Transsulphuration and methylation of homocysteine in control and mutant human fibroblasts

The ability of human skin-fibroblasts in monolayer culture to carry out transsulphuration and remethylation of homocysteine has been tested. The conversion of homocyst(e)ine to cyst(e)ine and methionine was studied in control and mutant cells by incubation for 16 h with l-[³⁵S]homocystine. Labelled cysteic acid and methionine sulphone were found in hydrolysates of oxidized cell proteins. The quantities found were dependent on the time of incubation and were used as a measure of cyst(e)ine and methionine formation, respectively. In control cells, labelled cyst(e)ine and labelled methionine were found. In cystathionine β-synthase-deficient cell lines, labelled cyst(e)ine formation was reduced, while labelled methionine formed was similar to that of controls, indicating the role of transsulphuration in the formation of cyst(e)ine observed in control cells. In a 5,10-methylenetetrahydrofolate reductase-deficient cell line, labelled methionine formation was reduced, indicating the role of N-5-methyltetrahydrofolate-requiring methylation of homocysteine in the formation of methionine observed in control cells.     

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

Summary The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat¹²⁵I-atrial natriuretic peptide_(1–28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.     

Transplanted interleukin-4--secreting mesenchymal stromal cells show extended survival and increased bone mineral density in the murine femur

Background

Mesenchymal stromal cell (MSC)–based therapy has great potential to modulate chronic inflammation and enhance tissue regeneration. Crosstalk between MSC-lineage cells and polarized macrophages is critical for bone formation and remodeling in inflammatory bone diseases. However, the translational application of this interaction is limited by the short-term viability of MSCs after cell transplantation.

Somatic embryogenesis of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> (L.): Histological and ultrastructural changes in seedling hypocotyl explant

Summary Structure and ultrastructure changes that occurred during tissue culture of upper explants of hypocotyl (adjacent to cotyledons) of 10-d-old seedlings of Gentiana cruciata were studied. The explants were cultured on Murashige and Skoog induction medium supplemented with 1.0 mg l⁻¹ dicamba +0.1 mg l⁻¹ naphthaleneacetic acid +2.0 mg l⁻¹ benzyladenine +80.0 mg l⁻¹ adenine sulfate. The initial response of the explant and callus formation were ultrastructurally analyzed during the first 11 d of culture. After 6–8 wk, various methods were employed to collect evidence of indirect somatic embryogenesis. After 48 h of culture, the earliest cell response was cell division of epidermis and primary cortex. There were numerous disturbances of karyo- and cytokinesis, leading to formation of multinuclear cells. With time, the divisions ceased, and cortex cells underwent strong expansion, vacuolization and degradation. About the 6th day of culture, callus tissue proliferated and the initial divisions of vascular cylinder cells were observed. Their division appeared normal. Cells originating from that tissue were small, weakly vacuolated, with dense cytoplasm containing active-looking cell organelles. Numerous divisions occurred in the vascular cylinder, which led to its expansion and the formation of embryogenic callus tissue. During the 6–8th wk of culture, in the proximal end of the explant, masses of somatic embryos were formed from outer parts of intensively proliferating tissue.     

Transdifferentiation of outgrowth cells and cultured epithelial cells from swine trachea

Summary The morphologic and functional properties of explant out-growth cells and epithelial cells isolated from swine trachea epithelium by proteolysis were examined. A mixed population of ciliated, serous, and basal cells, obtained from out-growths, from proteolysis of trachea epithelium, and from unattached explants in organ culture, all yielded cell cultures that were composed almost entirely of mucus-secreting cells. When the cells were grown in primary or secondary culture on a modified collagen matrix in supplemented HAM:DMEM (1:1) medium they expressed a mucus-secreting phenotype with numerous mucus granules at various stages of maturation and incorporated [³H]GlcN and³⁵SO₄ into secreted mucin glycoproteins. Results obtained in these studies suggest that extensive transdifferentiation of ciliated and serous cells to mucus-secreting cells occurs after the release and during subsequent attachment and culture. Ciliated cells containing mucus granules were seen in various stages of cilia resorption. Basal cells containing mucus granules were also frequently observed. The number of mucus-secreting cells and the synthesis of mucin glycoproteins increased dramatically with time of attachment and culture, whereas cell proliferation, population doubling time of 72 h, and incorporation of [³H]thymidine into DNA increased much more slowly. The number of mucus-secreting cells correlated closely with the level of secretion of mucin glycoproteins. Taken collectively, these studies help to elucidate the transdifferentiation process, which dramatically increases the number of mucus-secreting cells after disruption and release of epithelial cells from swine tracheobronchial epithelium. A similar mechanism involving disruption of the extracellular matrix may be involved in the stimulation of hypersecretion of mucus and mucin glycoproteins by chemical and infections irritants.     

