[Actin localization in the nuclei of two-cell mouse embryos].

In this study, preimplantation mouse embryos were used as a new model for investigation of actin distribution in the nuclei and identification of functional forms of intranuclear actin. Combination of direct detection of actin by fluorescent-conjugated falloidin and DNase I with the method of indirect immunofluorescence was applied as an integrated approach to study localization of actin in the nuclei of two-cell mouse embryos. Monomeric actin and two oligomeric forms of actin were detected in the nuclei, and each of these forms demonstrated its own pattern of distribution. Oligomeric actin recognized by antibodies to C-terminal domain of actin was associated with condensed chromatin as well as with metaphase chromosomes and chromatin of second polar body. Monomeric actin and another oligomeric form recognized by antibodies to N-terminal domain were revealed in the area of dispersed chromatin localization.

Nuclear structure in early mouse embryos: A comparative ultrastructural and immunocytochemical study with special emphasis on the "2-cell block in vitro".

The embryos from many outbred and inbred strains of mice are arrested at the late 2-cell stage when cultured in vitro in simple culture media. This phenomenon is referred to as the "2-cell block in vitro". The ultrastructural morphology of the nuclei of the blocked embryos is not yet well described. In the present paper we documented the results of a comparative study on the nuclei of mouse embryos, both normally developing and arrested at the 2-cell stage. The blocked embryos maintain the morphological integrity of their nuclei. Main nuclear domains (nucleolus precursor bodies, interchromatin granule clusters, perichromatin granules, and perichromatin fibrils), typical for the control embryos, are observed in the blocked ones. A number and morphological characteristics of these nuclear substructures are not changed significantly in the blocked embryos. At the same time, RNA polymerase II and pre-mRNA splicing factors are redistributed in the nucleus of the blocked embryos. Although something goes to show that nuclear organization of the blocked embryos differ from that of the control, we could not reveal in the blocked embryos distinct signs of degeneration which might characterize aged or dying cells. Our data confirm a peculiar functional state of the 2-cell blocked embryos.

[Dynamics of morphofunctional organization and developmental potential of mouse embryos in the state of "two-cell block in vitro"].

Vital observation in combination with electron microscopy and immunocytochemistry was used for studying structural organization and developmental potential of BALB/c mouse embryos after cleavage cessation at a two cell stage caused by the "two-cell block in vitro" phenomenon. Modification of structure and viability of embryos was followed for 2 days from the time of cleavage arrest. Several hours before cleavage arrest, changes in mitochondrian distribution were noticed in embryos, no other disturbances in structural organization of blastomeres being obvious. Embryos, whose development was arrested for 24 h, remained viable and demonstrated some morphological changes similar to those seen in normally developing embryos of the same age. Towards the end of a 48 h block period some embryos died, the surviving embryos remained morphologically intact and metabolically active. At the same time, the nuclei of the latter frequently displayed chromatin condensation near the nuclear membrane, which is similar to the pattern of chromatin reorganization in the nuclei of early apoptotic cells. Our results support a concept on the "two-cell block in vitro" phenomenon as a specific functional state of embryos, and well compare with data on a partial realization by blocked embryos of the developmental program.

[Changes in the distribution of mitochondria in mouse embryos blocked at the two-cell stage].

Changes in the distribution of mitochondria in the two-cell mouse embryos preceding the developmental arrest in vitro, caused by a genetically determined "two-cell block in vitro" or genisteine treatment, were examined vitally using the mitochondrial-specific probe rhodamine 123 and conventional fluorescence microscopy. In the former case, serious disturbances in the localization of mitochondria appeared already from the middle of two-cell stage, long before the time corresponding to the 2nd cleavage division. Comparison of the behavior of mitochondria in the embryos successfully developing between the one- and two-cell stages and that in the embryos that ceased to cleave suggests that the developmental arrest was accompanied by aggregation of the mitochondria into clusters. There are many such clusters unlike in the cytoplasm of normally developing embryos. Intracellular localization of clusters observed in the genisteine-treated embryos differed radically from that observed in the embryos blocked in vitro at the two-cell stage.

[Characteristic of structural and functional organization of two-cell mouse embryos exposed to inhibitors of cell proliferation]. / Osobennosti strukturno-funktsional'noi organizatsii dvukhkletochnykh zarodyshei myshi pri vozdeistvii nekotorykh ingibitorov kletochnoi proliferatsii.

