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[37] T e c h n i q u e s for S t u d y i n g D e v e l o p m e n t o f N o r m a l M a m m a r y E p i t h e l i a l Ceils in O r g a n C u l t u r e
By YALE J. TOPPER, TAKAMI OKA, and BARBARAK. VONDERttAAR The mammary gland is a useful model system for the study of developmental biology. Normal differentiation of mammary epithelial cells can be simulated in vitro using chemically defined media. This facilitates control of the cellular environment and eliminates certain problems of analysis that are inherent in experiments performed in vivo. Furthermore, the fully developed cells produce specific milk products which are convenient markers of functional differentiation. The purpose of this chapter is to describe procedures that have been used for the culture of mammary gland of the mouse and rat and to provide pertinent information on, and references to, analytical techniques used in the assessment of mammary gland development. The methods to be described are intended primarily for the use of investigators who wish to study the murine mammary system, specifically. Developmental Characteristics in Vivo
As a prelude to discussion of culture procedures, it is useful to outline some of the characteristics of the mouse mammary epithelium at various stages of development in vivo. These are summarized in the table. Mammary Gland Culture
Developmental Stage o] Donor Animal In the organ culture system epithelial cells in mammary explants from nonlactating mice are stimulated to differentiate and produce milk products in the presence of the appropriate hormones. However, the kinetics of response varies depending upon the developmental stage of the donor animal. For example, the cells in the gland of virgin C 3 H / H e N animals are initially unresponsive to mitogens such as insulin and serum, and acquire responsiveness after about 24 hours in culture. 1,~ This results in delayed cell-proliferation and elaboration of milk products. In general, the duration of the unresponsiveness period is fairly constant for 3- to 5month-old C 3 H / H e N mice. However, various environmental factors, 1 S. Friedberg, T. Oka, and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 67, 1493 (1970). : T. Oka and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 69, 1693 (1972).
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such as crowding, frequency of handling, bedding, can alter the length of this period, so that occasionally partial responsiveness is observed in freshly isolated tissue, and shortened lag periods occur. Also, the length of the lag period may v a r y as a function of the age and strain of the animal. We house no more than 5 mice in one cage (10.5 }( 6.5 inches) in an animal room which is illuminated from 8:00 AM to 5:00 PM. Since mouse m a m m a r y development is rapid during pregnancy, the study of tissue from pregnant animals requires proper staging. This can be done on the basis of the morphological characteristics of the fetuses, 3 or by counting days after the appearance of the vaginal plug. Generally, use of first-pregnancy animals is recommended.
Culture Media
Chemically defined synthetic media have been used successfully for organ culture of m a m m a r y gland. Medium 199, Trowell T8, Weymouth MB752/1, and N C T C 109, supplemented with Hanks' or Earle's salt mixture, are among those used. The composition of these media are given in Biology D a t a Handbook. 4 Complex supplements, such as blood sera and lactalbumin hydrolyzate, have been employed in particular investigations. 2,~-7 Generally, penicillin G is added to a final concentration of 35 ~g/ml. Sterilization of the media is effected by passage through Millipore filters (0.45 ~m) contained in Swinnex-13 filter units attached to syringes.
Hormones
Addition of insulin, a glucocorticoid, and either prolactin or placental lactogen to the culture system promotes the conversion of nonsecretory epithelial cells, in explants of m a m m a r y tissue derived from animals in various developmental stages, into secretory cells. Partial development can be effected by addition of incomplete complements of these hormones? ,9 Beef and porcine insulin are about equipotent. The most frequently used glucocorticoid is hydrocortisone, but a number of others 3H. Gruneberg, J. Hered. 34, 88 (1943). 4 Biology Data Handbook (P. L. Altman and D. S. Dittmer, eds.), p. 530. FASEB. Bethesda, Maryland, 1964. G. C. Majumder and R. W. Turkington, Endocrinology 88, 1506 (1971). 6B. I. Balinsky, Trans. Roy. Soc. Edinburgh 62, 1 (1950). 7F. J. A. Prop, Pathol. Biol. 9, 640 (1961). E. S. Mills and Y. J. Topper, J. Cell Biol. 44, 310 (1970). ~T. Oka and Y. J. Topper, J. Biol. Chem. 246, 7701 (1971).
446
EXOCRINE TmSUE
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have also been shown to have the requisite biological activity.TM Both ovine and bovine prolactin are effective; human placental lactogcn is about 80% as active, on a weight basis, as prolactin in those instances in which it has been tested. 1~ Although these hormones have been shown to produce their characteristic effects when used at levels between 10-9 and 10-8 M, ~,~2they are usually employed at considerably higher concentrations, i.e., 5 ~g/ml each. Appropriate amounts of freshly prepared stock solutions of each hormone are added directly to Medium 199 before sterilization. Insulin stock: 1 mg per 0.5 ml of 5 X 10-3 HC1 (2 t~g/~l). Prolactin stock: 1 mg per 0.5 ml of l0 -4 M NaOH (2t~g/td). Hydrocortisone stock: 1 mg per 0.2 ml of anhydrous ethanol (5 ~g/td). When systems to which hydrocortisone is added are to be compared to systems without added glucocorticoid, a corresponding amount of ethanol should be added to the latter. The final concentration of alcohol in the media should be no more than 0.5%.
Preparation o] Siliconized Lens Paper In most of the studies on mammary gland explants, the tissue is supported at the surface of the medium. A common support is lens paper, which is siliconized to enable it to float. Prior to siliconization the paper is suspended in ethyl ether for 30 minutes and then the ether is removed by aspiration. This is done 3 times. The same procedure is then performed with 95% ethyl alcohol. Finally, the paper is washed 4 times for 15-minute intervals with quartz-distilled water, and dried at 37 °. Siliconization is accomplished by submerging the dried paper in Siliclad (Clay Adams)-quartz-distilled water (l:1000) for 10 minutes at room temperature. After removal of the Siliclad solution by aspiration, excess silicone is eliminated by treatment with water as described above. Again the papers are dried at 37 ° . A convenient type of container for culture of the explants is the sterile and disposable Falcon microdiffusion dish (Catalog No. 7004) whose dimensions are 65 X 15 mm. Accordingly, the dry lens paper is cut into pieces corresponding in size to that of the inner well of these dishes. The pieces are placed in covered, glass petri dishes and heated at 150° for 1 hour in a dry oven. This serves two purposes. The lens paper is sterilized, and the impregnated silicone is modified so that it now permits the paper to float. L°R. W. Turkington, W. G. Juergens, and Y. J. Topper, Endocrinology 80, 1139 (1967). 11R. W. Turklngton and Y. J. Topper, Endocrinology 79, 175 (1966). 12F. E. Stockdale, W. G. Juergens, and Y. J. Topper, Develop. Biol. 13, 266 (1966).
