Tropomyosin in preimplantation mouse development: identification, expression, and organization during cell division and polarization

Tropomyosin is an actin-binding cytoskeletal protein which has been extensively characterized in a variety of cell types and tissues, with the exception of very early developmental stages during which cellular polarization first occurs. We have identified five polypeptides in mouse preimplantation conceptuses which show many of the characteristics of tropomyosin. They form the major portion of the heat-stable cytoskeletal protein fraction of blastomeres and have the characteristic isoelectric and SDS-PAGE migration characteristics on 1-D and 2-D gels. All five polypeptides were synthesized in late 2- and 4-cell, and all 8-cell stages, with three of the five polypeptides showing lower synthetic levels in fertilized eggs and early 2-cell conceptuses. These heat-stable proteins showed specific differences from proteins isolated from mouse 3T3 fibroblasts by the same method, namely higher Mr isoforms were not represented, also some of the isoforms can be labeled by incorporation of [14C]proline. The cellular distribution of tropomyosin in early stage conceptuses was examined using monoclonal and affinity-purified polyclonal antibodies. Tropomyosin becomes associated both with the blastomere cortex postfertilization and with the cleavage furrow during cytokinesis. The interphase cortical association is uniform until the 8-cell stage, when tropomyosin becomes associated with the developing apical pole and is excluded from the basolateral cortex. This polar localization is inherited along with the pole at the 8- to 16-cell division, but experiments in which cell division is artificially prolonged show that tropomyosin localization does not represent a permanent marking of the pole. We conclude that the early mouse conceptus contains a unique and specific set of tropomyosins which respond to polarizing signals.     

Ultrastructure and dynamics of the transosomes of the follicular epithelium of avian ovaries

Specific organelles in follicular cells of the avian ovaries - so called transosomes - are described. Transosomes are formed in the follicular cell cytoplasm under the cytoplasmic membrane and consist of a dense layer 100-150 A in thickness and granules 110-250 A in diameter evenly distributed on its inner surface and containing r-RNA. The main property of transosomes is to move from one follicular cell to another or to an oocyte having separated from numerous processes of follicular cells and being surrounded by two additional membranes. In the ooplasm the transosome takes part in the development of primordial yolk granules. It is supposed that transosomes bring about a transmission of somatic follicular cells to the occyte which is necessary for the initial stages of vitellogenesis.     

Ultrastructure of developing substantia nigra in humans

Electron microscopy of the maturing neurons and developing and maturing synapses in the substantia nigra of 14 human embryos/foetuses of 8-24 weeks of gestation are reported. At 8 weeks, cells were immature with very little cytoplasm and cellular organelles. Contact sites of processes appeared more electron dense than the other areas. At 12 weeks, many of the cells had acquired more cytoplasm and cellular organelles and could be identified as neurons. Asymmetric synapses with clear, round synaptic vesicles also were identifiable at this age. Such synapses, first to appear in the developing substantia nigra, are reported to be formed by recurrent collateral nigro-striatal fibres. Substance P fibres from the striatum also are contributing to this type of synapse. At 15-16 weeks, not only was the number of such synapses increased, but many appeared morphologically mature. Symmetric synapses having clear round vesicles along with a few dense core vesicles also appeared at this stage, suggesting striatal input. By 24 weeks of gestation, most of the neurons had cytological features comparable to that of the mature neurons. There was an increase in the total number of synapses and the individual variety from 15 to 24 weeks of gestation. The present study indicates that synaptogenesis starts at 8 weeks and continues beyond 24 weeks of gestation.     

