Quantitative and ultrastructural analysis of the chondriome in ovogenesis and embryogenesis of the sea urchin Paracentrotus lividus. 2. Growth and proliferation of mitochondria in embryogenesis.

The dynamics of structural changes of the chondriome in the early development of the sea urchin Paracentrotus lividus was studied. Mature eggs and embryos at various stages of cleavage were used for quantitative and ultrastructural analysis based on computerized 3D reconstruction from serial ultrathin sections. The following structural transformations of the chondriome were shown to occur in the course of embryogenesis: (i) 15 min after fertilization, mitochondrial clusters disintegrate, and mitochondrial division is induced. At the stage of two blastomeres the population of mitochondria increases twofold; (ii) the mitochondria divide by means of the contraction of both outer and inner membranes. The forming furrow divides the "parental" mitochondrion into two equal "daughter" parts; (iii) at the four-cell stage the division ceases, and mitochondria start to grow, so that the mitochondrial length increases; (iv) cell differentiation further stimulates elongation of rod-shaped mitochondria, and the ratio of rod-shaped to spherical mitochondria changes; (v) in an unfertilised egg, the mitochondria are in a condensed form; after fertilisation all the mitochondria acquire a conventional form. Modern concepts of chondriome proliferation in eukaryotic cells are discussed.

Spatial localization of chromosome-nuclear envelope interaction sites.

In the interphase nucleus chromosomes are tightly associated with the nuclear envelope (NE) through special granular chromatin particles termed anchorosomes. It remains unclear whether anchorosomes represent constant nuclear structures, persisting throughout the cell cycle, or they appear only in the interphase during the formation of contacts between the chromosomes and NE. In other words, whether specific NE interaction sites do exist in chromosomes or any region can form anchorosome. In this work, we used micrononucleated PK cells, in which almost every micronucleus (MN) is formed by a single chromosome. The spatial distribution and quantitative characteristics of the anchorosomal layer in MN was studied using stereological analysis and three-dimensional computer reconstruction. It was shown that in cells with about 30 MN, the total surface area of NE reaches about 355 microm2, whereas in normal mononuclear cells it is 110 microm2. Hence, the NE surface increases 3-fold during MN formation. In contrast to normal cells, only 80% of the NE surface in MN is covered with anchorosomes, i.e., the total surface area of the anchorosomal layer increases by a factor of 2.5. The 3D reconstruction has demonstrated highly random distribution of anchorosome-free zones, the distribution patterns varying in individual MN. These findings are thought to be evidence for the existence of a limited number of specific chromosomal sites potentially capable of forming contacts with NE.

Quantitative and ultrastructural analysis of the chondriome in ovogenesis and embryogenesis of the sea urchin Paracentrotus lividus.

The dynamics of chondriome in the ovogenesis of the sea urchin Paracentrotus lividus was studied. Growing oocytes 20-30, 50-60 and 90-100 microm in diameter ("small", "medium-sized" and "large", respectively) and mature eggs were used for the ultrastructural and stereological analysis of mitochondria. Linear parameters of mitochondria (length and thickness) were measured on 3-D reconstructions of serial ultrathin sections using the software developed in the laboratory. The following transformations of chondriome structure were shown to occur during ovogenesis: (1) the number of mitochondria (MT) increases with the growth of cytoplasmic compartment; (2) the modal length of MT increases from 0.5 microm in small oocytes to 1 microm in large ones and decreases again to 0.5 microm in the egg; this process is accompanied by changes in the relative number of spherical MT which decreases in medium-sized oocytes and subsequently rises again in the egg; (3) in medium-sized oocytes, dumbbell-shaped MT appear first, the number of these MT reaching the maximum to the stage of large oocytes. In mature eggs, the dumb-bell-shaped MT are absent; (4) in small and medium-sized oocytes, the orthodox conformation of MT is observed, in contrast to MT with a condensed matrix in large oocytes and eggs; (5) in mature eggs, mitochondrial clusters containing 10 to 20 MT of various size are formed. Based on the data obtained, we suggested that during ovogenesis of the sea urchin, specific differentiation of the chondriome is induced which leads to the increase in the quantity of MT via multiple division acts, while restricting the MT growth and variability of their shape.

The dynamics of structural modifications of mitochondria at the early stages of sea urchin embryonic development.

The organization of the chondriome and the ultrastructure of mitochondria have been studied in eggs and embryos of the sea urchin Paracentrotus lividus. The egg chondriome is characterized by an arrangement in well-delimited clusters. Analysis of mitochondrial clusters on electron micrographs of ultrathin serial sections shows two kinds of mitochondria of different shapes, the rod-shaped and the spherical. The egg mitochondria have a dense matrix and a well-ordered arrangement of cristae which, in rod-shaped variety, are perpendicular to the major axis. Cell division is accompanied by significant changes in intracellular distribution of mitochondria and in their structure. At the stage of 2-4 blastomeres, the clusters break up and numerous mitochondrial rods show signs of fragmentation; most of the observable mitochondria are of spherical shape. At the same time, the matrix becomes less dense, and the orderly arrangement of the cristae disappears. From the blastula to the gastrula stage, the observed modifications are reversed: the number of spherical-shaped mitochondria decreases, while that of the rod-shaped increases; the diameter of the latter is almost equal to the initial diameter of the spherical forms, the matrix becomes dense again and the cristae resume their orderly arrangement.