Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling

DNA labeling by ³H-thymidine in vitro and antiglobulin-¹³¹I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow.Bone marrow cells from CBA mice previously injected repeatedly with ³H-thymidine for 1–84 hr were exposed to ¹³¹I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The ³H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show ³H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% ³H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin-¹³¹I at a concentration of 1.0 μg/ml became labeled with ³H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in ³H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions.The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.     

MIGRATION OF SMALL LYMPHOCYTES IN ADULT MICE DEMONSTRATED BY PARABIOSIS

Parabiotic BALB/C mice were used to study the traffic of small lymphocytes in immunological mature but unchallenged mice. By giving ³H-thymidine (³H-TdR) injections to only one member (A) of a pair by preventing the escape of the radioactive isotope to the other member (B), the kinetics of newly-formed cells was followed. Less than 10% labelled small lymphocytes were found in the peripheral lymphoid tissues of both A and B members, while the thymusses and bone marrows of A members showed labelling percentages up to 70% in this period. Hardly any labelled cells gained entrance into the thymus while a detectable number was found in the bone marrows of B members. Results from pairs set up to follow migration of long-lived lymphocytes revealed that labelled cells detected 4–5 weeks after injections were equilibrated between the peripheral tissues and the bone marrows of the partners. Very few labelled cells were seen in the thymic medulla and none were observed in the thymic cortex, germinal centres or medullary cords of lymph nodes from any B member. It was concluded that short-lived small lymphocytes are formed primarily in the thymus and bone marrow and the migration of these cells is limited in adult animals. Furthermore, the vast majority of long-lived small lymphocytes are freely recirculating, and these cells gain entrance to and are normal residents in the bone marrow.     

MALL LYMPHOCYTE POPULATIONS IN THE MOUSE BONE MARROW

Autoradiography and scintillation counting have been used for evaluation of lymphocyte turnover and life span in the bone marrow, peripheral blood and thoracic duct lymph of BALB/C mice. It was shown that the bone marrow contained two populations of small lymphocytes. One population was labelled 100% after 3–4 days of intensive injections of ³H-thymidine and constituted about 75% of the lymphocytes. The remaining 25% of the lymphocytes turned over at a much slower rate comparable to the rate of increase in labelled small lymphocytes of the thoracic duct. More than 10% of the small lymphocytes of the bone marrow were found to be unlabelled after 10 days of intensive injections of ³H-thymidine. Nine weeks after giving ³H-thymidine for 30 consecutive days, 8·6% of the small lymphocytes in the bone marrow remained labelled. The mean grain counts of cells in this population were comparable to those of thoracic duct lymphocytes at corresponding times. About 90% of the peripheral blood lymphocytes were found to have a slow turnover and a long life span.     

DISTRIBUTION OF 3H-THYMIDINE IN ARABIDOPSIS VEGETATIVE MERISTEMS AFTER 5-IODODEOXYURIDINE TREATMENT

Arabidopsis thaliana vegetative meristems, growing under short photoperiods, respond to the application of IUdR by precocious floral morphogenesis. ³H-thymidine was used to label cells active in DNA synthesis and study the effect of analogue application on the amount and distribution of DNA synthesis throughout the meristem during 78 hr subsequent to IUdR treatment. Photomicrographs, autoradiographs, and composite plots of label distribution in transparent superimposed sequential sections revealed a non-uniform distribution of labelling in control vegetative meristems, which typically contained a central and axial core incorporating little ³H-thymidine and a peripheral flanking tunica region which contained densely labelled cells. The ratio of labelled cells in the peripheral region to labelled cells in the central region was about 10:1 in the controls. In meristems pretreated with IUdR there was a brief suppression of ³H-thymidine incorporation during 6–12 hr after treatment, followed by two waves of enhanced incorporation in the peripheral region, and a progressive increase in the frequency of labelled cells in the central core of the meristem. After 78 hr the frequency of labelled cells in the central core of IUdR-treated meristems was 8-fold higher than in untreated meristems, and the frequency in the peripheral regions was about the same in both IUdR and control series. The enhancement in amount and uniformity of DNA synthesis after temporary inhibition by IUdR parallels the normal enhancement which is observed when vegetative meristems are transferred to long photoperiods causing floral induction.     

