Rate of loss of tritiated thymidine label in basal cells in mouse epithelial tissues

A subpopulation of epithelial cells which retains a tritiated thymidine label (termed label-retaining cells, LRCs) has been previously demonstrated in skin and oral mucosae of mice and hamsters. To examine the rate of decrease in the number of LRCs and the changes in degree of labelling, young mice were labelled with tritiated thymidine and the rate at which label was diluted from basal keratinocytes assessed for up to 90 days. The number of LRCs in each tissue examined decreased from 15 to 90 days after labelling with the epidermal tissues maintaining a higher percentage of LRCs than the oral mucosae. Grain counts for LRCs in each tissue at each time period indicated that the number of silver grains overlying LRCs also decreased with time. The observed decrease in numbers of LRCs and the change in their degree of labelling with time suggest that such cells divide slowly, a property associated with stem cells.     

Rate of Loss of Tritiated Thymidine Label In Basal Cells In Mouse epithelial tissues

A subpopulation of epithelial cells which retains a tritiated thymidine label (termed label-retaining cells, LRCs) has been previously demonstrated in skin and oral mucosae of mice and hamsters. to examine the rate of decrease in the number of LRCs and the changes in degree of labelling, young mice were labelled with tritiated thymidine and the rate at which label was diluted from basal keratinocytes assessed for up to 90 days. the number of LRCs in each tissue examined decreased from 15 to 90 days after labelling with the epidermal tissues maintaining a higher percentage of LRCs than the oral mucosae. Grain counts for LRCs in each tissue at each time period indicated that the number of silver grains overlying LRCs also decreased with time. the observed decrease in numbers of LRCs and the change in their degree of labelling with time suggest that such cells divide slowly, a property associated with stem cells.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

CELL POPULATION KINETICS OF AN OSTEOGENIC TISSUE · II

A study of the cell kinetics on the actively growing periosteal surface of the femur of rabbits aged 2 weeks has been continued. A single injection of tritiated thymidine was given and the rabbits killed from 1 hour to 4 days after injection. The grain count spectra of the different cell types, pre-osteoblast, osteoblast, and osteocyte, have been compared at different times after injection. The results showed evidence for the uptake of thymidine in nuclei which is not associated with cell division. A small percentage of osteoblasts was initially labeled at 1 hour and there was evidence that the majority of these had not divided by 3 or 4 days after injection. Some thymidine-labeled cells had also become osteocytes without division. Furthermore, it appeared that a considerable fraction of the initially labeled pre-osteoblasts did not divide. The S period for the pre-osteoblasts and osteoblasts was measured using a double-labeled thymidine technique.     

Gill infection of Leporinus macrocephalus Garavello & Britski, 1988 (Osteichthyes: Anostomidae) by Henneguya leporinicola n. sp. (Myxozoa: Myxobolidae). Description, histopathology and treatment

Piau?us (Leporinus macrocephalus), were raised in 300 m2 ponds (density of 10 fish/m2) presenting asphyxia signals and daily mortality of 27 fishes. Specimens with 8-cm total body length, were collected for necropsy. Mucus of body surface and pieces of organs were collected and examined microscopically, in wet mounts, stained or in histological sections. The smears examination showed the presence of several spores in the secondary lamellae of the gill filaments, identified as Henneguya leporinicola n.sp (Myxozoa: Myxobolidae). Histopathological study showed epithelial hyperplasia and fulfilling of the spaces between the secondary lamellae, congestion and telangiectasia sinusoidal. It was also observed hyperplasia of the goblet cells and several cysts of parasite with 70.3 microns diameter. Such cysts were situated among the secondary lamellae, covered or not by the hyperplasic epithelium. With this diagnostic, three applications of formalin solution 10 ml/m3 were carried out. Fifteen days after that, fish were examined again to ascertain whether the treatment was efficient on disease caused by the protozoa. The tissue alterations present in the gills after the treatment were just a moderate sinusoidal congestion and a slight epithelial hyperplasia on the base of the secondary lamellae.     

