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The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis
Biol Cell (1992) 75, 211-216
211
© Elsevier, Paris
Original article
The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis Kralj Metka, Pipan Nada Institute of Cell Biology, Medical Faculty, University of Ljubljana, Lipiceva 2, 61000 Ljubljana, Slovenia (Received 18 December 1991 ; accepted 29 July 1992)
Summary - Functional relations between exocytotic vesicle membranes, plasmalemma and milk fat globule membranes (MFGM) were studied during the final stages of mouse mammary gland differentiation, in the gland during full lactation and in the postpartum gland in which the synthesis of secretory products was partly inhibited by application of 2-Br-ct-ergokryptin. Analysis of ultrathin sections, freeze-fracture replicas, scanning electron microscopy and application of a cytochemical marker filipin showed that the apocrine secretion of lipid globules was closely related to the exocytosis of milk proteins. During the last days of gestation the secretion of lipid globules resulted from many exocytotic events of the secretory vesicles that accumulated and fused around the cytoplasmic lipid droplets. Seldom the lipid droplet protruded partly into the gland lumen and a part of its surface became covered with the apical plasmalemma. Although apical plasmalemma became more important in the formation of MFGM in the postpartum period, we could still confirm a direct contribution of secretory vesicle membranes to the final detachment of the lipid globule. The application of 2-Br-=-ergokryptin hindered the apocrine secretion of the lipid globules and a situation similar to the situation in the prepartum gland was observed. apocrine secretion / mammary gland
milk fat globule membrane / exocytosis
Introduction Emphasised membrane dynamics can be observed in the mammary alveolar cells during gland differentiation. In completely differentiated cells, it results in numerous membrane fusions between secretory vesicles, a great number of exocytotic events and formation of milk fat globule membrane (MFGM) [7, 8, 13, 23]. Final cell differentiation is attained under the influence of lactogenic hormones through two successive processes. Structural differentiation is completed during the gestation period when the secretion of cell products remains low [6]. Endomembraneous surfaces for the synthesis of secretory products are greatly increased and this differentiation phase corresponds to the hormonal activation of genes coding for milk proteins and enzymes for lipid synthesis [24, 25, 35]. Functional differentiation level is accomplished in the postpartum period, when secretion of milk constituents is not inhibited any more by progesterone [2, 18]. Apocrine secretion of lipid globules coated with a membrane is specific for the mammary gland. Due to the constant loss of membranes from the alveolar cell very extensive membrane synthesis and traffic can be expected in the differentiated cells in which synthesis and secretion of lactose, milk proteins and lipids take place. Both exocytosis and apocrine secretion are present in the same cell and at the same time but it is not clear whether they are obligatory connected. There are two basic models that try to explain how the lipid globules are secreted, and consequently, what the origin of MFGM is [20]. In many morphological studies, secretion of lipid globules is described as progressive extrusion of a lipid globule which becomes enveloped with the apical plasmalemma and finally pinches off [5, 15, 17,
28, 29]. According to this model, MFGM should be homologous only with the plasmalemma and no direct interactions between exocytosis and the apocrine lipid secretion were necessary. On the other hand, some studies of MFGM properties show a certain biochemical homology between the MFGM and the membranes of Golgi apparatus and secretory vesicles [12, 19, 33]. These results indicate that the membranes of secretory vesicles and Golgi apparatus can be important for the formation of MFGM. Because these are biochemical and not structural studies they can not directly explain the way the enveloping of the lipid globule with the plasmalemma and membranes of secretory vesicles takes place. The importance of interactions between different endomembranes, plasmalemma and the lipid droplet in the course of apocrine secretion in the mammary gland remains undefined [9, 14]. Wooding [39] proposed that a part of MFGM can originate from the plasmalemma and the other part from the fused secretory vesicles that accumulate around the lipid droplet. When these secretory vesicles fuse with the plasmalemma the lipid globule detaches from the secretory cell. It also was mentioned that the normal exocytotic process was necessary for normal milk secretion and that colchicine treatment could decrease milk production [26, 37]. We tried to demonstrate the existence of a permanent relationship between the exocytosis and the apocrine secretion of the lipid globules by the ultrastructural analysis of the mammary gland secretory cells in different stages of differentiation. The occurrence of simple and compound exocytosis and lipid secretion have been studied in the mouse mammary gland at the end o f gestation when the gland already completes its structural and functional
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The role of exocytosis in apocrine secretion differentiation [22] while the synthesis of the secretory products and their secretion are slowed down and thus can be observed more easily. This situation has been compared to the synthesis and secretion of milk in the postpartum period when both processes are very intense. It has been compared also to the situation when the normal lactation has again been slowed down by application of 2-Br-aergokryptin that reduces prolactin secretion.
Materials and methods Mice of Albany strain in day 14 and 19 of gestation and day 1 and 9 postpartum were used. Animals were kept at standard laboratory conditions. To partially suppress lactation some lactating raice were treated with a single dose of 0.5 mg of 2-Br-~-ergokryptin dissolved in 5°70 ethanol and their litter has been removed on day 9 postpartum, 24 h before removal of the gland [1].
Transmission electron microscopy Tissue was fixed in fresh, ice-cold mixture of 1.25°/0 glutaraldehyde and 2% OsO4 and 0.5% tannic acid [21] for 1 h and postfixed in 207o water solution of OsO 4 for 4 h [10]. After a quick rinse in distilled H20 the tissue sample was put in I o7o aqueous uranyl acetate overnight, dehydrated and embedded in Epon. Ultrathin sections were stained with uranyl acetate for 5 min and lead citrate for 1-2 min and examined under a Jeol T8 electron microscope.
Scanning electron microscopy Epon tissue blocks were prepared as for transmission electron microscopy. By means of semithin sections an appropriate part with transected alveoli was chosen under a light microscope. Epon was dissolved from this part with 1°7o NaOH in absolute ethanol [3]. Critical point dried blocks were mounted on preparation holders, vacuum coated with gold in a Sputter Coater (Polaron) and observed in Jeol JSM 840A scanning electron microscope.
Freeze-fracturing The tissue was fixed for 2 h in a mixture of 4°/0 paraformaldehyde and 2070glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2), rinsed in 0.1 M cacodylate buffer, soaked with 30070 glycerol in 0.1 M cacodylate buffer for 2 h, frozen in freon and cooled with liquid nitrogen. Freeze-fracture replicas, prepared in freezeetching device Balzers were cleaned in 5% Na-hypochlorite overnight and then in a 2:1 mixture of chloroform and methanol [31]. Some tissue samples were incubated in 0.02070 filipin (Sigma) in 0.2 M cacodylate buffer with 2% DMSO, before being freezefractured [11].