STEM CELL KINETICS IN THE L 5222 RAT LEUKAEMIA

The behaviour of normal haemopoietic stem cells of the bone marrow during the development of the acute transferable rat leukaemia, L 5222, has been investigated. The granulopoietic committed stem cells, measured by the in vitro colony technique, showed a marked decrease to less than half normal levels. Pluripotent stem cells included in the small lymphocytes of the bone marrow, and labelled with ³H-thymidine by the complete labelling method, showed only a modest decrease in number and an unchanged labelling intensity. The results suggest that in this leukaemia the pluripotent stem cells may be affected in such a way that they are unable to react by proliferation to the depletion of the succeeding cell compartments. This might be due to inhibition by leukaemic cells or to a disturbed feedback regulation between the committed and pluripotent stem cell compartments.     

Selective adsorption of immune lymphocytes, synthesizing DNA, on corresponding target cells]

Lymphocytes of inbred mice immunized with allogenic tumour cells were labelled in vitro or in vivo by 3H-thymidine, washed out and incubated with target cells in the presence of "cold" thymidine. A fraction of labelled small and medium lymphocytes was demonstrated by autoradiography to be absorbed rapidly and specifically onto the corresponding target cells. When the non-labelled immune lymphocytes were preincubated with target cells for 2 hours, and 3H-thymidine was added to the medium at various time periods, the percentage of labelled small and medium lymphocytes which adhered to the target cells appeared to be reduced in the course of the incubation, with the target reaching the initial value after an 8-hour contact. Small and medium immune lymphocytes which synthesize DNA are supposed to be the effector killer fraction which do not transform into blasts during the interaction with the target cells.     

KINETICS OF ERYTHROPOIETIN STIMULATED PROLIFERATION OF ERYTHROID PROGENITORS IN THE SPLEEN

The effect of RBC transfusion and erythropoietin (EPO) on the proliferation of immature erythrocyte progenitors was studied in the spleens of RBC transfused, lethally irradiated mice injected with bone marrow. Transfusion decreased expansion of the progenitors and slowed their proliferation: the mean cycle time as measured by per cent labelled mitosis (PLM) on the third day after injection of bone marrow was 10.7 hr in transfused as compared to 5.6 hr in non-transfused mice. One injection of five units of erythropoietin on day 2 decreased the mean cycle time to 7.3 hr in transfused mice and increased expansion of the progenitor cells. The effects of erythropoietin on cell proliferation were prompt: a significant increase of incorporation of ³H-TdR into DNA occurred within 2 hr of injection. Erythroblasts were absent from the spleens of transfused, irradiated bone marrow injected mice; however, erythroblasts appeared by 72 hr and 48 hr following EPO injection either 2 days or 5 days after transplantation respectively. Increased uptake of radioactive iron in spleen after erythropoietin injection preceded the appearance of erythroblasts by 2 and 1 days when erythropoietin was injected either 2 or 5 days after marrow transplantation respectively. The increase in cellular proliferation induced by erythropoietin in transfused irradiated mice injected with bone marrow equivalent to 0.35 femoral shaft was manifested as an increase of the total DNA content in the spleen by 119 μg (11.9 × 10⁶ cells) within 48 hr of injection. The cellular increment produced by EPO injection on day 5 to mice given 0.05 femoral shaft consisted mainly of undifferentiated mononuclear cells, most of which were labelled, with erythroblasts comprising only one quarter of the increment. Erythropoietin inactivated by mild acid hydrolysis failed to increase cellular proliferation.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.     

Proteases as mitogens : The effect of trypsin and pronase on mouse and human lymphocytes

Treatment of mouse spleen lymphocytes with trypsin (from 0.1 to 1.0 μg/ml) was found to cause a significant stimulation of the incorporation of ³H-thymidine. When spleen cells from nude (congenitally athymic) mice were incubated with trypsin in the absence of serum for 3 days, very high levels of incorporation were noted (stimulation index of 10 to 20). Trypsin was without effect on thymic lymphocytes of the mouse but caused significant activation of human peripheral blood lymphocytes. The stimulatory effect of trypsin was a consequence of its enzymatic activity. Prolonged treatment with pronase also caused small but significant increases in the incorporation of labelled thymidine (stimulation index of 2 to 4) into the thymic and splenic lymphocytes of the mouse and into human lymphocytes. The evidence suggests that trypsin stimulates the B-derived lymphocytes.     

