Subdivision of the mitotic cycle into eleven stages, on the basis of the chromosomal changes observed in mouse duodenal crypt cells stained by the DNA-specific Feulgen reaction.

The Feulgen reaction has been utilized to localize DNA in nuclei throughout the cycle of mouse duodenal crypt cells using Epon-embedded 1 micron thick sections. The observed changes indicate that the 12.3 h long mitotic cycle of these cells can be subdivided into eleven stages, seven of which take place during the interphase. Computer measurements of Feulgen-stained nuclei and previous radioautographic studies indicate that DNA synthesis begins during stage I and ends during stage IV. The staining pattern shows no distinctive feature in the nuclei of the 1.5 h long stage I. Thereafter, marked changes occur during the rest of the interphase--that is during the 6.3 h that precede karyokinesis and the 3.5 h that follow it. Thus, at stage II the background of the nuclei darkens; at stage III, there appear stained threads interpreted as densifying chromosomes and dots interpreted as chromomeres, both of which thicken from 0.2 to 0.4 micron; at stage IV they further thicken to about 0.5 micron and at stage V, to about 0.7 micron. At this stage, which approximately corresponds to prophase, the intensely stained, discrete dots are localized within the less intensely stained sausage-shaped threads. As the breakup of the nuclear envelope introduces stage VI, whose early part corresponds to prometaphase, the intensely stained dots become close to one another within the threads and eventually fuse. The staining of the threads thus intensifies, and, by the late part of the stage that corresponds to metaphase, they have become the homogeneously dense metaphase chromosomes. At stage VII, the anaphase chromosomes reach each pole where they associate into a compact mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of apical tubules in endocytosis in nonciliated cells of the ductuli efferentes of the rat: a kinetic analysis.

The apical region of nonciliated cells of the ductuli efferentes of the rat contains tubular coated pits (TCP) connected to the apical plasma membrane, apical tubules (AT) which occasionally show a partial coat, and endosomes which are often continuous with one or more apical tubules. To investigate the formation and fate of TCP and AT, a quantitative analysis was performed on the labeling indices of these structures at various time intervals (0.5-120 min) after a single injection of a tracer, cationic ferritin (CF), into the lumen of the rete testis. The labeling indices of both TCP and AT exhibited similar cyclical patterns, first reaching a peak at 25 min, then dropping to a minimum at 35 min, then rising to a second peak at 60 min. Since TCP were well labeled at 30 sec while AT were not, the tracer must rapidly enter TCP and thence AT. However, since tracer was virtually absent from the lumen by 30 min, it was not possible to reconcile the second peak of labeling index of TCP and AT by this mechanism. In another experiment, rats were injected once as before, injected again at 30 min, and then sacrificed at 30 min following the second injection. The results from this experiment showed that the labeling index of TCP and AT did not drop but was similar to that of the 60-min peak after a single injection. The interpretation is that there was recycling of tracer, which had already migrated from TCP to AT to endosomes, back to the apical plasma membrane via apical tubules. Moreover, when rats were injected once, injected again at 30 min, and sacrificed 3 min following the second injection, the labeling index for TCP and AT was significantly higher (P less than .05) than at the 30-min time interval after a single injection, indicating that recycled apical tubules were functionally capable of binding further CF. Morphological observations on images of transition between TCP and AT and the fact that AT were often found connected to endosomes suggest that TCP detach from the cell surface to give rise to AT, which in turn fuse to form endosomes. The kinetic analysis demonstrates in quantitative terms that a portion of the AT, which fuses to form endosomes, recycles back to the apical plasma membrane and contributes to the formation of new TCP.

Cyclic endocytic activity and kinetics of lysosomes in Sertoli cells of the rat: a morphometric analysis.

