Locations of the 5S rRNA genes (rDNA) are usually considered to be helpful, as generally invariable chromosome markers, especially if chromosome banding is not possible. The genes have been defined in about 90 invertebrate species, and were found linked to other multigene families in genomes of some nematodes and arthropods (Drouin and Moniz de Sa, 1995; Barzotti et al., 2000). They were also found co-localized by FISH with major rDNA clusters (NORs) and telomeric repeats in some annelids and molluscs (Vitturi et al., 2002, 2004). Here we report for the first time the chromosomal localization of 5S rDNA in a species of echinoderms, Paracentrotus lividus, which, compared to other echinoderms, has an exceptionally low chromosome number, 2n = 36 (Boveri, 1902). Its karyotype is composed of one pair of large submetacentric chromosomes, a pair of sex-specific heteromorphic chromosomes, and 16 pairs of subtelocentric chromosomes (Lipani et al., 1996). Materials and methods

Individual mitochondria which form the chondriom of eucaryotic cells are highly dynamic systems capable of fusion and fragmentation. These two processes do not exclude one another and can occur concurrently. However, fragmentation and fusion of mitochondria regularly alternate in the cell cycle of some unicellular and multicellular organisms. Mitochondrial shapes are also described which are interpreted as intermediates of their "equational" division, or fission. Unlike the fragmentation, the division of mitochondria, especially synchronous division, is also accompanied by segregation of mitochondrial genomes and production of specific "dumbbell-shaped" intermediates. This review considers molecular components and possible mechanisms of fusion, fragmentation, and fission of mitochondria, and the biological significance of these processes is discussed.

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules—chromomeres, chains of chromomeres—chromonemata, aggregates of chromomeres—blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei.

The influence of actin and tubulin cytoskeletons on the shape, division and on intracellular motility of mitochondria was studied in eggs and embryos of the sea urchin Paracentrotus lividus. Depolymerization of actin filaments and microtubules was induced by specific inhibitors as cytochalasin D (CytD) and colcemid respectively. The quantitative analysis of the mitochondrial population shows that: 1) the chondriome of an egg consists of numerous (about 15,000) discrete mitochondrial clusters uniformly distributed throughout the cytoplasm, each cluster containing 10 to 20 mitochondria of spherical or rod-like shape; 2) fertilization induces cluster break-down and mitochondrial division within 15 min after insemination; at 100 min after fertilization mitochondria become evenly distributed throughout the cytoplasm and the population of mitochondria doubles; 3) in embryos obtained from eggs inseminated after treatment with CytD clusters break-down and mitochondriokinesis are blocked; 4) when added 15 min after insemination, CytD uncouples coordinated invagination of outer and inner membranes in dividing mitochondria thus bringing about abnormal mitochondriokinesis; 5) the treatment of the eggs with colcemid does not affect the normal embryonic mitochondriokinesis.

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.

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. in the identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein γ-tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes was obtained from the loach spermatozoa. These centrosomes contained γ-tubulin, although they had lost their ability to induce the polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and then on microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising means to identify new proteins in the mammalian centrosome.

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. for identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein gamma-tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes has been obtained from the loach spermatozoa. These centrosomes contained gamma-tubulin although they lost their ability to induce polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and on then microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising object for identification of new proteins of the mammalian centrosome.

The dynamics of structural and functional organization of the nucleolus in the oocytes of P. lividus is described. At the late stages of oogenesis the nucleolus is composed of two main components, namely the peripheral zone (PZ) and the central zone (CZ) which are spatially separated. This two-component structure of the nucleolus is formed, at early stages of oogenesis, by stepwise segregation of the fibro-granular component and by its migration to the nucleolar periphery. Absence of morphologically distinct fibrillar centers and dense fibrillar component in nucleoli of both somatic cells and oocytes makes it possible to classify the nucleoli of P. lividus as 'noncanonical' type. Based on detailed morphological and cytochemical analysis the following molecular interpretation of nucleolar ultrastructure in oocytes of P. lividus is proposed: 1) the PZ, containing RNP-positive granules 15 nm in size, but lacking Ag-NOR proteins and BrU incorporation, can be considered a structural equivalent of the granular component of 'typical' nucleoli; 2) the CZ, which is the site of incorporation of RNA precursors, contains intranuclear DNA, RNP-fibers and accumulates Ag-NOR proteins, corresponds to both FC and DFC of 'typical' nucleoli; 3) nucleolar growth during oogenesis, leading to the 1000-fold increase of nucleolar volume, seems to be correlated with the stockpiling of nonfunctioning mature preribosomal particles which will be utilized during embryogenesis.

