Low temperature ultramicroincineration of thin-sectioned tissue.

Low temperature ultramicroincineration was employed to determine the morphological localization of "structure-bound" mineral and/or metallic elements within biological cells at the electron microscope level. This technique chemically removes organic material from thin sections of tissues with reactive, excited oxygen instead of heat as used in a furnace. The remaining ash representing the mineral/metallic ultrastructure of cells is advantageous for ultrastructural studies because incineration without applying heat is less destructive than the burning associated with high temperatures. This low temperature incineration method was applied to thin-sectioned avian shell gland mucosa, a calcium transporting system, as a sample tissue. The results include: recognition of many subcellular organelles in the ash patterns, identification of dense, ash-containing granules (possibly organic-metallic complexes) in epithelial cells which may be involved in calcium transport, description of ashed erythrocytes and collagen, comparison of ashed glutaraldehyde fixed tissue with and without osmium postfixation, description of lead-stained cells after ashing, demonstration that ash preservation is dependent upon section thickness, illustration of the fine resolution obtainable because the ash residues remain relatively near their in situ origins, discussion of technical problems in this relatively new field, and demonstration of the presence of Ca and P in the ash with electron microprobe X-ray analysis.

The intracellular distribution of calcium in the mucosa of the avian shell gland.

The intracellular distribution of calcium has been studied in the mucosa of the avian shell gland, a tissue which transports large quantities of calcium during discrete time intervals. Ca(45) was administered to hens either in a single dose followed by sacrifice 5 min later or in repeated doses over an extended period followed by sacrifice 2 hr or 24 hr after the last injection. Subcellular fractions were isolated by differential centrifugation and analyzed for Ca(45). The Ca(45) was located principally in the particulate fractions; the concentration (CPM Ca(45)/mg N) was highest in the mitochondrial fraction. Comparisons of (1) the Ca(45) distribution in shell gland cells with that of liver cells, (2) the alterations which occur due to the phase of the egg laying cycle, (3) the effects due to the time elapsed since the last injection of Ca(45), and (4) the Ca(45) distribution of the short term experiments with that of the long term experiments revealed that the mitochondrial fraction of the shell gland appeared to be active in the movement of calcium. The microsomal fraction showed increased values in CPM Ca(45)/mg N when calcification was occurring, which may indicate that the subcellular components of this fraction have a role in calcium transport. The nuclear and supernatant fractions did not seem to be involved in the transport process. The implications of these results concerning the manner by which calcium may be controlled on a cellular level in this system are discussed.

Characterization of isolated and cultured chick osteoclasts: the effects of acetazolamide, calcitonin, and parathyroid hormone on acid production.

The effects of acetazolamide, calcitonin (CT), and parathyroid hormone (PTH) on acid production in isolated osteoclasts has been investigated. Osteoclasts were isolated from the endosteum of 3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with acetazolamide (10 x 4 M and 10(-7) M), CT (1 mU/ml and 0.31 mU/ml) and PTH (6.5 U/ml and 0.40 U/ml) for 1, 3, 6, and 18 hr. The cells were stained with acridine orange and the intensity of fluorescence measured by a light microscope photometer. Acetazolamide treatment resulted in a steady decline in intracellular acidity, suggesting that carbonic anhydrase plays a major role in acid production in isolated osteoclasts. Treatment with PTH produced a decline in acidity at 1 hr, followed by a peak at 3 hr and then a decline at 6 and 18 hr. The transient increase in acidity may be due to activation of carbonic anhydrase by PTH. Calcitonin treatment also resulted in a decline in cell acidity which was similar, but less pronounced than that resulting from acetazolamide treatment. These results indicate that calcitonin may mediate osteoclast activity by alterations in intracellular acid production.

Characterization of the cytoskeleton of isolated chick osteoclasts: effect of calcitonin.