Native ferritin was injected into the rete testis of rats, and seminiferous tubules infused with the tracer were collected 6 h later and prepared for electron microscopic analysis. As a result of internalization of the tracer by Sertoli cells, label was found within 12-66% of the secondary lysosomes, depending on the stage of the cycle of the seminiferous epithelium. The Zeiss MOP-3 instrument was used on selected electron microscope photographs to measure a number of morphometric parameters. Applying appropriate formulae and a computerized program, it was possible to determine the absolute numbers of labeled and unlabeled secondary lysosomes per Sertoli cell for each one of the 14 stages of the cycle. Knowing the duration of these stages, it was also possible to evaluate the turnover kinetics and life span of lysosomes for each stage of the cycle. The percentage of ferritin-labeled lysosomes, regarded as an index of the endocytic activity of Sertoli cells, remained low in stages II to VIII, increased abruptly during stage IX, stayed high during stages X to XIV, and decreased to a low level during stage I of the following cycle. Correspondingly, the turnover of secondary lysosomes was relatively slow and their life span relatively long during stages II through VIII, while the turnover of lysosomes was faster and their life span shorter during stages X through XIV-I of the cycle. During stage IX, there was a sharp drop in the number of lysosomes per Sertoli cell associated with a fast rate of disappearance and a remarkably short life span of less than 4 h for the lysosomes. These features, characteristic of stage IX, are explained by the rapid fusion of lysosomes with residual bodies, which are phagocytosed by Sertoli cells at this particular stage of the cycle. The accelerated endocytosis taking place during stages IX through XIV of the cycle may explain the reduction of the surface area of the adluminal plasma membrane of Sertoli cells as well as the reduction in volume of the tubular lumen observed during these stages. Thus, the demonstrated cyclic endocytic activity of Sertoli cells and several other cyclical events taking place within seminiferous tubules correlate well.

Analysis of gland cell behavior: appendix to the dynamic histology of the antral epithelium in the mouse stomach.

The origin, route of migration, and ultimate fate of mucous gland cells in mouse antral units have been described in the preceding article. Three regions of the mucous gland, consisting of gland neck cells, mid-gland cells, and gland base cells, respectively were defined; and the renewal events in each of these regions were studied. In this article, the data obtained by Lee and Leblond (1985) were subjected to a mathematical analysis to obtain additional information about the rate of loss of cells. Calling each anatomical division a compartment, and utilizing the available histometric knowledge concerning the number of cells and the turnover of labeled cells in each compartment, a set of equations was developed, assuming a steady state, for which solutions yielded the number of cells lost from each compartment per unit time.

Assessment of resolution by half distance values for tritium and radioiodine in electron microscopic radioautographs using Ilford L4 emulsion developed by "solution physical" or D-19b methods.

Half distance values for electron microscopic (EM) radioautographs with the isotopes 3H and 125I were determined using Ilford L4 emulsion processed with either fine grain, solution physical development, or filamentous grain, chemical development with D-19b. 3H- and 125I-line sources, obtained by cutting perpendicular sections from sections of 3H-labeled methacrylate or 125I-labeled thyroid glands, were processed for EM radioautography. The distribution of silver grains around a line source was determined by measuring their distance from the source in photographs of EM radioautographs. The number of silver grains per unit distance from the line source was plotted on graphs and half distance values were calculated. With solution physical development, the half distance value was 74 nm for 3H and 80 nm for 125I; whereas with D-19 b development it was 187 nm for 3H and 157 nm for 125I. Since solution physical development produced a reduction of about 50% in the half distance values for both isotopes, it is concluded that the production of fine grain by this method provides better resolution for EM radioautography than filamentous grain development with D-19b.

Quantitation and resolution in electron microscope radioautography.

Electron microscopy can resolve structures with accuracy of the order of 0.2-0.3 nm, but the radioautographic technique is able to locate radioactive label with much less certainty, at best 50 nm. This difference in resolution is what creates the problem of quantitation in electron microscope radioautographs.

Polypeptide hormone binding sites in vivo: initial localization of 125I-labeled insulin to hepatocyte plasmalemma as visualized by electron microscope radioautography.

The rationale of the specific-binding assay was applied to the detection of the liver insulin receptor in vivo. Quantitative electron microscope radioautography indicated that, 3 min after an intraportal injection, 125I-insulin was exclusively located to the hepatocyte plasmalemma.

Renewal of the epithelium in the descending colon of the mouse. IV. Cell population kinetics of vacuolated-columnar and mucous cells.

