Studies on the sea urchin egg nucleus: its isolation, structure, physical properties and DNA content

Abstract

Present day cell theory considers deoxyribonucleic acid (DNA) and the genetic substance of the cell to be synonymous. If this is in fact true, then there should be no stage during the life history of an animal or plant when DNA is not present in the cell and, more specifically, in the nucleus. Investigations of a large variety of cells have always, with one important exception, found DNA in the nucleus. This DNA is present in amounts proportional to the number of chromosomes in the cells of anyone organism. For example, the haploid sperm of the mouse has half (1.68 arbitrary units) the DNA found in diploid liver nuclei (3.34 units). Polyploid liver nuclei have DNA in amounts proportional to their chromosome number (6.77 units for tetraploid and 13.2 units for octoploid nuclei) (Swift 1950). One exception to this rule is the nucleus of the mature haploid egg. Here, it is often very difficult and sometimes impossible to demonstrate the presence of any DNA. In the past the only means for making such measurements has been through the use of histochemical methods such as the Feulgen stain. There has, until now, been no way to isolate nuclei (except by micromanipulation) and measure their DNA content directly. 2 When the DNA content is measured for the whole egg (for example, sea urchin eggs) a large amount is found, usually more than 30 times the haploid amount (see Table V). This much DNA can not all be accounted for as nuclear DNA and thus the eggs of all animals investigated so far are unique in having cytoplasmic DNA or at least DNA fragments. Therefore measurement of whole cell DNA (a common technique used for somatic cells), when applied to the egg, gives no indication of how much DNA the nucleus contains. The results of Marshak and Marshak (see Chapter IX), which represent the most complete analysis of sea urchin DNA to date, lead to the surprising conclusion that the sea urchin egg contains no DNA at all. They conclude that the DNA measured by other investigators is in the form of low molecular weight fragments and is therefore not genetically significant. Marshak and Marshak also used whole eggs as analytical material. Thus the present situation in the published literature is: the sea urchin egg pronucleus is not easily stained by the Feulgen reagent, and sometimes not at all; analysis of whole eggs indicates large amounts of DNA which must, at least in part, be cytoplasmic. This DNA has been found by Marshak and Marshak to consist of low molecular weight fragments and can not be genetically significant. Therefore there is no proof that the nucleus contains DNA. Whether or 3 not it does, determines whether or not DNA could be the genetic material of the cell. The research carried out in this thesis was designed to conclusively answer this question. To do this, a method for measuring the DNA content of the nucleus alone had to be developed. This could be done 1) by utilizing single isolated nuclei which could, in theory, be analyzed by a method using radioactive isotopes (see Chapter VIII), or 2) by analyzing large numbers of purified isolated nuclei. Both these methods were thoroughly investigated. Another method, by which the presence of DNA could be conclusively shown, would be through the use of the Feulgen stain accompanied by the necessary controls. A method for controlled Feulgen staining of sea urchin eggs is described in Chapter X. In several cases an investigation of problems not directly related to the topic of this thesis was required. For example, in order to use the local sea urchins it became necessary to know where they could be found, their spawning season and, in the case of the three urchins of the family Centrechinidae, it was necessary to work out a means for easily identifying them. This work is included here in the hope it will be of use to other investigators who may want to utilize the Hawaiian sea urchins. During the investigation leading to the method for isolating sea urchin egg nuclei described in Chapter IV, a number of interesting observations were made on the nature of the isolated nucleus and its nucleoplasm. These observations are considered in Chapters IV and VII. They give further insight into the nature of the sea urchin nucleus over that obtained by workers using whole eggs. The major part of the work described in this thesis was carried out at the Hawaii Marine Laboratory on Coconut Island in Kaneohe Bay, Oahu.