EPIDERMAL REGENERATION AFTER CELLOPHANE TAPE STRIPPING OF HAIRLESS MOUSE SKIN

After repeated applications of cellophane tape to the dorsal skin of hairless mice, the proliferative response in the treated epidermis was estimated by three different methods. The mitotic rate was determined in the interfollicular epidermis using the Colcemid technique, and the DNA synthetic activity was estimated after ³H-thymidine injection by counting labelled interfollicular cells in autoradiographs and by determining the specific activity of epidermal DNA. An initial 40–50% inhibition of DNA synthesis and mitosis was followed by an increase in the labelling index and the mitotic rate 8–10 hr after tape stripping. By 24 hr, peak values 5–6 times the controls were attained for both parameters. The labelling index and the mitotic rate were nearly normal at 3–4 days, but a second small peak was seen on day 5. Normal values were found on days 6 and 8. A similar pattern of response was found biochemically, but the peak of DNA specific activity was much broader and the extent of the increase was only about half as great as the increase in the labelling index. Possible reasons for these differences are discussed.     

Circulating T and B lymphocytes of the mouse. I. Migratory properties

Studies on the identity of thoracic duct lymphocytes (TDL⁴) from normal and T cell-depleted mice indicated that as many circulating B lymphocytes were produced by healthy T cell-depleted mice as normal mice. Proportions of T and B cells from the thoracic duct of CBA mice changed markedly during the first 4 days of drainage from 82% T cells and 16% B cells at 12 hr to approximately equal proportions of both classes after 3 days. In absolute terms, T cells were mobilized rapidly by thoracic duct drainage and B cells very slowly. Histologically, this was reflected in a rapid depletion of the T cell-dependent areas of the lymphoid organs. The B cell-dependent areas, in contrast, became depleted of lymphocytes only after drainage for a week or more.The homing properties of circulating lymphocytes were investigated using TDL from normal and T cell-depleted mice as relatively pure sources of T and B cells, respectively. Four hours after injection of ⁵¹Cr-labeled T and B cells, a large proportion of both cell classes were found in the spleen. By 24 hr, many T cells had left the spleen and appeared in the lymph nodes. Such redistribution by B cells, however, was minimal.Intravenously injected T and B cells, labeled with tritiated uridine (³HU), localized specifically in the T and B cell-dependent areas, respectively, of the lymphoid tissues.³HU-labeled T cells were found to recirculate rapidly from blood to lymph. Labeled B cells, in contrast, recirculated only very slowly.     

DYNAMICS OF LEUKOCYTE MIGRATION INTO THE MOUSE ASCITES TUMOR

There is a marked increase in the number of peritoneal leukocytes (lymphocytes, monocytes and granulocytes) during the growth of Ehrlich ascites tumor in mice. No local proliferation (as indicated by a labeling at 1 hr following a single ³H-TdR injection) was observed in the normal peritoneal leukocytes or those in the ascites tumor, except for a very minor labeling of some tumor macrophages. Kinetics of peritoneal leukocytes was studied with a series of twelve injections of ³H-thymidine (20 μCi every 8 hr) in normal mice as well as mice injected with 10⁶ tumor cells i.p. 2 hr after the last ³H-TdR injection. Animals were sacrificed at intervals up to 6 days. Granulocyte labeling in the blood as well as peritoneal space was near 100% in both groups of animals at all the intervals. Temporal changes in the labeling of lymphocytes (from 10% at 0 day to 22% at day 6), and monocytes (from 20% at 0 day to 57% at day 6) were identical in the blood and peritoneal space of normal animals, indicating a free exchange of cells between these compartments. Higher labeling indices than those in the controls were attained in the blood of tumor-bearing hosts (viz 40% for lymphocytes and 80% for monocytes at 6 days) suggesting an increased turnover of these cells in the circulation. In addition, peritoneal mononuclear cells of tumor-bearing mice showed even a higher labeling than those in the blood (viz 65% for lymphocytes and 92% for monocytes at 6 days) indicating a selective migration and/or retention of newly formed cells within the tumor, in contrast to a random migration into the normal peritoneal cavity. Furthermore, an identical labeling of macrophages to that of monocytes within the tumor indicated a short monocyte-macrophage transition. The preferential accumulation of young mononuclear cells into the tumor may be of functional importance.     