213
Results
Ultrastructure of alveolar cells in prepartum period Main structural differential features and accumulation of secretory products (proteins, lipids) were observed already on day 14 of gestation (fig 1). At the same time the amount o f lipid droplets in the gland lumina was found to be minimal. Spatial associations between cytoplasmic lipid droplets and secretory vesicles were not frequent. Exocytosis of single casein micelles could rarely be observed and almost no lipid secretion was detected. Apical cell surfaces were rather flat, sometimes covered with microvilli but lipid droplets never pushed apical plasmalemma into the lumen. Lipid droplets were generally small, numerous and accumulated in the apical cytoplasmic region where some secretory granules with casein micelles could also be observed. After day 19 of gestation accumulation of secretory products in the gland cells and the establishing of secretory function were observed. The size of cytoplasmic lipid droplets and secretory vesicles was greatly increased and only one lipid droplet was present in most cells. Secretory vesicles usually contained more than one casein micelle and appeared to be dilated due to lactose accumulation. Frequently, they could be found opposed to the lipid droplet forming a m e m b r a n e bound cisterna that separated the cytoplasm and the lipid droplet (fig 2). Such big secretory vesicle or cisterna could open into the gland lumen by exocytosis, either on the top or on the lateral side of the lipid droplet (figs 3, 4). Thus the cavity around the lipid droplet connects to the gland lumen and the lipid droplet, covered with a thin rim of cytoplasm and the membrane gradually separates from the rest of the cytoplasm. Frequent spatial associations between the cytoplasmic lipid droplet and the secretory vesicles were observed, resulting in larger patches of membranes associated with the lipid droplet. In these membrane patches, cholesterol could be demonstrated by filipin (fig 5).
Ultrastructure of alveolar cells in the postpartum period Normal lactation On the first day postpartum the alveolar ceUs acquire their final functional differentiation stage and merocrine and apocrine secretion of secretory products was intensified. Secretory vesicles adjoining a cytoplasmic lipid droplet were frequent. Cytoplasmic distribution of secretory vesicles and the
Figs 1-8. 1. Apical region of the mammary gland cell. Secretory vesicles with single casein micelles (arrows) do not fuse and only occasionally they are located near the cytoplasmic lipid droplet (LD) (arrowheads). Day 14 of gestation, x 36000.2. Direct contact between the cytoplasmic lipid droplet (LD) and a membrane-bound cisterna (arrows) which was formed by fusion of secretory vesicles (LU, gland lumen). Day 19 of gestation, x 33000. 3. By exocytosis of the secretory vesicle (arrow) very close to the cytoplasmic lipid droplet (LD), the lipid droplet acquires a patch of membrane that contributes to the formation of the milk fat globule membrane (LU, gland lumen). Day 19 of gestation, x 25000.4. A membrane bound groove (arrows) comunicating with the gland lumen (LU). Milk fat globule membrane (arrow-heads) is forming synchronously with the separation of the lipid droplet (LD). Day 19 of gestation. x 12500.5. Freeze-fracture replica of the mammary gland cell. Part of the surface of the lipid droplet (LD) is heavily labelled with filipin (arrowheads), similar to the membrane of the secretory vesicle (arrow). Day 19 of gestation, x 14500. 6. Cytoplasmic lipid droplet (LD) protruding into the gland lumen (arrowhead). Secretory vesicles accumulate around the lipid droplet (arrows). Day 1 postpartum, x 19000.7. A membrane-bound cisterna (arrows) separates the cytoplasmic lipid droplet (LD) from the cytoplasm in the apical cell region. Lipid droplet is partly covered with the membrane of the cisterna. Part of the milk fat globule (MFG) is seen in the gland lumen (LU). Day 9 postpartum, x 11200. 8. The lipid droplet (LD) is protruding far into the gland lumen (LU). Large portion of its surface is covered with the apical plasmalemma (arrowheads). Secretory vesicles accumulate in the cell apex between the lipid droplet and the rest of the cytoplasm (arrows). Day 9 postpartum, x 4000.
214
K Metka, P Nada
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Figs 9, 10. 9. Scanning micrograph of the mammary gland alveolus on the first day postpartum. Big, crater-like depressions corresponding to the prints of lipid droplets are present in the apical cell surfaces (arrows). × 1200.10. Apical region of the mammary gland cell after the treatment of the lactating animal with a low dose of 2-Br-a-ergokryptin. Apical cell surface is flat (arrowheads) and secretory vesicles are scattered through the cytoplasm (arrows). The lipid droplet (LD) remains inside the cell (LU, gland lumen). Application of 2-Br-a-ergokryptin on day 9 postpartum. × 11500.
points of contact between plasmalemma and secretory vesicles determined the size and position of the lipid droplet. When a large lipid droplet filled the apical cytoplasmic region almost completely and pushed the apical plasmalemma into the gland lumen, the secretory vesicles accumulated at its basal side and exocytosis could take place only on the basis of the lipid droplet (fig 6). The cells in the gland in full lactation were not essentially different. The secretory vesicles accumulated near the cytoplasmic lipid droplet and could also contribute to the formation of MFGM. Sometimes the membranebound cavities similar to those observed before parturition were present near the cytoplasmic lipid droplet (fig 7). The lipid droplet sometimes protruded far into the gland lumen so that a series of secretory vesicles were present only at its base (fig 8). Scanning electron microscopy of alveoli from the lactating mammary gland showed frequent crater-like depressions in the apical cell surface of the alveolar cells, Usually one such depression per cell was present (fig 9). These depressions'were covered with a membrane and their dimensions were in the range of the smaller milk fat globules (0.5-1.0 gm). According to their size and position we concluded that they were prints of the detached milk fat globules.
Suppressed lactation When the synthesis of secretory products in normally lactating mammary gland has been decreased by the application of 2-Br-a-ergokryptin the alveolar ceils became similar to the cells in prepartum period. We could observe that vesicle fusions were much less frequent but the vesicles were still dilated due to lactose accumulation and could contain more casein micelles (fig 10). The apical cell surface was fiat, exocytosis was rare and the lipid droplets were prevented from protruding into the gland lumen.