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MAMMARY GLAND ORGAN CULTURE
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Preparation o] Tissues
The explantation technique employed is determined by the developmental stage of the donor animal, and by the particular interests of the investigator. Thus, in some instances the whole gland is cultured as a unit, while in others the gland is cut into smaller pieces before explantation. Embryonic Rudiments. Fetal mouse and rat mammary rudiments are detectable by day 13 of gestation. The effects of androgens on whole rudiments in vitro have been studied by Kratochwil. 13 Ceriani TM has reported that whole fetal rat rudiments can be stimulated to produce casein in the presence of the appropriate hormones. Whole Glands ]rom Postnatal Mice. For studies on lobuloalveolar development it is advantageous to maintain the epithelial architecture intact. To this end, Prop 7 aDd Ichinose and Nandi '~ have developed a method for the culture of whole thoracic mammary glands from 3-weekold mice. Gland Fragments. Most frequently the abdominal glands are used in organ culture studies. Limitations in the rate of diffusion of gases and components from the media necessitate the use of small explants prepared from these glands. TM Using instruments sterilized in 70% ethanol and rinsed in boiling water, the glands are isolated aseptically after the animals have been killed by cervical dislocation. Isolation is facilitated by placing the animal on a dissection board, ventral surface up, and securing this position with pins through the appendages. After carefully shaving the abdomen and thighs with a sharp razor blade, loose hair is removed by swabbing with a wet cloth. Four incisions, through the skin only, are then made with a scissors. One is in the middle of the abdomen from the genitalia to the diaphragm. Another intersects the first at its upper extremity, completely transversing the ventral surface. Incisions are then made from tile lower end of the median cut diagonally across each thigh. Skin flaps can then be peeled from the abdominal wall with forceps. These are pinned to the dissection board, exposing the mammary glands on the inner skin surface. Lymph nodes, one or two in number, are extirpated with forceps from each gland. The glands are then excised with forceps and scissors, removing as much connective tissue as possible, and placed in a sterile microdiffusion dish containing 1-2 ml of sterile medium 199. It is desirable that the explants to be prepared contain approximately 12K. Kratochwil, or. Embryol. Exp. Morphol. 25, 141 (1971). 14R. L. Ceriani, Develop. Biol. 21, 530 (1970). 1.~R. R. Ichinose and S. Nandi, Science 145, 496 (1964). lgj. j. Elias, Proc. Soc. Exp. Biol. Med. 101, 500 (1959).
448
EXOCatNE TISSU~
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equal numbers of epithelial cells. Since the extent to which the ductal-alveolar system extends into the mammary fat pad is a function of both the species and the developmental state of the animal, preparation of equivalent explants from a given animal may require selective utilization of certain portions of the gland. Mature virgin and multiparous nonpregnant mice (approximate weight per gland, 100-150 mg) : discard peripheral portion, constituting about 20% of the gland. Pregnant (approximate weight per gland, 80-100 rag) and lactating (approximate weight per gland, 200-300 rag) mouse gland: use entire gland. Mature virgin, multiparous nonpregnant, and 1-15-day pregnant rat gland: use inner core, constituting about 30% (250-350 mg) of the entire gland. Late pregnant and lactating rat gland: use entire gland. These precautions will assure that explants from a given type of animal will contain approximately equal numbers of epithelial cells. Base-line activities and quantitative responses to hormones may vary somewhat from animal to animal. In experiments in which it is necessary to use pooled tissue from more than one animal it is desirable to employ equal numbers of explants from each animal in every determination. The situation is different with the abdominal gland of the 3-week-old mouse, however. In this animal the epithelial cells are concentrated in a small area at the level of the nipple. For operational reasons it is desirable to use all the explants derived from one gland for each determination. The contralateral gland may serve as a control. The explants are commonly prepared directly in the microdiffusion dish in which the glands and medium 199 were placed. The tissue is gently cut into small pieces (0.5-1.0 mg wet weight). Remove as much cormective tissue as possible. Transfer of the explants to the culture dishes which have been preloaded with 2 ml of medium and siliconized lens paper is facilitated by first aspirating away most of the medium 199 in which the explants were prepared. Using a fine forceps, the explants may then be transferred to the floating lens paper, taking care not to carry over too much medium with the tissue fragments. The number of explants needed for each experimental point is determined by the parameter being studied. Up to about 25 explants can be accommodated in each dish.
Maintenance o] Cultures Incubation of the explants is carried out in a closed system. The microdiffusion dishes are stacked in heavy-duty polycarbonate boxes containing an open water container for maintenance of high humidity. Each
[37]
MAMMARY GLAND ORGAN CULTURE
449
box has, in addition to a hinged top, a elosable opening at either end for introduction of 5% C02-95% air. The gas mixture is first bubbled through sterile distilled water, and is then permitted to flow into the box for a predetermined time at a predetermined rate such that the pH of the media is brought into range 7.3-7.6. This equilibration may take up to an hour after the gas flow has been stopped and the box has been sealed. The temperature is maintained at 37 ° in a water-jacketed incubator. Media should be replaced at least every 48 hours. Old media may be removed by aspiration, using sterile Pasteur pipettes, and 2 ml of fresh media introduced with sterile pipettes. The lens papers are easily refloated under these conditions. In some cases it is desirable to transfer the lens paper containing the explants to a fresh dish preloaded with medium. Gassing is then repeated. To terminate the culture, the lens paper is removed from the dish and gently blotted to remove adhering medium. Explants are then carefully collected with a fine forceps.
Responses of Mammary Epithelial Cells Morphological Methods Whole Mounts. Whole mounts prepared from mammary gland according to the method of Lyons~7 provide insight into the architecture of the epithelial component of the whole tissue. Preparations from animals in different developmental states dramatically illustrate the changing ratios of epithelial cells to mammary fat, and the increased branching of the epithelial tree, as maturation proceeds. Histology. The preparation, fixation, and staining of secretions from cultured explants have been performed by using standard histological techniques. TM Examination of histological sections, prepared for light microscopy, from explants has provided information not obtainable by other methods. Insulin-induced increase in the number of epithelial cells per average alveolar cross section has been determined, s Also, alterations in the spatial arrangement of the alveolar cells and in the size of alveolar lumina consequent to hormone treatment have been reported. ~2 Additional observations made on such sections have supplemented determinations made by other methods. Secretory material in the lumina is stainable with eosin. Increased nucleic acid content in the cytoplasm is reflected by enhanced basophilic character. 12 Electron Microscopy. Visualization of most cellular organelles, how17W. R. Lyons, R. E. Johnson, R. D. Cole, and C. H. Li, in "The Hypophyseal Growth Hormone" (R. W. Smith, O. H. Gaebler, and C. N. H. Long, eds.), p. 461. McGraw-Hill (Blakiston), New York, 1955.