We investigated the effects of calcitonin (CT) and parathyroid hormone (PTH) on the distribution of actin, tubulin, vimentin, and on cell size in cultured chick osteoclasts. In addition, we studied the effects of colchicine on intracellular acidity. Osteoclasts were isolated from the endosteum of 2-3-week chick tibias and were maintained under culture conditions for 5 days. The cells were treated with CT for 30 min or PTH for 60 min and were observed after immunocytochemical staining of cytoskeletal proteins. In untreated cells, actin was found in both a filamentous and a punctate staining pattern, with indented or invaginated regions free of punctate spots. The tubulin distribution in untreated cells was characterized by a pattern of microtubules radiating from the cell center and running parallel to the cell edge. Vimentin staining was usually localized to the perinuclear area. There were no changes in cytoskeletal element distribution or morphology attributable to PTH treatment. Osteoclasts treated with CT were more irregularly shaped, contained more retraction fibers, and were more rounded, with a denser array of cytoskeletal elements in the cell center. In addition, the mean area of the CT-treated cells was significantly less than that of the untreated cells. The actin distribution after CT treatment was still characterized by both a filamentous and a punctate pattern. After CT treatment, vimentin staining appeared more centrally localized than in untreated cells and tubulin staining revealed microtubules which now extended to the retracted cell margin. These results indicate that isolated osteoclasts respond to CT by significant morphological changes which are reflected in the distribution of the major cytoskeletal elements. Disruption of the microtubular system by colchicine treatment also resulted in an initial increase in intracellular acidity, suggesting the involvement of microtubules in the movement of acid-laden vesicles to the exterior.

Carbonic anhydrase localization by light and electron microscopy: a comparison of methods.

The histochemical localization of carbonic anhydrase by Hansson's cobalt-salt method was compared with immunocytochemical localization in the proventriculus (glandular stomach), the chorioallantoic membrane, and in articular and growth-plate cartilages from the domestic hen. Numerous differences were observed. Staining was positive by Hansson's method and negative by immunocytochemistry for the submucosal glands of the proventriculus, articular cartilage cells, resting and proliferating cells of the growth plate, nuclei, and intercellular spaces. Red blood cells stained faintly and inconsistently by Hansson's method. Both methods were in agreement for the cytoplasm of the surface mucosal cells of the proventriculus, the cytoplasm of the villus cavity cells in the chorioallantoic membrane, and in hypertrophic cells of growth-plate cartilage. Acetazolamide usually inhibited the histochemical reaction, even in those sites that, according to other methods, did not contain enzyme. Consequently, acetazolamide inhibition appears to be an unreliable control for the histochemical reaction.

Ultrastructural localization of carbonic anhydrase in the chorioallantoic membrane by immunocytochemistry.

Carbonic anhydrase was localized in the chick embryo chorionic ectoderm at the ultrastructural level by immuno-cytochemistry. Preembedding staining of whole tissue was performed. The enzyme was present in the cytoplasm, on the membranes of apical vesicles, and on the membranes of microvilli in villus cavity cells, cells that may be involved in acid secretion and subsequent dissolution of the egg shell. In sinus covering cells, the enzyme is solely in the cytoplasm. The location of the enzyme in the thin cytoplasmic arms of the sinus covering cells is consistent with the role in nonrespiratory CO2 release.

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis.

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.

Identification of carbonic anhydrase in chick growth-plate cartilage.

Carbonic anhydrase has been localized by immunocytochemistry in the cells and territorial matrix of the hypertrophic and calcifying zones of chick growth-plate cartilage. Adjacent epiphyseal and articular cartilage were not stained. By biochemical assay the activity levels were 61.3, 1.8, 34.7, and 703.3 units/g wet weight for growth plate, epiphyseal/articular cartilage, spongiosa, and blood, respectively. The role of the enzyme in growth plate is discussed.

Autoradiographic localization of carbonic anhydrase in the developing chorioallantoic membrane.