Discussion By its biochemically heterogeneous product and complex way of secretion, the mammary gland differs considera-
bly from the other mammalian glands. We applied different methods of ultrastructural analysis in order to study relations between different membranes in the cell and to find potential correlations between membrane interactions, synthesis of secretory products and their secretion. Synthesis of milk proteins and lipids is present already in the prepartum period, while the synthesis of lactose is still blocked by high concentrations of progesterone [34] which inhibit also microtubule polymerization, polarized transport of secretory vesicles and their normal exocytosis [18, 26]. Decrease of progesterone levels after parturition correlates with deblocking o f lactose synthesis and the maturation of secretory vesicles, their frequent fusions and exocytosis [36]. It is well established that secretion of milk proteins and lactose takes place by exocytosis [13], but it is less clear what role the membranes of secretory vesicles and plasmalemma play in lipid secretion [14, 22]. The secretion of lipid globules is functionally related to the increased rate of exocytosis, despite the fact that the lipids do not accumulate in secretory vesicles [40]. Molecular analysis could not confirm absolute homology of protein and phospholipid composition of MFGM and plasmalemma [4, 21, 29]. The results of our morphological analysis of mammary cells during lactogenesis indicated a strong connection between the exocytosis of proteins and lactose and apocrine secretion of lipids. Obvious spatial interactions of cellular lipid droplets with secretory vacuoles in functionally differentiated cells and filipin labelling of the membranes on the surface of the cytoplasmic lipid droplets showed that the membranes of secretory vacuoles were involved in the gradual separation of the lipid droplet from the cytoplasm. Membranes of both the secretory vacuoles and the plasmalemma could be incorporated into the MFGM of the secreted lipid globule. When secretory products accumulated in the cell, numerous interactions between the endomembranes occurred and resulted in the fragmentation of the apical cell region. The majority of these cell fragments contained a lipid droplet. The role of the membranes of secretory vesicles in the formation of MFGM was especially pronounced when the synthesis of secretory products was not balanced with their secretion and when
The role of exocytosisin apocrine secretion the apical plasmalemma was relatively stable (day 19 of gestation). Our observations in the prepartum mammary gland, when the exocytosis of secretory vesicles was slowed down by high progesterone concentrations, were in agreement with the results reported in the experiments with the infusion of colchicine into the mammary gland [16, 26, 30]. It has been reported that colchicine considerably reduced the amount of milk and that the secretory products accumulated inside the gland cells. A strong tendency to the lining up of the secretory vesicles near the cytoplasmic lipid droplet has been observed, and the phospholipid composition of MFGM became more similar to the phospholipid composition of the membranes of the secretory vesicles [38]. We can conclude from our observations that in the situation of obstructed exocytosis the amount of membranes contributed to MFGM by the secretory vesicles was greater relative to the amount of plasmalemma than in the case of normal lactation. In the postpartum period the lipid synthesis increased so that the droplet quickly reached the apical cell region and the plasmalemma. At the same time the rate of exocytosis also increased, the apical cell surface could become unproportionally enlarged and the lipid droplet could push it in front of itself into the lumen so that the secretory vesicles could assemble only at the free surface of the lipid droplet facing the rest of cytoplasm. We consider the crater-like depressions in the apical cell surfaces revealed by scanning electron microscopy of the lactating mammary gland also as an additional indication that the detachment of lipid globules takes place independently of the degree to which the lipid droplet protrudes from the cell surface and that the lipid globule can detach before it completely pinches off. A similar situation has already been described [32], but as the main interest of the study lay in the cell contacts, the morphology of the apical plasmalemma has been mentioned only briefly. These depressions are covered with membrane and from our point of view they are prints of the lower part of the lipid droplet that has been surrounded by a cisterna formed by fusion of secretory vesicles at the time o f apocrine lipid secretion. The importance of exocytosis for providing sufficient plasmalemma surface on one side and final detachment of the lipid globule on the other side was demonstrated also in our experiments with 2-Br-~-ergokryptin in postpartum lactation period. The overall morphological situation of the gland cells became very similar to the situation in the prepartum period. The lipid secretion has not been completely stopped but the protrusion of the lipid droplet into the lumen could only rarely be seen and accumulation of secretory vesicles around the cytoplasmic lipid droplet was observed. On the basis of our results we suggest that the exocytosis of secretory vesicles that separate the lipid droplet from the majority of the cytoplasm is necessary for the final separation of the lipid globule into the gland lumen. The secretory vesicles directly provide the membranes for at least a part of MFGM and for the apical plasmalemma at the time of lipid globule detachment. The suggested mechanism can also explain why the phospholipid composition of MFGM in the situation of inhibited secretion, either by colchicine in lactation [30] or by progesterone in prepartum period, resembles more the membranes of the secretory vesicles than the plasmalemma. The ultrastructural studies of the mammary alveolar cells during functional differentiation support the hypothsis that apocrine secretion occurs as the result of a very
215
intensive exocytosis and fragmentation of the apical cell region where the lipid droplet is located. The share of the plasmalemma and of the membranes of the secretory vesicles in the formation of MFGM is variable and depends mainly on the intensity of exocytosis. Very intensive exocytosis during lactation in the postpartum period provides a surplus of apical plasmalemma that can be secreted together with the lipid globule, while the membranes of secretory vesicles become more important in the formation of MFGM when the exocytosis is hindered.
Acknowledgments We wish to thank prof dr Kristijan Jezernik for helpful discussion and help in the freeze-fracturing technique, and dr Majda Pgeni~nik for help in the scanning electron microscopy.