450
EXOCRINE TISSUE
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ever, requires electron microscopy. Details of the methodology employed in studies on mammary gland have been presented. 8,1s-21 Morphological effects of insulin, hydrocortisone, and prolactin on explants from midpregnancy C 3 H / H e N mice have been studied, 8 using a multiple incubation technique. 22
Biochemical Methods The mammary gland contains two major cell-types, epithelial and fat. The proportions of these vary widely as a function of the developmental state of the animal. Changes in the synthesis of certain products, such as casein and a-lactalbumin, obviously reflect response of the epithelial cells. In other instances one cannot directly ascertain whether a response of explants is ascribable to the component epithelial or fat cells. The following techniques have been helpful in making this distinction. The small clump of epithelial cells concentrated in the nipple area can be removed from the 3-week-old animal by cauterization. 23 As the animal matures this gland remains essentially devoid of epithelial cells, and explants prepared from it can be used to determine fat-cell responses. The unoperated abdominal gland can be used as a contralateral control. Epithelial cells, free from fat cells but contaminated with a small percentage of connective tissue cells, can be prepared by treating finely minced mammary gland or explants with crude eollagenase obtained from Worthington. 1,24 The mince is suspended in a 0.15% solution of collagenase in Medium 199 containing 4% bovine serum albumin, adjusted to pH 7.4. The suspension is shaken at 37 ° for 40 minutes. At intervals of about 5 minutes the suspension is aspirated into Pasteur pipettes. Use of a series of these with progressively smaller bore-size aids in the dispersion of the epithelial cells and their separation from fat cells. Fibrous tissue which adheres to the tips of the pipettes can be removed. After centrifugation (200 g; room temperature for 2 minutes) the epithelial pellet is washed 4 times with Medium 199. Residual fibrous tissue can be removed mechanically during this series of resuspensions and centrifugations. The yield of epithelial cells is about 90%, based on thymidine3H-labeled DNA. 1, W. Bargman and A. Knoop, Z. Zelllorsch. Mikrosk. Anat. 49, 344 (1959). 1, K. H. ttollman, J. Ullraslruct. Res. 2, 23 (1959). 2, K. K. Sekhri, D. R. Pitelka, and K. B. DeOme, J. Nat. Cancer Inst. 39, 459 (1967). ,1 S. R. Wellings, R. A. Copper, and E. M. Rivera, J. Nat. Cancer Inst. 36, 657 (1966). 22 R. W. Turkington, D. H. Lockwood, and Y. J. Topper, Biochim. Biophys. Acla 148, 475 (1967). 2*K. B. DeOme, L. J. Faulkin, It. A. Bern, and P. B. Blair, Cancer Res. 19, 515 (1959). 2, E. Y. Lasfargues, Anal. Rec. 127~ 117 (1957).
[37]
MAMMARY GLAND ORGAN CULTURE
451
The collagenase technique has been applied in many studies in which the tissue was first cultured as explants. Determinations on the residual pellet provide direct information on the response of the epithelial cells. Use of this method as a means of preparing epithelial cells which are to be cultured subsequently has been less successful. The isolated cells appear to have limited ability, compared to the cells within explants, to respond to the hormones. Cell Proli]eration. The epithelial cells in mammary explants derived from animals in different physiological states make DNA and divide under the influence of insulin, 25 various blood sera, "~and epidermal growth factor. 2~ The cells from mature C3H/HeN virgin mice and SpragueDawley rats are insensitive to insulin and serum factor(s) in terms of DNA synthesis. 1,~ They are also insensitive to insulin in terms of transport of a-aminoisobutyric acid (AIB), and the synthesis of glucose-6phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase. 1 After about 24 hours of culture the explants acquire these sensitivities. No exogenous hormones are required for the acquisition of these sensitivities. Cells in explants from mid-pregnancy mouse and late-pregnancy rat mammary gland, on the other hand, respond to these agents promptly after isolation. The proliferative response can be quantitated in a number of ways: 1. The extent of epithelial DNA synthesis can be determined by comparing the epithelial DNA content of freshly isolated explants with that after culture, using the method of Dische 27 as modified by Burton. ~s This determination is performed on the cell-pellet obtained after treating the mouse tissue with collagenase. Minimum tissue requirement: 40-60 mg mid-pregnancy ; 60-75 mg mature virgin. 2. DNA synthesis can also be estimated by incorporation of [~H]thymidine into an acid-insoluble fraction. Since fat cells do not make DNA under the conditions used 1: treatment with collagenase is not necessary. Tissue requirement: 4-8 rag. 3. In some instances it may be desirable to know which epithelial cells within explants have synthesized DNA. Such information can be obtained by autoradiography of histological sections prepared from [ :H] thymidine-labeled tissue. 1~ 4. The number of cells which are dividing at any given time during culture can be estimated by counting mitotic figures in histological sections prepared from explants. The results are usually expressed as mitotic in25F. E. Stockdale and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 56, 1283 (1966). 2~R. W. Turkington, Exp. Cell Res. 57, 79 (1969). 2~Z. Dische, Mikrochemie 8, 4 (1930). ~sK. Burton, Biochem. J. 61~, 315 (1956).
452
EXOCRINE TISSUE
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dices, i.e., the ratio of the number of cells in mitosis (M) to the total number of cells counted. The total number of cells which have entered M during a particular period can be determined by culturing in the presence of a mitotic inhibitor such as colchicine. The ratio of mitotic figures to total cells counted reflects the percentage of cells which have entered M during the culture period in question. 5. The increase in the number of cells can be directly determined, after treatment of the tissue with collagenase, by counting the suspended epithelial cells in a hemacytometer. 29 Counting is facilitated by brief treatment of the cell suspension with 0.5% trypsin solution (GIBCO) to prevent clumping. Minimum tissue requirement: 75 mg, mid-pregnancy mouse; 100 rag, mature virgin. An important consideration in differentiation is whether or not certain developmental processes are dependent upon cell proliferation. Inhibitors of proliferation are useful tools for attacking this question experimentally. Agents which arrest cells in the mitosis (M) phase of the cell-cycle are usually not satisfactory because cells in M have a reduced general capacity for macromolecular synthesis2 °,31 Agents which prevent cells from entering the DNA-synthesis phase (S), such as lfl-D-arabinosylcytosine and fluorodeoxyuridine, can be fruitfully employed. Under these circumstances one may regard the inhibited cells as normal G1 cells if proper controls are unaffected. In order to minimize the possibility of toxic side effects the smallest concentration of agent giving maximal inhibition should be used; this may vary as a function of the hormones added, and the developmental state of the donor animal. It is also necessary to establish that the DNA-inhibitor does not affect the developmental process in question when the agent is added to postmitotic cells. 32 Assay of Products Made Uniquely by Mammary Gland. The formation of the following secretory products is a reliable indication that the mammary epithelial cells have attained a highly differentiated state. Development of these functional capacities may not be synchronous in vitro. 29 1. Casein. Casein is a family of phosphoproteins. The isolation and characterization of the material synthesized by mammary explants is facilitated by incorporation of 32Pi or labeled amino acids added to the culture system. After suitable labeling periods the phosphoproteins are precipitated by the addition of Ca 2÷ and rennin to the soluble fraction 29B. K. Vonderhaar, I. S. Owens, and Y. J. Topper, J. Biol. Chem. 248, 467 (1973). 3o H. Pan and S. Penman, Science 168, 135 (1970). ,1 D. Martin, Jr., G. M. Tomkins, and D. Granner, Proc. Nat. Acad. Sci. U.S. 62, 248 (1969). ~ I. S. Owens, B. K. Vonderhaar, and Y. J. Topper, J. Biol. Chem. 248, 472 (1973).