Development, glycolytic activity, and viability of preimplantation mouse embryos subjected to different periods of glucose starvation

After compaction, the preimplantation mouse embryo switches to a glucose-based metabolism, whereas for the 2- to 4-cell stage embryo, glucose can be inhibitory. In this study, we investigated the adaptability of preimplantation embryos to different periods of glucose starvation by culturing in vitro fertilized (IVF) and in vivo-fertilized 1-cell OF1 mouse embryos. Blastocysts obtained from exposure to glucose starvation for different periods of time were examined for the number of cells in the trophectoderm and inner cell mass, and for glycolytic activity and viability. A high percentage of blastocysts was obtained when 1-cell embryos fertilized in vitro or in vivo were cultured in M16 until the 2-cell stage, were transferred to M16 without glucose (M16-G) until the 4- or 8-cell stage, and then were transferred to fresh M16-G. When in vivo-fertilized 1-cell embryos were cultured to the 2-cell stage and then left in M16, less than 5% formed blastocysts compared to 26% of those transferred into M16-G. Blastocysts obtained when in vivo-fertilized 1-cell embryos were left in M16-G after the 2-cell stage, however, showed a significantly elevated glycolytic activity compared to those transferred to fresh M16 or M16-G medium at the 4- or 8-cell stage. Interestingly, even though these embryos displayed elevated glycolytic activity, they did not exhibit differences in the numbers of inner cell mass and trophectoderm cells or in viability compared to embryos cultured according to other protocols. Blastocysts from all cultured protocols had a significantly lower total cell number and a lower trophectoderm, but not inner cell mass, cell number compared to blastocysts developed in vivo. This study documents the metabolic adaptability of the preimplantation embryo by highlighting its ability to proceed with development and retain viability when challenged with glucose starvation at different periods.     

Identification of 4(-) states in the 14C(p,n)14N reaction at 135 MeV

The tadpole larva of solitary ascidians has 40 notochord cells in its tail. Of these cells, 32 in the anterior and middle part of the tail are derived from the A-line blastomeres, while 8 in the posterior part of the tail originate from the B-line blastomeres. Previous experiments involving continuous dissociation of daughter blastomeres from the first cleavage to the 110-cell stage suggested that cellular interactions may be involved in the formation of notochord cells. In the present study, the presumptive-notochord blastomeres isolated from the 32-cell embryos did not develop features of notochord. These results suggest that cellular interactions may be required for the fate specification of notochord, that is to say, notochord formation occurs as a result of inductive interaction between blastomeres. In order to confirm the involvement of induction in the determination of notochord and to identify the inducer blastomeres, the presumptive-notochord blastomeres at the 32-cell stage were coisolated or recombined with one of the surrounding blastomeres in a series of experiments. The results suggested that, for the A-line precursors, notochord differentiation occurs as the result of an inductive influence from vegetal blastomeres that include the presumptive-endoderm blastomeres and the presumptive-notochord blastomeres themselves. It was also suggested that induction of notochord is complete by the 64-cell stage and that inductive interactions have to be initiated before the decompaction of blastomeres during the 32-cell stage. Ascidians are Urochordata and are closely related to vertebrates. In vertebrates, it is well known that inductive interactions play a crucial role in the determination of notochord. It appears, therefore, that induction of notochord is common throughout the phylum Chordata.     

Hematopoiesis in the yolk sac of several representatives of the Sauropsida]

Hemopoiesis in the yolk sac of chicken and crocodile embryos was studied at different developmental stages. Primary intravascular erythropoiesis is closely related to the formation of sanguineous islands appearing in the zone of the yolk growth in the visceral mesoderm. With the development of folds enlarging the surface of the yolk resorbtion, around the vessels running within the folds there appear foci of primary granulopoiesis. The process begins in the paravasal mesenchyma which is gradually disguised by hemopoietic cells (in chicken embryos--at the stage of 8 days, in crocodiles--23 days of incubation). The granulopoiesis continues in chicken embryos during 1/3 of the incubation period, in crocodiles--during 2/3 of the incubation time. The leukopoiesis foci are developed more intensively in crocodiles as well at the size of the yolk sac folds. Leukocytic accumulations disappear in the crocodile sac after hatching (38-40 cm). The change of primary erythropoiesis for the secondary one in the chicken is preceded by the appearance of megaloblastic forms of erythrocytes with the compact homogeneous cytoplasm. They are absent from crocodiles.     