References 1 Akers RM, Bauman DE, Goodman GT, Capuco AV, Tucker HA (1981) Prolactin regulation of cytological differentiation of mammary epithelial cells in periparturient cows. Endocrinology 109, 31-40 2 Assairi L, Delouis C, Gaye P, Houdebine LM, OllivierBousquet M, Denammur R (1974) Inhibition by progesterone of the lactogenic effect of prolactin in the pseudo pregnant rabbit. Biochem J 144, 245-252 3 Cajander SB (1986) A rapid and simple technique for correlating light microscopy, transmission and scanning electron microscopy of fixed tissues in Epon blocks. JMicrosc 143, 265-274 4 Calberg-Bacq CM, Frant;ois C, Gosselin L, Osterrieth PM, Rentier-Delrue F (1976) Comparative study of the milk fat globule membrane and the mouse mammary tumor virus prepared from the milk of an infected strain of Swiss albino mice. Biochim Biophys Acta 419, 458-478 5 Dowben RM, Brunner JR, Philpott DE (1976) Studies on milk fat globule membranes. Biochim Biophys Acta 135, 1-10 6 Dulbecco R, Henaham M, Armstrong B (1982) Cell types and morphogenesis in the mammary gland. Proc Natl Acad Sci USA 79, 7346-7350 7 Dylewski DP, Keenan TW (1983) Compound exocytosis of casein micelles in mammary epithelial cells. Eur J Cell Biol 31, 114-124 8 Dylewski DP, Keenan TW (1987) Ball and socket figures between secretory vesicles in mammary epithelial cells. Protoplasma 138, 65-68 9 Dylewski DP, Dapper CH, Valivullah HM, Deeney JT, Keenan TW (1984) Morphological and biochemical characterization of possible intracellular precursors of milk lipid globules. Eur J Cell Biol 35, 99-111 10 Franke WW, Krien S, Brown RM Jr (1969) Simultaneous glutaraldehyde-osmium tetroxide fixation with postosmication. Histochemie 19, 162-164 11 Ginsbach C, Fahimi HD (1987) Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs. Histochemistry 86, 241-248 12 Huggins JW, Trenbeath TP, Chesnut RW, Carothers Carraway CA, Carraway KL (1980) Purification of plasma membranes of rat mammary gland. Exp Cell Res 126, 279-288 13 Hurley D, Hwang S, Rocha V (1989) Casein accumulation in distended rough endoplasmic reticulum of collagen gelcultivated mouse mammary epithelia. J Cell Physiol 141, 135-141 14 Kanno C (1990) Secretory membranes of the lactating mammary gland. Protoplasma 159, 184-208 15 Keenan TW, Sasaki M, Eigel WN, Morre D J, Franke WW, Zulak IM, Bushway AA (1979) Characterization of a secre-
216
16
17 18 19
20
21
22 23 24 25
26
27
K Metka, P Nada tory vesicle-rich fraction from lactating bovine mammary gland. Exp Cell Res 124, 47-61 Knudson CM, Stemberger BH, Patton S (1978) Effects of colchicine on ultrastructure of the lactating mammary cell: Membrane involvement and stress on the Golgi apparatus. Cell Tissue Res 195, 169-181 Linzell JL, Peaker M (1971) Mechanism of milk secretion. Physiol Rev 51, 564-597 Loizzi RF (1985) Progesterone withdrawal stimulates mammary gland tubulin polymerisation in pregnant rats. Endocrinology 116, 2543-2547 Martel-Pradal MB, Got R (1972) Pr6sence d'enzymes marqueurs des membranes plasmiques, de l'appareil de Golgi et du reticulum endoplasmique dans les membranes des globules lipidiques de lait maternel. FEBS Lett 21,220-222 Mather IH (1987) Proteins of the milk fat globule membrane as markers of mammary epithelial ceils and apical plasma membrane. In: The mammary gland. Development, regulation, and function (Neville MC, Daniel CW, eds) Plenum Press, New York London, 217-276 Mather IH, Jarasch ED, Bruder G, Heid HW, Mepham TB (1984) Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. I. Radiolabelling of membrane and secretory proteins. Exp Cell Res 151, 208-223 Mepham TB (1987) Physiology o f lactation. Open University Press, Milton Keynes, Philadelphia Morre JD, Kartenbeck J, Franke WW (1979) Membrane flow and interconversions among endomembranes. Biochim Biophys Acta 559, 71-152 Muldoon TG (1987) Prolactin mediation o f estrogen-induced changes in mammary tissue estrogen and progesterone receptors. Endocrinology 121, 141-149 Nakhasi HL, Quasba PK (1979) Quantitation of milk proteins and their mRNAs in rat mammary gland at various stages of gestation and lactation. J Biol Chem 254, 6016-6025 Nickerson SC, Smith J J, Keenan TW (1980) Role of microtubules in milk secretion - action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells. Cell Tissue Res 207, 361-376 Nickerson SC, Akers RM, Weinland BT (1982) Cytoplasmic organization and quantitation of microtubules in bovine mammary epithelial cells during lactation and involution. Cell Tissue Res 223, 421-430
28 Patton S, Fowkes FM (1967) The role of plasma membrane in the secretion of milk fat. J Theor Biol 15, 274-281 29 Patton S, Keenan TW (1975) The milk fat globule membrane. Biochim Biophys Acta 415, 273-309 30 Patton S, Stemberger BH, Knudson CM (1977) The suppression of milk fat globule secretion by colchicine: an effect coupled to inhibition of exocytosis. Biochim Biophys Acta 499, 404-410 31 Peixoto de Menezes A, Pinto da Silva P (1978) Freezefracture observations of the lactating rat mammary gland. J Cell Biol 76, 767-778 32 Pitelka DR, Hamamoto ST, Duafala JG, Nemanic MK (1973) Cell contacts in the mouse mammary gland. I. Normal gland in postnatal development and the secretory cycle. J Cell Biol 56, 797-818 33 Powell JT, Jarlfors U, Brew K (1977) Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk. J Cell Biol 72, 617-627 34 Quirk S J, Gannell JE, Funder JW (1988) ~t-Lactalbumin production by rat mammary gland: prepartum emergence of resistance to glucocorticoids and progestins. Mol Cell Endocrinol 58, 183-189 35 Rillema JA, Etindi RN, Cameron CM (1986) Prolactin actions on casein and lipid biosynthesis in mouse and rabbit mammary gland explants are abolished by p-bromphenacyl bromide and quinacrine, phospholipase A 2 inhibitors. Horm Metab Res 18, 672-674 36 Sasaki M, Keenan TW (1978) Membranes of mammary gland. XV. 5-thio-D-glucose decreases lactose content and inhibits secretory vesicle maturation in lactating rat mammary gland. Exp Cell Res I l l , 413-425 37 Sokka TK, Patton S (1983) In vivo effects of colchicine on milk fat globule membrane. Biochim BiophysActa 731, 1-8 38 Stemberger BH, Walsh RM, Patton S (1984) Morphometric evaluation of lipid droplet associations with secretory vesicles, mitochondria and other components in the lactating cell. Cell Tissue Res 236, 471-475 39 Wooding FBP, Peaker M, Linzel JL (1970) Theories of milk secretion: Evidence from the electron microscopic examination of milk. Nature 226, 762-764 40 Jaczek M, Keenan TW (1990) Morphological evidence for an endoplasmic reticulum orgin of milk lipid globules obtained using lipid-selective staining procedures. Protoplasma 159, 179-182
211
© Elsevier, Paris
Original article
The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis Kralj Metka, Pipan Nada Institute of Cell Biology, Medical Faculty, University of Ljubljana, Lipiceva 2, 61000 Ljubljana, Slovenia (Received 18 December 1991 ; accepted 29 July 1992)
