Ultrastructural autometallography: a method for silver amplification of catalytic metals.

The autometallographic technique involves application of a silver bromide-containing emulsion on the surface of ultrathin sections placed on grids that are subsequently exposed to a photographic developer. In tissue sections from animals treated intravitally with gold, silver, or mercury compounds, accumulations of the metals are visualized by autometallography and can be used for quantitative studies. After amplification, sections can be stained with lead citrate and uranyl acetate. Using autometallography, particles of colloidal gold dispersed in a film of gelatin showed a time-dependent growth and were gradually amplified up to 3.5-fold after 15 min of development. Hence the method may prove useful tracing colloidal gold particles in sections with low particle density, and be a powerful tool for revealing metals in biological tissues.

Light microscopic visualization of colloidal gold on resin-embedded tissue.

RNase labeled with colloidal gold was used as a model for the present technique evolved for the light microscopic localization of gold-labeled substances in semithin resin-embedded sections. Tissue sections placed on glass slides were treated with the gold-enzyme complex and subsequently exposed to a photographic developed containing silver lactate. During the development gold particles are encapsulated in growing shells of metallic silver and gradually made visible in the light microscope. The amplification method can be applied to paraffin-embedded and frozen sections as well. This technique may prove useful as a supplement to studies utilizing colloidal gold or silver as markers normally used at the electron microscopic level.

Silver enhancement of tissue mercury: demonstration of mercury in autometallographic silver grains from rat kidneys.

The autometallographic silver enhancement method has been applied increasingly to detect trace amounts of mercury in preparations of biological tissue. It has, however, been difficult to establish the presence of a core of mercury within the silver grain by direct methods such as energy dispersive X-ray analysis. In the present work, a sample of autometallographic silver grains was prepared from kidneys of rats exposed to mercury in the drinking water. Frozen sections from the kidneys were silver-enhanced and subsequently all organic material was removed by enzymatic digestion. The remaining pellet of silver grains was analyzed by proton-induced X-ray emission (PIXE) and mercury was demonstrated in an amount of 0.1-0.5% compared to silver. In addition, it was demonstrated that two pools of catalytic mercury compounds exist, probably corresponding to sulfide- and selenium-bound mercury.

A new method for the isolation of ultrastructurally preserved glomeruli.

A method is described for the isolation of kidney glomeruli using centrifugation in a discontinuous Ficoll gradient. The method, applied to rats, rabbits, pigs and man, yields a glomerular fraction of high purity with a tubular contamination of normally less than 2%. From observations by light microscopy on epoxy resin-embedded fractions, one-third to one-fourth of the glomeruli had suffered only slight damage during isolation. In the electron microscope these best preserved glomeruli showed a close morphologic similarity to those of the intact tissue and preliminary experiments have indicated that they are well-suited for further studies of the in vitro behavior of isolated, viable glomeruli.

Origin of outgrowth from isolated glomeruli in culture.

The primary cellular outgrowth from isolated glomeruli was identified by examinations of the ultrastructural changes occurring in the three glomerular cell types during culture and by light and electron microscopic autoradiographic preparations of isolated glomeruli incubated with 3H-thymidine. It is demonstrated that the visceral epithelial cells constitute the majority of cells in the primary glomerular outgrowth and that another cell type probably originates from the mesangial cells. A method is described to obtain rather pure cultures of proliferating visceral epithelial cells suitable for further studies of their metabolic properties.

Cellular outgrowth from isolated glomeruli. Origin and characterization.

