiol.100:1415±1422.ACKNOWLEDGMENTS17.K.Hoshino(1980).Mammary transplantation and its histogen-esis in mice.In A.Yokoyama,H.Mizuno,and H.Nagasawa,(eds.),Physiology of Mammary Glands,University Park Press, We thank Constantine Landos,National CancerBaltimore,pp.163±228.Institute for the kind gift of antibody to perilipin.The18.K.Hoshino(1967).Transplantability of mammary gland inbrown fat pads of mice.Nature213:194±195.work of M.C.Neville and J.Monks was supported19. 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