Carbonic anhydrase was localized in the chorioallantoic membrane with labeled inhibitor autoradiography of 3H-acetazolamide at 11, 14 and 19 days of incubation. At 11 days carbonic anhydrase was present in low amounts only in the undifferentiated ectoderm cells. At 14 and 19 days, the enzyme was found in increased amounts in all three germ layers of the chorioallantois. In the chorionic ectoderm the villous cavity cells contained the highest level of carbonic anhydrase. This finding lends support to the theory of H+ production to solubilize the CaCO3 of the egg shell. Sinus covering cells showed a considerable lower concentration of the enzyme than did villous cavity cells. Carbonic anhydrase in these cells may be multifunctional, assisting in calcium transport, subserving HCO3- transport from egg shell to blood, and supporting gaseous exchange. In the allantoic endoderm carbonic anhydrase was found in granule-rich cells and might be involved in the transport of Na+ and Cl- ions from allantoic fluid into the blood. The enzyme in the undifferentiated endoderm cells may have a respiratory function. In the mesoderm carbonic anhydrase was detected in the endothelium and pericytes of blood vessels where it is interpreted to support respiratory function.

Frozen thin-sections of rapidly forming bone: bone cell ultrastructure.

Frozen thin-sections of fresh medullary bone, which had been calcifying for 2 to 4 days, were prepared and examined in the electron microscope. The bone was obtained from male Japanese quail treated with estradiol valerate. Large numbers of 200-800 A electron dense granules, which consisted of 50-75 A subparticles, were seen within mitochondria. A population of electron-dense particles slightly smaller than ribosomes was also observed. Most granules were not present after conventional fixation, dehydration, embedding, sectioning and staining. Oother structures which were visible, also on the basis of their intrinsic electron density, were nuclei with regions which resembled condensed chromatin, ribosomes and rough endoplasmic reticulum. By the use of osmium tetroxide vapor staining, inner and outer membranes and cristae of mitochondria were delineated. Other membranous components were not detected. Since very little loss or dislocation of cell components can occur during the preparation of frozen thin-sections, the micrographs obtained may be a more accurate representation of cell ultrastructure.

Cytology and autoradiography of estrogen-induced differentiation of avian endosteal cells.

The endosteal reaction, the initial step in the formation of medullary bone, was investigated in femurs of estrogen-treated male Japanese quail. Morphologically, the endosteal cells were in an undifferentiated state until 30 h after estrogen treatment and showed characteristics resembling those of resting cells. Many preosteoblasts were seen on the endosteum at 33 h, whereas mitotic figures and fully differentiated osteoblasts were recognized at 36 h after estrogen. The mitotic figures were observed among the preosteoblasts on the endosteum. Autoradiographs showed that the number of endosteal cells labeled by [3H]thymidine injected 1 h before sacrifice was maximal 27 h after the estrogen administration and decreased markedly by 30 h. When a single injection of [3H]thymidine was given at 26 h after estrogen, the highest percent of labeled endosteal cells was observed 1 h later (27 h after estrogen). Labeled preosteoblasts and osteoblasts were observed at 7 h (33 h after estrogen) and 10 h (36 h after estrogen), respectively. Our results show that under the influence of estrogen, endosteal cells are induced to maximally synthesize DNA about 27 h after estrogen. These cells appear to modulate into preosteoblasts in about 6 h and then divide via mitosis to become osteoblasts within an additional 3 h. The development of medullary bone induced by estrogen occurs in a sequential and predictable manner, which makes it a useful system for studying basic problems on bone cell differentiation.

Matrix vesicles in newly synthesizing bone observed after ultracryotomy and ultramicroincineration.

Matrix vesicles were observed in femurs of 8-day-old chick embryos prepared by ultracryotomy. Some of the sections were subjected to ultramicroincineration. The unfixed tissues never came into contact with solutions, and thereby artifacts due to dissolution, redistribution, or rearrangement of the mineral constituents were avoided. In the osteoid, electron dense objects with the size and appearance of matrix vesicles were seen, although limiting membranes were not visible. After ultramicroincineration the vesicles were observed to contain small crystals and a less dense amorphous mineral material which may te the precursor of bone mineral. In addition, a ring of ash enclosed the crystalline and amorphous mineral and appeared to occupy the position of the vesicle membrane as seen in conventionally prepared material.

Nucleoside incorporation as a function of hormone levels during the early phases of estrogen-induced genesis of medullary bone in the Japanese quail, Coturnix coturnix.

