Milk lipid globule precursor release from endoplasmic reticulum reconstituted in a cell-free system.

Lipid droplet precursors of milk lipid globules are believed to be derived from elements of endoplasmic reticulum in milk-secreting mammary epithelial cells. Endoplasmic reticulum isolated from mammary gland was able to generate small droplets morphologically resembling microlipid droplet precursors of milk lipid globules. Droplet generation was time and temperature dependent and required a cytosol fraction of Mr greater than 10,000. Droplet generation was enhanced by, but did not require, addition of nucleoside triphosphates, fatty acids, coenzyme A, glycerol-3-phosphate, and dithiothreitol. Microlipid droplets generated in this cell-free system were enriched in triacylglycerols and resembled microlipid droplets formed within mammary epithelial cells in polar lipid and polypeptide composition. Endoplasmic reticulum immobilized onto nitrocellulose retained activity in generation of putative microlipid droplets, and this immobilization method provided a facile means for separation of the donor from the generated products.

Distribution of terminal transferases of acylglycerol synthesis in cell fractions from lactating mammary gland.

Cytoplasmic lipid droplets and microlipid droplets, intracellular precursors of milk lipid globules, had little ability to incorporate radioactivity from glycerol 3-phosphate or palmitoyl-CoA into triacylglycerols. The limited incorporation of these precursors by micro- and cytoplasmic lipid droplets from rat and cow mammary gland was into phospholipids primarily. Acyltransferases catalyzing incorporation of glycerol 3-phosphate into acylglycerols were concentrated in a relatively high buoyant density class of rough microsomes. Palmitoyl-CoA-sn-1,2-diacylglycerol acyltransferase activity was distributed heterogeneously among fractions obtained by equilibrium density gradient fractionation of mammary homogenates. Observations suggest that terminal steps of acylglycerol synthesis are localized primarily in rough endoplasmic reticulum of milk secreting mammary epithelial cells. There appears to be a heterogeneous distribution of acyltransferases along the reticular network.

Microlipid droplets in milk secreting mammary epithelial cells: evidence that they originate from endoplasmic reticulum and are precursors of milk lipid globules.

Microlipid droplets, structures with diameters less than 0.5 micron, resemble larger cytoplasmic lipid droplets of milk secreting mammary epithelial cells in triacylglycerol core and surface coat composition. Previously, evidence was obtained that microlipid droplets fuse with and support growth of cytoplasmic lipid droplets, which are immediate precursors of large milk lipid globules. Morphological observations suggested that microlipid droplets may also be secreted directly from mammary epithelial cells, yielding the very small lipid globules of milk. The secretion mechanism, which involves envelopment of triacylglycerol droplets in apical plasma membrane, appeared to be the same for microlipid droplets as for larger cytoplasmic lipid droplets. Microlipid droplets appeared to originate by blebbing from cisternae of endoplasmic reticulum. By immunogold cytochemical localization and by immunological identification of electrophoretically separated polypeptides, endoplasmic reticulum, micro- and cytoplasmic lipid droplets, and milk lipid globules had a number of common polypeptides. Kinetics of incorporation of radiolabeled palmitate or glycerol into triacylglycerols and phospholipids were consistent with a possible endoplasmic reticulum origin of microlipid droplets and with the view that microlipid droplets may be secreted directly from the cell or may fuse with cytoplasmic lipid droplets.

Centrioles in the mammary epithelium of the rat.

Through serial thin-section analysis of rat mammary epithelial cells, the number of centrioles per cell and their intracellular location were determined. In all developmental stages (e.g. virginal, pregnant, lactation, involution), each epithelial cell contained a single centriole that was located in the apical region. Centrioles were 200-220 (mean = 210) nm in transverse section, and exhibited the typical 9 X 3 'pinwheel' configuration of microtubules. In longitudinal section, centrioles were 330-380 (mean = 360) nm in length. Each centriole was surrounded by a homogeneous pericentriolar matrix. During mitosis in pregnant animals, centrioles were paired at the nuclear poles and oriented at right angles (90 degrees) to each other. At the completion of mitosis a single diplosome (pair of centrioles) was associated with each interphase nucleus. Because all postmitotic cells contained only a single centriole, it was assumed that one of the two diplosomal centrioles had disintegrated. There appeared to be a correlation between centriole location and cell polarity. When centrioles were located near the apical plasma membrane, epithelial cells exhibited polarity. However, when centrioles were associated with the nuclear poles during mitosis, epithelial cells were typically apolar. These observations suggest that centrioles may function as determinants in cell polarity.

Morphological and biochemical characterization of possible intracellular precursors of milk lipid globules.

