Histo- and cytophysiology of the lactating mammary gland of the African elephant (Loxodonta africana).

The lactating mammary gland of the African elephant (Loxodonta africana) has been studied with a panel of morphological techniques focusing on (1) the functional changes during the secretory process, (2) proliferative process [by application of proliferating cell nuclear antigen (PCNA) immunohistochemistry] and apoptotic phenomena [by use of the TUNEL technique] in the individual lobules, and (3) components of milk and milk-fat-globule membrane. In the lactating gland, the lobules are variably differentiated; within a lobule, however, the alveoli are usually similarly differentiated. The morphology of their alveoli suggests a classification of the lobules into types 1-3. Lobules of type 1 are composed of immature tubular alveoli with mitotic figures and numerous PCNA-positive nuclei; advanced type 1 alveoli contain abundant glycogen and specific secretory granules. Lobules of type 2 are further subdivided. In type 2a lobules, the epithelial cells of the alveoli form tall apical protrusions, which in part are occupied by small lipid droplets and which are pinched off in an apocrine fashion. The number of lysosomes varies considerably. Type 2b is the most common type, with striking basal membrane foldings, abundant rough endoplasmic reticulum cisterns, large Golgi apparatus, numerous mitochondria, lipid droplets, and protein vesicles with 30- to 90-nm-wide casein micelles. The lipid droplets are pinched off with minimal amounts of cytoplasm. Type 2c is composed of alveoli with a cuboidal epithelium and few signs of secretory activity. Increasing expression of peanut-agglutinin-binding sites parallels the maturation and differentiation of the glandular cells. Type 3 lobules are marked by numerous TUNEL-positive nuclei and large lipid droplets and are apparently degenerating structures. Cytokeratin (CK) 14 is usually present in the myoepithelial cells; CK 19 and CK 7 mark ductal and immature alveolar epithelia. Milk protein content varies between 2.6% and 6.3%, and casein micelles range from 35 to 90 nm in diameter. The diameter of intra-alveolar milk fat globules ranges from 5 to 25 micrometer and the membranes bear a filamentous surface coat composed of membrane-anchored mucins; gel-electrophoretic analysis of these mucins from different individuals demonstrates the presence of mucin MUC 1, which is expressed with considerable genetic heterogeneity.

High pressure effects on the colloidal calcium phosphate and the structural integrity of micellar casein milk. Part 1. High pressure dissolution of colloidal calcium phosphate in heated milk systems.

Results of this study confirm that high temperature (118 degrees C/15 min) and high pressure (400 MPa/5 min) processing of skim milk, skim milk ultrafiltration and ultracentrifugation fractions, and model milk salt solutions cause dramatic shifts in their colloidal and soluble Ca phosphate equilibrium that affect their pH, dissolved Ca content, turbidity, and casein micelle microstructure. The relations between high temperature and high pressure processing-induced changes in the colloidal and soluble Ca phosphate equilibrium were evaluated in raw, pasteurized, and high temperature treated skim milk, ultrafiltration retentate and permeate of pasteurized skim milk, clear ultracentrifugation infranatant of pasteurized skim milk, and synthetic milk ultrafiltrates with and without lactose or Ca. The magnitude of the pH and dissolved Ca shifts caused by high temperature and high pressure processing was a function of casein micelle concentration. Ultrafiltration permeate exhibited the most drastic shits in pH and dissolved Ca contents due to high temperature and high pressure processing. Although high temperature processing reduced the pH of ultrafiltration permeate from 6.59 to 6.03 and the dissolved Ca from 100% to 58%, high pressure processing reversed both of these changes. These changes in high temperature and high pressure processed milk, milk fractions, and model milk salt solutions were related to microstructural changes in the casein micelles as revealed by electron microscopy.

Histochemical and biochemical observations on milk-fat-globule membranes from several mammalian species.

A specific secretory product of the lactating mammary gland are triglyceride fat globules which are enveloped by a very complex membrane, the milk fat globule membrane (MFGM). In different mammalian species (man, rhesus monkey, horse, goat, sheep, cow, grey seal, camel, alpaka) the glycoproteins of this membrane have been analyzed by gel electrophoresis, Western blotting and lectin histochemistry. A remarkable intra- and interspecific variability of these glycoproteins has been detected pointing to so far unknown physiological adaptions, which may play a role in the intestine of the new born. High molecular weight glycoproteins, with a very high degree of glycosilation have been found only in primates, horse and camel; the MFGMs of the true ruminants (cow, sheep, goat) are characterized by specific glycoproteins of a lower molecular weight range.