Summary - Functional relations between exocytotic vesicle membranes, plasmalemma and milk fat globule membranes (MFGM) were studied during the final stages of mouse mammary gland differentiation, in the gland during full lactation and in the postpartum gland in which the synthesis of secretory products was partly inhibited by application of 2-Br-ct-ergokryptin. Analysis of ultrathin sections, freeze-fracture replicas, scanning electron microscopy and application of a cytochemical marker filipin showed that the apocrine secretion of lipid globules was closely related to the exocytosis of milk proteins. During the last days of gestation the secretion of lipid globules resulted from many exocytotic events of the secretory vesicles that accumulated and fused around the cytoplasmic lipid droplets. Seldom the lipid droplet protruded partly into the gland lumen and a part of its surface became covered with the apical plasmalemma. Although apical plasmalemma became more important in the formation of MFGM in the postpartum period, we could still confirm a direct contribution of secretory vesicle membranes to the final detachment of the lipid globule. The application of 2-Br-=-ergokryptin hindered the apocrine secretion of the lipid globules and a situation similar to the situation in the prepartum gland was observed. apocrine secretion / mammary gland
milk fat globule membrane / exocytosis
Introduction Emphasised membrane dynamics can be observed in the mammary alveolar cells during gland differentiation. In completely differentiated cells, it results in numerous membrane fusions between secretory vesicles, a great number of exocytotic events and formation of milk fat globule membrane (MFGM) [7, 8, 13, 23]. Final cell differentiation is attained under the influence of lactogenic hormones through two successive processes. Structural differentiation is completed during the gestation period when the secretion of cell products remains low [6]. Endomembraneous surfaces for the synthesis of secretory products are greatly increased and this differentiation phase corresponds to the hormonal activation of genes coding for milk proteins and enzymes for lipid synthesis [24, 25, 35]. Functional differentiation level is accomplished in the postpartum period, when secretion of milk constituents is not inhibited any more by progesterone [2, 18]. Apocrine secretion of lipid globules coated with a membrane is specific for the mammary gland. Due to the constant loss of membranes from the alveolar cell very extensive membrane synthesis and traffic can be expected in the differentiated cells in which synthesis and secretion of lactose, milk proteins and lipids take place. Both exocytosis and apocrine secretion are present in the same cell and at the same time but it is not clear whether they are obligatory connected. There are two basic models that try to explain how the lipid globules are secreted, and consequently, what the origin of MFGM is [20]. In many morphological studies, secretion of lipid globules is described as progressive extrusion of a lipid globule which becomes enveloped with the apical plasmalemma and finally pinches off [5, 15, 17,
28, 29]. According to this model, MFGM should be homologous only with the plasmalemma and no direct interactions between exocytosis and the apocrine lipid secretion were necessary. On the other hand, some studies of MFGM properties show a certain biochemical homology between the MFGM and the membranes of Golgi apparatus and secretory vesicles [12, 19, 33]. These results indicate that the membranes of secretory vesicles and Golgi apparatus can be important for the formation of MFGM. Because these are biochemical and not structural studies they can not directly explain the way the enveloping of the lipid globule with the plasmalemma and membranes of secretory vesicles takes place. The importance of interactions between different endomembranes, plasmalemma and the lipid droplet in the course of apocrine secretion in the mammary gland remains undefined [9, 14]. Wooding [39] proposed that a part of MFGM can originate from the plasmalemma and the other part from the fused secretory vesicles that accumulate around the lipid droplet. When these secretory vesicles fuse with the plasmalemma the lipid globule detaches from the secretory cell. It also was mentioned that the normal exocytotic process was necessary for normal milk secretion and that colchicine treatment could decrease milk production [26, 37]. We tried to demonstrate the existence of a permanent relationship between the exocytosis and the apocrine secretion of the lipid globules by the ultrastructural analysis of the mammary gland secretory cells in different stages of differentiation. The occurrence of simple and compound exocytosis and lipid secretion have been studied in the mouse mammary gland at the end o f gestation when the gland already completes its structural and functional
+.+
"- Is'+
.+-
..
,+i-,,+~+..
,+++z, Iml,'vlW~
P • I.
+++,•. ' +,,,.
•
•
I-C3
+*+
11,
+;.4e.,....,,~ i ~41,1. J
°++,.
~J
t~
D+
The role of exocytosis in apocrine secretion differentiation [22] while the synthesis of the secretory products and their secretion are slowed down and thus can be observed more easily. This situation has been compared to the synthesis and secretion of milk in the postpartum period when both processes are very intense. It has been compared also to the situation when the normal lactation has again been slowed down by application of 2-Br-aergokryptin that reduces prolactin secretion.
Materials and methods Mice of Albany strain in day 14 and 19 of gestation and day 1 and 9 postpartum were used. Animals were kept at standard laboratory conditions. To partially suppress lactation some lactating raice were treated with a single dose of 0.5 mg of 2-Br-~-ergokryptin dissolved in 5°70 ethanol and their litter has been removed on day 9 postpartum, 24 h before removal of the gland [1].
Transmission electron microscopy Tissue was fixed in fresh, ice-cold mixture of 1.25°/0 glutaraldehyde and 2% OsO4 and 0.5% tannic acid [21] for 1 h and postfixed in 207o water solution of OsO 4 for 4 h [10]. After a quick rinse in distilled H20 the tissue sample was put in I o7o aqueous uranyl acetate overnight, dehydrated and embedded in Epon. Ultrathin sections were stained with uranyl acetate for 5 min and lead citrate for 1-2 min and examined under a Jeol T8 electron microscope.
Scanning electron microscopy Epon tissue blocks were prepared as for transmission electron microscopy. By means of semithin sections an appropriate part with transected alveoli was chosen under a light microscope. Epon was dissolved from this part with 1°7o NaOH in absolute ethanol [3]. Critical point dried blocks were mounted on preparation holders, vacuum coated with gold in a Sputter Coater (Polaron) and observed in Jeol JSM 840A scanning electron microscope.
Freeze-fracturing The tissue was fixed for 2 h in a mixture of 4°/0 paraformaldehyde and 2070glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2), rinsed in 0.1 M cacodylate buffer, soaked with 30070 glycerol in 0.1 M cacodylate buffer for 2 h, frozen in freon and cooled with liquid nitrogen. Freeze-fracture replicas, prepared in freezeetching device Balzers were cleaned in 5% Na-hypochlorite overnight and then in a 2:1 mixture of chloroform and methanol [31]. Some tissue samples were incubated in 0.02070 filipin (Sigma) in 0.2 M cacodylate buffer with 2% DMSO, before being freezefractured [11].