The structure of a “noncanonical” nucleolus of vitellogenic oocytes in the sea urchin Paracentrotus lividus was studied using the inhibitor of transcription actinomycin D. In the control cells, the nucleolus consists of two separated structural subdomains: the dense fibrillar-granular peripheral area and the fibrillar central area. The nucleolus did not contain subdomains corresponding to the fibrillar center and dense fibrillar component of “typical” nucleoli. After treatment with actinomycin D, numerous argyrophilic granules appeared in the karyoplasm, the intranucleolar DNA became compact, and the nucleolar material was segregated into two or three separated zones, the residual peripheral area being the densest and largest. Lesser zones had a decreased electron density and contained argyrophilic proteins and, apparently, the nucleolar organizer material. These results suggest that, for normal rRNA expression and processing, the presence of structural subdomains in the nucleolus, such as fibrillar complexes and a dense fibrillar component, is not essential.

Activated oocytes of Xenopus laevis were injected with centriole preparations isolated either from spermatozoa of leach fish Misgurnus fossilis or form rat liver. These injections induced the development of cytasters in the ooplasm and egg cleavage. Electron microscopic study of cytasters was made at the stage that corresponded to interphase between first and second cleavage divisions. This study revealed in cytasters singleton centrioles surrounded by pericentriolar material and numerous microtubules. Surprisingly, the ultrastructure of centrioles in cytasters corresponded to that of juvenile, newly formed vertebrate centrioles, whereas the injected preparations contained only adult mature centrioles. We suggested that xenogenic centrioles injected to Xenopus laevis oocytes could dissolve after formation of centrioles made from molecules of oocyte origin. A special mechanism that eliminates male centrioles after egg fertilization. is speculated. (C) 1999 Wiley-Liss, Inc.

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.

It is known that in oocytes of P. lividus the nucleus contains a single giant nucleolus of unusual structure where intensive rRNA synthesis occurs. However, the questions of structural and functional relationships between the nucleolar compartments and the sites of active rRNA transcription still remain open. In the present work, we studied the chromatin organization in the nucleoli of P. lividus oocytes using Feulgen's and osmium amine staining procedures. Our results indicate that nucleolar chromatin of small (immature) oocytes differs from that in large mature oocytes. At the early stage of development, the DNA filamentous network 0.2-0.5 microm in diameter is formed in the nucleoli. Profound changes in the structure of the nucleolar DNA are observed in the course of oogenesis. Thus, at the late stage of development, the nucleolar chromatin forms characteristic ring-like structures, which indicates a non-uniform distribution of active ribosomal genes. A model of structural organization of the P. lividus nucleolus is discussed.

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 chondriome changes in oogenesis of the sea urchin Paracentrotus lividus were studied by electron microscopy. An oocyte‐enriched fraction obtained by gonad mechanical dissociation without protease treatment was used. The shape, size and arrangement of mitochondria (Mt) in cells were quantitatively analysed on the basis of data from reconstruction experiments, with serial sections performed using a specific computer program. At all stages of oogenesis, the chondriome was shown to consist of rod‐shaped Mt of various lengths and also of small amounts of globular Mt about 0.3 μm in diameter. Chondriome transformation during oogenesis is shown to involve the following processes: (1) a 64‐fold increase in number of Mt, with the ratio of cytoplasm to Mt volume quite constant in the course of oogenesis; (2) an increase in length of Mt to a maximum of 1.54 μm in medium oocytes and successive considerable mitochondrial division; (3) changes in Mt ultrastructure; and (4) a clustering of Mt. In a mature egg, the modal value of Mt length was reduced and, unlike the oocytes, was more homogeneous, and the Mt were completely clustered.

Treatment of the isolated nuclei with polyanions (PA) (heparin and dextran-sulfate) at the PA/DNA ratio of about 1 in the 0.15-0.5 M ammonium acetate solution leads to rearrangement of the chromatin structure and formation of compact globular particles (GP) 40-70 nm in diameter, bound by fibrils of variable thickness (2-10 nm). GP formation is accompanied by a loss of nucleosomal periodicity in DNA organization. However, no considerable loss of the DNA contents has been found. At the PA/DNA ratio of more than 2, destruction of the DNP-network and aggregation of GP in larger globular structures are observed. The same effect is observed under the nuclease treatment. Under this condition nuclei lose a considerable part of DNA, but retain histones supposedly as histone-PA aggregates. Therefore the joint effect of PA and salt on nuclei at certain conditions may lead to formation of artefact structures.

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.