Glycoprotein filament removal from human milk fat globules by heat treatment.

Freeze-etch electron microscopy was applied to milk fat globules to observe surface details. A remarkable array of filaments, approximately 0.5 micron in length, was seen on human, but not bovine, globules. Heating human globules removed the filaments that were identified as high molecular weight glycoproteins by freeze-etch and gel electrophoretic analysis of the heating medium. Extraction of these globule glycoproteins was slight at 60 degrees C for one minute but substantial and tending to plateau at 80 degrees C for one minute. Such heat-induced alterations of the milk fat globule surface provide an alternative or additional explanation to milk lipase inactivation as the cause of reduced milk fat absorption from heated milk by the preterm infant.

Structural, histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles.

Horse milk fat globules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 microns. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealed by its binding of various lectins. In addition, the glycocalyx binds cationized ferritin, which indicates the presence of negative electric charges. Gel electrophoresis revealed the presence of high-molecular-weight glycoproteins in the MFG membrane of horse milk. Such glycoproteins are also present in human MFG membranes but are absent in the bovine MFGs. The casein micelles in horse milk are relatively large, their average volume-surface diameter being about 200 nm.

A comprehensive study of the relationship between size and protein composition in natural bovine casein micelles.

Casein micelles of bovine skimmed milk were fractionated by permeation chromatography on porous glass (CPG-10, 50 nm followed by CPG-10, 300 nm) at 30 degrees C. Micelles were pooled in eight eluant fractions and their size distribution was determined by electron microscopy. The composition of casein in the eight fractions was determined by quantitative hydroxyapatite chromatography. Micelle size decreased progressively with increasing elution volume, and volume-to-surface average diameter ranged from 154 nm in fraction 1 to 62 nm in fraction 8. Concurrently there was a decrease in relative proportions of alpha s- and beta-caseins and a large enrichment of kappa-casein, which changed from 4.1% total casein in fraction 1 to 12.1% total casein in fraction 8. At least half the decrease in alpha s-casein proportions was attributed to the alpha s1-casein component, but the data also suggested a decline in proportions of alpha s2-casein in the smallest micelle fractions. A plot of kappa-casein fractional content versus micelle surface-to-volume ratio gave a straight line (correlation coefficient from linear regression 0.98) from which an average kappa-casein surface coverage of 1.5 m2/mg or 47.3 nm2/molecule was obtained. If a constant surface coverage for kappa-casein is assumed, the parameters of the linear equation predict that micelle voluminosity is inversely related to micelle diameter, being approximately 30% larger in fraction 8 compared to fraction 1.

Internalization of ferritin-concanavalin A by the lactating mammary cell in vivo.

Ferritin-concanavalin A (Fer-Con A) was used to label the apical plasma membrane of the lactating cell to determine whether membrane internalization takes place. Rat glands were infused in vivo via the teat with 0.2 mg of Fer-Con A in 0.2 ml tris buffer (pH 7.0) containing 0.1% trypan blue, the latter acting as a marker of the infusate. Tissues were obtained from separate animals 5, 10 and 60 min postinfusion. Fer-Con A was seen in alveolar lumina bound to the outer surfaces of apical plasma membrane, microvilli and milk fat globules. It was observed within lactating cells on the inner membrane surfaces of endocytotic vesicles, Golgi cisternae, and secretory vesicles containing casein micelles, and in multivesicular bodies and lysosomes. Internalization of the ferritin-lectin conjugate into casein-containing secretory vesicles was detectable in the 5-min postinfusion tissue. Lysosomes were the only structures in control tissue that contained particles bearing some resemblance to Fer-Con A. The data provide evidence that apical plasma membrane is internalized and distributed to a number of intracellular compartments.

Access and distribution of exogenous substances in the intercellular clefts of the rat adenohypophysis.

In model experiments with the use of horseradish peroxidase (HRP), two pathways of transport of substances to the adenohypophysis were studied, as well as the distribution of the tracer in the latter organ. The first pathway allows the tracer to penetrate from the intercellular milieu of the median eminence below the meningeal sheath covering the adenohypophysis to the surface of the pituitary gland. The second pathway transports the tracer via the capillaries of the hypophysial portal circulation to the interior of the glandular parenchyma. These results show (i) that the meningeal sheath establishes a barrier between the hemal milieu of the pituitary and the hemal milieu of the general circulation, and (ii) that the tracer reaching the adenohypophysis via both routes is found in the intercellular clefts of the glandular parenchyma only to a limited extent. By means of conventional electron microscopy, intercellular contacts between hormone-producing adenohypophysial cells are observed resembling focal tight junctions. Between the membranes of entwined processes of stellate cells, only small maculae adhaerentes are found. Freeze-etch studies on unfixed adenohypophyses reveal zonulae occludentes between the durafacing layers of the meningeal sheath and focal maculae occludentes between parenchymal cells. Additional tissue-culture experiments with adenohypophysial cells directly exposed to HRP reveal a gradual cessation of the labeling process in the intercellular clefts in accord with the observations from the in-vivo experiments, as well as intercellular focal tight junctions between individual hormone-producing cells.