The incorporation of 3H-thymidine by isolated decapsulated glomeruli from pigs was studied up to 5 days after explantation by light and electron microscopic autoradiography. The visceral epithelial cells (VEC) were the only cell type to be labeled during the first 2.5 days, showing that this tracer can be applied as a specific marker for VEC in decapsulated glomeruli. From the third day, label was incorporated by another cell type, probably mesangial cells. Labeling of endothelial cells could not be observed. Isolated glomeruli were then incubated for 2.5 days with 3H-thymidine in order to label the VEC, and after 5 to 6 days the primary outgrowth of cells appeared. Light and electron microscopic autoradiography performed in situ showed that the majority of cells in the primary outgrowth consisted of labeled cells, thus showing their origin from the VEC. The VEC in culture appeared as very large, irregular cells with a slow proliferation rate. After 9 days of culture another cell type was seen in the outgrowth (type II cells), probably derived from the mesangial cells. This cell grew in multilayers and had a close similarity to smooth muscle cells in culture, with thick bundles of microfilaments in the periphery of the cell and an abundant intercellular matrix. A third cell type (type III) with a regular angulated, "epitheloid" shape was rarely seen in the outgrowths. Because 1 to 2% of the isolated glomeruli were encapsulated, the parietal epithelial cell is suggested as the mother cell for this cell type; this is also thought to be true because the parietal epithelial cell showed 3H-thymidine incorporation in autoradiographs of intact glomeruli.

Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin?

Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of [3H]thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of [3H]thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with [3H]thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium. Type I cells proliferated for approximately two weeks around the glomerular explant and then reached steady state while type II cells showed only very limited proliferative capacity. Furthermore, rapidly proliferating cells of supposed mesangial origin (type III cells) grew out later from isolated rat glomeruli. Thus, the present results suggest that outgrowths from rat glomeruli contain three types of cells which can be identified on basis of structure and growth characteristics as visceral and parietal epithelial cells and mesangial cells, while endothelial cells do not proliferate.

Export of protein from the osteoclast as studied by electron microscopic autoradiography.

The present electron microscopic autoradiographic study includes a quantitative analysis of osteoclasts in vitro using tritiated leucin as a protein tracer. A significant increase in the grain density over the ruffled border and the underlying resportion zone was demonstrated two hours post pulse whereas the grain density of the remaining cytoplasm was relatively constant. This indicates a transport of newly synthesized protein from the osteoclast to the extracellular resorption zone. Earlier histochemical and biochemical experiments suggest that the exported protein may represent lysosomal enzymes to be used in the extracellular bone degradation.

Uptake of peroxidase by calcitonin inhibited osteoclasts.

The present electron microscopic histochemical study demonstrates that osteoclasts from calcitonin treated bone are able to take up organic macromolecules even though the ruffled border has disapppeared. The absorption occurs around the entire periphery of the osteoclast, but the amount of absorbed peroxidase seems to be reduced in comparison with that of untreated cells. It is concluded that the effect of calcitonin on osteoclasts is primarily a cessation of the exocytosis and the concomitant disappearance of the ruffled border.

Renal enlargement: comparative autoradiographic studies of 3H-thymidine uptake in diabetic and uninephrectomized rats.

Incorporation of 3H-thymidine into renal cortical tissue has been studied by light microscopic autoradiography in streptozotocin-diabetic rats, uninephrectomized rats, uninephrectomized diabetic rats, insulin-treated diabetic rats and control rats. The percentage of labelled cortical nuclei (the labelling index) was determined separately in glomeruli, proximal tubules and distal tubules after 2, 4 and 6 days on autoradiographs from 1 micron thick plastic embedded sections. The incorporation of thymidine in glomerular nuclei was consistently low (less than 1%) and no differences were found between the control and experimental groups. In both proximal and distal tubules an increase in thymidine incorporation was seen on day 2 followed by a decline on days 4 and 6. The maximal labelling on day 2 in proximal tubules was 9.1% in the uninephrectomized diabetic group, 3.7% in the diabetic group and 1.4% in the uninephrectomized group. In distal tubules the corresponding values were 5.2, 3.5 and 1.1%. The increase in kidney weight after 6 days was 83, 62 and 37%, respectively. Estimates of the net increase in the number of cortical tubular cells in the different experimental groups showed that the kidney enlargement followed different patterns with respect to the extent of cellular hyperplasia and hypertrophy. The kidney growth in uninephrectomized diabetic rats was dominated by tubular cellular hyperplasia, in the diabetic group hyperplasia and hypertrophy participated to approximately the same extent, whereas cellular hypertrophy was most pronounced in the uninephrectomized animals. Nuclear labelling in the insulin-treated diabetic rats was not different from that of control rats and consequently a hyperplastic effect of streptozotocin can be ruled out.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficiency of autometallographic detection of mercury in the rat kidney.

