Studies on lymphocytes: XV. Analysis of the in vivo division cycle of large lymphoid cells in calf thoracic duct using combined microspectrophotometry and autoradiography

Abstract

From previously reported labeled mitosis curves, the majority of proliferating large lymphoid cells in the thoracic duct of the calf have a T G of about 5 h. The division cycle in vivo of these elements was further investigated by combining microspectrophotometry and autoradiography of a sample taken 20 min following i. v. injection of 3H-thymidine. Compared with small lymphocytes, unlabeled large lymphoid cells with a DNA content near the 2 c mode showed approx. 4% higher mean, and a greater coefficient of variation of, Feulgen absorbance values per nucleus. In contrast to small lymphocytes, they exhibited a frequency distribution with regard to DNA content which diverged from normality with skewness to the right. Large lymphoid cells with a relative DNA content of 1.9–2.3 c showed a significantly smaller mean nuclear surface area in smears than elements with a relative DNA content of 3.7-3.1 c although there was a considerable overlap between the two groups of cells with regard to size. The sum of labeled and unlabeled large lymphoid cells per standard class of DNA content ( x ± 0.2 c) was greatest in a class around the 2 c mode, and smallest in classes of from 3.0 to 3.8 c DNA content. All large blasts with a relative DNA content of between 2.5 and 3.8 c were labeled indicating that throughout the major part of S phase there was no appreciable interruption in nuclear DNA synthesis. The median grain count per labeled large lymphoid blast was lowest in cells with a DNA content near the 2 c and 4 c modes, respectively, and highest in elements with a DNA content approximating 3.6 c. The inverse relative number of large lymphoid cells per class of DNA content paralleled satisfactorily the median grain count per labeled cell in the same class. These results are interpreted as indicating that in this particular cell system in vivo (1) unlabeled blasts with a DNA content near the 2 c mode are comprised of both elements in G 1 and some in early S phase; (2) nuclear size increases as cells develop from G 1 to G 2 phase although the nuclear surface area of an individual lymphoid cell in smears does not permit classification with regard to subphases of the division cycle; (3) throughout the major part of S phase overall nuclear DNA synthesis rate is rather adequately reflected by incorporation of 3H-thymidine suggesting that for the latter, possible changes during S phase of endogenous precursor pool sizes and/or activities of enzymes instrumental in incorporation of 3H-thymidine into DNA are of minor importance; (4) integrated nuclear DNA synthesis rate is highest in the second half and most probably lowest at both extremes of the S period.