DNA TURNOVER IN ADRENAL MEDULLARY CELLS OF DIFFERENT STRAINS OF RATS AND ITS ENHANCEMENT AFTER INTERMITTENT EXPOSURE TO COLD

In Italico and Wistar rats maintained at room temperature 1% of nuclei of cells in the adrenal medulla incorporate ³H-thymidine. Following intermittent exposure to cold, the numbers incorporating increase to 10% and 7% in Italico and Wistar strains respectively. Only in the Wistar strain does the increase occur during actual exposure.
Assuming S to be 6–8 hr the labelling indices actually determined indicate a turnover time of DNA of 21–29 days. Since mitotic indices in experimental and control animals were found to be 0.004% without colchicine treatment and 0.009% after 3 hr of colchicine treatment, the turnover times of cells were calculated to be 1388–3125 days. To correlate mitotic indices and labelling indices, an S period of 400 hr would have to be assumed. It is concluded therefore that: (i) most of the observed labelling of DNA is due to metabolic turnover of DNA and only a small proportion represents pre-mitotic synthesis, (ii) differences in the rates of DNA synthesis found during exposure to cold in Italico and Wistar rats respectively are sufficient to account for the differences in reduction of DNA previously found between the two strains under these experimental conditions.     

II. DIFFERENCES IN ADRENAL MEDULLA NUCLEAR DNA CONTENT AMONG RATS OF DIFFERENT STRAINS FOLLOWING INTERMITTENT EXPOSURE TO COLD

The amount of DNA per nucleus in the adrenal medulla cells of four different strains of rats (Wistar, Sprague-Dawley, Long-Evans, and Italico) is determined both under control conditions and after 300 hr of intermittent exposure to cold. The adrenal medulla nuclei of the four strains of rats contain the same amount of DNA; however, the loss of DNA observed after the same experimental treatment differs markedly in the different strains. The loss is small in Wistar and Sprague-Dawley rats (8–13%), larger in Long-Evans rats (20%) and still larger in Italico rats (45%). The DNA loss in Wistar rats increases if the animals are fed the same diet as the Italico rats, and the DNA loss in Italico rats is reduced if the animals are fed the same diet as the Wistar rats. The different behavior of the four strains is discussed in terms of turnover of DNA.     

A RADIOAUTOGRAPHIC STUDY WITH H3-THYMIDINE ON ADRENAL MEDULLA NUCLEI OF RATS INTERMITTENTLY EXPOSED TO COLD

A considerable decrease (24 to 40%) of DNA content per nucleus previously observed in the adrenal medulla of rats exposed intermittently to cold is followed by restoration to normal and supranormal values. This phenomenon has now been studied by use of H³-thymidine, which was given to normal rats, to rats exposed to cold, and to animals brought to room temperature after cold exposure. In the first two conditions, no significant labeling of nuclei was observed. In the third, labeling took place clearly in the 1st 3 days. The grain counts showed that the early labeled nuclei had more grains than those labeled later, indicating differences in the rate of DNA synthesis. A statistically significant correlation was found, on the same nuclei, between amount of Feulgen dye and number of grains. It is concluded that net synthesis of DNA takes place in the phase of recovery from cold. This fact is not related to cell division, as no mitoses could ever be detected, but rather to the cold-induced loss of DNA. Clear demonstration is thus given of a marked variation in the amount of DNA per nucleus in relation to the functional conditions of adrenal medulla cells.     

AN ANALYSIS OF DNA LOSS AND SYNTHESIS IN THE RAT ADRENAL MEDULLA NUCLEI UPON COLD STIMULATION

The peculiar changes previously observed in DNA content of rat adrenal medulla cell nuclei upon intermittent cold exposure (15 hr at +4°C followed by 9 hr at room temperature) have been further studied with the aid of Feulgen histophotometry and H₃-thymidine radioautography. The amount of DNA decreases progressively with increasing length of cold exposure until 300 hr (-32%). Later a rapid change takes place, whereby DNA content per nucleus returns to values which are slightly, but consistently lower than normal. At termination of a period of cumulative exposure to cold, an analysis of a whole-day experimental cycle shows that the DNA decrease is due to loss of DNA during cold exposure and that DNA synthesis occurs upon return to room temperature. The balance between these two processes can be divided into three stages: (a) loss of DNA up to 300 hr of cumulative cold exposure; (b) marked increase in DNA by 350 hr; (c) oscillation around zero or slightly negative at 400 hr and beyond. These variations are due to: (1) the extension of DNA synthesis into the period of cold exposure as clearly demonstrated by radioautography (stage b), and (2) a later still greater DNA loss (stage c) which partly offsets the increased synthesis. A complex pattern of adaptation of the adrenal medulla cells, as regards DNA content, to the repetitive cold stimulus is thus demonstrated.     