Within milk secreting mammary epithelial cells, immediate precursors of milk lipid globules existed as relatively large (diameter greater than 1 micron) cytoplasmic lipid droplets. The periphery of these lipid droplets was characterized by an electron-dense, granular material which lacked unit membrane character. Cytoplasmic lipid droplets which retained surface coat material were isolated from lactating cow and rat mammary glands. Surface coat material on isolated droplets was composed of polar lipids and proteins. In enzyme activities, this surface material was distinct from plasma membrane and endomembranes. In polar lipid composition, surface material on cytoplasmic lipid droplets was similar to milk lipid globule membrane or was intermediate between endoplasmic reticulum and milk lipid globule membranes. A previously unrecognized group of small structures (diameter less than 0.5 micron), which resembled cytoplasmic lipid droplets in matrix and surface coat appearance, was observed and characterized. These structures were isolated and found to contain large amounts of triacylglycerols, which closely resembled triacyl/glycerols of cytoplasmic lipid droplets in fatty acid composition. Surface coat materials of these small structures and of cytoplasmic lipid droplets were similar in enzymatic, polypeptide and polar lipid composition. Morphological evidence that these small structures may fuse with cytoplasmic lipid droplets was obtained. These small structures, for which we propose- the name micro lipid droplets, may provide triacylglycerols to support growth of cytoplasmic lipid droplets.

Three-dimensional ultrastructure of the Golgi apparatus in bovine mammary epithelial cells during lactation.

The three-dimensional ultrastructure of the Golgi apparatus in milk secreting epithelial cells of bovine mammary gland was explored. From computer-aided reconstructions of serial thin sections, it was determined that the Golgi apparatus was composed of a single set of stacked cisternae. The three-dimensional shape of the dictyosome varied from cell to cell, but the overall shape was that of a hollow cone, cylinder, or bowl. The cis and trans surfaces of the dictyosome were arranged in three-dimensional space such that the cis face was located on the outer surface of the hollow structure and the trans face on the inner surface. The cytoplasmic channel (secretory channel) that traversed the longitudinal axis of the hollow dictyosome contained secretory vesicles. Densely stacked cisternae of rough endoplasmic reticulum surrounded the dictyosome, and microvesicles appeared to fuse with, or bud from, cisternae of both organelles. These findings suggest that Golgi apparatus of the lactating epithelial cell is highly organized and that the Golgi apparatus and secretory channel are essentially an independent compartment within the cell.

Protein secretion from rat mammary epithelial cells during pregnancy.

Aspects of the apparent origin, maturation, and secretion of proteins in mammary epithelial cells of the pregnant rat were investigated using standard transmission electron microscopy. Epithelial cells from prelactating animals, sacrificed at intervals up to 1 day ante partum, contained protein-filled vesicles which appeared to arise from two separate intracellular sources, (1) Golgi apparatus dictyosomes, and (2) dilated cisternae of rough endoplasmic reticulum. Vesicles which appeared to originate in the Golgi apparatus were small (means = 0.28 micron diameter), contained a single protein particle, and appeared to release their content exocytotically into the lumen. Vesicles which appeared to originate in the endoplasmic reticulum were larger (means = 3.2 micron diameter), contained numerous protein particles, and appeared to be released from cells with their outer membrane. Post-exocytotic endoplasmic reticulum-derived vesicles in alveolar lumina appeared to be the 'nuclei' of structures previously described as colostrum cells or corpuscles of Donn é. Results from quantitative ultrastructural analysis of mammary tissue from pregnant animals suggested that the intracellular disposal mechanism for turnover of secretory protein may not be of major importance.

Compound exocytosis of casein micelles in mammary epithelial cells.

Ultrastructure of lactating bovine and rat mammary epithelial cells was studied with emphasis on secretory vesicle interactions. In the apical zone of the cell, adjacent secretory vesicles formed ball and socket configurations at their points of apposition. Similar configurations were formed between plasma membrane and secretory vesicle membrane. These structures may be formed by the diffusion of water between vesicles with different osmotic potentials. Frequently, vesicular chains consisting of 10 or more linked secretory vesicles were observed. Prior to the exocytotic release of casein micelles, adjacent vesicles fused through fragmentation of the ball and socket membrane. These membrane fragments and the casein micelles appeared to be secreted into the alveolar lumen after passing from one vesicle into another and finally through a pore in the apical plasma membrane. Emptied vesicular chains appeared to collapse and fragmentation of their membrane was observed. Based on these observations, we suggest that most vesicular membrane does not directly contact or become incorporated into the plasma membrane during secretion of the nonfat phase of milk.

Effects of centrophenoxine on lipofuscin in the retinal pigment epithelium of old mice.

The effects of centrophenoxine on the retinal pigment epithelium (RPE) of 17 month old female mice have been studied. Animals were injected subcutaneously for 3 months (60 injections) with the drug (0.1 mg/g of body wt) daily in 0.1 M phosphate buffered saline at pH 7.0. The morphological changes in the pigment layers of the retina of both eyes were studied by light and electron microscopy and the lipofuscin pigment was demonstrated by its autofluorescence and ultrastructural characteristics. There was a significant reduction of the lipofuscin pigment in the treated animals, but the melanin pigment remained unchanged. The lipofuscin granules also appeared less osmiophilic and showed a greater preponderance of membranes and vacuoles. Although the precise mechanism of action of the drug is not clear, an increased protective function of the pigment epithelium by the drug has been suggested.