The role of myonuclei in muscle regeneration: An in vitro study

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or “freed” as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H³-thymidine and their ultimate fate, either as “free” myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.     

Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system

Here, we evaluated the efficiency of shoot multiplication of Vanilla planifolia Jacks. ex Andrews using solid medium, partial immersion, and a temporary immersion system (TIS) to improve micropropagation in this species. Clusters of shoots were cultivated in vitro using Murashige and Skoog (MS) medium supplemented with 9.55 μM benzyladenine (BA) and 100 mL L^?1 coconut water. For the TIS, a RITA^® system was used and three immersion frequencies were evaluated (every 4, 8, and 12 h) with an immersion time of 2 min. After 30-d culture, the TIS produced the maximum multiplication rate (14.27 shoots per explant) when using an immersion frequency of 2 min every 4 h, followed by the partial immersion system (8.64 shoots per explant), and solid medium (5.80 shoots per explant). Next, the effect of the volume of culture medium per explant was also evaluated for TIS. The most suitable volume of culture medium for shoot formation was 25 mL per explant, which increased the rate of multiplication to 17.54 shoots per explant. Root initiation was 90% successful in TIS using half-strength MS medium supplemented with 0.44 μM naphthaleneacetic acid (NAA) and an immersion frequency of 2 min every 4 h. With this system, the shoot multiplication rate increased threefold compared to that obtained with solid medium. In addition, this system produced good results for the transplantation and acclimation (90% of survival) of in vitro-derived plants. These results offer new options for large-scale micropropagation of vanilla.     

Explants of porcine coronary artery in culture: A paradigm for studying the influence of heparin on vascular wall cell proliferation

Explant cultures of porcine coronary artery provided a coculture model, used as a paradigm of arterial wall in contact with vascular prosthesis which allowed the study of spatial and temporal changes in cell phenotype. First cells emerging from the explant had an endothelial phenotype monitored by cytoimmunostaining. Percentages of anti-smooth muscle α-actin labelled cells were assessed at early and late phase by flow cytofluorometric analysis to control the effect of heparin. At 100 μg ml^-1, no effect on α-actin labelled cell growth has been detected. This result contrasted with the inhibition of monolayer cell cultures. At 500 μg ml^-1, the proliferation of smooth muscle cells was reduced. This explant system should be useful for testing drugs susceptible to interfere with restenosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quantitative analysis of composite umbilical cord tissue health using a standardized explant approach and an assay of metabolic activity

Background

Umbilical cord (UC) tissue can be collected in a noninvasive procedure and is enriched in progenitor cells with potential therapeutic value. Mesenchymal stromal cells (MSCs) can be reliably harvested from fresh or cryopreserved UC tissue by explant outgrowth with no apparent impact on functionality. A number of stem cell banks offer cryopreservation of UC tissue, alongside cord blood, for future cell-based applications. In this setting, measuring and monitoring UC quality is critical.

Analysis of rat adrenal cells in primary culture separated by velocity sedimentation

Summary Cell separation was used to follow the fate of the cortical cells of the adrenal gland in primary culture, and to assess some of the changes that occur as cells adapt to culture conditions. Primary cultures of rat adrenal gland were dissociated with trypsin and separated by velocity sedimentation at unit gravity. After two days in culture, cells showed a reproducible sedimentation profile consisting of two classes of cells with mean sedimentation rates of 5.8 and 2.1 mm/h, and a third sedimentation peak consisting mainly of nuclei at 0.5mm/h. All populations continued to incorporate ³H-thymidine in relatively constant proportion throughout the culture period, but the relative number of cells in the 2.1 mm/h peak increased two-fold in the last few days of primary culture. Cells labelled in primary culture, but separated after an additional 5 days in secondary culture had lost proportionately more labelled cells from the 5.8 mm peak. The results suggest that cells of the 2.1 mm peak survive longer in culture in a post-replicative condition.Work reported in this paper was performed while the author was a Research Fellow of the National Cancer Institute of Canada in the laboratory of Dr. N. Auersperg, Cancer Research Centre, University of British Columbia, Vancouver, B.C., Canada     

Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells

Background and Aims

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula.

Methods

Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression.

Key Results

Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature.