DNA Turnover in Adrenal Medullary Cells of Long Evans Rats

The nuclei of adrenal medullae of Long Evans rats, exposed intermittently to cold for a total of 300 h, showed a 20% decrease of their DNA content. This decrease is less than that found in similarly treated Italico rats (45%).
When ³H-thymidine was injected into Long Evans rats 6 h after the end of 300 h of intermittent cold exposure, incorporation of the label in the nuclei was approximately half of that found in Italico rats subjected to the same treatment. If the rats were re-exposed to cold, a decrease of labelling with a biological half-life of 17 days was observed; in animals kept at room temperature the half-life was 27 days.
Analysis of the DNA turnover in the adrenal medulla of Long Evans rats compared with Italico rats, shows that the smaller loss induced by cold exposure in Long Evans rats can be attributed to a reduced response to cold rather than to a slower DNA turnover under normal conditions or to a lower level of synthesis.     

CHANGES IN THE DNA CONTENT OF ADRENAL MEDULLA NUCLEI OF RATS INTERMITTENTLY EXPOSED TO COLD

In the adrenal medulla of rats exposed intermittently to cold (+4°C) for 100 and 300 hours, a considerable decrease (24 to 40 per cent) of the DNA content per nucleus was observed, followed by restoration to normal or above normal values within 10 days after the withdrawal of the stimulus. The findings were obtained with a scanning integrating histophotometer, and confirmed by microinterferometric investigations (on the basis of the measurement of total dry mass of nuclei isolated in aqueous medium before and after treatment with DNase) and by microchemical determinations, combined with the count of the nuclei in the homogenates. The observed decrease of DNA content cannot be attributed to errors of the methods used, nor to consequences of cellular degeneration. The available evidence seems to indicate a real decrease rather than a change in the state of a part of DNA in the nucleus in vivo whereby it becomes extractable by aqueous solutions. The restoration cannot be due to mitotic processes, which were actually never detected even with the use of colchicine, since the adrenal medulla cells in the adult rat are known to be irreversible, postmitotic cells. A correlation between the functional activity of the adrenal medulla cells and the content or state of DNA in their nuclei is demonstrated.     

THYROID PROLIFERATIVE POTENTIAL AS A FUNCTION OF AGE

The effect of a goitrogenic stimulus on thyroid weight and thyroid cell ³HTdR labeling of Sprague-Dawley rats varying from 2 to 40 weeks of age was determined. Propylthiouracil ad libitum in drinking water produced a spurt in follicle cell labeling index and thyroid weight evident after 24 hr for all age groups. The increase in labeling index reached a peak at 5–7 days and then decreased to a level a few times greater than that of the normal unstimulated thyroid. The tritiated thymidine labeling index for thyroid follicle cells and the effect of PTU thereon was determined for August male rats of 3 days to 12 weeks of age. In the older rats, the follicle cell labeling index rose to 5–6% after 4–5 days of PTU treatment and then slowly fell to about 1%, For the unstimulated control rat of comparable age, the labeling index was about 0.1%. At all ages the thyroid showed a rapid response to PTU. Examination of the time sequence of mitotic labeling showed that the DNA synthesis period was 7.5 hr for normal 2-week-old rats and for 10–12-week-old rats that had received PTU for 4 days. There was no second wave of labeled mitoses in either group during the 48-hr interval studied. From the curve of thyroid weight vs time on PTU and from the labeled mitoses curve, inferences regarding the minimum fraction of proliferating follicle cells in the stimulated ‘adult’rat thyroid were made.     

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.     

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.     

AN AUTORADIOGRAPHIC AND MORPHOLOGICAL STUDY OF MOUSE BONE MARROW LITTORAL CELLS DURING AND AFTER TREATMENT WITH URETHANE

This study demonstrates that the labeling index of mouse marrow littoral cells can be markedly altered as a result of treatment with ethyl carbamate (urethane). Young C57/BL mice were given daily intraperitoneal urethane injections for periods up to 6 days. Following treatment each day, as well as daily over a 10 day recovery period, a group of animals was administered tritiated thymidine every 3 hr over a 24 hr period and sacrificed ½ hr after the last injection. Marrow was embedded in epon and 0·5 μm sections cut for autoradiographic and light microscopic analysis. A thirty-five-fold increase in the percentage labeled littoral cells was observed after three injections of urethane. The labeling index of littoral cells fluctuated during the treatment period and during the 10 day recovery period. The labeling data are discussed in relation to the possible effects of urethane on the cell cycle of mouse marrow littoral cells; the morphological sequela to urethane treatment and the nossibilitv that littoral cells mav act as stem cells.     

