Immunocytochemical Evidence for Golgi Vesicle Involvement in Milk Fat Globule Secretion

Abstract

The exact mechanism of secretion of the milk fat globule (MFG) from the mammary secretory cell is still controversial. We have previously suggested close involvement of Golgi vesicles in this process. This paper provides direct immunocytochemical evidence that butyrophilin is present in the Golgi stack and vesicles in ovine and caprine mammary glands. We suggest that it is the butyrophilin in the Golgi vesicle membrane that forms the specific association with the adipophilin on the lipid surface in the cytoplasm. Exocytosis of the associated Golgi vesicle will then initiate the process of MFG secretion. Further exocytosis of associated Golgi vesicles will continue and complete the process. Areas of the plasmalemma that have butyrophilin delivered by previous non-lipid associated Golgi exocytoses may also contribute to the process of forming the milk fat globule membrane (MFGM).

Introduction

Despite numerous investigations over many years, there is still no consensus as to the mechanism of secretion of the milk fat globule (MFG) or the structure of the milk fat globule membrane (MFGM) after secretion. The individual proteins and phospholipids involved have been comprehensively investigated, and butyrophilin (BTN), xanthine oxidase (XO), adipophilin (ADP/Plin2) are generally accepted as the building blocks of the MFGM; however, the ways in which they interact to form the MFGM are still controversial (McManaman et al. 2002; Heid and Keenan 2005; Chong et al. 2011; Mather 2011).

We have recently demonstrated that the biochemical structure of BTN is consistent with processing via the Golgi system (Jeong et al. 2013). This would support the electron microscopical observations of Wooding (1971, 1977) that, in all species examined, a significant area of Golgi secretory vesicle membrane associates with the perimeter of the intracellular apical lipid droplet to preform patches of a structure identical to the MFGM around the newly secreted, alveolar MFG. This general observation was reinforced by the finding that, under certain conditions in the goat mammary gland, the MFGM could form intracellularly by the fusion of lipid-associated Golgi vesicles with each other without the participation of the apical plasmalemma (Wooding 1975). This produces a large intracellular vacuole containing a MFG bounded by a MFGM.

The conventional fusion of the Golgi vesicles with the plasma membrane would allow for the “patches” of membrane associated with the lipid to aggregate into the continuous unit membrane separated from the lipid by a uniform, 15-nm of material that is continuous with the cytoplasm.

If two Golgi vesicles separated by a cytoplasmic “bleb” fuse simultaneously with the plasma membrane, this would produce the “signet ring” MFGM, which can be found in a small variable percentage in all milks so far investigated (Wooding 1977). This scenario takes into account all of the EM observations in a variety of species—cow, ewe, goat and rodents—as long as the material is fixed by aldehyde perfusion as soon as practical after the death of the animal. Any fixation can produce artefacts; but if the perfusion produces uniform fixation with resolvable membranes, the results are always more reliable than those achieved with immersion fixation. Immersion fixation must produce a variety of fixation quality from the edge to the middle of the block, and this seems especially true for lipid-laden tissue. Immersion fixation can produce useful corroborative evidence but any micrographs from either fixation must show resolvable unit membranes if it is to be used to support a mechanism for MFGM formation.

BTN has previously been localized with immunocytochemistry to the apical plasmalemma of the mammary cell in several mammals and around the MFG in the alveolus (Mather and Jack 1990, 1993; Heid and Keenan 2005), but no one has been able to demonstrate any cytoplasmic label indicating the route to the plasmalemma.

We now have more direct evidence for the involvement of the Golgi vesicles with BTN synthesis and transport using Light Microscope (LM) immunocytochemistry. Using an antibody raised in rabbits against a mouse BTN peptide on sections of perfused sheep or goat mammary gland, we obtained an identical supranuclear localization to that produced by a rabbit anti-casein antibody (Uejyo et al. 2015). Casein is generally accepted to be processed in the Golgi and secreted via the Golgi vesicles (Beery et al. 1971; Linzell and Peaker 1971; Wooding 1977; Mather 2011). Other BTN antibodies sometimes show indications of such a localization but have never showed such reproducibility or uniformity.

Materials & Methods

Electron Microscopy

Mammary tissue from four lactating goats, five ewes and two cows was either excised immediately after death by barbiturate overdose and cut into small cubes in glutaraldehyde fixative, or fixed initially by perfusion via the mammary artery (goat, ewes). All processing was carried out at room temperature

All animal work was carried out in accordance with the Animals (Scientific Procedures) Act 1986.