Composition and ultrastructure of calcium phosphate-citrate complexes in bovine milk systems.

The present studies show that the colloidal calcium phosphate of cows' milk has a (Ca + Mg)/Pi ratio of 1.67 (+/- 0.10; n = 22) and contains citrate, Mg and Zn at molar ratios to Ca averaging 0.05, 0.03 and 0.003, respectively. The composition of the natural colloidal phosphate of milk is similar to the precipitates formed by neutralization of ultrafiltrates obtained from acidified milks, and to that of the calcium phosphate-enriched fraction produced by extensive enzymic hydrolysis of the casein micelles in milk. Examination by electron microscopy of these artificial preparations of milk calcium phosphate revealed in both a very fine and uniform substructure which consisted of granules having an average, true diameter of approx. 2.5 nm. The size and shape of these tiny granules closely resemble the morphologies reported for the colloidal phosphate particles in native casein micelles, as well as for the subunits of amorphous calcium phosphate observed during calcification in other biological systems such as mitochondria and bone.

Induction of high titer mouse--antihuman spermatozoal antibodies by liposome incorporation of spermatozoal membrane antigens.

This is the first report of induction of high titer mouse-antihuman spermatozoal antibodies using a spermatozoal antigen incorporated in liposomes. The peptide antigens that were incorporated into pure phosphatidylcholine liposomes are known to react with the naturally occurring antibodies in sera of sterile patients. Electron microscopy and the complement-dependent glucose release tests indicated that the peptides were entrapped in the aqueous phase between the lipid bilayers in the liposome vehicles. This is in contrast to most other liposomal antigens that are expressed on the outer lipid surface. In view of the nontoxic nature of the liposomes these results could be of some interest in immunological fertility regulation trials with appropriate antigens in an allogeneic system.

Amorphous calcium phosphate in casein micelles of bovine milk.

The calcium phosphate remaining after hydrazine deproteination of casein micelles isolated from bulk skim milk exhibits under the electron microscope a very fine and uniform granularity being formed by small subunits with a true diameter of approximately 2.5 nm. This material, which is about 10 percent by weight citrate, termed calcium phosphate citrate (CPC) complex, also contains Mg and Zn at molar ratios of 0.03 and 0.003 respectively. Radial distribution function (RDF) and infrared analyses show that CPC is a Mg-containing amorphous calcium phosphate (ACP) similar to synthetic and cytoplasmic ACP. presence of CPC in casein micelles as an amorphous colloid bonded with phosphoproteins provides the means for storing in milk large amounts of Ca (16 mM) and Pi (10 mM) in a readily utilizable form but at a higher ion concentration than found in biological solutions.

Ultrastructural changes in lactating tissue related to the suppression of milk secretion by concanavalin A.

The plant lectin, concanavalin A (Con A) suppresses milk secretion when infused into the mammary gland or when incubated with lactating tissue in vitro. Toward defining its mode of action, we infused Con A into rat and goat mammary glands via the teats and observed effects on lactating cells. Lectin dosages were 2 and 25 mg per gland for rats and goat, respectively. Tissue samples were taken 1 and 3 h post infusion for rats and at 24 h for the goat. Control and Con A-treated tissues were observed by light microscopy and by both thin section and freeze fracture electron microscopy. In comparison to controls, Con A-treated tissues of both species exhibited alveoli with enlarged cells and relatively empty lumina; cells were distended with secretory vesicles and fat droplets. Apical plasma membranes of lectin-affected cells of the rat displayed a marked reduction in the number of microvilli, and exhibited an atypical branching and folded structure. Morphometry was employed to quantitate changes in cell and secretory product parameters in both rat and goat tissue. Microtubule numbers and distribution did not appear to be altered by Con A but considerable changes were noted in the arrangement of microfilaments associated with the secretory surface of lectin-treated epithelial cells. Various related ultrastructural changes and the role of Con A in perturbing the microfilament system are discussed.