Cell renewal in the epidermis of the loach Misgurnus anguillicaudatus (Cypriniformes)

Cell kinetics of the epidermal cells of normal juvenile loach ( Misgurnus anguillicaudatus ) were studied with autoradiography. Fish were labelled with single tritiated thymidine injections and killed at regular time intervals. Three cell types are identified by light microscopy, namely the epithelial cells, the club cells and the mucous cells. Epithelial cells are the only cell type that is involved in cell proliferation and, like the epithelial cells in the epidermis of other teleosts, proliferation of these cells occurs at all epidermal layers. The club cells and the mucous cells seem to be differentiated from the epithelial cells. Based on the time-course study of the labelling index and the grain count halving method, the generation time of the epithelial cells is estimated to be 4 days. From the labelling index of double injections, the duration of the S phase is determined as 8.3 h. Significant cell loss from the outermost layer and cell translocation from the lower layer to the upper layer within 4 days are inferred from the fluctuations of the labelling index curve. The renewal of these cells in the tissue seems rapid in comparison to the epidermis of terrestrial vertebrates.     

Mode of Filamentous Growth of Leucothrix mucor in Pure Culture and in Nature, as Studied by Tritiated Thymidine Autoradiography

Mode of growth of Leucothrix mucor filaments was measured by autoradiography with tritiated thymidine. Studies were performed on L. mucor in pure cultures in free suspension, as an epiphyte of pure cultures of the red alga Antithamnion sarniense, and as an epiphyte of red algae in the sea. Statistical analyses of the distribution of growing cells was done by use of the nonparametric One-Sample Runs Test and a Cluster analysis adapted from quadrat analyses of plant ecologists. No evidence of preferential growth at base or tip of L. mucor filaments was obtained in any of these studies. However, in nature, but not in the laboratory, there were regions of L. mucor filaments which were nongrowing or dormant. Such nongrowing regions could incorporate tritiated glucose.     

Ultrastructural autoradiographic and immunocytochemical analysis of setae formation and keratinization in the digital pads of the gecko Hemidactylus turcicus (Gekkonidae,Reptilia)

The modified subdigital scales of some lizards allow them to climb vertical surfaces. This is due to the action of millions of tiny setae present in the digital pads. Setae are mainly composed of beta-keratin which may have some modality of aggregation similar to that of barbs and barbules of feathers. Keratins and associated proteins are involved in the organization of setae. The formation of setae in the climbing pad lamellae of the gecko Hemidactylus turcicus has been analyzed under the electron microscope after injection of tritiated histidine and immunocytochemistry for a chick scale beta-keratin. Setae are made up of dense and pale filaments, both oriented along the longer axis of setae. Beta-keratin is present in the oberhautchen layer and in the growing setae which are highly modified oberhautchen cells. Most of the immunolabeling concentrated in the central part of setae. This cross-reactivity suggests that some epitopes in chick beta-keratin are also present in gecko setae. Four hours after injection of tritiated histidine, the labeling is localized over setae, in particular in the dense filaments and less in the pale filaments. Some labeling is also seen in the keratinaceous material present in the cytoplasm of clear cells, which are believed to mold setae. The present observations suggest that both beta-keratin and denser matrix proteins, possibly incorporating histidine, are packed into growing setae. These proteins may be mixed to form pale and dense filaments oriented along the longer axis of setae, a pattern resembling that of barb and barbule cells of feathers. The role of matrix material in the orientation of the deposited beta-keratin during setal outgrowth is discussed with the problem of barb and barbule differentiation in avian feathers.     

