In the adrcnal medulla of rats cxposcd intermittently to cold (+4°C) for 100 and 300 hours, a considerable decrease (24 to 40 per cent) of the DNA contcnt per nuclcus was observed, followcd by restoration to normal or above normal values within l0 days aftcr the withdrawal of the stimulus. Thc findings were obtalncd with a scanning integrating histophotometer, and confirmed by microintcrfcrometric investigations (on the basis of the measurement of total dry mass of nuclci isolatcd in aqucous mcdium before and after treatment with DNasc) and by microchemical determinations, combined with the count of thc nuclei in the homogcnates. Thc obscrved decrease of DNA contcnt cannot bc attributed to crrors of the methods uscd, nor to consequences of cellular degeneration. The available cvidcncc sccms to indicatc a real dccrease rather than a change in the statc of a part of DNA in the nucleus in vivo whereby it becomes cxtractablc by aqueous solutlons. The restoration cannot be due to mitotic processes, which werc actually never dctected even with thc use of colchicine, since the adrenal medulla cclls in the adult rat arc known to bc irreversible, postmitotic cells. A correlation betwccn the functional activity of the adrcnal mcdulla ceils and the content or state of DNA in their nuclci is demonstrated.
A method for the determination of the DNA content of isolated nuclei of different ploidy has been developed. It is based on measurement of the nuclear dry mass, with an integrating microinterferometer, before and after DNase treatment . The values found are slightly low, because, as indicated by biochemical determinations, consistently 5 % to 8 % of DNA is not extracted by DNase under these conditions . The average DNA values thus obtained for diploid and tetraploid nuclei of adult rat liver are 7 .7 and 15 .6 pg (10 -12 g), respectively .
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.
The peculiar changes previously observed in DNA content of rat adrenal medulla cell nuclei upon intermittent cold exposure (15 hr at -4-4°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.
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.
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