Conclusions

The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.Key words: Callus, dedifferentiation, leaf veins, Medicago truncatula, pluripotency, procambium, protoplasts, reactive oxygen species, SERK, somatic embryogenesis, stem cells, totipotency     

An autoradiographic and cytophotometric study of oogenesis in a pulmonate snail,Planorbarius corneus

Summary The spatial and temporal patterns of macromolecular syntheses in oocytes and somatic auxiliary cells of the snail Planorbarius corneus have been investigated by autoradiography and cytophotometry. Oogenesis has been divided into three stages, comprising early meiosis up to diplotene (stage I), previtellogenetic growth phase (stage II), and vitellogenesis (stage III). No DNA synthesis was found in any oocyte stage. In stage-I oocytes, only nucleoli were found labelled with ³H-uridine. Oocyte nuclei of stage II and III actively synthesize RNA in nucleoli and chromosomes. The most intense incorporation of uridine in chromatin probably occurs during the previtellogenesis — vitellogenesis transition period during which cytological findings suggest well developed lampbrush chromosomes. RNA synthesis in amphinucleoli of stage-III oocytes is restricted to basophilic nucleolar parts, whereas acidophilic parts (protein bodies) neither synthesize nor store RNA. During vitellogenesis oocytes incorporate amino acids into yolk platelet proteins. Radioactive proteins are found in yolk platelet precursors 5 h after injection of the tracer and in yolk platelets 3 h thereafter. The labelling pattern suggests that oocytes synthesize certain hitherto unidentified yolk components. No evidence for the participation of follicle cells in synthesis and transport of vitellogenic proteins has been obtained from autoradiography. Cytological findings suggest an important role for these cells in oogenesis. They are highly active in RNA and protein synthesis. Cellular differentiation is accompanied by polyploidization of the nuclei which attain a highest DNA content of 256 c. Polyploidization probably occurs in incremental steps as indicated by complete endomitotic chromosomal cycles. Autoradiographs show that, during vitellogenesis, oocytes do not incorporate significant amounts of glucose, and only certain follicle cells were labelled with glucose, probably indicating the synthesis of glycogen.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

Abstract. The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm^-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 /iCi tritiated thymidine ([³H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [³H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a sixfold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10–12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [³H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [³H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Long-term culture of human esophageal explants and cells

Human esophageal epithelium obtained from intermediate autopsies (<12 h) was maintained as cell and explant cultures. In order to develop a serum-free, defined media culture model, several medias and additives were evaluated. The viability and differentiation of the epithelial cells cultured with serum-free, Keratinocyte Growth Media (KGM, Clonetics Co., San Diego, CA) was improved over that of esophageal cells and explants cultured in either serum-supplemented CMRL 1066 (OCM), serum-free additive-supplemented CMRL 1066, or cimetidine-supplemented CMRL 1066. The KGM component EGF was determined to be trophic for esophagus cells on the basis of findings of increased ³H-TdR tabelling in KGM cultures when compared to control cells grown in KGM without EGF (KBM). The morphologic pattern of the cytoskeletal proteins actin, keratin, and vimentin were characterized in isolated cell populations. The intermediate filaments, keratin, and vimentin were co-expressed in these epithelial cells. Esophageal explant viability, differentiation, and outgrowth from 15 cases were also evaluated in dishes coated with basement membrane associated proteins. Explants cultured in these dishes were equally well-preserved and differentiated. There were no significant differences in the explant histology when there was protein coating of the culture dishes, although one case showed improved outgrowth with laminin coating. A main advantage for using this culture system is that the same medium (KGM) can be used for both the culture of explants and isolated epithelial cells. Future applications of this model include determining: (1) the effect different concentrations of EGF and calcium in the media will have on esophageal proliferation and differentiation, and (2) the role of different basement membrane associated proteins on the plating efficiency of either isolated or outgrowth epithelial esophageal cells.This is publication #2544 from the Pathobiology Laboratory.     

Characteristics of the chemostat culture of murine leukemia L 1210 cells

Mouse leukemia L 1210 cells were cultivated under glucose limitation in a chemostat. More than 20 steady-states were established over 9 different dilution rates ranging from 0.20 day⁻¹ (cell doubling time 83 h) to 2.0 days⁻¹ (cell doubling time 8.3 h). The steady-states were characterized by: a constant cell number, constant cell volume, constant concentrations of DNA, RNA, and L-lactate (in the culture supernatant), a constant percentage of cells labelled by autoradiography, and constant rate of incorporation of [³H]TdR, [³H]uridine, and ¹⁴C-labelled amino acids into cellular acid-precipitable material. Individual steady-states were maintained for periods up to 600 h continuous operation of the chemostat. A maximum output of 66.4 × 10⁶ cells/h was obtained at a dilution rate of 1.3 day⁻¹. The glucose substrate constant was determined as 0.0063 mg/ml. The relationships between dilution rate and the steady-state cell concentration, glucose concentration, and output of L 1210 cells from the chemostat, were in general agreement with the theoretical curves. It was found that the principles of continuous culture derived from the study of microorganisms are to a large extent applicable to the cultivation of animal cells.     

