Nuclear changes in the course of development of the sea urchin studied by means of Hale staining

Abstract

The nuclear changes occurring in the course of the early development of Paracentrotus lividus have been studied by means of the ferric acetate staining method of Hale. If the sections of Carnoy fixed eggs or embryos are subjected to the Hale procedure, very few interphase nuclei become stained. Treatment of the sections with a buffer or a buffered trypsin solution prior to the Hale procedure may induce a staining of the interphase nuclei, which varies according to pH of the buffer, stage of development and position in the embryo, in a way, shown in Text-Figs 1–3. During the early development, the interphase nuclei remain unstained even after pretreatment. At an early blastula stage (6 hr after fertilization), the interphase nuclei become Hale positive, following a pretreatment with 0.0025 per cent trypsin (Figs 1c, d). Only at the stage of free swimming blastulae (14 hr after fertilization), pretreatment with acetate buffer gives a certain staining of the interphase nuclei, a staining that then increases in intensity and becomes less pH dependent during the following stages of development (Figs 2 e-j). The prophase chromosomes are not directly stained by the Hale reagent, but staining may be induced in any stage of development by pretreatment with buffer at pH above 3, or with trypsin within the pH range of activity of this enzyme. At pH values above 3, the meta- and anaphase chromosomes become stained without any pretreatment of the sections. At early blastula stage, the Hale staining of the interphase nuclei is not influenced by exposure to DNase or RNase. If, however, the sections are pretreated with trypsin and thereafter exposed to a mixture of DNase and RNase, the staining capacity of the interphase nuclei is abolished. In stages from the late blastula stage onwards, DNase decreases or abolishes the Hale reaction without any previous pretreatment. RNase may affect, but does not alone abolish the staining. The inference from the data is that Fe 3+ in the Hale reagent competes with nuclear proteins for reactive sites in the nucleic acids. These sites may correspond mainly to the phosphate groups. In the interphase nuclei, the proteins block the access of Fe 3+ to the phosphate groups with a strength varying according to the circumstances defined above. In prophase nuclei, the effect of the proteins is weaker and the Hale staining is therefore easily provoked upon pretreatment. In the meta- and anaphase chromosomes, Fe 3+ eliminates without pretreatment the action of proteins. The Hale staining of interphase nuclei is different in different embryonic regions and it often seems to reflect different intensities in synthetic activity. The propensity of interphase nuclei for Hale staining seems to parallel their nucleolar activity and consequently their production of ribosomal RNA. It may also parallel certain changes in pattern of messenger RNA formation during development.