SOME MORPHOLOGIC AND KINETIC STUDIES OF THE DEVELOPING ERYTHROID CELLS OF THE COMMON GOLDFISH, CARASSIUS AURATUS

Developing erythroid cells of the goldfish Carassius auratus were obtained from kidney prints and from smears of the peripheral blood. All preparations were stained with the May-Grunwald Giemsa technique. Developing cells were divided into six different stages. The criteria used to stage the cells were degree of chromatin condensation, degree of basophilia, nuclear:cytoplasmic ratio, and cell shape. The morphology of the maturation sequence for erythroid cells in this organism was similar to that found by other workers in other non-mammalian vertebrates. Fish received intraperitoneal injections of tritiated thymidine, tritiated uridine or tritiated leucine so that the stages involved in DNA synthesis, RNA synthesis and protein synthesis could be determined by means of autoradiography. For the tritiated thymidine studies the per cent labeled cells per stage from four different series receiving 0.5, 1.0, 3.0 or 6.0 μCi/g body weight were pooled, since subjecting the average per cent labeled cells per stage at the lowest and at the highest dosages to Student's t-test showed no significant differences. In all four series the fish were killed 2 hr, 12 hr and daily, 1–8 days post-injection. The ³H-TdR studies showed that stages I-IV were engaged in DNA synthesis; they also showed that about 5 days were required for the stage V cell to become a mature erythrocyte (stage VI cell). Tritiated uridine was injected at a dosage of 5.0 μCi/g body weight and animals were killed 1/2, 1, 3 and 6 hr post-injection. Grain counts showed that stages I-IV are engaged in RNA synthesis and that the rate of this synthesis decreased as maturation proceeded. Tritiated leucine was administered at a dosage of 5.0 μCi/g body weight, and fish were killed 45 min and 3 hr post-injection. Grain counts indicate that stages I-V are engaged in the synthesis of protein (assumed to be globin). The fact that DNA and RNA synthesis ceased with stage IV cells while protein synthesis continued into stage V cells indicated that the mechanism responsible for protein synthesis in stage V cells was produced at an earlier stage and was self-sustained for about 5 days.     

CELL PROLIFERATION IN THE RAT KIDNEY INDUCED BY FOLIC ACID

The changes in proliferative activity of tubular epithelial cells of the rat kidney following a single injection of folic acid (250 mg/kg body weight) have been studied. Autoradiography with tritiated thymidine revealed a large increase in numbers of labelled cells, beginning at about 18 hr, in each of the three kidney zones examined. In the cortex the maximum increase in labelling index (16 times normal) was found at 36 hr whereas that of the outer medulla (34 times normal) occurred at 24 hr; there was no clearly defined peak in the inner medulla, values of up to 36 times normal being found between 24 and 96 hr. These changes were followed several hours later by similar changes in mitotic index in the corresponding zones. All the indices, except the mitotic index of the inner medulla, had returned to normal by 6 days. Comparison of the curves of labelling index and mitotic index in each zone indicated that the number of cells induced to synthesize DNA was approximately similar to the number of cells which subsequently underwent mitosis. A large increase was also found in the specific activity of DNA extracted from homogenates of whole kidneys from folic acid-injected rats, again using tritiated thymidine as label. The increase began at about 18 hr, reached a maximum of 16 times normal at 32 hr and returned to normal by 6 days. These changes were similar to those of labelling index in the cortical zone.     

THE EFFECT OF CYTOSINE ARABINOSIDE IN VITRO ON AGAR COLONY FORMING CELLS AND SPLEEN COLONY FORMING CELLS OF C57BL MOUSE BONE MARROW

This study reports the effect of cytosine arabinoside in culture on two classes of bone marrow progenitor cells in C57BL mice, agar colony forming cells (ACU) and spleen colony forming cells (CFU). Both normal cells and rapidly proliferating cells were studied. The results show that in normal mice, 23 % of ACU but only 7 % of CFU are killed following 1 hr incubation with the drug. With longer periods of incubation, the survival of ACU in the controls is poor, and the results for the drug-treated cultures suggest that the cells are held up in cycle. In continuously irradiated mice, the proportion of ACU and CFU killed after 1 hr incubation with drug is increased to 43–54%, confirming previous results that these cells are proliferating more rapidly than in normal mice. In mice treated with myerlan, 54 % of ACU are killed by 1 hr in vitro exposure to cytosine arabinoside, again confirming that ACU are rapidly proliferating. However, the proportion of CFU killed is lower (23 %). These results are compared with other studies of the effect of cytosine arabinoside in vivo and also with thymidine suicide in the same strain of mice. The results show that cytosine arabinoside has the same effect as tritiated thymidine, and also that the proportion of CFU killed by these agents in vitro is lower than when the agents are injected in vivo. It is suggested that the conditions in culture have an adverse effect on CFU, which cease DNA synthesis, and are protected from the killing effect of cytosine arabinoside and tritiated thymidine. Since cytosine arabinoside in vitro has an effect similar to tritiated thymidine in vitro on bone marrow progenitor cells in C57BL mice, in vitro incubation with cytosine arabinoside could be an alternative method to thymidine suicide for measuring differences in cell proliferation rate.     