Morphological and electrophysiological properties of centrifuged stratified Xenopus oocytes

To separate and concentrate various cytoplasmic organelles in wild type and albino Xenopus oocytes, defolliculated cells were loaded on a Ficoll-400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10,000 g for 5 min at 20 degrees C. The cells became pear-shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100-300 microns, and the greenish protein yolk at the bottom. To determine the cellular constituents, particularly of the transparent zone, electron microscopy was performed. The transparent zone was found to contain (from animal to vegetal) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged stratified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. Centrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The resting membrane potentials of centrifuged oocytes were between -35 and -65 mV, and the membrane resistances were in the 500 k omega to 1 M omega range. Under voltage clamp conditions, the oocytes exhibited Ca(2+)-activated Cl- currents with biphasic kinetics and spontaneous oscillations of these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suitable preparation to study the contribution of various cellular organelles to the propagation of second messengers in the cytosol.     

Comparison of monolayer and bilayer plates used in antibiotic assay

Development of the rat embryo is arrested at the 2-cell stage in vitro in the presence of inorganic phosphate (Pi). Rat embryos were affected by exposure to 1.19 mM KH2PO4 in modified hamster embryo culture medium-1 at the late 2-cell stage only. When exposure durations were 6 h, embryos whose exposure timings were prior to cleavage had a reduced rate of development to the blastocyst stage (2-8%) when compared with embryos with no exposure to Pi (97%, P < 0.05). When exposure durations were 18 h, all embryos were arrested at the 2- to 4-cell stage. These timings would correspond to the G2 to M phase of the second cell cycle. Maturation-promoting factor (MPF), which is regulated by a phosphorylation cascade, controls cell division, and its kinase activity is necessary in order for the cell to enter the M phase. However, the histone H1 kinase activity levels and the patterns of the state of phosphorylation of cdc2 were the same in blocked and non-blocked embryos. Because MPF was active in blocked embryos, the developmental block in rat 2-cell embryos caused by phosphate was not due to MPF activity or its phosphorylation cascade.     

Increased functional load on mouse kidney proximal tubule epithelial cells causes changes in nucleolar 3-D architecture

Ultrastructural 3-D analysis of nucleolar architecture and Ag-NOR protein distribution in mouse kidney-cortex proximal-tubule epithelium has been performed. A principal scheme of structural changes of the nucleolus and organization of its components during the intensification of pre-rRNA synthesis (dynamic model of a nucleolus) based on computer spatial modelling has been advanced. According to the nucleolar composition, three groups of cells, which differ from each other by rRNA synthesis, are defined in normal kidney. Most nephron proximal-section cells (about 52%) are characterized by lower activity of RNA synthesis. Such kind of cells are defined as group I (nucleolar diameter 0.7-1.5 microm) and always contain resting, ring-shaped or close to ring-shaped dense nucleoli, which have 2 or 3 fibrillar centers. Nucleoli of group II cells (about 37%, nucleolar diameter 1.5-2.5 microm) have a higher level of activity, contain 4-7 fibrillar centers, and their structural organization is close to reticulated forms due to the first indications of vacuolar network (identified as prereticulated nucleoli). The most active cells of group III (about 11%, nucleolar diameter 2.5-3.5 microm) include cells with typical reticulated nucleoli with a well expressed vacuolar network and numerous fibrillar centers (18-22). Increased functional load of the epithelium caused by unilateral nephrectomy and diuretic (4-chlor-H [2-furylmethyl] 5-sulphamyl-antranic acid) injection changed the proportion of the different cell groups: group I decreased (about 25%), whereas groups II and III increased (about 8% and 17%, respectively). The increase of nucleolar activity first causes a deformation of the individual fibrillar centers as well as complication and growth of their surface. Further, a progressive fragmentation of the fibrillar centers and the growth of their total volume is observed. The complication and growth of the total volume of Ag-positive zones is another indication of the nucleolar activation. The vacuolar system develops by a gradual fusion of small isolated cavities into a united vacuolar network. Nucleoli with 2-7 fibrillar centers are considered to be intermediate forms reflecting successive stages of its activation or inactivation: from the resting ring-shaped nucleolus via transient stages of increasing functional activity to the active reticulated nucleoli and vice versa. The observed differences in the nucleolar ultrastructure are regarded as evidence of the functional heterogeneity of cell populations within one functional segment of nephron.     