POST-MITOTIC AGE OF MONONUCLEAR CELLS MIGRATING INTO TA-3(St) SOLID TUMORS

Subcutaneous transplantation of 5 × 10⁶ TA-3(St) mammary carcinoma cells into A/J mice produced rapidly growing tumors that showed a substantial accumulation of lymphocytes, monocytes and macrophages. This must have resulted primarily from an influx of circulating cells, since none of the cell types exhibited any significant local proliferation as indicated by 1 hr ³H-TdR uptake. The post mitotic age of the various leukocyte types in circulation of normal and tumor bearing animals, and those appearing within the tumors, was evaluated by a repeated ³H-TdR labeling protocol designed to label newly formed leukocytes. Tumor transplantation (or injection of an equivalent volume of saline in control animals) was preceded by thirteen ³H-TdR injections (12.5 μCi every 8 hr) and followed by eight more injections at equivalent intervals. Animals were serially sacrificed at 5-14 days after tumor transplantation or saline injection. Total lymphocyte labeling in the blood during these intervals was higher in tumor bearing mice (approximately 25% between 5 and 12 days) than in the saline injected controls (approximately 20%), indicating that tumor transplantation resulted in an increase in the proportion of young lymphocytes in circulation. More significantly, a much higher labeling (57-60% at 5-12 days) was exhibited by lymphocytes isolated from the tumors, indicating selective migration (and/or retention) of newly formed lymphocytes within the tumor. This finding applied to lymphocytes in all size categories. Although blood monocyte labeling in the control and experimental animals was similar (e.g. 83% at day 5 and 52% at day 14), significantly higher labeling (e.g. 93% and 70% at the respective intervals) was exhibited by monocytes isolated from tumors. This suggested a preferential accumulation of young monocytes at the tumor site. An identical macrophage labeling pattern compared to that shown by monocytes within the tumor indicated a rapid monocyte-macrophage transition. Since these findings in the present strain-specific solid tumor are similar to those previously obtained in this laboratory in the strain-nonspecific Ehrlich ascites tumor, the phenomenon of selective localization of newly formed mono-nuclear leukocytes appears to be a general occurrence in transplanted murine tumors.     

CONSTANCY OF DNA CONTENT IN ADRENAL MEDULLA NUCLEI OF COLD-TREATED RATS

Reports of changes in DNA content of certain types of cells following exposure to conditions of stress has led to the suggestion that two kinds of DNA may be present. One is genetic DNA, and the other is called "metabolic" DNA. In a further attempt to investigate the possibility of this phenomenon, determinations of DNA content were made on Feulgen-stained nuclei of adrenal glands and kidneys in cold-treated rats. Feulgen-stained nuclei were measured by two-wavelength microspectrophotometry. Particular attention was given to the handling of the smears in hydrolysis and staining. Mean values of Feulgen-DNA contents in a total of 720 nuclei demonstrated (a) a constancy of DNA content within 2% in individual nuclei both in adrenal medulla and kidney cortex, (b) no more than an average of 2% difference in DNA content between control and experimental nuclei, and (c) no more than an average of 1.5% difference in DNA content between normal kidney cortex nuclei and normal adrenal medulla nuclei. These results confirm the view that the more precise the measurement, the more accurately the constancy rule is obeyed. Moreover, there is no support for the concept of a metabolic DNA in the rat adrenal medulla.     

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 effects of enalapril maleate and cold stress exposure on tyrosine hydroxylase activity in some rat tissues

Enalapril is a highly specific and competitive inhibitor of angiotensin-I converting enzyme (ACE) and thus belongs to the category of ACE inhibitors. The beneficial effects of ACE inhibitors appear to result primarily from the suppression of the plasma renin-angiotensin-aldesterone system. This study was designed to detect the effects of enalapril maleate and cold stress on tyrosine hydroxylase (TH) activity in adrenal medulla, heart and hypothalamus in rat. In cold stress treatment (exposed to 8 degrees C cold for 48 h) TH activity was found to be raised significantly (p < 0.05) in adrenal medulla, hypothalamus and heart tissues. In the adrenal medulla, hypothalamus and heart tissues, TH activity of enalapril maleate treated rats (10 mg kg(-1) body weight) group was not raised significantly (p > 0.05). Following intraperitoneal injection of enalapril maleate (10 mg kg(-1) body weight) the rats were exposed to 8 degrees C cold for 48 h. After cold stress and enalapril maleate treatment no statistically significant change in tyrosine hydroxylase activity was detected in adrenal medulla, hypothalamus or heart (p > 0.05). The results of our studies show that enalapril maleate blocks the effect of cold stress on the regulation of TH activity.     