Observations on the endogenous stages of Eimeria crandallis in domestic lambs (Ovis aries)

Lambs reared coccidia-free were inoculated orally with various numbers of sporulated oocysts of E. crandallis and were killed between 1 and 22 days after inoculation; tissues were examined histologically. Sporozoites were seen 1, 2 and 3 days after inoculation (DAI) in crypt epithelial cells in the mid-jejunum. Infected cells migrated into the lamina propria where the parasite within them developed into a firstgeneration meront containing about 250,000 merozoites at 10 DAI. A second generation of meronts was seen at 10–12 DAI, each containing up to about 10 merozoites, situated mainly at the bases of crypts in the jejunum and ileum but also in the caecum. From 11 DAI pro-gamonts were seen which were enveloped by the host cell nucleus and which divided in synchrony with the host cell for an undetermined number of generations. Mature gamonts began to develop from them by 16 DAI. Oocyst output began at 16 DAI and rose to a peak at about 22 DAI. Up to 10⁸ oocysts were produced per oocyst inoculated. They showed wide variation in size and colour.     

Observations on the endogenous stages of Eimeria crandallis in domestic lambs (Ovis aries)

Lambs reared coccidia-free were inoculated orally with various numbers of sporulated oocysts of E. crandallis and were killed between 1 and 22 days after inoculation; tissues were examined histologically. Sporozoites were seen 1, 2 and 3 days after inoculation (DAI) in crypt epithelial cells in the mid-jejunum. Infected cells migrated into the lamina propria where the parasite within them developed into a firstgeneration meront containing about 250,000 merozoites at 10 DAI. A second generation of meronts was seen at 10–12 DAI, each containing up to about 10 merozoites, situated mainly at the bases of crypts in the jejunum and ileum but also in the caecum. From 11 DAI pro-gamonts were seen which were enveloped by the host cell nucleus and which divided in synchrony with the host cell for an undetermined number of generations. Mature gamonts began to develop from them by 16 DAI. Oocyst output began at 16 DAI and rose to a peak at about 22 DAI. Up to 10⁸ oocysts were produced per oocyst inoculated. They showed wide variation in size and colour.     

Life Cycle of Eimeria christenseni Levine,Ivens & Fritz, 1962 from the Domestic Goat,Capra hircus L.1

The endogenous development of Eimeria christenseni was studied in 10 two- to four-week-old kids inoculated with 10⁶-10⁷ sporulated oocysts. They were killed at intervals from two to 26 days after inoculation, and their tissues were examined for endogenous stages of the coccidian by light microscopy. Such stages were found in the small intestine and mesenteric lymph nodes. In the sexual cycle, two generations of meronts were found. The first generation developed in endothelial cells of lacteals in the jejunun and ileum and mesenteric lymph nodes, and mature meronts were first seen 14 days after inoculation. The second generation developed in epithelial cells of the glands of Lieberkuehn in the jejunum and ileum and in mesenteric lymph nodes, and its mature meronts were first seen by 16 days. Sexual stages were present mostly in epithelial cells of the tips and sides of the villi and less frequently in crypt cells of the jejunum and ileum. Mature macrogametes and microgamonts and oocysts were also first seen by 16 days. The prepatent period was 17 (14-23) days; the patent ranged from 8 to more than 30 days. Sporulation time was 3-4 days at 30°C. E. christenseni was found to be pathogenic, kids inoculated with 1-5 × 10⁵ sporulated oocysts exhibited the following signs: severe diarrhea, anorexia, polydipsia, poor hair coat, and extreme weakness. They recovered about a month later, but their growth rates appeared to be lower than those of uninoculated animals kept under the same conditions. One kid died 20 days after inoculation with 10⁷ oocysts.     