Fate of H2-activated T lymphocytes in syngeneic hosts: II. Residence in recirculating lymphocyte pool and capacity to migrate to allografts

T.TDL—a purified population of lymph-borne H2-activated T lymphocytes—were transferred to syngeneic mice to examine their capacity to remain in the recirculating lymphocyte pool (RLP). Experiments with cells labelled with ³H-thymidine (³HTdR) or ⁵¹Chromium showed that although a considerable proportion of T.TDL joined the RLP (i.e., were mobilizable through a thoracic duct fistula) for several days after transfer, most of the cells soon left the pool. This applied whether the cells were transferred to normal mice or to “B” mice. Normal thoracic duct lymphocytes, by contrast, joined the RLP for long periods post-transfer.Studies with ³HTdR-labelled T.TDL showed that a small number of heavily labelled cells remained in the RLP for at least 3 months. Experiments with the θ-antigen as a cell marker suggested that a further small proportion of cells underwent division at some stage after transfer and then rejoined the RLP in expanded numbers.T.TDL showed a tendency to home to specific allografts of either skin, tumor cells or lymphoid cells. Although homing was specific it was very limited in extent.     

ESTIMATION OF THE MIGRATION OF THORACIC DUCT LYMPHOCYTES TO NON-LYMPHOID TISSUES

The distribution of radioisotopes in tissues was measured following i.v. injection of labelled thoracic duct lymphocytes into syngeneic rats. The rate of elution of an isotope from the labelled cells and the subsequent fate of the eluted isotope were shown to be the most important factors limiting the usefulness of such isotopes for measuring cell localization particularly in non-lymphoid tissues. Comparison of labelling procedures using [³H] and [¹⁴C]uridine, [³H] and [¹⁴C]leucine, [⁷⁵Se]-L-selenomethionine, [^99mTc]sodium pertechnetate and [⁵¹Cr]sodium chromate in vitro and [³H]thymidine in vivo showed that ⁵¹Cr had the fewest disadvantages in the present context. Using ^5ICr-labelled cells, the radioactivity was measured in a wide range of non-lymphoid tissues, and estimates of cell traffic were obtained. In skin, for example, the results indicate a cell flux in the range of 10⁴-10⁵ lymphocytes/gm/hr. Evidence is presented which suggests that the early substantial localization of labelled cells in the lung is not an artefact due to sequestration or embolization of traumatized cells but probably reflects a slow intravascular transit time through this capillary bed. The primary lymphoid organs, thymus and bone marrow were shown to include a subpopulation of lymphocytes which belong to the recirculating pool. The thymus always contained a greater concentration of radioactivity at 24 hr than all non-lymphoid tissues except liver and kidney (approx. 0-1% of the recirculating lymphocyte pool) and the bone marrow was capable of temporarily accepting a substantial proportion (approx. 25%) of the injected cells.     

Can cells extruded from denervated skeletal muscle become circulating potential myoblasts?

Summary The hypothesis that satellite cells which leave denervated skeletal muscle might become circulating potential myoblasts which could participate in myogenesis in distant sites in the body has been tested.Sixteen mice had one hindlimb denervated and were given 7 daily injections of ³H-thymidine (³H-Tdr). One day later extensor digitorum longus muscle isografts from unlabelled mice were inserted into each hindlimb. As controls, the procedure was repeated in 6 non-denervated labelled mice. Fourteen days after their insertion, isografts in denervated mice contained many labelled myotubes with a labelling index of 55±4% (mean±SEM). In the control isografts in non-denervated mice, 38±4% of myotube nuclei were labelled. The results show that either labelled cells, or ³H-Tdr, had transferred from the host to isografts in both cases. The probability of ³H-Tdr reutilization was demonstrated in regenerating livers of 8 similarly labelled mice, where 34±3% hepatocytes adjacent to crush lesions were labelled after 14 days. This conclusion was reached because only 2–3% of normal hepatocytes incorporate ³H-Tdr under these conditions and this population is inadequate to provide sufficient labelled precursor cells for the large numbers of labelled regenerated hepatocytes. Therefore, it was concluded that ³H-Tdr reutilization is the most likely explanation for labelled myotube nuclei in the muscle isografts (rather than movement of labelled precursor cells), and that additional label for reutilization had been derived from breakdown of labelled cells in denervated muscle. The data do not support the hypothesis of a circulating precursor for skeletal muscle cells.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Tritiated thymidine autoradiographic study on the origin and renewal of argentaffin cells in the pyloric gland of hamsters