[37]
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of the tissue? 3 If no further fractionation is to be performed, carrier bovine casein is added prior to homogenization. If the precipitate is to be resolved into separate casein polypeptides, carrier mouse casein, rather than bovine casein, is added prior to homogenization? 4,3~ The precipitate contains casein and other phosphoproteins. Although not all this material is authentic casein, :2 measurement of its isotope content represents a fairly reliable and rapid method for determining the relative rate and extent of casein synthesis by explants. More precise quantitation of casein synthesis is accomplished by electrophoresis of the labeled phosphoproteins on starch gels or polyacrylamide gels. 3~,~6 Individual casein polypeptides can be visualized by staining or autoradiography. Tile individual casein bands can then be cut out and counted. Minimum tissue requirement: 4-8 mg per determination. The synthesis of casein by different systems within a given experiment may often be compared by expressing results as count per minute per unit wet weight tissue. However, in some instances it is necessary to express results as counts per minute per unit of epithelial D N A ? ~ Similar considerations apply to the study of other functional parameters. 2. a-Lactalbumin, a-Lactalbumin is tile B-protein component of the lactose synthetase system?; The ability of a-lactalbumin to enable the A-protein of lactose synthetase to utilize glucose as an acceptor of the galactose moiety of UDP-Gal, with consequent formation of lactose, is the basis for its assay. A modification 29 of the method of Brew ~8 has been used for determination of a-lactalbumin activity in homogenates from cultured explants. Minimum tissue requirements: 50-80 mg of mature virgin or 30-40 mg mid-pregnancy tissue homogenized in 300 ~l of buffer; 20-30 mg of lactation tissue. 3. Lactose. The incorporation of radioactive galactose into lactose during culture '~9 has also been used to study the control of lactose synthetase activity. ~° 4. F a t t y acids. Up to 70% of the fatty acids in rabbit milk trigly~3W. G. Juergens, F. E. Stockdale, Y. J. Topper, and J. J. Elias, Proc. Nat. Acad. Sci. U.S. 54, 629 (1965). 34R. W. Turkington, W. G. Juergens, and Y. J. Topper, Biochim. Biophys. Actc~ 111, 573 (1965). 3~A. E. Voytovich and Y. J. Topper, Science 158, 1326 (1967). 3~R. W. Turkington and M. Riddle, Endocrinoloqy 84, 1213 (1969). ~TU. Brodbeck, W. L. Denton, N. Tanahashi, and K. E. Ebner, J. Biol. Chem. 242, 1391 (1967). 3SK. Brew, T. C. Vanaman, and R. L. Hill, Proc. Nat. Acad. Sci. U.S. 59, 491 (1968). ~J. C. Bartley, S. Abraham, and I. L. Chaikoff, I. Biol. Chem. 241, 1132 (1966). 4. R. D. Palmiter, Biochem. J. 113, 409 (1969).
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cerides is comprised of octanoic and decanoic acids. The hormone dependence of the synthesis of these characteristic products by explants from rabbit mammary gland has been studied. 41 Other Metabolic Activities o] Mammary Gland in Vitro. Other functional parameters of mouse mammary gland which have been studied by the organ culture method, in attempts to understand more fully the development of this tissue, are listed: 1. Accumulation of a-aminoisobutyric acid 1,42 2. Glucose uptake and to¢al fatty acid synthesis 43,44 3. Synthesis of glucose-6-phosphate dehydrogenase and gluconate-6phosphate dehydrogenase~,2,4~ 4. RNA synthesis 1:,46-4s 5. Formation of rough endoplasmic reticulum. 8,9 6. DNA polymerase activity 49 7. Synthesis and acetylation of histones ~°-~2 411. A. Forsyth, C. R. Strong, and R. Dils, Biochem. J. 129, 929 (1972). 42T. Oka and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 68, 206'6 (1971). 4.~R. L. Moretti and S. Abraham, Biochim. Biophys. Acta 124, 280 (1966). 44R. Mayne and J. M. Barry, J. Endocrinol. 46, 61 (1970). ~D. P. Leader and J. M. Barry, Biochem. J. 113, 175 (1969). '6R. W. Turkington and O. T. Ward, Biochim. Biophys. Acta 174, 291 (1969). 4~M. R. Green and Y. J. Topper, Biochim. Biophys. Acta 204, 441 (1970). ~sM. R. Green, S. L. Bunting, and A. C. Peacock, Biochemistry 1O, 2366 (1971). 4~D. H. Lockwood, A. E. Voytovich, F. E. Stockdale, and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 58, 658 (1967). W. F. Marzluff, Jr., K. S. McCarty, and R. W. Turkington, Biochim. Biophys. Acta 190, 517 (1969). W. F. Marzluff, Jr. and K. S. McCarty, J. Biol. Chem. 245, 5635 (1970). :~ P. Hohmann and R. D. Cole, J. Mol. Biol. 58, 533 (1971).
[38] A n d r o g e n M e t a b o l i s m a n d R e c e p t o r A c t i v i t y in P r e p u t i a l G l a n d s a n d K i d n e y s o f N o r m a l a n d Androgen-Insensitive tfm Rats and tfm/y Mice 1
By C. WAYNE BARDIN, LESLIE P. BULLOCK, and IRENE MOWSZOWICZ2
In recent years, many laboratories have examined the pretranscriptional events involved in testosterone activation of target cells, and the 1Supported in part by PHS Grant No. HD05276. 2On leave of absence from Faculte de M~decine, Pitid-Salpetri~re, Service de Biochimie, Paris.