Fixation was performed for 45 min using 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, with 2% sucrose. The tissue was washed briefly in buffer, postfixed first in 1% osmium tetroxide in 0.1 M veronal buffer, pH 7.2, for 30 min, then in 5% aqueous uranyl acetate for 2 hr followed by ethanol dehydration and embedding in araldite. Sections were cut on an LKB Ultrotome, stained with uranyl acetate and lead hydroxide and observed in an AEI EM6B microscope operated at 60 kV.

Light Microscope Immunocytochemistry

Tissue was fixed by immersion or perfusion in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2, or in 4% formaldehyde plus 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. No osmium was used.

Dehydration and embedding was performed at room temperature for araldite resin. Semi-thin 1–2-µm sections were cut from the araldite blocks and picked up onto coverglass squares coated with APES. Resin was removed from semi-thin araldite sections by incubation in sodium ethoxide solution (15 g NaOH pellets in 15 ml absolute alcohol) for 15 min followed by alcohol and water washes.

For immunocytochemistry, the coverslip squares were floated section side down on drops of antibody followed by immunogold colloid (Goat anti-rabbit G4; Jackson Immunoresearch Labs, West Grove, PA), then intensified with silver reagent (Aurion; Wageningen, Netherlands) followed, if necessary, by 0.05% fast green counterstain.

The following primary antibodies were used: rabbit anti-β-casein (ab91167, Abcam; 1:3000), rabbit anti-MCT-1 (monocarboxylate transporter isoform 1; Halestrap and Price 1999, 1:100), rabbit anti-glucose transporter 1 (Baldwin et al. 1982; 1:1000), rabbit anti-lactoferrin (ab15811, Abcam; 1:100), rabbit anti-CD36 (1:100, a gift from Prof. M. Febbraio (Case Western Reserve University, Cleveland); see Silverstein and Febbraio 2009).

Three antibodies were gifts from Prof. I. Mather (University of Maryland, College Park): rabbit anti-butyrophilin 1A1 (bovine whole molecule; 1:1000), rabbit anti-butyrophilin1 A1 (peptide from mouse butyrophilin, 1:1000) and goat anti-adipophilin (Plin 2) (1:3000); see Mather 2000 for references.

Two GM 130 antibodies (cis-Golgi body marker) were purchased: Mouse anti-GM130 (610822; BD Transduction Laboratories, San Diego, CA) and rabbit anti-GM 130 (ab52649; Abcam). Antibodies were used with or without prior heat-induced antigen retrieval (sections were treated for 3 × 2 min at 95°C in 0.1 M NaCitrate buffer, pH 6).

Immunocytochemical controls, in which the primary antibody was omitted and replaced with buffer or a non-specific antibody at the same concentration, were carried out routinely alongside the experimental samples. Controls showed an insignificant level of labelling.

Western Blotting

Mammary tissue from a ewe in full lactation was sampled and immediately frozen in liquid nitrogen. Milk from the same ewe was spun to concentrate the cream, which was then resuspended in 50 times by volume phosphate-buffered saline at 37°C. The sample was respun, and the washed cream was frozen in liquid nitrogen.

Mammary tissue and cream were ground to a fine powder using a mortar and pestle. Protein from mammary gland or cream powder was extracted in RIPA buffer (50 mM Tris pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA and 1× Complete protease inhibitor (Roche Applied Science; Indianapolis, IN), 1 mM Na₃VO₄, and 1 mM NaF). Protein concentration was assessed with the BCA Protein Assay (Thermo Fisher Scientific; Waltham, MA) and equal amounts of protein (20 μg) were denatured and resolved on SDS–polyacrylamide gels. Immunoblotting was performed using standard techniques and antibody detection was achieved with enhanced chemiluminescence reagent (ECL; GE Healthcare, Buckinghamshire, UK). The primary antibody used was a rabbit anti-BTN 1A1 (a gift from Prof. I. Mather, 1:5,000).

