DNA changes involved in the formation of metaphase chromosomes,as observed in mouse duodenal crypt cells stained by osmium-ammine I. New structures arise during the S phase and condense at prophase into “chromomeres,” which fuse at prometaphase into mitotic chromosomes

Background: In the hope of understanding how chromosomes condense at mitosis, we took advantage of a subdivision of the cell cycle into 11 stages to examine the changes in DNA taking place during the stages preceding the emergence of metaphase chromosomes. Methods: To identify DNA changes, pieces of mouse duodenum were fixed in formaldehyde, and sections of the rapidly dividing cells of the crypts were stained by the osmium-ammine method, which is specific for the detection of DNA in the electron microscope. Results: Throughout the cell cycle, DNA is present in nucleofilaments composed of rows of 11-nm-wide nucleosomes. At stage I, during which the DNA-synthesizing or S phase of the cell cycle begins, some of the nucleofilaments are compacted in the heterochromatin accumulations associated with nuclear envelope and nucleoli, while the others are scattered in the nucleoplasm where they appear either “free” or “attached” to the heterochromatin. This DNA distribution is similar to that observed in the noncycling cells examined. After the beginning of the S phase, “free” nucleofilaments are seen to assemble into structures composed of compacted nucleofilaments and referred to as “aggregates”; these make their appearance at stage II and increase in size through stage III up to the end of S during stage IV. Meanwhile, the heterochromatin associated with nuclear envelope and nucleoli expands toward the nucleoplasm in the form of protrusions referred to as “bulges,” which gradually enlarge during stages III and IV, while the heterochromatin shrinks and eventually vanishes. On average, a total of 1,171 aggregates and bulges are formed in the nucleus during the S phase. At the apparition of stage V, which corresponds approximately to prophase, aggregates and bulges are rapidly gathered into an average of 288 spheroidal bodies referred to as “chromomeres.” These are connected to one another by nucleofilamentous bridges in such a way as to be lined up in rows. The formation of rows of chromomeres represents in the electron microscope the prophasic condensation observed in the light microscope. Finally, during stage VIa, which corresponds to prometaphase, the chromomeres approach one another within each row, make contact, and coalesce to become the 40 chromosomes of the mouse, which during stage VIb are organized in the equatorial plate of metaphase. Conclusions: The condensation of metaphase chromosomes occurs in three main steps. The first and longest takes place during the S phase, as nucleofilaments are assembled into aggregates, while the heterochromatin give rise to bulges. The brief second step occurs toward the beginning of prophase, when the numerous aggregates and bulges are congregated into a limited number of chromomeres, which are lined up in rows. The third step takes place during the brief prometaphase, when the chromomeres of a row coalesce into a mitotic chromosome. © 1995 Wiley-Liss, Inc.