MAMMARY GLAND ORGAN CULTURE
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[37] T e c h n i q u e s for S t u d y i n g D e v e l o p m e n t o f N o r m a l M a m m a r y E p i t h e l i a l Ceils in O r g a n C u l t u r e
By YALE J. TOPPER, TAKAMI OKA, and BARBARAK. VONDERttAAR The mammary gland is a useful model system for the study of developmental biology. Normal differentiation of mammary epithelial cells can be simulated in vitro using chemically defined media. This facilitates control of the cellular environment and eliminates certain problems of analysis that are inherent in experiments performed in vivo. Furthermore, the fully developed cells produce specific milk products which are convenient markers of functional differentiation. The purpose of this chapter is to describe procedures that have been used for the culture of mammary gland of the mouse and rat and to provide pertinent information on, and references to, analytical techniques used in the assessment of mammary gland development. The methods to be described are intended primarily for the use of investigators who wish to study the murine mammary system, specifically. Developmental Characteristics in Vivo
As a prelude to discussion of culture procedures, it is useful to outline some of the characteristics of the mouse mammary epithelium at various stages of development in vivo. These are summarized in the table. Mammary Gland Culture
Developmental Stage o] Donor Animal In the organ culture system epithelial cells in mammary explants from nonlactating mice are stimulated to differentiate and produce milk products in the presence of the appropriate hormones. However, the kinetics of response varies depending upon the developmental stage of the donor animal. For example, the cells in the gland of virgin C 3 H / H e N animals are initially unresponsive to mitogens such as insulin and serum, and acquire responsiveness after about 24 hours in culture. 1,~ This results in delayed cell-proliferation and elaboration of milk products. In general, the duration of the unresponsiveness period is fairly constant for 3- to 5month-old C 3 H / H e N mice. However, various environmental factors, 1 S. Friedberg, T. Oka, and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 67, 1493 (1970). : T. Oka and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 69, 1693 (1972).
444
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such as crowding, frequency of handling, bedding, can alter the length of this period, so that occasionally partial responsiveness is observed in freshly isolated tissue, and shortened lag periods occur. Also, the length of the lag period may v a r y as a function of the age and strain of the animal. We house no more than 5 mice in one cage (10.5 }( 6.5 inches) in an animal room which is illuminated from 8:00 AM to 5:00 PM. Since mouse m a m m a r y development is rapid during pregnancy, the study of tissue from pregnant animals requires proper staging. This can be done on the basis of the morphological characteristics of the fetuses, 3 or by counting days after the appearance of the vaginal plug. Generally, use of first-pregnancy animals is recommended.
Culture Media
Chemically defined synthetic media have been used successfully for organ culture of m a m m a r y gland. Medium 199, Trowell T8, Weymouth MB752/1, and N C T C 109, supplemented with Hanks' or Earle's salt mixture, are among those used. The composition of these media are given in Biology D a t a Handbook. 4 Complex supplements, such as blood sera and lactalbumin hydrolyzate, have been employed in particular investigations. 2,~-7 Generally, penicillin G is added to a final concentration of 35 ~g/ml. Sterilization of the media is effected by passage through Millipore filters (0.45 ~m) contained in Swinnex-13 filter units attached to syringes.
Hormones
Addition of insulin, a glucocorticoid, and either prolactin or placental lactogen to the culture system promotes the conversion of nonsecretory epithelial cells, in explants of m a m m a r y tissue derived from animals in various developmental stages, into secretory cells. Partial development can be effected by addition of incomplete complements of these hormones? ,9 Beef and porcine insulin are about equipotent. The most frequently used glucocorticoid is hydrocortisone, but a number of others 3H. Gruneberg, J. Hered. 34, 88 (1943). 4 Biology Data Handbook (P. L. Altman and D. S. Dittmer, eds.), p. 530. FASEB. Bethesda, Maryland, 1964. G. C. Majumder and R. W. Turkington, Endocrinology 88, 1506 (1971). 6B. I. Balinsky, Trans. Roy. Soc. Edinburgh 62, 1 (1950). 7F. J. A. Prop, Pathol. Biol. 9, 640 (1961). E. S. Mills and Y. J. Topper, J. Cell Biol. 44, 310 (1970). ~T. Oka and Y. J. Topper, J. Biol. Chem. 246, 7701 (1971).
446
EXOCRINE TmSUE
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have also been shown to have the requisite biological activity.TM Both ovine and bovine prolactin are effective; human placental lactogcn is about 80% as active, on a weight basis, as prolactin in those instances in which it has been tested. 1~ Although these hormones have been shown to produce their characteristic effects when used at levels between 10-9 and 10-8 M, ~,~2they are usually employed at considerably higher concentrations, i.e., 5 ~g/ml each. Appropriate amounts of freshly prepared stock solutions of each hormone are added directly to Medium 199 before sterilization. Insulin stock: 1 mg per 0.5 ml of 5 X 10-3 HC1 (2 t~g/~l). Prolactin stock: 1 mg per 0.5 ml of l0 -4 M NaOH (2t~g/td). Hydrocortisone stock: 1 mg per 0.2 ml of anhydrous ethanol (5 ~g/td). When systems to which hydrocortisone is added are to be compared to systems without added glucocorticoid, a corresponding amount of ethanol should be added to the latter. The final concentration of alcohol in the media should be no more than 0.5%.
Preparation o] Siliconized Lens Paper In most of the studies on mammary gland explants, the tissue is supported at the surface of the medium. A common support is lens paper, which is siliconized to enable it to float. Prior to siliconization the paper is suspended in ethyl ether for 30 minutes and then the ether is removed by aspiration. This is done 3 times. The same procedure is then performed with 95% ethyl alcohol. Finally, the paper is washed 4 times for 15-minute intervals with quartz-distilled water, and dried at 37 °. Siliconization is accomplished by submerging the dried paper in Siliclad (Clay Adams)-quartz-distilled water (l:1000) for 10 minutes at room temperature. After removal of the Siliclad solution by aspiration, excess silicone is eliminated by treatment with water as described above. Again the papers are dried at 37 ° . A convenient type of container for culture of the explants is the sterile and disposable Falcon microdiffusion dish (Catalog No. 7004) whose dimensions are 65 X 15 mm. Accordingly, the dry lens paper is cut into pieces corresponding in size to that of the inner well of these dishes. The pieces are placed in covered, glass petri dishes and heated at 150° for 1 hour in a dry oven. This serves two purposes. The lens paper is sterilized, and the impregnated silicone is modified so that it now permits the paper to float. L°R. W. Turkington, W. G. Juergens, and Y. J. Topper, Endocrinology 80, 1139 (1967). 11R. W. Turklngton and Y. J. Topper, Endocrinology 79, 175 (1966). 12F. E. Stockdale, W. G. Juergens, and Y. J. Topper, Develop. Biol. 13, 266 (1966).