On the size of small protein particles determined by electron microscopy of unidirectionally shadowed freeze-etched preparations.

The influence exerted by the thickness of the deposited metal layer and the ionic strength of the solution on the apparent size of particles of bovine serum albumin in unidirectionally shadowed freeze-etch preparations of spray-frozen specimens was investigated. It appeared that the size increase due to shadowing is nearly twice the thickness of the deposited metal layer. Apparent particle size was shown to increase linearly with the inverse square root of the ionic strength of the solution. At ionic strength 0.001 the particles appeared about 30% larger than at infinite ionic strength.

Composition and size distribution of bovine casein micelles.

Chromatography of glutaraldehyde-fixed skim-milk on controlled-pore glass (CPG-10, 300 nm) gave three micellar fractions whose averaged diameters, measured by electron microscopy, decreased progressively with increasing elution volume. Casein micelles with diameters up to 680 nm were detected. The casein composition of the same fractions from unfixed skim-milk was determined. As the fraction elution volume increased, kappa-casein varied from 7.7 to 11.4% of total casein, giving alpha s/kappa ratios of 6.1, 4.7 and 3.3. A plot of kappa-casein content versus micelle surface-to-volume ratio for skim-milk and the column fractions approximated to a straight line. Re-calculation of the published results from two other studies also gave linear relationships between kappa-casein content and surface area for artificial micelles. The three regression lines thus obtained had small intercepts. It was concluded that the data indicated the same fundamental structure for casein micelles, with a predominant surface location for kappa-casein, whether the micelles are natural or artificial and whether they are aggregated by Ca2+ alone or by Ca2+ together with calcium phosphate-citrate complex.

Freeze-fracture studies of cytoplasmic inclusions occurring in experimental lipidosis as induced by amphiphilic cationic drugs.

The ultrastructure of cytoplasmic inclusions, which characterize experimental lipidosis as induced by several amphiphilic cationic drugs, was studied by means of freeze-fracturing and thin-sectioning. Retinal and adrenal tissues of rats chronically treated with high oral doses of chlorphentermine were used. In thin sections the cytoplasmic inclusions, which were previously shown to represent lysosomes overloaded with polar lipids, exhibit lamellated or lattice-like internal patterns. The present freeze-fracture observations are interpreted as to indicate that the lamellated inclusions contain polar lipids in the lamellar phase, whereas those with lattice-like patterns contain polar lipids in a hexagonal phase.

On the size of monomers and polymers of beta-casein.

The size and number average molecular weight have been determined for beta-casein monomers and polymers from electron micrographs using the freeze-etching procedure with spray-frozen specimens. For the spherical beta-casein monomers we found a mol. wt of 22600 and a diam. about 10 nm, which compared quite well with data obtained from ultracentrifugation, light scattering and viscosity measurements. Polymer sizes were in agreement with molecular weight determinations from ultracentrifugation and light scattering, assuming that the volume and weight of the particles are proportional.

The functional and structural border between the CSF- and blood-milieu in the circumventricular organs (organum vasculosum laminae terminalis, subfornical organ, area postrema) of the rat.

The present study continues a previous investigation on the median eminence (EM) (Krisch et al., 1978). In rats with high levels of neurohormones (LHRH, vasopressin) a limited immunohistochemical labeling of perivascular tanycyte processes can be observed surrounding capillaries in the marginal region of the organum vasculosum laminae terminalis (OVLT) and in the inner part of the subfornical organ (SFO). This labeling extends from the perivascular space a short distance along the tanycyte processes. By conventional electron microscopy and by freeze-etching, tight junctions are demonstrated at a distance from the capillary lumen which corresponds to the borderline of the immunohistochemical labeling of perivascular tanycyte processes in light microscopic preparations. The tight junctions are arranged in several parallel and helical rows and correspond to those found in the median eminence. Consequently, the immunohistochemical labeling the OVLT and in the SFO marks the intercellular cleft. In the circumventricular organs the immunostaining labels the extension of the perivascular space characterized by the hemal milieu. The perivascular space is separated off by tight junctions from the CSF-milieu of the adjacent neuropil. Furthermore, the present study demonstrates tight junctions in the marginal region of the area postrema (AP) between the perivascular processes of the tanycytes.

Freeze fracture studies on the annelid septate junction.

Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80--120 A, the distance between two particles (centre to centre) is 150--250 A. Additionally striking rows of large particles (long diameter 150--200 A). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100--150 A, centre to centre distance approx. 250 A) whereas the particles of the extracellular face (EF, mean diameter 200-250 A) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.