Results

The uniformity of the ultrastructure of milk secretion has been clearly shown previously to involve Golgi vesicle association with the cytoplasmic lipid (Wooding 1977), and this is reinforced in Fig. 1A from the ewe mammary gland. All five apical lipid droplets are surrounded by rosettes of Golgi vesicles. Figures 1B–1F illustrate, in a variety of species, the consistent formation of a flattened area of Golgi vesicle membrane paralleling the lipid contour. The unit membrane is separated only by a 100–150 Å cytoplasmic gap from the single line of electron dense—probably lipoprotein—material around the lipid globule. Figure 1G shows the structure of the MFGM immediately after secretion; this would be the structure of the alveolar-facing membrane around the partly secreted lipid in Fig. 1H.

Figure 1.

Electron Micrographs illustrating the Golgi vesicle–lipid droplet association in mammary cells in full lactation. (A) Ewe mammary gland. All of the apical lipid cytoplasmic lipid droplets (1–5) show associated Golgi vesicles (arrowheads). Golgi bodies and their vesicles (ga) are extensive; the vesicles are usually identified by their casein content. (B–D) Details of the Golgi vesicle – lipid association in (B) ewe, (C) mouse, and (D) cow. This illustrates the uniformity of the cytoplasmic gap between the vesicle membrane and the lipid contour, which is equivalent to the structure around the recently secreted alveolar Milk Fat Globule (MFG), as seen in (G). (E-F) Rat mammary gland. (E) Typical Golgi vesicle–lipid association, showing distortion of the Golgi vesicles to follow the contour of the lipid. (F) Initiation of MFG secretion by exocytosis of a Golgi vesicle also associated with a cytoplasmic lipid droplet. (G) Horse mammary gland. High magnification of the structure of the recently secreted MFG membrane (MFGM). (H) Ewe mammary gland. MFG nearly released, exocytosis of the remaining associated Golgi vesicles (arrowheads) at its base would continue and finally complete the process. Scale (A) 5 µm, (B–D, F) 200 nm, (E) 2 µm, (G) 50 nm, (H) 1 µm. Abbreviations: A, alveolus; c, capillary; L, basal lipid; n, nucleus of mammary cell; nm, nucleus of myoepithelial cell.

The casein-containing Golgi vesicles are programmed to fuse with the apical plasma membrane as seen, for example, in Fig. 1F. Continued exocytosis of the adjacent lipid-associated vesicles in Fig. 1F would form a nascent MFGM facing the alveolus leading, eventually, to the lipid droplet seen in Fig. 1H, with its associated Golgi vesicles ideally positioned to exocytose and release the MFG into the alveolus.

What is the basis for the association between the Golgi vesicles and the lipid? One of the components of the lipoprotein boundary around the lipid in the mammary cell and alveolus is adipophilin (Plin 2), as can be seen in Fig. 3E. Figures 3C, 4, 5 and 6 show in ewe and goat that the Golgi area and vesicles can be labelled with an antibody to BTN. The specificity of this antibody is confirmed by the western blot results in Fig. 8. Identification of the site of the BTN label as Golgi area plus vesicles is confirmed by the similar localization, on adjacent sections, of casein (Fig. 3B) and lactoferrin (Fig. 3D), proteins known to be processed via the Golgi (Linzell and Peaker 1971; Beery et al. 1971; Wooding 1977). Also, the supranuclear label and the label surrounding the lipid droplets is in the identical position to that shown with electron microscopy for the mammary cell Golgi body and vesicles, as seen in Fig. 1A and 1H.

Figure 3.

Light microscope (LM) immunocytochemistry of ewe mammary gland. Adjacent sections of the same alveolus (labeled as “A”). (A) Butyrophilin is localized with an antibody against bovine butyrophilin. Label is restricted to the alveolar plasmalemma (arrow) and the alveolar milk fat globule (MFG, arrowheads). (B) Casein localized to the Golgi area and around apical lipid drops (arrows) as well as to diffuse areas in the alveolus. (C) Butyrophilin is localized with an antibody against mouse butyrophilin. Label is restricted to the Golgi area and around apical lipid drops (arrows). A few alveolar MFGs are also labelled (arrowheads). (D) Lactoferrin is localized to the Golgi area and around apical lipid drops (arrows). The apical plasmalemma shows some labeling but the alveolar MFGs show none. (E) Adipophilin labels all the lipid droplet surfaces, both cytoplasmic (arrows) and alveolar (arrowheads). Scale, 50 µm.

Figure 4.