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Preparation o] Tissues
The explantation technique employed is determined by the developmental stage of the donor animal, and by the particular interests of the investigator. Thus, in some instances the whole gland is cultured as a unit, while in others the gland is cut into smaller pieces before explantation. Embryonic Rudiments. Fetal mouse and rat mammary rudiments are detectable by day 13 of gestation. The effects of androgens on whole rudiments in vitro have been studied by Kratochwil. 13 Ceriani TM has reported that whole fetal rat rudiments can be stimulated to produce casein in the presence of the appropriate hormones. Whole Glands ]rom Postnatal Mice. For studies on lobuloalveolar development it is advantageous to maintain the epithelial architecture intact. To this end, Prop 7 aDd Ichinose and Nandi '~ have developed a method for the culture of whole thoracic mammary glands from 3-weekold mice. Gland Fragments. Most frequently the abdominal glands are used in organ culture studies. Limitations in the rate of diffusion of gases and components from the media necessitate the use of small explants prepared from these glands. TM Using instruments sterilized in 70% ethanol and rinsed in boiling water, the glands are isolated aseptically after the animals have been killed by cervical dislocation. Isolation is facilitated by placing the animal on a dissection board, ventral surface up, and securing this position with pins through the appendages. After carefully shaving the abdomen and thighs with a sharp razor blade, loose hair is removed by swabbing with a wet cloth. Four incisions, through the skin only, are then made with a scissors. One is in the middle of the abdomen from the genitalia to the diaphragm. Another intersects the first at its upper extremity, completely transversing the ventral surface. Incisions are then made from tile lower end of the median cut diagonally across each thigh. Skin flaps can then be peeled from the abdominal wall with forceps. These are pinned to the dissection board, exposing the mammary glands on the inner skin surface. Lymph nodes, one or two in number, are extirpated with forceps from each gland. The glands are then excised with forceps and scissors, removing as much connective tissue as possible, and placed in a sterile microdiffusion dish containing 1-2 ml of sterile medium 199. It is desirable that the explants to be prepared contain approximately 12K. Kratochwil, or. Embryol. Exp. Morphol. 25, 141 (1971). 14R. L. Ceriani, Develop. Biol. 21, 530 (1970). 1.~R. R. Ichinose and S. Nandi, Science 145, 496 (1964). lgj. j. Elias, Proc. Soc. Exp. Biol. Med. 101, 500 (1959).
448
EXOCatNE TISSU~
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equal numbers of epithelial cells. Since the extent to which the ductal-alveolar system extends into the mammary fat pad is a function of both the species and the developmental state of the animal, preparation of equivalent explants from a given animal may require selective utilization of certain portions of the gland. Mature virgin and multiparous nonpregnant mice (approximate weight per gland, 100-150 mg) : discard peripheral portion, constituting about 20% of the gland. Pregnant (approximate weight per gland, 80-100 rag) and lactating (approximate weight per gland, 200-300 rag) mouse gland: use entire gland. Mature virgin, multiparous nonpregnant, and 1-15-day pregnant rat gland: use inner core, constituting about 30% (250-350 mg) of the entire gland. Late pregnant and lactating rat gland: use entire gland. These precautions will assure that explants from a given type of animal will contain approximately equal numbers of epithelial cells. Base-line activities and quantitative responses to hormones may vary somewhat from animal to animal. In experiments in which it is necessary to use pooled tissue from more than one animal it is desirable to employ equal numbers of explants from each animal in every determination. The situation is different with the abdominal gland of the 3-week-old mouse, however. In this animal the epithelial cells are concentrated in a small area at the level of the nipple. For operational reasons it is desirable to use all the explants derived from one gland for each determination. The contralateral gland may serve as a control. The explants are commonly prepared directly in the microdiffusion dish in which the glands and medium 199 were placed. The tissue is gently cut into small pieces (0.5-1.0 mg wet weight). Remove as much cormective tissue as possible. Transfer of the explants to the culture dishes which have been preloaded with 2 ml of medium and siliconized lens paper is facilitated by first aspirating away most of the medium 199 in which the explants were prepared. Using a fine forceps, the explants may then be transferred to the floating lens paper, taking care not to carry over too much medium with the tissue fragments. The number of explants needed for each experimental point is determined by the parameter being studied. Up to about 25 explants can be accommodated in each dish.
Maintenance o] Cultures Incubation of the explants is carried out in a closed system. The microdiffusion dishes are stacked in heavy-duty polycarbonate boxes containing an open water container for maintenance of high humidity. Each
[37]
MAMMARY GLAND ORGAN CULTURE
449
box has, in addition to a hinged top, a elosable opening at either end for introduction of 5% C02-95% air. The gas mixture is first bubbled through sterile distilled water, and is then permitted to flow into the box for a predetermined time at a predetermined rate such that the pH of the media is brought into range 7.3-7.6. This equilibration may take up to an hour after the gas flow has been stopped and the box has been sealed. The temperature is maintained at 37 ° in a water-jacketed incubator. Media should be replaced at least every 48 hours. Old media may be removed by aspiration, using sterile Pasteur pipettes, and 2 ml of fresh media introduced with sterile pipettes. The lens papers are easily refloated under these conditions. In some cases it is desirable to transfer the lens paper containing the explants to a fresh dish preloaded with medium. Gassing is then repeated. To terminate the culture, the lens paper is removed from the dish and gently blotted to remove adhering medium. Explants are then carefully collected with a fine forceps.
Responses of Mammary Epithelial Cells Morphological Methods Whole Mounts. Whole mounts prepared from mammary gland according to the method of Lyons~7 provide insight into the architecture of the epithelial component of the whole tissue. Preparations from animals in different developmental states dramatically illustrate the changing ratios of epithelial cells to mammary fat, and the increased branching of the epithelial tree, as maturation proceeds. Histology. The preparation, fixation, and staining of secretions from cultured explants have been performed by using standard histological techniques. TM Examination of histological sections, prepared for light microscopy, from explants has provided information not obtainable by other methods. Insulin-induced increase in the number of epithelial cells per average alveolar cross section has been determined, s Also, alterations in the spatial arrangement of the alveolar cells and in the size of alveolar lumina consequent to hormone treatment have been reported. ~2 Additional observations made on such sections have supplemented determinations made by other methods. Secretory material in the lumina is stainable with eosin. Increased nucleic acid content in the cytoplasm is reflected by enhanced basophilic character. 12 Electron Microscopy. Visualization of most cellular organelles, how17W. R. Lyons, R. E. Johnson, R. D. Cole, and C. H. Li, in "The Hypophyseal Growth Hormone" (R. W. Smith, O. H. Gaebler, and C. N. H. Long, eds.), p. 461. McGraw-Hill (Blakiston), New York, 1955.