Summary The origin and renewal of the argentaffin cells in the pyloric glands of hamsters were studied by flash, cumulative and pulse labelling autoradiography with ³H-thymidine. The argentaffin cells were identified by the Diazo Method using Fast Red B Salt.By flash labelling autoradiography, it was shown that the argentaffin cells located from the middle to the lower level of the pyloric mucosa were not labelled with ³H-thymidine, indicating that this cell type has no proliferative activity. On the 10th and the 20th day of cumulative labelling, 31% and 63% of the argentaffin cells in the gland were found to be labelled, respectively. The labelled argentaffin cells were concentrated in the upper part of the gland (around the region of the isthmus), and no label was found over nuclei of the cells at the lowermost level of the gland. These labelled cells were shown to undergo a downward migration in the days following pulse labelling. They were replaced by unlabelled (and weakly or very weakly labelled) cells which arose at the region of the isthmus. The argentaffin cells in the pyloric gland are thought to arise from epithelial precursor cells at the region of the isthmus.The labelled argentaffin cells in the gland were found to decrease in number almost exponentially after pulse labelling. This indicates that the life span of argentaffin cells is not fixed, but their renewal conforms to the random loss system. The half time of turnover of this cell population was 15 days on average.Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan     

DIURNAL VARIATION IN THE LABELING INDEX OF MOUSE EPIDERMIS: A DOUBLE ISOTOPE AUTORADIOGRAPHIC DEMONSTRATION OF CHANGING FLOW RATES

A diurnal rhythmicity in the labeling index was observed in the epidermis of hairless mice, injected with either ¹⁴C- or ³H-thymidine, at different times during a 24 hr period. A modified autoradiographic technique, using ¹⁴C- and ³H-thymidine and two overlying emulsion layers, makes it possible to clearly differentiate synthesizing cells which are singly labeled with either carbon-14 or tritium, and cells labeled with both isotopes. At various times during a 24 hr period, hairless mice were injected with thymidine-2-¹⁴C and colcemid, followed at 2 or 3 hr by a second injection of ³H-thymidine. The labeling indices were calculated for the ¹⁴C- and ³H-thymidine injection times. These labeling indices were consistent with the control, single isotope, labeling indices and exhibited the same diurnal rhythm. Cells singly labeled with ³H- or ¹⁴C-thymidine have either started or completed DNA synthesis during the interval between the two injections. Flow rates into and out of DNA synthesis, throughout the 24 hr period, can be calculated from these singly labeled cells. The flow rates varied rhythmically throughout the day and paralleled changes in the labeling indices. The influx and efflux flow rates, at all times measured, were not equal. The influx flow rate was reflected in the efflux rate at a time later equal to the duration of S. By means of these flow rates, the per cent of cells in DNA synthesis was calculated for each hour during a 24 hr period. The resulting labeling index curve matches the observed 24 hr diurnal rhythm in labeling indices. By extension of these flow rates through mitosis, the resulting mitotic index curve is comparable to the reported 24 hr diurnal rhythm in mitotic indices.     

‘3H-testosterone distribution and binding in RAT thymus cells in vivo’

Summary Autoradiography and biochemical investigations showed that [³H]-testosterone where injected intraperitoneally into male white rats was incorporated rapidly into thymus lymphocytes. Thymic cortex contained more silver grains than medulla, and larger lymphocytes were more labelled than medium or small lymphocytes.Cytosol fraction of thymus cells labelledin vivo with [³H]-testosterone, contained the largest quantity of labelled hormone. A 4S cytosol fraction binds [³H]-testosterone. This could be separated by Sephadex chromatography or by linear sucrose gradient centrifugation. Nuclear extract contained also a small quantity of the labelled hormone.