The sequence of changes in RNA synthesis during the early phases of genesis of medullary bone induced in male Japanese quail by estrogen treatment was studied by 3H-uridine uptake. Analyses of plasma estrogen and testosterone were done by radioimmunoassay at 12, 24, 38 and 61 h. A dose of 5 mg kg-1 estradiol-17 beta was found to stimulate the same 3H-uridine uptake 15 h after hormone treatment as a dose of 20 mg kg-1 of estradiol valerate. The uptake of 3H-uridine rose as the dose of estradiol-17 beta increased. Plasma estrogen levels, which were highest 12 h after injection, declined sharply during the next 12 h, returning to control levels between 38 and 61 h. Testosterone levels declined after estrogen administration and remained below control values at all time points. Following estrogen administration, 3H-uridine uptake declined from control values for the first 8 h. Twelve hours after hormone administration control levels were again reached, with maximum 3H-uridine uptake occurring 16 h after hormone treatment. The 16-h maximum was followed by a steady decline to below control levels at 20, 24 and 28 h, the time at which the experiment was discontinued. Maximum 3H-uridine uptake following estrogen stimulation is similar to that observed for the stimulated immature rat uterus.

Estrogen-induced sequential changes in avian bone metabolism.

Medullary bone was induced in male Japanese quail by administration of estradiol valerate. An increase in the organic weight of the femur was observed by 36 h after estrogen and an increase in ash weight was observed by 96 h. A complex sequence of metabolic changes in the femur occurred after estrogen treatment. A large increase in uptake of 3H-proline associated with enhanced collagen formation began 36 h after estrogen and reached a peak 3.5 times the control rate at 60 h. The onset of mineralization of the newly formed bone matrix, as determined by 45Ca uptake, began about 24 h after onset of collagen synthesis indicating that synthesis of the bone matrix and its mineralization occur sequentially. Large increases in the rates of uptake of 3H-uridine and 3H-thymidine occurred prior to the onset of medullary bone formation and appear to reflect the differentiation of osteogenic precursors to bone-forming cells.

Ultrastructural observations of amorphous bone mineral in avian bone.

Mineral from medullary bone of three avian species was examined with the electron microscope in order to clarify the ultrastructure of amorphous bone mineral (ABM) in a mineralized tissue. Powders from freeze-dried bone revealed bone mineral with morphology and behavior identical to synthetic amorphous calcium phosphate (ACP). These powders exhibited spherically shaped particles 180--400 A in diameter with uniform electron density when viewed at low beam intensity. Thin sections of embedded freeze-dried bone also revealed spherically shaped particles 100--350 A in diameter with electron beam sensitivity characteristic of ACP. Regions of bone mineral with irregular outline and morphology were observed which closely resemble the discoidal form of synthetic ACP. More electron-dense spherical particles (150 A in diameter) could be seen budding from these regions. Some of these buds exhibited electronlucent centers characteristic of AMB. The inorganic nature of these features of bone mineral was confirmed using ultramicroincineration. Preliminary exploration of a freeze-substitution technique showed spherical bone mineral particles which were similar in morphology to those observed in freeze-dried samples. A limited degree of preservation of cellular material was observed using this freeze-substitution technique.

In vitro synthesis of proteoglycans associated with medullary bone in Japanese quail.

A short-term incubation system was used to study proteoglycan synthesis during the early stages of medullary bone formation in estrogen-treated male Japanese quail. The proteoglycans were separated by chromatography on a DEAE Bio-Gel A column eluted with a 400-ml 0-1 M NaCl gradient. The profile from uninjected control birds showed a single peak, whereas profiles from estrogen-treated birds showed development of another peak. Incorporation of [35S]sulfate into the estrogen-induced proteoglycan increased most dramatically between 25 and 37 h after hormone treatment. The estrogen-induced proteoglycan has a Kav = 0.65 on Sepharose CL-4B, an average buoyant density of 1.50 g/ml, and contains keratan sulfate as its constituent glycosaminoglycan. The second proteoglycan has a Kav = 0.52 on Sepharose CL-4B, an average buoyant density of greater than or equal to 1.7 g/ml, and has chondroitin sulfate as it major glycosaminoglycan. It may also contain some heparin or heparan sulfate. The results support the usefulness of the incubation system for studying the dynamics of bone matrix production.