Proliferation and migration of cells in the vacuolated-columnar and mucous cell lines were studied in the descending colon of adult female mice given a single injection or a continuous infusion of 3H-thymidine and killed at various intervals from one hour to 12 days. This investigation was carried out using one mum-thick Epon sections which were radioautographed after staining with the periodic acid-Schiff technique and iron-hematoxylin. In the normalized crypts with ten equal segments, labeled vacuolated cells at one hour after injection of 3H-thymidine were encountered in the lower four segments and in decreasing numbers in segments 5 through 7. From the percent labeled cells in segments of the crypt, the birth rate and fluxes of cells were computed. Moreover, it was found that a cell in the vacuolated-columnar cell line would undergo three mitotic cycles on the average from its birth at the cryptal base to its extrusion from the surface; of these three cycles, the last one which took place from segment 3 to segment 7 appeared to be a changeover from dividing cells to non-dividing cells, in accordance with the "slow cut-off" model of Cairnie et al. ('65b). Mucous cells located in segments 1 through 6 of the crypt were capable of incorporating 3H-thymidine and thus capable of undergoing mitosis. However, the rate of turnover of mucous cells based on proliferative rate was found to be much lower than the rate of turnover of mucous cells based on the transit time in the non-dividing segments of the crypt. Since there was a concomitant overproduction of cells in the vacuolated cells and newly formed mucous cells in the lower portion of the crypt, it was concluded that some vacuolated cells would give rise to mucous cells. This putative transformation occurred in the lower four segments of the crypt. Mucous cells which were formed by transformation would migrate upward along the cryptal wall and accumulate more mucus in the theca; in doing so, they would undergo two divisions, on the average, before they became non-dividing mucous cells. In ascending the cryptal walls, both vacuolated-columnar cells and mucous cells appeared to migrate at a similar speed; they moved much slower at the base of the crypt and accelerated toward the upper portion of the crypt, but they migrated at a constant speed in the non-dividing segments of the crypt.

[Thyroid pigments in Gunn rats]. / Observations sur le pigment thyroïdien chez les rats de Gunn

The structural and histochemical characteristics of the thyroid pigment in homozygous Gunn rats were examined. The pigment occurs in the form of numerous yellow granules in the cytoplasm of the follicular cells. The focal depositions of the pigment were also seen in the central part of the luminal colloid. However, the pigment granules were not present in the light or parafollicular cells. The main histochemical properties of the pigment are that it is basophilic, PAS-positive, acid-fast and reducing toward alcaline silver nitrate and ferricyanide. Besides, it was easily bleached with oxidizing agents. It was deduced that the pigment was a lipofuscine. Ultrastructurally, pigment bodies are characterised by an electron dense content, and a smooth surface single limiting membrane. They resembled lysosomes or peroxisomes. It was concluded that the pigment granules visible at the light microscopic level resulted frim the accumulation of the pigment substance in the preexisting lysosomes.

Radioautographic study of in vivo and in vitro incorporation of fucose-3H into thyroglobulin by rat thyroid follicular cells.

The incorporation of fucose-(3)H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-(3)H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-(3)H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-(3)H. At 3-5 min following fucose-(3)H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.

Radioautography of noradrenaline-14-C in atopic dermatitis.

Since storage-time of administered noradrenaline in the skin of patients with atopic dermatitis may be prolonged, it would be of interest to demonstrate the site of uptake of noradrenaline-(14)C in atopic dermatitis as compared with other eczematous and normal skins. Two adult patients with longstanding atopic dermatitis, a patient with contact dermatitis to nickel and one with normal skin, were studied. Identical sites in the four patients were injected intradermally with 0.02 mug. DL-noradrenaline-7-(14)C acetate. An 8-mm. punch biopsy of the injected site was performed 24 hours later. Radioautographs were developed between three and 199 days, according to the technique of Kopriwa and Leblond.(2) At 199 days, the number of grains in atopic dermatitic skin was greater than in contact dermatitis or normal skin. There was a concentration of grains over arrectores pilorum muscles and the upper one-third of the epidermis of atopic skin. Grains were also visible in proximity to arteriolar walls. There were few grains visible in the control sections. The results confirm earlier studies suggesting that atopic dermatitic skin retains noradrenaline longer than other dermatoses. Noradrenaline concentrates in the arrectores pilorum muscles and the upper epidermis. These findings may explain the cutis anserina (goose-flesh) appearance in atopic dermatitis.