Nucleo-cytoplasmic exchange of non-histone proteins in amphibian embryos

As a basis for understanding the role of non-histone proteins in nuclear differentiation, we have identified one period during embryogenesis when intense accumulation of non-histones occurs in nuclei of Rana pipiens. We then demonstrated, experimentally, the loss of non-histones from nuclei after transplantation into enucleated eggs. ³H-tryptophan or ³H-lysine was injected into blastocoeles of mid-blastulae and into archenterons of late gastrulae; embryos were subsequently studied autoradiographically. Nuclei of animal hemisphere cells from blastulae accumulated only small amounts of ³H-tryptophan within 3 h, whereas a large accumulation occurred in endodermal nuclei of gastrulae as early as 1 h, and after 3 h 95.9% ( ) of the nuclei were densely labelled. Significant accumulation of ³H-lysine occurred in the majority of nuclei of both types within 3 h (blastulae ; gastrulae ). Controls, involving RNase or boiling TCA, demonstrated that the ³H-amino acids have been incorporated mainly into proteins. Endodermal nuclei labelled either with ³H-tryptophan or ³H-lysine after a 3 h incubation were transplanted singly into enucleated eggs. Autoradiograms demonstrated that most non-histones leave the nucleus during its reprogramming in the egg cytoplasm prior to first cleavage; whereas other types of proteins labelled with ³H-lysine remain for the most part in the nucleus. Cytochemical studies indicated that some of the non-histones which leave transplanted nuclei are acidic proteins; whereas some of those proteins which remain in the nucleus are histones.In addition to the above findings, the results of these studies demonstrate the feasibility in the future of studying the nucleocytoplasmic migration of different kinds of macromolecules in a developmental metazoan system and determining their roles in the establishment of nuclear differentiation.     

Growth characteristics of human esophageal epithelial cells in primary explant and serial culture

Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to 22 h after death) was not viable. When mucosal specimens (1.5 mm²) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48 h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8 and no growth at 6.2. Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate compared to platic. The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium. Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared in Clin. Res. 30: 93A; 1982.     

Oosome formation during in vitro oogenesis inBradysia tritici (syn.Sciara ocellaris)

Summary The development of follicles fromBradysia tritici (syn.Sciara ocellaris) during in vitro culture was studied. When follicles are isolated from 12-h-old females and placed in Robb's R-14 medium, their nurse cells regress with the same kinetics as in vivo and a histologically normal oosome forms at the posterior pole of the oocyte. Protein synthesis during in vitro development was studied by labelling follicles for 15 min and culturing them in vitro until the oosome had formed (28 h after eclosion of the donor). The time-course of protein labelling was defined by studying the incorporation kinetics of³H-amino acids into TCA-precipitable material; 50% of the radioactivity in the follicles was incorporated into TCA-precipitable material in less than 30 min. Autoradiographs of follicles labelled at different stages of oogenesis always showed a labelled oosome even if the labelling period was hours before oosome formation. These results indicate that the synthesis of oosome material starts long before the oosome forms at the end of vitellogenesis. Oosome formation can be inhibited by colchicine (20 g/ml) and is, therefore, likely to be dependent directly or indirectly on microtubule function.     

In vitro modulation of differentiation by calcium in organ cultures of human and murine epithelial tissue

Summary The differentiation of epithelial tissue in organ cultures of murine buccal mucosa, various human oral mucosa, and human newborn foreskin was found to be dependent on the calcium concentration of the culture media. In low calcium medium (≤0.07 mM) epithelial differentiation was inhibited. The original stratifying layers separate and can be removed, producing a destratified explant. Histologically such an explant consits of a dorsal epithelial layer of basal keratinocytes resting on an intact basal lamina with subjacent stroma. At 0.01 mM calcium, the epithelial layer was one to two cells thick whereas at 0.07 mM it could be three or more layers in thickness with the most superficial cells being spread over the underlying cells. In addition to differentiation, keratinocyte migration over the sides of the explant (epiboly) and epithelial proliferation as determined by [³H]thymidine autoradiography were reduced by culture in low calcium medium. Redifferentiation occurs upon return to normal calcium levels (1.8 mM); addition of hydrocortisone to low calcium media was found to facilitate this redifferentiation. This investigation was supported by NIH Grant CA29255 from the National Cancer Institute, PHS/DHHS, and by NIH Grant RR01219 supporting the New York State High-Voltage Electron Microscope as a National Biotechnology Resource, awarded by the Division of Research Resources, PHS/DHHS.     

Labelling of the chromatid body by [3H]uridine in rat pachytene spermatocytes

In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with [³H]uridine in the pachytene spermatocytes. The chromatoid body becomes labelled when the cells are first labelled for 2 h in the presence of [³H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor α-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.