450
EXOCRINE TISSUE
[37]
ever, requires electron microscopy. Details of the methodology employed in studies on mammary gland have been presented. 8,1s-21 Morphological effects of insulin, hydrocortisone, and prolactin on explants from midpregnancy C 3 H / H e N mice have been studied, 8 using a multiple incubation technique. 22
Biochemical Methods The mammary gland contains two major cell-types, epithelial and fat. The proportions of these vary widely as a function of the developmental state of the animal. Changes in the synthesis of certain products, such as casein and a-lactalbumin, obviously reflect response of the epithelial cells. In other instances one cannot directly ascertain whether a response of explants is ascribable to the component epithelial or fat cells. The following techniques have been helpful in making this distinction. The small clump of epithelial cells concentrated in the nipple area can be removed from the 3-week-old animal by cauterization. 23 As the animal matures this gland remains essentially devoid of epithelial cells, and explants prepared from it can be used to determine fat-cell responses. The unoperated abdominal gland can be used as a contralateral control. Epithelial cells, free from fat cells but contaminated with a small percentage of connective tissue cells, can be prepared by treating finely minced mammary gland or explants with crude eollagenase obtained from Worthington. 1,24 The mince is suspended in a 0.15% solution of collagenase in Medium 199 containing 4% bovine serum albumin, adjusted to pH 7.4. The suspension is shaken at 37 ° for 40 minutes. At intervals of about 5 minutes the suspension is aspirated into Pasteur pipettes. Use of a series of these with progressively smaller bore-size aids in the dispersion of the epithelial cells and their separation from fat cells. Fibrous tissue which adheres to the tips of the pipettes can be removed. After centrifugation (200 g; room temperature for 2 minutes) the epithelial pellet is washed 4 times with Medium 199. Residual fibrous tissue can be removed mechanically during this series of resuspensions and centrifugations. The yield of epithelial cells is about 90%, based on thymidine3H-labeled DNA. 1, W. Bargman and A. Knoop, Z. Zelllorsch. Mikrosk. Anat. 49, 344 (1959). 1, K. H. ttollman, J. Ullraslruct. Res. 2, 23 (1959). 2, K. K. Sekhri, D. R. Pitelka, and K. B. DeOme, J. Nat. Cancer Inst. 39, 459 (1967). ,1 S. R. Wellings, R. A. Copper, and E. M. Rivera, J. Nat. Cancer Inst. 36, 657 (1966). 22 R. W. Turkington, D. H. Lockwood, and Y. J. Topper, Biochim. Biophys. Acla 148, 475 (1967). 2*K. B. DeOme, L. J. Faulkin, It. A. Bern, and P. B. Blair, Cancer Res. 19, 515 (1959). 2, E. Y. Lasfargues, Anal. Rec. 127~ 117 (1957).
[37]
MAMMARY GLAND ORGAN CULTURE
451
The collagenase technique has been applied in many studies in which the tissue was first cultured as explants. Determinations on the residual pellet provide direct information on the response of the epithelial cells. Use of this method as a means of preparing epithelial cells which are to be cultured subsequently has been less successful. The isolated cells appear to have limited ability, compared to the cells within explants, to respond to the hormones. Cell Proli]eration. The epithelial cells in mammary explants derived from animals in different physiological states make DNA and divide under the influence of insulin, 25 various blood sera, "~and epidermal growth factor. 2~ The cells from mature C3H/HeN virgin mice and SpragueDawley rats are insensitive to insulin and serum factor(s) in terms of DNA synthesis. 1,~ They are also insensitive to insulin in terms of transport of a-aminoisobutyric acid (AIB), and the synthesis of glucose-6phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase. 1 After about 24 hours of culture the explants acquire these sensitivities. No exogenous hormones are required for the acquisition of these sensitivities. Cells in explants from mid-pregnancy mouse and late-pregnancy rat mammary gland, on the other hand, respond to these agents promptly after isolation. The proliferative response can be quantitated in a number of ways: 1. The extent of epithelial DNA synthesis can be determined by comparing the epithelial DNA content of freshly isolated explants with that after culture, using the method of Dische 27 as modified by Burton. ~s This determination is performed on the cell-pellet obtained after treating the mouse tissue with collagenase. Minimum tissue requirement: 40-60 mg mid-pregnancy ; 60-75 mg mature virgin. 2. DNA synthesis can also be estimated by incorporation of [~H]thymidine into an acid-insoluble fraction. Since fat cells do not make DNA under the conditions used 1: treatment with collagenase is not necessary. Tissue requirement: 4-8 rag. 3. In some instances it may be desirable to know which epithelial cells within explants have synthesized DNA. Such information can be obtained by autoradiography of histological sections prepared from [ :H] thymidine-labeled tissue. 1~ 4. The number of cells which are dividing at any given time during culture can be estimated by counting mitotic figures in histological sections prepared from explants. The results are usually expressed as mitotic in25F. E. Stockdale and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 56, 1283 (1966). 2~R. W. Turkington, Exp. Cell Res. 57, 79 (1969). 2~Z. Dische, Mikrochemie 8, 4 (1930). ~sK. Burton, Biochem. J. 61~, 315 (1956).
452
EXOCRINE TISSUE
[37]
dices, i.e., the ratio of the number of cells in mitosis (M) to the total number of cells counted. The total number of cells which have entered M during a particular period can be determined by culturing in the presence of a mitotic inhibitor such as colchicine. The ratio of mitotic figures to total cells counted reflects the percentage of cells which have entered M during the culture period in question. 5. The increase in the number of cells can be directly determined, after treatment of the tissue with collagenase, by counting the suspended epithelial cells in a hemacytometer. 29 Counting is facilitated by brief treatment of the cell suspension with 0.5% trypsin solution (GIBCO) to prevent clumping. Minimum tissue requirement: 75 mg, mid-pregnancy mouse; 100 rag, mature virgin. An important consideration in differentiation is whether or not certain developmental processes are dependent upon cell proliferation. Inhibitors of proliferation are useful tools for attacking this question experimentally. Agents which arrest cells in the mitosis (M) phase of the cell-cycle are usually not satisfactory because cells in M have a reduced general capacity for macromolecular synthesis2 °,31 Agents which prevent cells from entering the DNA-synthesis phase (S), such as lfl-D-arabinosylcytosine and fluorodeoxyuridine, can be fruitfully employed. Under these circumstances one may regard the inhibited cells as normal G1 cells if proper controls are unaffected. In order to minimize the possibility of toxic side effects the smallest concentration of agent giving maximal inhibition should be used; this may vary as a function of the hormones added, and the developmental state of the donor animal. It is also necessary to establish that the DNA-inhibitor does not affect the developmental process in question when the agent is added to postmitotic cells. 32 Assay of Products Made Uniquely by Mammary Gland. The formation of the following secretory products is a reliable indication that the mammary epithelial cells have attained a highly differentiated state. Development of these functional capacities may not be synchronous in vitro. 29 1. Casein. Casein is a family of phosphoproteins. The isolation and characterization of the material synthesized by mammary explants is facilitated by incorporation of 32Pi or labeled amino acids added to the culture system. After suitable labeling periods the phosphoproteins are precipitated by the addition of Ca 2÷ and rennin to the soluble fraction 29B. K. Vonderhaar, I. S. Owens, and Y. J. Topper, J. Biol. Chem. 248, 467 (1973). 3o H. Pan and S. Penman, Science 168, 135 (1970). ,1 D. Martin, Jr., G. M. Tomkins, and D. Granner, Proc. Nat. Acad. Sci. U.S. 62, 248 (1969). ~ I. S. Owens, B. K. Vonderhaar, and Y. J. Topper, J. Biol. Chem. 248, 472 (1973).