The autometallographic silver enhancement method is a method for subcellular localization of some heavy metals, such as mercury. However, no quantitative estimate has been made of the amount of mercury demonstrated by the method. In this study, pellets of autometallographic silver grains were prepared from unfixed kidney slices of rats exposed i.p. to mercury chloride containing trace amounts of 203Hg. The slices were silver-enhanced, and subsequently all organic material was removed by enzymatic digestion. During all stages of the experiment the solutions and tissue were gamma-counted. The analysis showed that the final pellets contained approximately 30% of the mercury compared to that found in the slices prior to development and that the mercury was probably located in lysosomes.

Autometallography: tissue metals demonstrated by a silver enhancement kit.

In biological tissue, minute accumulations of gold, silver, mercury and zinc can be visualized by a technique whereby metallic silver is precipitated on tiny accumulations of the two noble metals, or on selenites or sulphides of all four metals. In the present study a silver enhancement kit, primarily intended for the amplification of colloidal gold particles, has been used to demonstrate these catalytic tissue metals. Sections from animals exposed intravitally to aurothiomalatate, silver lactate, mercury chloride, sodium selenite or perfused with sodium sulphide were subjected to a commercial silver enhancement kit (IntenSE, Janssen Pharmaceutica). It was found that the kit performs adequately to the silver lactate gum arabic developer and to the photographic emulsion technique. The kit can be used as a silver enhancement medium for the demonstration of zinc by the Neo-Timm and selenium methods and for demonstration of gold, silver, and mercury in tissues from animals intravitally exposed to these metals. It can also be used for counterstaining silver treated osmium fixed tissues embedded in plastic.

Characterization of a human ovarian teratocarcinoma-derived cell line.

A cell line (PA I), derived from human ovarian teratocarcinoma cells, was obtained by culturing ascitic fluid cells from a patient with recurrence of malignant ovarian teratoma. During early passages the cultured cells showed a variable morphology, a long doubling time, and a low plating efficiency (2%). After about 50 passages in vitro, a cell population which was more homogeneous and resembled embryonal carcinoma cells were obtained. These cells had a shorter doubling time (26 h), and increased plating efficiency (77%). The early-passage cells were aneuploid (P 24) whereas the late-passage cells had a normal diploid karyotype with one balanced translocation between chromosomes No. 15 and No. 20 (P 224). Details of the karyotype suggest that the cells are heterozygous, i.e. derived from a stage before the first meiotic division. One of the two X chromosomes were inactive, and the cells expressed HLA antigens (A28 and B12), and beta 2-microglobulin. Expression of F9 antigen, characteristic of two-cell and later preimplantation embryos, was absent, while expression of PCC4 antigen, expressed also by blastocysts, was present. This finding suggests that the line might express some embryonic characteristics. The PA I cell line maintained in monolayer cultures showed several characteristics of malignant cells. The proportion of malignant cells increased with successive passages in vitro. The late-passage cells represented a fairly homogenous population of malignant cells similar to embryonal carcinoma cells. Late-passage PA I cells, when seeded under conditions that prevented attachment of cells to the substratum, formed embryoid bodies consisting of an inner core of cells similar to embryonal carcinoma cells, surrounded by a rind of endoderm-like cells. These two cell layers were separated by a basement membrane-like structure containing fibronectin. The core embryonal carcinoma cells expressed high alkaline phosphatase activity whereas the endoderm-like cells had low alkaline phosphatase activity. Embryoid bodies seeded on an adhesive substratum formed polycystic structures divided by layers of epithelial-like cells and containing extracellular fibrils similar to collagen type I or III. In these cultures, further limited differentiation into endoderm-like, epithelial-like cells and pigmented cells was observed. Morphological differenciation of undifferentiated PA I cells into endoderm-like cells in monolayer cultures could be obtained by treatment with BrdUrd or by plating in low serum concentration and at low density. Cells with characteristic fibrillar distribution of fibronectin and actin microfilament bundles were then observed, indicating formation of cells lacking properties of malignant cells. As indicated by these results, the PA I cell line, in spite of a limited capacity to differentiate in vitro, shares some of the properties of mouse teratocarcinoma cell lines and might therefore serve as a useful model for studies on some developmental mechanisms in human cells.