Histopathological alterations and induction of cytochrome P-450 1A in the liver and gills of the gilthead seabream (Sparus aurata) exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated in the seabream Sparus aurata specimens. Liver presented hepatocytic alterations, with an increase of lipid droplets and glycogen granules. Ultrastructural modifications of hepatocytes included RER fractionation, glycogen augmentation, as well as a rise in the number of lipid droplets, vacuoles and secondary lysosomes. In the gills, secondary lamellar epithelium showed hyperplasia, hypertrophy and lamellar fusion on the edge of the filaments. At the end of the exposure period (1 pg1(-1) TCDD for 20 days), some organelles in epithelial cells of the secondary lamellae and the tubular system of the chloride cells appeared altered. In the liver of TCDD-exposed specimens, immunoreactive cytochrome P-450 1A was concentrated close to the cytoplasmic and nuclear membranes, and positive granules were also evident throughout cytoplasm of the hepatocytes. Significant cytochrome P-450 staining was especially evident in endothelium of the hepatic vascular system. At the beginning of the exposure (1 pg 1(-1) TCDD, for 5 and 10 days), cytochrome P-450 immunostaining was observed in the cytoplasm of scarce hepatic cells and after 20 days of treatment, specific immunostained cytoplasmic granules were detected in most hepatocytes. In gills of TCDD-treated specimens, pillar-endothelial cells showed a cytochrome P-450 1A immunostaining concentrated close to the base of gill filaments and dispersed through the gill lamellae. There was also significant cytochrome staining of the endothelium of the branchial vascular system. However, no cytochrome immunoreactivity was observed in epithelial-respiratory cells.     

THE PROLIFERATIVE STATUS OF INTESTINAL EPITHELIAL CLONOGENIC CELLS: SENSITIVITY TO S PHASE SPECIFIC CYTOTOXI CAGENTS

High concentrations of tritiated thymidine and cytosine arabinoside (Ara-C) have been used to selectively kill cells in the crypts of Lieberkuhn that are synthesizing DNA. The effect of these agents on the number of regenerating microcolonies seen 3-J days after a range of radiation doses indicates that a majority of the clono-genic cells are proliferating rapidly and that the slowly proliferating cells at the base of the crypt do not represent the whole clonogenic population.     

The proliferative status of intestinal epithelial clonogenic cells: sensitivity to S phase specific cytotoxic agents.

High concentrations of tritiated thymidine and cytosine arabinoside (Ara-C) have been used to selectively kill cells in the crypts of Lieberkuhn that are synthesizing DNA. The effect of these agents on the number of regenerating microcolonies seen 3 1/2 days after a range of radiation doses indicates that a majority of the clonogenic cells are proliferating rapidly and that the slowly proliferating cells at the base of the crypt do not represent the whole clonogenic population.     

Scanning electron microscopy of the gills of a freshwater catfish,Rita Rita

Variations in the gross morphology and surface architecture of the gill filaments and secondary lamellae of a freshwater catfish (Rita rita) have been investigated using scanning electron microscopy. Heterogeneity of the gill has been correlated with the distribution of lamellar water-flow at different regions of a gill filament. Higher lamellar water flow (cc/pore/cmH₂O/sec) was estimated for the middle region of the filaments. The filaments are covered with epithelial cells whose surface is provided with well-developed microridges. The lamellae are generally covered with microvillous epithelial cells. The variations in surface architecture of the gill filaments and secondary lamellae have been correlated with their probable functions.     

Deoxyribonucleic Acid synthesis in root cap cells of cultured roots of convolvulus

Isolated cultured roots of Convolvulus arvensis L. were incubated in 0.2 microcurie per milliliter methyl-³H-thymidine for 14 hours, for 64 hours, or for 14 hours followed by transfer to fresh nutrient medium without tritiated thymidine. Autoradiographs of serial, longitudinal sections of roots which were continuously incubated with tritiated thymidine showed that cells of the root cap columella did not undergo DNA synthesis after their formation from the root cap initials. In roots pulse-labeled with tritiated thymidine, the movement of labeled cells through the root cap columella was followed. Labeled cells were displaced at a constant rate of 72 microns per day over a period of 6 to 9 days before they were sloughed off from the root cap. The specialized role of the root cap cells in relation to their distinctive metabolism and longevity is discussed.     

Progenitor cells of the definitive erythroid series in the chick embryo

Chick embryos, developing in ovo, were treated with methyllabeled tritiated thymidine at 3 days. Definitive erythroid cells isolated from such embryos at 6 days had incorporated tritiated thymidine into nuclear DNA. Progenitor cells for the definitive erythroid cell series appear, therefore, to exist in the embryo prior to the initiation of definitive erythropoiesis and are capable of DNA synthesis.