Ewe mammary gland. Closely adjacent sections of the same alveolus (labeled as “A”), with different levels of the same capillary marked by an asterisk. The 0.5–5-µm white circular areas in the cytoplasm of the mammary cells are lipid droplets, most obvious in (A). In the apical cytoplasm of cells in 4b, there are populations of even smaller white circular areas (double arrowheads) which by analogy with Fig 1a are most likely Golgi areas. This conclusion is reinforced by the fact that similar areas on Figs 4a (casein) and 4c (mouse butyrophilin) label positively. Arrows denote the same lipid droplet in at least two of the sections. Arrowheads in 4b and 4c indicate an MFG in the process of secretion and an MFG free in the alveolus. The bovine butyrophilin antibody labels only the apical plasmalemma but the mouse antibody only labels the cytoplasmic structures. Nuclei can occasionally be identified (curved arrows) Inset on Fig 4a is the cell immediately below taken at x100, the information available and the resolution are not significantly improved. Scale bar same for all, 50µm.

Figure 5.

Ewe mammary gland. Details of the mouse butyrophilin antibody localization, which, by analogy with Figure 1A (electron microscopy) and Figure 4A (casein localization), shows a strong label in the apical cytoplasmic Golgi areas. The arrows indicate lipid droplets surrounded by Golgi vesicles; arrowheads indicate two milk fat globules in the process of secretion; asterisks show basal lipid droplets with no adjacent Golgi vesicles. sa, small artery; c, capillary. Scale, 8 µm.

Figure 6.

Goat mammary gland. Closely adjacent sections of the same alveolus (labeled as “A”), with different levels of the same capillary marked by an asterisk. The micrographs show equivalent localization to Golgi areas, with mouse butyrophilin (A) and casein (C), as seen in the ewe samples (Figure 5). With bovine butyrophilin (B), the cytoplasmic Golgi vesicles are free of label (arrowheads) although the apical plasmalemma is heavily labeled. Neither casein nor mouse butyrophilin produce any significant label on the apical plasmalemma. Scale, 50 µm.

Figure 8.

Western blotting for butyrophilin (M_r 70 Kd), showing that butyrophilin is present in both mammary tissue and cream samples.

Unfortunately this Golgi localization could not be additionally confirmed using two commercial antibodies to a Golgi protein, GM130, as neither produced any localization in this immunocytochemical system.

This BTN localization can only be shown with a rabbit anti-mouse BTN peptide, and is restricted to the ewe and goat mammary tissue. None of the other BTN antibodies show this localization. Previous papers using rabbit anti-bovine BTN protein or peptide show a localization only to the apical membrane and the alveolar MFG (Franke et al. 1981). This is corroborated by our results with such a bovine antibody on adjacent sections to those using the anti-mouse BTN in Figs. 3, 4 and 6.

The immunocytochemical method used here, with de-resinated 1–2-µm sections, is unusual but has been validated in a number of studies (see Groos et al. 2001). To confirm its validity here, Figs. 2 and 3 show that the localizations on adjacent sections of ewe mammary gland of glucose transporter 1, monocarboxylic acid transporter 1, CD36 (Fatty acid transporter), ADP, casein and lactoferrin are no different from those shown previously (Uejyo et al. 2015; Tachebe et al. 2009) for the rodent and bovine glands. The de-resinated section method has the advantage of providing a much better resolution than the normal 5–10-µm wax sections (see Figure 2 in Groos et al. 2001).

Figure 2.

Light microscope (LM) immunocytochemistry of ewe mammary gland. Adjacent sections of the same alveolus (labeled as “A”), with the asterisk marking different levels of the same capillary on each section. (A) Glucose transporter 1; (B) Monocarboxylic acid transporter 1; (C) CD36/Fatty acid transporter all show the same localization to the mammary cell basolateral plasmalemma (arrows). The capillary endothelium can also be seen on the CD36 stained section (arrowheads). (D) Control, no antibody. Scale, 50 µm.

Close examination of sections from mammary glands of guinea pig, rat, wildebeest and zebra showed no cytoplasmic localization with the anti-mouse BTN antibody (results not shown); although, in bovine and mouse, there is the occasional indication of label adjacent to apical cytoplasmic lipid (Fig. 7A, 7B).

Figure 7.