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Results
Ultrastructure of alveolar cells in prepartum period Main structural differential features and accumulation of secretory products (proteins, lipids) were observed already on day 14 of gestation (fig 1). At the same time the amount o f lipid droplets in the gland lumina was found to be minimal. Spatial associations between cytoplasmic lipid droplets and secretory vesicles were not frequent. Exocytosis of single casein micelles could rarely be observed and almost no lipid secretion was detected. Apical cell surfaces were rather flat, sometimes covered with microvilli but lipid droplets never pushed apical plasmalemma into the lumen. Lipid droplets were generally small, numerous and accumulated in the apical cytoplasmic region where some secretory granules with casein micelles could also be observed. After day 19 of gestation accumulation of secretory products in the gland cells and the establishing of secretory function were observed. The size of cytoplasmic lipid droplets and secretory vesicles was greatly increased and only one lipid droplet was present in most cells. Secretory vesicles usually contained more than one casein micelle and appeared to be dilated due to lactose accumulation. Frequently, they could be found opposed to the lipid droplet forming a m e m b r a n e bound cisterna that separated the cytoplasm and the lipid droplet (fig 2). Such big secretory vesicle or cisterna could open into the gland lumen by exocytosis, either on the top or on the lateral side of the lipid droplet (figs 3, 4). Thus the cavity around the lipid droplet connects to the gland lumen and the lipid droplet, covered with a thin rim of cytoplasm and the membrane gradually separates from the rest of the cytoplasm. Frequent spatial associations between the cytoplasmic lipid droplet and the secretory vesicles were observed, resulting in larger patches of membranes associated with the lipid droplet. In these membrane patches, cholesterol could be demonstrated by filipin (fig 5).
Ultrastructure of alveolar cells in the postpartum period Normal lactation On the first day postpartum the alveolar ceUs acquire their final functional differentiation stage and merocrine and apocrine secretion of secretory products was intensified. Secretory vesicles adjoining a cytoplasmic lipid droplet were frequent. Cytoplasmic distribution of secretory vesicles and the
Figs 1-8. 1. Apical region of the mammary gland cell. Secretory vesicles with single casein micelles (arrows) do not fuse and only occasionally they are located near the cytoplasmic lipid droplet (LD) (arrowheads). Day 14 of gestation, x 36000.2. Direct contact between the cytoplasmic lipid droplet (LD) and a membrane-bound cisterna (arrows) which was formed by fusion of secretory vesicles (LU, gland lumen). Day 19 of gestation, x 33000. 3. By exocytosis of the secretory vesicle (arrow) very close to the cytoplasmic lipid droplet (LD), the lipid droplet acquires a patch of membrane that contributes to the formation of the milk fat globule membrane (LU, gland lumen). Day 19 of gestation, x 25000.4. A membrane bound groove (arrows) comunicating with the gland lumen (LU). Milk fat globule membrane (arrow-heads) is forming synchronously with the separation of the lipid droplet (LD). Day 19 of gestation. x 12500.5. Freeze-fracture replica of the mammary gland cell. Part of the surface of the lipid droplet (LD) is heavily labelled with filipin (arrowheads), similar to the membrane of the secretory vesicle (arrow). Day 19 of gestation, x 14500. 6. Cytoplasmic lipid droplet (LD) protruding into the gland lumen (arrowhead). Secretory vesicles accumulate around the lipid droplet (arrows). Day 1 postpartum, x 19000.7. A membrane-bound cisterna (arrows) separates the cytoplasmic lipid droplet (LD) from the cytoplasm in the apical cell region. Lipid droplet is partly covered with the membrane of the cisterna. Part of the milk fat globule (MFG) is seen in the gland lumen (LU). Day 9 postpartum, x 11200. 8. The lipid droplet (LD) is protruding far into the gland lumen (LU). Large portion of its surface is covered with the apical plasmalemma (arrowheads). Secretory vesicles accumulate in the cell apex between the lipid droplet and the rest of the cytoplasm (arrows). Day 9 postpartum, x 4000.
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LU
Oo
tl II 0 ! I
LD
Figs 9, 10. 9. Scanning micrograph of the mammary gland alveolus on the first day postpartum. Big, crater-like depressions corresponding to the prints of lipid droplets are present in the apical cell surfaces (arrows). × 1200.10. Apical region of the mammary gland cell after the treatment of the lactating animal with a low dose of 2-Br-a-ergokryptin. Apical cell surface is flat (arrowheads) and secretory vesicles are scattered through the cytoplasm (arrows). The lipid droplet (LD) remains inside the cell (LU, gland lumen). Application of 2-Br-a-ergokryptin on day 9 postpartum. × 11500.
points of contact between plasmalemma and secretory vesicles determined the size and position of the lipid droplet. When a large lipid droplet filled the apical cytoplasmic region almost completely and pushed the apical plasmalemma into the gland lumen, the secretory vesicles accumulated at its basal side and exocytosis could take place only on the basis of the lipid droplet (fig 6). The cells in the gland in full lactation were not essentially different. The secretory vesicles accumulated near the cytoplasmic lipid droplet and could also contribute to the formation of MFGM. Sometimes the membranebound cavities similar to those observed before parturition were present near the cytoplasmic lipid droplet (fig 7). The lipid droplet sometimes protruded far into the gland lumen so that a series of secretory vesicles were present only at its base (fig 8). Scanning electron microscopy of alveoli from the lactating mammary gland showed frequent crater-like depressions in the apical cell surface of the alveolar cells, Usually one such depression per cell was present (fig 9). These depressions'were covered with a membrane and their dimensions were in the range of the smaller milk fat globules (0.5-1.0 gm). According to their size and position we concluded that they were prints of the detached milk fat globules.
Suppressed lactation When the synthesis of secretory products in normally lactating mammary gland has been decreased by the application of 2-Br-a-ergokryptin the alveolar ceils became similar to the cells in prepartum period. We could observe that vesicle fusions were much less frequent but the vesicles were still dilated due to lactose accumulation and could contain more casein micelles (fig 10). The apical cell surface was fiat, exocytosis was rare and the lipid droplets were prevented from protruding into the gland lumen.
