Electron microscopic and time lapse studies of mitosis in cultured rat hepatocytes

Abstract

Primary cultures of adult rat hepatocytes in a serum-free medium were observed by time lapse cinematography to proceed through mitotis and cytokinesis. An ultrastructural study of these cultures is presented with electron micrographs of each stage of mitosis and cytokinesis. The cultured hepatocytes begin to enter prophase about 48 hr after plating and proceed through mitosis in approximately 70 min not including cytokinesis. During this time, they remain somewhat flattened and joined to neighboring cells rather than rounding up. Both mononucleate and binucleate hepatocytes proceed through mitosis. Some mononucleate cells do not undergo cytokinesis, resulting in the formation of binucleate cells. In binucleate hepatocytes, both nuclei proceed through prophase simultaneously. Usually a single mitotic spindle with a large metaphase plate containing chromosomes from both nuclei is observed. Cytokinesis frequently occurs in binucleate hepatocytes which have a single mitotic spindle. Some binucleate cells form tripolar or 4-polar metaphase plates. In tripolar metaphases, some cells do not divide, resulting in multinucleate cells, whereas others undergo cytokinesis yielding three mononucleate cells or one binucleate and one mononucleate cell. Two mitotic spindles located perpendicularly to each other with microtubules intertwining have been observed in 4-polar metaphases. In this latter case, no cytokinesis has been observed. This study shows that both mononucleate and binucleate adult rat hepatocytes cultured in a serum-free medium in the presence of epidermal growth factor not only synthesize DNA, but progress through mitosis and often cytokinesis.

Occurrence and possible consequences of multipolar mitoses in primary cultures of adult rat hepatocytes

“…Since the mitotic spindles of both nuclei are oriented in the same plane a maximum interaction is also provided enhancing the probability for multipolar mitotic stages to occur. The different degrees of interaction between bipolar spindles of binucleated cells observed and time-lapse studies by Sattler et al (1989) confirm this suggestion.…”

Citron Kinase Is a Cell Cycle-dependent, Nuclear Protein Required for G2/M Transition of Hepatocytes

“…Liver Perfusion-Single cell suspensions of hepatocytes were prepared with a modified two-step collagenase perfusion method (24). Cell viability was Ͼ90% as measured by trypan blue exclusion.…”

“…Liver samples removed during surgery were saved as time 0 quiescence controls. Animals were euthanized by CO 2 asphyxiation at the following hourly intervals after PH: 20,22,24,26,28, and 30 h. Portions of livers were snap frozen in liquid nitrogen and stored at Ϫ80°C for protein extraction. A sham operation, including laparotomy and mobilization of liver without tissue removal, was performed as a control.…”

Measurement of ploidy and cell proliferation in the rodent liver.

“…In the guinea pig, the process of polyploidization is limited to the production of a small fraction of binucleated hepatocytes (1 7), and in the hamster a small population of tetraploid cells also appears (17), but in rats and mice polyploidization affects the majority of hepatocytes and is a progressive phenomenon that continues throughout the life of the animal (11)(12)(13)(14). In rats the rate of polyploidization slows markedly by the age of about 10 weeks and the liver consists mainly of tetraploid cells (2 x 2n and 4n) and some octoploids (2 x 4n and 8n), but in the mouse the phenomenon progresses to a greater extent, and higher multiples of the diploid DNA content may be observed as the animal ages.…”