A U T O R a D I O G R a P H I C S T U D Y of D N a S Y N T H E S I S a N D the Cell Cycle

Mice were injected intraperitoneally with 15 #c of H3-thymidine. The time course of the labeling in spermatogonia and spermatocytes was studied by using autoradiography on 5 # sections stained by the periodic acid-Schiff method and hematoxylin over a period of 57 hours after injection. Four generations of t~pe A (called AI, AII, Am, and Air), oneofintermediate, and one of type B spermatogonia occur in one cycle of the seminiferous epithelium. The average life span is about the same in all spermatogonia, i.e., about 27 to 30.5 hours. The average pre-DNA synthetic time, including the mitotic stages from metaphase through telophase and the portion of interphase preceding D N A synthesis, is also not very different, ranging between 7.5 and 10.5 hours. A remarkable difference exists, however, in the duration of DNA synthesis and of the post-DNA synthetic period. The average DNA synthetic time is very long and is highly variable in type B (14.5 hours), a little shorter and less variable in intermediate (12.5 hours) and AIv (t 3 hours) spermatogonia, and much shorter and very constant in Ani (8 hours), All and AI (7 to 7.5 hours) spermatogonia. Conversely, the average post-DNA synthetic time, corresponding essentially to the duration of the prophase, is short and very constant in type B (4.5 hours), longer and variable in intermediate (6 hours) and Ai r (8 hours) spermatogonia, and much longer and much more variable in AnI (11 hours), All and Ax (14 hours) spermatogonia. The premeiotic synthesis of DNA takes place in primary spermatocytes during the resting phase and terminates just before the visible onset of the meiotic prophase. Its average duration is 14 hours. No further synthesis of DNA takes place in later stages of spermatogenesis. I N T R O D U C T I O N Labeled thymidine, which is incorporated exclusively into DNA (8, 37), is a powerful tool for studying cell population kinetics and cell turnover. This label is bound firmly within the cells (there is no reduction of labeling in time due to exchange of tritium or turn-over of DNA), the label being subjected only to dilution following cell division (4, 13, 45, 46). Thus the labeling can be followed intact through one or more cell cycles by using autoradiographic techniques. In addition, the pool of labeled precursors is depleted within a very short time after administration, so that the complicating factor of a continuous labeling of the cells is lacking (4, 13, 39, 45). Autoradiographs made using tritium-labeled thymidine, furthermore, have the advantage of a on S etem er 0, 2017 jcb.rress.org D ow nladed fom very high resolving power because of the very low energy and consequently short range of t r i t i um/3 particles, the average range in tissues being less than 1 lZ (7). Tr i t ia ted thymidine has been used in the past few years by a n u m b e r of workers to study many problems in cell physiology (1, 5, 13, 19, 22, 23, 30, 31, 33, 36, 42, 4 n, 46, 48). The present research, which utilized these techniques, was under taken to study the cellular cycle and D N A synthesis in the spermatogonia and spermatocytes of the mouse. These problems have not been investigated as yet in the spermatogonia of m a m m a l i a n testis. I t has previously been shown tha t the meiotic synthesis of DNA in m a m m a l i a n testis occurs in resting pr imary spermatocytes jus t before the onset of meiotic prophase and tha t no synthesis of D N A takes place in the course of meiosis (3, 26, 43). O the r workers have estimated the dura t ion of the spermatogenesis or its parts (3, 9, 10, 34, 40, 41). I t has also been observed tha t in the mouse testis radia t ion from tr i t iated thymidine has no appreciable effect on survival of spermatogonia at the activities tha t are normal ly used in t racer experiments using the autoradiographic technique (14). M A T E R I A L A N D M E T H O D S (C3H o ~ X 101 9)F1 hybrid male mice, 10 to 13 weeks old, ranging in weight from 21 to 24 gm, were injected intraperitoneally with 15 ~c of Ha-thymidine per mouse and sacrificed by cervical dislocation at intervals ranging from 30 minutes to 57 hours after injection. Five mice were used for the 1 hour interval and three mice for each of the other times. Six mice served as non-radioactive controls. The specific activity of the tritiated thymidine (Schwarz BioResearch, Mount Vernon, New York) measured by the supplier was 1.9 curies per millimole. The testes were removed free of fat, and the tunica cut at one pole. They were then fixed for 24 hours in Orth fluid, washed for 20 hours in running water, embedded in paraffin, sectioned at 5 ~ and stained by the periodic acid-Schiff (PAS) technique. After staining autoradiograms were prepared with NTB2 nuclear track liquid emulsion (Kodak, Rochester, New York), and stored in light tight boxes in a refrigerator at 4°C for 21 days. At the end of the exposure period, they were processed in Kodak D-19 at 17 °(2 for 5 minutes, rinsed in tap water, and cleared in Kodak acid fixer for 8 minutes at the same temperature as the developer. Nuclear staining with Ehrlich's acid hematoxylin was carried out through the autoradiographic emulsion after processing. The seminiferous tubules of the testis were classified according to the description of Leblond and Clermont (18) and Oakberg (28) in twelve successive stages? A fixed number of tubules were scored for each of the 12 stages of the cycle, according to the frequency distribution given by Oakberg (29). Only those tubules cut in cross-section, as judged by their approximation to a circle, were scored. Counts were made of the number of labeled and unlabeled interphases, early and middle prophases, late prophases, metaphase-anaphases, and telophases in type A, intermediate, and type B spermatogonia. Only cells having more than four grains per nuclei were scored as labeled. The number of labeled and unlabeled resting primary spermatocytes and early leptotene nuclei were also scored at tubule stages VI I and VII I . Spermatogonia were classified as necrotic only when degenerative changes were obvious. No attempt was made to count the number of grains lying over the nuclei or to relate this number to the amount of H~-thymidine incorporated into the On the basis of the development of the acrosomic system of the head of the spermatids, as revealed by elective staining with the PAS method, the spermiogenesis in mammals can be accurately studied and subdivided into stages (18). Nineteen stages have been described in the rat (18) and sixteen in the mouse (28). Each stage of spermiogenesis is associated with definite types of spermatogonia and spermatocytes, so that the developmental sequences of spermatogonia to spermatocytes can be accurately identified and studied on the basis of the concomitant process of development of spermatids to spermatozoa. The first twelve stages in the mouse correspond to one "cycle" of the seminiferous epithelium, the cycle being defined as the "series of changes occurring between two successive appearances of the same cell association in an area" (18). Four such cycles occur during the entire duration of spermatogenesis in the mouse, from the "dorman t" type A spermatogonia to the mature spermatozoa (29). Spermatogonia are divided into three classes, type A, intermediate, and type B. The type A spermatogonia have an ovoid, pale, "dust-like" nucleus with a thin nuclear membrane. The type B spermatogonia have a smaller, spherical nucleus that stains more deeply due to the presence of coarse chromatin masses ("crust-like" spermatogonia) lying mainly against the inner surface of the nuclear membrane. The intermediate type spermatogonia are a transitional form between type A and type B spermatogonia, as shown by a thickening of the nuclear membrane caused by adherent chromatin flakes, a deepening of the nuclear staining and a gradual change in the shape of the nucleus from oval to round (2, 18). 2 THE JOURNAL OF CELL BIOLOGY " VOLUME 14, 1962 on S etem er 0, 2017 jcb.rress.org D ow nladed fom cell, because of the great variability among nuclei in the self-absorption of tritium flparticles in histological sections.