An autoradiographic and morphological study of mouse bone marrow littoral cells during and after treatment with urethane.

This study demonstrates that the labeling index of mouse marrow littoral cells can be markedly altered as a result of treatment with ethyl carbamate (urethane). Young C57/BL mice were given daily intraperitoneal urethane injections for periods up to 6 days. Following treatment each day, as well as daily over a 10 day recovery period, a group of animals was administered tritiated thymidine every 3 hr over a 24 hr period and sacrificed 1/2 hr after the last injection. Marrow was embedded in epon and 0-5 mum sections cut for autoradiographic and light microscopic analysis. A thirty-five-fold increase in the percentage labeled littoral cells was observed after three injections of urethane. The labeling index of littoral cells fluctuated during the treatment period and during the 10 day recovery period. The albeling data are discussed in relation to the possible effects of urethane on the cell cycle of mouse marrow littoral cells; the morphological sequela to urethane treatment and the possibility that littoral cells may act as stem cells.     

Upregulation of neuronal nitric oxide synthase mRNA and protein in adrenal medulla of water-deprived rats.

Experiments were performed to investigate whether adrenal neuronal nitric oxide synthase (nNOS) mRNA and protein expression are responsive to alterations in body volume. Using an RT-PCR technique, the relative quantities of nNOS mRNA as well as the tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA in the adrenals of water-deprived rats significantly increased from 12 hr to 4 days. In situ hybridization and immunohistochemical study showed that water deprivation activated nNOS mRNA and protein expression in the adrenal medulla. Four days after water deprivation, nNOS protein expression determined by Western blot significantly increased in the adrenal gland. Our results are the first to demonstrate that nNOS syntheses in the adrenal medulla are markedly increased in water-deprived rats. This study also indicates that the upregulation of nNOS synthesis of the adrenal medulla is associated with the activation of adrenal medullary function in the face of volume depletion.     

THE CELL POPULATION KINETICS AND RESPONSE TO AN S-PHASE SPECIFIC AGENT OF THREE TRANSPLANTABLE COLON TUMOR LINES

The relative cell population kinetics of three transplantable murine colon tumor lines (Colon 26, 36 and 38) with different histological and metastatic characteristics were studied in relation to the response of each line to an S-phase specific agent. The mean doubling times for the three lines between 0·1 and 1·0 g are similar (4·2 days) but marked differences are apparent in times to tumor appearance (0·1 g) and in median days to death. The length of the cell cycle is about one day and the length of the S-phase 10–11 hr for Colon 36 and 38. The length of the cell cycle in Colon 26 is difficult to estimate by conventional methods but probably exceeds 24 hr and the S-phase is 10–11 hr; [³H]TdR pulse labeling indices for Colon 36 and 38 decrease with time and tumor size from about 0·45 in 0·1 to 0·2 g tumors to about 0·33 at 3 g. The decrease in the [³H]TdR labeling index for Colon 26 is more pronounced (from about 0·38 at 0·1 g to 0·21 at 1·0 g). The shapes of the PLM curves and the [³H]TdR labeling index data are consistent with the observed sensitivity to an S-phase specific agent (Palmo-AraC, NSC 135962) in Colon 36 and the minimal response observed in Colon 26. Colon 38 is intermediate between Colon 36 and Colon 26 in kinetic properties and in response to the S-phase agent.     

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.     

Cell Proliferation In Emt6 Tumors Treated With Single Doses of X-Rays Or Hydroxyurea. I. Experimental Results

EMT6 mouse mammary tumors were treated in vivo with 5 mg/mouse of hydroxyurea (HU) or 300 rads of X-rays. the proliferation of the tumor cells was followed for 28 hr after treatment. Changes in the ³H-TdR labeling index, the mitotic index, the specific activity of the ³H-TdR-labeled DNA, and the proportion of suspended, clonogenic cells in the S phase of the cell cycle were examined and compared. Evidence was found for reassortment of the surviving cells in treated tumors into partially synchronous cohorts. the partial synchrony in the proliferation of the surviving cells was not accurately predicted by the changes in the labeling index and the mitotic index. the changes in DNA specific activity proved unacceptable as an indicator of cell proliferation in solid EMT6 tumors treated with low doses of radiation or HU.     

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.