Discussion By its biochemically heterogeneous product and complex way of secretion, the mammary gland differs considera-
bly from the other mammalian glands. We applied different methods of ultrastructural analysis in order to study relations between different membranes in the cell and to find potential correlations between membrane interactions, synthesis of secretory products and their secretion. Synthesis of milk proteins and lipids is present already in the prepartum period, while the synthesis of lactose is still blocked by high concentrations of progesterone [34] which inhibit also microtubule polymerization, polarized transport of secretory vesicles and their normal exocytosis [18, 26]. Decrease of progesterone levels after parturition correlates with deblocking o f lactose synthesis and the maturation of secretory vesicles, their frequent fusions and exocytosis [36]. It is well established that secretion of milk proteins and lactose takes place by exocytosis [13], but it is less clear what role the membranes of secretory vesicles and plasmalemma play in lipid secretion [14, 22]. The secretion of lipid globules is functionally related to the increased rate of exocytosis, despite the fact that the lipids do not accumulate in secretory vesicles [40]. Molecular analysis could not confirm absolute homology of protein and phospholipid composition of MFGM and plasmalemma [4, 21, 29]. The results of our morphological analysis of mammary cells during lactogenesis indicated a strong connection between the exocytosis of proteins and lactose and apocrine secretion of lipids. Obvious spatial interactions of cellular lipid droplets with secretory vacuoles in functionally differentiated cells and filipin labelling of the membranes on the surface of the cytoplasmic lipid droplets showed that the membranes of secretory vacuoles were involved in the gradual separation of the lipid droplet from the cytoplasm. Membranes of both the secretory vacuoles and the plasmalemma could be incorporated into the MFGM of the secreted lipid globule. When secretory products accumulated in the cell, numerous interactions between the endomembranes occurred and resulted in the fragmentation of the apical cell region. The majority of these cell fragments contained a lipid droplet. The role of the membranes of secretory vesicles in the formation of MFGM was especially pronounced when the synthesis of secretory products was not balanced with their secretion and when
The role of exocytosisin apocrine secretion the apical plasmalemma was relatively stable (day 19 of gestation). Our observations in the prepartum mammary gland, when the exocytosis of secretory vesicles was slowed down by high progesterone concentrations, were in agreement with the results reported in the experiments with the infusion of colchicine into the mammary gland [16, 26, 30]. It has been reported that colchicine considerably reduced the amount of milk and that the secretory products accumulated inside the gland cells. A strong tendency to the lining up of the secretory vesicles near the cytoplasmic lipid droplet has been observed, and the phospholipid composition of MFGM became more similar to the phospholipid composition of the membranes of the secretory vesicles [38]. We can conclude from our observations that in the situation of obstructed exocytosis the amount of membranes contributed to MFGM by the secretory vesicles was greater relative to the amount of plasmalemma than in the case of normal lactation. In the postpartum period the lipid synthesis increased so that the droplet quickly reached the apical cell region and the plasmalemma. At the same time the rate of exocytosis also increased, the apical cell surface could become unproportionally enlarged and the lipid droplet could push it in front of itself into the lumen so that the secretory vesicles could assemble only at the free surface of the lipid droplet facing the rest of cytoplasm. We consider the crater-like depressions in the apical cell surfaces revealed by scanning electron microscopy of the lactating mammary gland also as an additional indication that the detachment of lipid globules takes place independently of the degree to which the lipid droplet protrudes from the cell surface and that the lipid globule can detach before it completely pinches off. A similar situation has already been described [32], but as the main interest of the study lay in the cell contacts, the morphology of the apical plasmalemma has been mentioned only briefly. These depressions are covered with membrane and from our point of view they are prints of the lower part of the lipid droplet that has been surrounded by a cisterna formed by fusion of secretory vesicles at the time o f apocrine lipid secretion. The importance of exocytosis for providing sufficient plasmalemma surface on one side and final detachment of the lipid globule on the other side was demonstrated also in our experiments with 2-Br-~-ergokryptin in postpartum lactation period. The overall morphological situation of the gland cells became very similar to the situation in the prepartum period. The lipid secretion has not been completely stopped but the protrusion of the lipid droplet into the lumen could only rarely be seen and accumulation of secretory vesicles around the cytoplasmic lipid droplet was observed. On the basis of our results we suggest that the exocytosis of secretory vesicles that separate the lipid droplet from the majority of the cytoplasm is necessary for the final separation of the lipid globule into the gland lumen. The secretory vesicles directly provide the membranes for at least a part of MFGM and for the apical plasmalemma at the time of lipid globule detachment. The suggested mechanism can also explain why the phospholipid composition of MFGM in the situation of inhibited secretion, either by colchicine in lactation [30] or by progesterone in prepartum period, resembles more the membranes of the secretory vesicles than the plasmalemma. The ultrastructural studies of the mammary alveolar cells during functional differentiation support the hypothsis that apocrine secretion occurs as the result of a very
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intensive exocytosis and fragmentation of the apical cell region where the lipid droplet is located. The share of the plasmalemma and of the membranes of the secretory vesicles in the formation of MFGM is variable and depends mainly on the intensity of exocytosis. Very intensive exocytosis during lactation in the postpartum period provides a surplus of apical plasmalemma that can be secreted together with the lipid globule, while the membranes of secretory vesicles become more important in the formation of MFGM when the exocytosis is hindered.
Acknowledgments We wish to thank prof dr Kristijan Jezernik for helpful discussion and help in the freeze-fracturing technique, and dr Majda Pgeni~nik for help in the scanning electron microscopy.