[37]
MAMMARY GLAND ORGAN C U L T U R E
453
of the tissue? 3 If no further fractionation is to be performed, carrier bovine casein is added prior to homogenization. If the precipitate is to be resolved into separate casein polypeptides, carrier mouse casein, rather than bovine casein, is added prior to homogenization? 4,3~ The precipitate contains casein and other phosphoproteins. Although not all this material is authentic casein, :2 measurement of its isotope content represents a fairly reliable and rapid method for determining the relative rate and extent of casein synthesis by explants. More precise quantitation of casein synthesis is accomplished by electrophoresis of the labeled phosphoproteins on starch gels or polyacrylamide gels. 3~,~6 Individual casein polypeptides can be visualized by staining or autoradiography. Tile individual casein bands can then be cut out and counted. Minimum tissue requirement: 4-8 mg per determination. The synthesis of casein by different systems within a given experiment may often be compared by expressing results as count per minute per unit wet weight tissue. However, in some instances it is necessary to express results as counts per minute per unit of epithelial D N A ? ~ Similar considerations apply to the study of other functional parameters. 2. a-Lactalbumin, a-Lactalbumin is tile B-protein component of the lactose synthetase system?; The ability of a-lactalbumin to enable the A-protein of lactose synthetase to utilize glucose as an acceptor of the galactose moiety of UDP-Gal, with consequent formation of lactose, is the basis for its assay. A modification 29 of the method of Brew ~8 has been used for determination of a-lactalbumin activity in homogenates from cultured explants. Minimum tissue requirements: 50-80 mg of mature virgin or 30-40 mg mid-pregnancy tissue homogenized in 300 ~l of buffer; 20-30 mg of lactation tissue. 3. Lactose. The incorporation of radioactive galactose into lactose during culture '~9 has also been used to study the control of lactose synthetase activity. ~° 4. F a t t y acids. Up to 70% of the fatty acids in rabbit milk trigly~3W. G. Juergens, F. E. Stockdale, Y. J. Topper, and J. J. Elias, Proc. Nat. Acad. Sci. U.S. 54, 629 (1965). 34R. W. Turkington, W. G. Juergens, and Y. J. Topper, Biochim. Biophys. Actc~ 111, 573 (1965). 3~A. E. Voytovich and Y. J. Topper, Science 158, 1326 (1967). 3~R. W. Turkington and M. Riddle, Endocrinoloqy 84, 1213 (1969). ~TU. Brodbeck, W. L. Denton, N. Tanahashi, and K. E. Ebner, J. Biol. Chem. 242, 1391 (1967). 3SK. Brew, T. C. Vanaman, and R. L. Hill, Proc. Nat. Acad. Sci. U.S. 59, 491 (1968). ~J. C. Bartley, S. Abraham, and I. L. Chaikoff, I. Biol. Chem. 241, 1132 (1966). 4. R. D. Palmiter, Biochem. J. 113, 409 (1969).
454
EXOCRINE TISSUE
[38]
cerides is comprised of octanoic and decanoic acids. The hormone dependence of the synthesis of these characteristic products by explants from rabbit mammary gland has been studied. 41 Other Metabolic Activities o] Mammary Gland in Vitro. Other functional parameters of mouse mammary gland which have been studied by the organ culture method, in attempts to understand more fully the development of this tissue, are listed: 1. Accumulation of a-aminoisobutyric acid 1,42 2. Glucose uptake and to¢al fatty acid synthesis 43,44 3. Synthesis of glucose-6-phosphate dehydrogenase and gluconate-6phosphate dehydrogenase~,2,4~ 4. RNA synthesis 1:,46-4s 5. Formation of rough endoplasmic reticulum. 8,9 6. DNA polymerase activity 49 7. Synthesis and acetylation of histones ~°-~2 411. A. Forsyth, C. R. Strong, and R. Dils, Biochem. J. 129, 929 (1972). 42T. Oka and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 68, 206'6 (1971). 4.~R. L. Moretti and S. Abraham, Biochim. Biophys. Acta 124, 280 (1966). 44R. Mayne and J. M. Barry, J. Endocrinol. 46, 61 (1970). ~D. P. Leader and J. M. Barry, Biochem. J. 113, 175 (1969). '6R. W. Turkington and O. T. Ward, Biochim. Biophys. Acta 174, 291 (1969). 4~M. R. Green and Y. J. Topper, Biochim. Biophys. Acta 204, 441 (1970). ~sM. R. Green, S. L. Bunting, and A. C. Peacock, Biochemistry 1O, 2366 (1971). 4~D. H. Lockwood, A. E. Voytovich, F. E. Stockdale, and Y. J. Topper, Proc. Nat. Acad. Sci. U.S. 58, 658 (1967). W. F. Marzluff, Jr., K. S. McCarty, and R. W. Turkington, Biochim. Biophys. Acta 190, 517 (1969). W. F. Marzluff, Jr. and K. S. McCarty, J. Biol. Chem. 245, 5635 (1970). :~ P. Hohmann and R. D. Cole, J. Mol. Biol. 58, 533 (1971).
[38] A n d r o g e n M e t a b o l i s m a n d R e c e p t o r A c t i v i t y in P r e p u t i a l G l a n d s a n d K i d n e y s o f N o r m a l a n d Androgen-Insensitive tfm Rats and tfm/y Mice 1
By C. WAYNE BARDIN, LESLIE P. BULLOCK, and IRENE MOWSZOWICZ2
In recent years, many laboratories have examined the pretranscriptional events involved in testosterone activation of target cells, and the 1Supported in part by PHS Grant No. HD05276. 2On leave of absence from Faculte de M~decine, Pitid-Salpetri~re, Service de Biochimie, Paris.