Influence of the foetus, placenta and ovary on the mammotrophic activity of pregnant rat serum

Serum mammotrophic activity was assayed using an organ culture technique with histological endpoints. Using sera of 13 days pregnant rats with 1 to 18 conceptuses, activity was detectable even in the presence of 1 conceptus, but the activity with 1-4 conceptus(es) tended to be less than with 6-18. Serum of rats with 1-3 conceptus(es) was approximately 2-4 times less active than serum of rats with 14 conceptuses. Removal of the conceptuses on day 15 caused loss of mammotrophic activity of the serum, tested 4 days later. When the foetuses, the ovaries or both the foetuses and ovaries were removed on day 15 of pregnancy, mammotrophic activity was present in the serum collected 4 days later. Differences in activity between the treated groups were small. The mammotrophic activity was comparable to the activity of serum of untreated 15 days pregnant rats or 19 days sham-operated pregnant rats. Explanted single fragments of a 19 days pregnant rat placenta released activity into the medium. The placenta retained this capicity even when the foetus had been removed 4 days previously.     

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status

Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41 degrees C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37 degrees and 41 degrees C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.     

Localization of activin and follistatin proteins in the Xenopus oocyte

We found a binding protein for activin and follistatin in serum from female Xenopus laevis and identified it as vitellogenin, which is synthesized in the liver and transported into yolk platelets. Then, we investigated the localization of activin and follistatin proteins in early Xenopus oocytes (stage 6) by electron microscopic immunolabeling with gold colloidal particles. The protein molecules were found to be localized uniformly in oocyte yolk platelets, but not in other cytoplasmic organelles. These findings suggest a novel role of yolk platelets as a reservoir for inductive signals transported by vitellogenin in the differentiation and patterning of cells in Xenopus embryos.     

Bacq and Alexander Award lecture--chemical radioprotection: past, present, and future prospects

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 +/- 0.011 mM and 129.3 +/- 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 +/- 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 +/- 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture.     

Morphogenesis of degenerative changes predisposing dogs to rupture of the cranial cruciate ligament

The cranial cruciate ligaments (CCL) of 13 dogs with clinical signs of CCL rupture and those of 22 clinically healthy young beagle dogs for laboratory use were examined histopathologically and immunohistopathologically. The most constant changes at an early stage of degenerating ligament tissue in affected dogs were nuclear enlargement and perinuclear halo formation of fibrocytes followed by chondroid metaplasia. These changes were also frequent in apparently healthy young beagles kept under laboratory conditions. PAS and alcian blue positive substance accumulated around activated fibrocytes and within perinuclear halos. S-100 protein was also positive in these cells preceding the morphological change of chondroid metaplasia. Increased mitotic figures and Ki-67 positive cells showed the proliferating nature of these cells at a later stage. Alteration of extracellular matrices from dense collagen fiber type to those of cartilage tissue seemed to predispose dogs to rupture of the CCL along with a degradation in collagen fiber of the primary bundles. Collagen fiber bundles with a parallel fibrillar array never formed in the CCL with degraded primary bundles, whereas activated fibrocytes constantly underwent chondroid metaplasia. The pathogenic mechanism underlying chondroid metaplasia was thought to be nonspecific and attributable to an essential property of activated fibrocytes in the mature tendon tissue.