Cow and mouse mammary glands. With the mouse anti-butyrophilin antibody, there are occasional indications (arrows) of label around or close to the apical lipid in (B) cow and in (C) mouse. However, most sections show no significant cytoplasmic label; although, the apical plasmalemma is labeled in the cow (A). No label is found on the mouse sections with the bovine antibody (results not shown). Scale (A, B) 40 µm; (C) 50 µm.

Preliminary investigation of the mouse BTN antibody with EM immunocytochemistry of ewe mammary tissue showed similar indications of labeled Golgi vesicles but the membrane definition is too poor and the de-resinated thin sections too fragile to label and counterstain reliably.

Discussion

Our previous biochemical demonstration that BTN is processed through the Golgi in the mouse mammary cell (Jeong et al. 2013) is now reinforced by immunocytochemical evidence in this paper that localizes the BTN label for the first time to the area on sections that contain the Golgi and/or Golgi vesicles. However, this cytoplasmic localization is unequivocal only with a specific antibody on two particular species, sheep and goat. Western blotting confirms that this antibody recognizes BTN. In the cow and rodent mammary cells, there are no clear indications of any cytoplasmic label. However, with both BTN antibodies, label is seen on the MFGM around the MFGs in the alveolus and this is true of all species investigated so far. Label is also usually seen on the alveolar plasmalemma (Mather and Jack 1993; Heid and Keenan 2005).

This Golgi localization gives a rationale for the clustering of Golgi vesicles around apical MFGs observed in all species examined with the electron microscope so far (Wooding 1977). The association is strong enough to distort the initially spherical Golgi vesicle to form a localized flattened membrane area parallel to the MFG contour but always separated from it by a 15-nm gap that is continuous with the adjacent cytoplasm (see Fig. 1).

The direct involvement of the Golgi vesicles in both protein and lipid secretion is also supported by Patton’s papers (Patton et al. 1977; Knudson et al. 1978) using infusions of colchicine into the teats of rats and goat. This treatment inhibits milk secretion by blocking Golgi vesicle exocytosis.

BTN has a transmembrane sequence and the C-terminal portion of the molecule would protrude into the cytoplasm from the Golgi vesicle (Mather and Jack 1990, 1993; Jeong et al. 2013). Interaction of this C-terminal sequence with the ADP present around the contour of the lipid and the XO from the cytoplasm could provide the force necessary for the formation of the Golgi–lipid association. There is good biochemical evidence for a specific trimer formation between these three molecules in the MFGM (McManaman et al. 2002; Jeong et al. 2013) and this is the basis for several hypotheses for the formation of the MFGM but with BTN on the plasma membrane.

Antibodies to bovine BTN label the mammary cell apical plasma membranes in several species. The mammary Golgi vesicles are designed to fuse with the apical plasma membrane, irrespective of whether they are associated with lipid, so that some would deliver some BTN there directly, and some subsequently after associating with the apical lipid.

The different localizations with the different antibodies could be due to differences in epitopes and/or epitope availability. In sheep and goat with the anti-mouse antibody, the epitope is clearly available on the Golgi vesicles, weakly on the plasma membrane, and readily on the alveolar MFGM. The bovine antibody does not bind until the BTN is exposed to the luminal environment, so it is possible that the epitope is occupied with a cytoplasmic molecule. If the interaction among the BTN, XO and ADP molecules is the basis for the formation of the MFGM, this will potentially also affect epitope availability, especially because, in the alveolus of several species, the MFGM modifies to produce a quasicrystalline protein lattice in the space between the membrane and the lipid contour (Wooding 1977; Buchheim 1982, 1986; Buchheim et al. 1988), hypothetically where these three proteins are located.

How necessary the Golgi vesicle association with the lipid is in terms of total lipid secretion is difficult to judge. All species so far examined adequately with EM show this close association, and this clearly supplies the necessary components for the formation of the MFGM. Also, examples of the exocytosis of a lipid-associated Golgi vesicle to initiate, continue and complete secretion can readily be found on well-fixed tissues in the EM (Wooding 1977; Kralj and Pipan 1992), whereas images of the MFG being “enveloped” by the plasmalemma with no vesicles associated are, in our experience, far less frequent. It has been estimated (Mather 2011) that, in the typical mammary cell, far more membrane is available around Golgi vesicles than is required to envelop all lipid secreted by that cell. The results in this paper suggest that a significant amount of the MFGM is derived from Golgi vesicles directly associated with lipid prior to secretion.