Within the framework of studying the "2-cell block in vitro" phenomenon, two variants of inhibitory-induced stoppage of development at a two-cell stage were produced and analysed. Mimosine arrested the cleavage on the G1/S interface, and genistein at G2 stage of the second cell cycle. In the experimentally blocked embryos a detailed study was made of the ultrastructural organization of blastomeres and intracellular localization of mitochondria vitally stained with rhodamine 123. The light and electron microscope observations testify to the viability of the embryos within a 22-24 hour exposure to inhibitors. Adhesive contacts between blastomeres were seen to slack after the treatment with both the inhibitors, resp., but in particular after genistein treatment. At the ultrastructural level no significant destructive modifications in blastomere organization were noticed. The cytoplasm of the control and treated embryo cells displayed diffusely distributed sheets of intermediate filaments, morphologically looking immature mitochondria and numerous aggregated lipid inclusions. The nuclear morphology was similar in both cases. Mitochondria of the treated embryo cells kept their ability to accumulate rhodamine 123, which testifies to their functional activity. However, the character of mitochondrial intracellular distribution was seen to change from diffuse to clustered. Numerous mitochondria clusters were concentrated mainly in the perinuclear area of blastomeres. As in the control ones, in the treated embryos the position of the nuclei was visualized by ring-like concentrations of mitochondria in the central part of blastomeres; in mimosine-treated cells the "rings" were thickened and contained mitochondria clusters. In genistein-treated embryos, mitochondria form numerous tiny clusters uniformly distributed in the cytoplasm; the perinuclear "rings" are still present, though less distinct than in the control embryos. Thus, it may be concluded that although the inhibitory treatment of two-cell embryos truly modified the mitochondrial distribution in these, the eventual pattern of such changes differed considerably from that characteristic of embryos in the state of "2-cell block in vitro". These results support the view on the unique character of morphofunctional modifications that occur in the latter embryos.

[Segregation and integration of cytoplasmic and nuclear materials during cleavage and induced fusion of blastomeres of mouse early embryos]. / Segregatsiia i integratsiia tsitoplazmaticheskogo i iadernogo materiala pri droblenii i indutsirovannom sliianii blastomerov rannikh zarodyshei myshei.

Mouse embryos were analyzed vitally, on fixed total and chromosome preparation at the two-cell stage and during transition to the four-cell stage, upon polyethylene glycol-induced fusion of sister blastomeres and early cleavage of thus obtained somatic hybrids. Vital staining by Rhodamine 123 showed a diffuse distribution of functionally active mitochondria with a decreasing gradient towards the periphery of blastomeres, concentration in the zone of contact between the blastomeres, and as a ring in the center. A weakly fluorescent zone inside the mitochondrial "ring" corresponded to the interphase nucleus position, as followed from vital staining by Hoechst 33258 and analysis of fixed embryos stained by lacmoid. In the course of cleavage, the mitochondria aggregated in the zone of condensed chromosomes. The topography of mitochondria reflected the barrel shape of the spindle division similar in the intact blastomeres and products of fusion and allowed us to follow segregation of the cytoplasmic and nuclear materials among the daughter cells until the mitosis completion. During formation of the fusion products, the preserved cytoplasmic boundary was visualized by the location of mitochondria, thus suggesting the absence of intense changes in the cytoplasm during fusion of the blastomeres. The movement of mitochondrial "rings" marked the centripetal translocation of the nuclei. We did not observe formation of a common "ring" or fusion of the interphase nuclei. Association of the nuclear materials took place only at the metaphase stage. Sister blastomeres of the two-cell somatic hybrids, just as those of intact embryos, entered the subsequent cleavage division asynchronously. Cytogenetic analysis confirms formation of the tetraploid embryos as a result of induced fusion of sister blastomeres.

The behaviour of mitochondria and cell integration during somatic hybridisation of sister blastomeres of the 2-cell mouse embryo.

The capacity of sister blastomeres of mouse embryos for induced fusion changed during the 2-cell stage. It was at low level (24%) at the early 2-cell stage, increased and reached 98.5% at the middle 2-cell stage and fell sharply to 31% at the late 2-cell stage. At the time corresponding to the G2/M-phase of the cell cycle the blastomeres fused in only 8% of cases. Vital staining of 2-cell embryos by rhodamine 123 showed that the mitochondria were dispersed throughout the cytoplasm with a ring-like (around the nucleus) or spot-like (over the metaphase plate) concentration in the centre of each blastomere. At the periphery of blastomeres the mitochondrial content was low. The behaviour of the mitochondria reflected the subsequent events of structural and functional integration of the sister blastomeres under induced fusion: a discernible boundary between partners during 30 min after electrofusion or 1 h after fusion with polyethylene glycol; movement of the two 'rings' to the centre of the blastomere fusion products (BFP) to form one large bright 'spot' over the common metaphase plate; mitochondria outlining the shape of the spindle and connection between sister blastomeres until completion of the first mitosis of BFP. The data obtained suggest that fusion of the blastomeres does not lead to extensive changes in the hybrid cytoplasm and integration of nuclear material is taking place only at metaphase stage. Cytogenetic examination of BFP at the 2-cell stage confirmed reconstruction of the tetraploid embryos and found that sister blastomeres of such embryos could asynchronously enter the next cleavage division similarly to normal diploid 2-cell embryos.

[Necessity of individual approach to evaluating the level of fatigue during prolonged work in the mountains]. / O neobkhodimosti individualnogo podkhoda k otsenke stepeni utomleniia pri rabote v usloviiakh dlitelnoi vysokogornoi vakhty.

Capacity to the physical work and functional status were studied during the prolonged (1 year) work in the mountainous conditions (3,600 m above sea level). The fatigue grade and capacity level were proved to depend on individual adaptation to the mountainous conditions.