Since we had subdivided the cell cycle into 11 stages—four for mitosis and seven for the interphase—and since we had experience in detecting DNA in the electron microscope (EN) by the osmium-amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041–3044), we combined the two approaches for the analysis of DNA-containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde-fixed mouse duodenum were stained by osmium-amine for electron microscopic examination of the stages in the 12.3-hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi-thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA-specific Hoechst 33258 dye and examined in the fluorescence microscope. The DNA detected by osmium-amine is in the form of nucleofilaments, seen at high magnification as long rows of 11 nm-wide rings (consisting of stained DNA encircling unstained histones). At all stages of the cycle as well as in nondividing cells, nucleofilaments are of three types: ‘free,’ ‘attached’ to chromatin accumulations, and ‘compacted’ in all chromatin accumulations, the form of dense spirals within. At stage I of the cycle, besides free and attached nucleofilaments, compacted ones are observed in the three heterochromatin forms (peripheral, nucleolus-associated, clumped). Soon after the S phase begins, chromatin ‘aggregates’ appear, which are small at stage II, mid-sized at stage III, and large at stage IV. Chromatin ‘bulges’ also appear at stage III and enlarge at stage IV, while heterochromatins disappear. At stage V, aggregates and bulges accrete into ‘chromomeres,’ a process responsible for the apparent chromosome condensation observed at prophase. The chromomeres gradually line up in rows and, at stage VIa (prometaphase), approach one another within each row and coalesce to build up the metaphase chromosomes which are fully formed at stage VIb (metaphase). Daughter chromosomes arising at stage VII (anaphase) are eventually packed into a chromosomal mass at each pole of the cell. During stage VIII (telophase), the chromosomal mass is split into large chunks. In the course of the G1 phase, the chunks thin out to give rise to irregular ‘bands’ at stage IX, the bands are then cleaved into central and peripheral fragments at stage X, and finally the central fragments are replaced by free nucleofilaments and clumps at stage XI, while the peripheral fragments are replaced by peripheral heterochromatin. The “nucleoli” at stages I–III are associated with stained heterochromatin but otherwise appear as unstained lucent areas, except for weakly stained patches composed of histone-free DNA filaments. During stage IV, nucleoli lose patches and associated heterochromatin, while weakly lucent, pale vesicles appear within nucleoli and in the nucleoplasm. By the end of substage VIa, nucleoli generally disappear, while pale vesicles persist around the chromosomes appearing at substage VIb. At stages VIII and IX, the vesicles seem to become strongly lucent and, at stages IX and X, they associate and fuse to yield homogeneous lucent areas, the ‘prenucleolar bodies,’ which include histone-free DNA patches. During stage XI, groups of these bodies associate to give rise to nucleoli. In conclusion, the cell cycle DNA changes can be classified into 4 broad periods (Fig. 6): 1) Stage I is a 2-hr long interphase “pause,” during which the stained DNA shows no signs of either chromosome condensation or decondensation, while the overall nuclear pattern is similar to that in nondividing cell nuclei. Nucleoli are fully developed. 2) From stage II to VIa, the “chromosome condensation” period extends over about 7 hr, during which the events are interpreted as follows. Throughout the S phase (stages II–IV), newly-synthesized segments of nucleofilaments approach one another, adhere and thus build aggregates and later bulges on nuclear matrix sites. The process continues until, soon after S ends, that is in G2, every segment of nucleofilament has become part of aggregates and bulges which thus include wholly new nucleofilaments. Then, the bonds tying aggregates and bulges to the matrix are broken, leaving them free at prophase (stage V) to approach one another, adhere, and thus build chromomeres. At prometaphase (substage VIa), a last adhesion process unites the chromomeres of a row which thus build metaphase chromosomes. Meanwhile, nucleoli lose the histone free-DNA patches at stage IV and disappear at the end of stage VIa. 3) Stages VIb and VII constitute the “climax” of the cell cycle, a 20-min period during which metaphase chromosomes at the equatorial plate split into daughter chromosomes. 4) From stage VIII to XI, the “chromosome decondensation” period extends over about 2 hr during which progressive dissociation of the stage VIII chromosomal mass eventually yields free and attached nucleofilaments as well as the three heterochromatins. Meanwhile lucent vesicles fuse into prenucleolar bodies which finally coalesce into nucleoli. Anat. Rec. 252:426–443, 1998. © 1998 Wiley-Liss, Inc.
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Aug 1, 2000
Celia Antonio + 6
