Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

Abstract

A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear membrane, nuclear pore complexes, and decondensed chromatin etc. Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei. Western-blotting results showed that lamin L II was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin L III .

In vitro nuclear reconstitution could be induced in a plant cell‐free system

“…The reason might be that there are much more nutrients (such as proteins) stored in Xenopus egg extract than in carrot cell extract, which only could support a small amount of sperm undergoing nuclear reconstitution. In Xenopus egg extract, the newly reassembled nuclei could maintain the conformation for more than 8 h [10]. However, we did not observe intact newly reassembled nuclei in carrot cell extract after incubation for 8 h. This result also indicated that there is a correspondingly small quantity of storage nutrition in the plant cell-free system, and there was a contrast between its holding ability to reassembled nuclei and Xenopus extract.…”

“…The newly reassembled nuclei in the plant cell‐free system had the typical characteristics of normal nuclei, such as a double‐layered nuclear envelope, nuclear pores and a nucleosome. However, only about 20% of the sperm could be induced to undergo the nuclear reconstitution in this cell‐free system, which differs from Xenopus extract, in which about 40% or more sperm formed new nuclei [10]. The reason might be that there are much more nutrients (such as proteins) stored in Xenopus egg extract than in carrot cell extract, which only could support a small amount of sperm undergoing nuclear reconstitution.…”

“…The sperm, purified from Xenopus , was highly purified for the experiment. Since it had been proven that the induction of reassembled nuclei in an animal cell‐free system was independent of the source and length of exterior DNA [9–12], the nuclear reconstitution in a carrot cell‐free system using highly purified Xenopus sperm chromatin could partially answer the question of whether a cell‐free system based on plant cell extract could induce nuclear reconstitution, and if the process of nuclear reconstitution in a plant cell‐free system was similar to the process in the animal cell extracts.…”

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

“…Since Lokha and Maisui first demonstrated that demembranated sperm chromatin can be induced to perform nuclear reassembly in a cell-free system from amphibian eggs components in 1983 [6], a great deal of works followed later suggested that nuclear assembly in vitro can be successfully induced around exogenous DNA or chromatin derived from animals [11], plants [9] and bacteria [7]. Thus, nuclear assembly is independent of the species from which DNA was purified.…”

“…Interphase extracts can support nuclear assembly and transport, whereas mitotic extracts can disassemble nuclei. Since 1989, in our laboratory, we have successfully got the nuclei assembled in vitro around exogneous naked DNA from animals [8], plants [9], rDNA [10] and demembranated sperm chromatin [11]. Meanwhile, we have done some research concerning the mechanism of nuclear envelope reconstitution [12].…”