References 1 Akers RM, Bauman DE, Goodman GT, Capuco AV, Tucker HA (1981) Prolactin regulation of cytological differentiation of mammary epithelial cells in periparturient cows. Endocrinology 109, 31-40 2 Assairi L, Delouis C, Gaye P, Houdebine LM, OllivierBousquet M, Denammur R (1974) Inhibition by progesterone of the lactogenic effect of prolactin in the pseudo pregnant rabbit. Biochem J 144, 245-252 3 Cajander SB (1986) A rapid and simple technique for correlating light microscopy, transmission and scanning electron microscopy of fixed tissues in Epon blocks. JMicrosc 143, 265-274 4 Calberg-Bacq CM, Frant;ois C, Gosselin L, Osterrieth PM, Rentier-Delrue F (1976) Comparative study of the milk fat globule membrane and the mouse mammary tumor virus prepared from the milk of an infected strain of Swiss albino mice. Biochim Biophys Acta 419, 458-478 5 Dowben RM, Brunner JR, Philpott DE (1976) Studies on milk fat globule membranes. Biochim Biophys Acta 135, 1-10 6 Dulbecco R, Henaham M, Armstrong B (1982) Cell types and morphogenesis in the mammary gland. Proc Natl Acad Sci USA 79, 7346-7350 7 Dylewski DP, Keenan TW (1983) Compound exocytosis of casein micelles in mammary epithelial cells. Eur J Cell Biol 31, 114-124 8 Dylewski DP, Keenan TW (1987) Ball and socket figures between secretory vesicles in mammary epithelial cells. Protoplasma 138, 65-68 9 Dylewski DP, Dapper CH, Valivullah HM, Deeney JT, Keenan TW (1984) Morphological and biochemical characterization of possible intracellular precursors of milk lipid globules. Eur J Cell Biol 35, 99-111 10 Franke WW, Krien S, Brown RM Jr (1969) Simultaneous glutaraldehyde-osmium tetroxide fixation with postosmication. Histochemie 19, 162-164 11 Ginsbach C, Fahimi HD (1987) Labeling of cholesterol with filipin in cellular membranes of parenchymatous organs. Histochemistry 86, 241-248 12 Huggins JW, Trenbeath TP, Chesnut RW, Carothers Carraway CA, Carraway KL (1980) Purification of plasma membranes of rat mammary gland. Exp Cell Res 126, 279-288 13 Hurley D, Hwang S, Rocha V (1989) Casein accumulation in distended rough endoplasmic reticulum of collagen gelcultivated mouse mammary epithelia. J Cell Physiol 141, 135-141 14 Kanno C (1990) Secretory membranes of the lactating mammary gland. Protoplasma 159, 184-208 15 Keenan TW, Sasaki M, Eigel WN, Morre D J, Franke WW, Zulak IM, Bushway AA (1979) Characterization of a secre-
216
16
17 18 19
20
21
22 23 24 25
26
27
K Metka, P Nada tory vesicle-rich fraction from lactating bovine mammary gland. Exp Cell Res 124, 47-61 Knudson CM, Stemberger BH, Patton S (1978) Effects of colchicine on ultrastructure of the lactating mammary cell: Membrane involvement and stress on the Golgi apparatus. Cell Tissue Res 195, 169-181 Linzell JL, Peaker M (1971) Mechanism of milk secretion. Physiol Rev 51, 564-597 Loizzi RF (1985) Progesterone withdrawal stimulates mammary gland tubulin polymerisation in pregnant rats. Endocrinology 116, 2543-2547 Martel-Pradal MB, Got R (1972) Pr6sence d'enzymes marqueurs des membranes plasmiques, de l'appareil de Golgi et du reticulum endoplasmique dans les membranes des globules lipidiques de lait maternel. FEBS Lett 21,220-222 Mather IH (1987) Proteins of the milk fat globule membrane as markers of mammary epithelial ceils and apical plasma membrane. In: The mammary gland. Development, regulation, and function (Neville MC, Daniel CW, eds) Plenum Press, New York London, 217-276 Mather IH, Jarasch ED, Bruder G, Heid HW, Mepham TB (1984) Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. I. Radiolabelling of membrane and secretory proteins. Exp Cell Res 151, 208-223 Mepham TB (1987) Physiology o f lactation. Open University Press, Milton Keynes, Philadelphia Morre JD, Kartenbeck J, Franke WW (1979) Membrane flow and interconversions among endomembranes. Biochim Biophys Acta 559, 71-152 Muldoon TG (1987) Prolactin mediation o f estrogen-induced changes in mammary tissue estrogen and progesterone receptors. Endocrinology 121, 141-149 Nakhasi HL, Quasba PK (1979) Quantitation of milk proteins and their mRNAs in rat mammary gland at various stages of gestation and lactation. J Biol Chem 254, 6016-6025 Nickerson SC, Smith J J, Keenan TW (1980) Role of microtubules in milk secretion - action of colchicine on microtubules and exocytosis of secretory vesicles in rat mammary epithelial cells. Cell Tissue Res 207, 361-376 Nickerson SC, Akers RM, Weinland BT (1982) Cytoplasmic organization and quantitation of microtubules in bovine mammary epithelial cells during lactation and involution. Cell Tissue Res 223, 421-430
28 Patton S, Fowkes FM (1967) The role of plasma membrane in the secretion of milk fat. J Theor Biol 15, 274-281 29 Patton S, Keenan TW (1975) The milk fat globule membrane. Biochim Biophys Acta 415, 273-309 30 Patton S, Stemberger BH, Knudson CM (1977) The suppression of milk fat globule secretion by colchicine: an effect coupled to inhibition of exocytosis. Biochim Biophys Acta 499, 404-410 31 Peixoto de Menezes A, Pinto da Silva P (1978) Freezefracture observations of the lactating rat mammary gland. J Cell Biol 76, 767-778 32 Pitelka DR, Hamamoto ST, Duafala JG, Nemanic MK (1973) Cell contacts in the mouse mammary gland. I. Normal gland in postnatal development and the secretory cycle. J Cell Biol 56, 797-818 33 Powell JT, Jarlfors U, Brew K (1977) Enzymic characteristics of fat globule membranes from bovine colostrum and bovine milk. J Cell Biol 72, 617-627 34 Quirk S J, Gannell JE, Funder JW (1988) ~t-Lactalbumin production by rat mammary gland: prepartum emergence of resistance to glucocorticoids and progestins. Mol Cell Endocrinol 58, 183-189 35 Rillema JA, Etindi RN, Cameron CM (1986) Prolactin actions on casein and lipid biosynthesis in mouse and rabbit mammary gland explants are abolished by p-bromphenacyl bromide and quinacrine, phospholipase A 2 inhibitors. Horm Metab Res 18, 672-674 36 Sasaki M, Keenan TW (1978) Membranes of mammary gland. XV. 5-thio-D-glucose decreases lactose content and inhibits secretory vesicle maturation in lactating rat mammary gland. Exp Cell Res I l l , 413-425 37 Sokka TK, Patton S (1983) In vivo effects of colchicine on milk fat globule membrane. Biochim BiophysActa 731, 1-8 38 Stemberger BH, Walsh RM, Patton S (1984) Morphometric evaluation of lipid droplet associations with secretory vesicles, mitochondria and other components in the lactating cell. Cell Tissue Res 236, 471-475 39 Wooding FBP, Peaker M, Linzel JL (1970) Theories of milk secretion: Evidence from the electron microscopic examination of milk. Nature 226, 762-764 40 Jaczek M, Keenan TW (1990) Morphological evidence for an endoplasmic reticulum orgin of milk lipid globules obtained using lipid-selective staining procedures. Protoplasma 159, 179-182