Requirement of cell division for muscle actin expression in the primary muscle cell lineage of ascidian embryos

The role of cell division in the expression of muscle actin and its relationship to acetylcholinesterase (AChE) development was examined in cleavage-arrested embryos of the ascidian Styela. Muscle actin expression was detected by two-dimensional gel electrophoresis of radioactively labelled proteins and by in situ hybridization with a cDNA probe, whereas AChE activity was assayed by enzyme histochemistry. In the majority of cases, muscle actin expression was first detected in embryos arrested after the 16-cell stage. Some embryos showed muscle actin expression after arrest at the 8-cell stage, however, muscle actin mRNA did not accumulate in embryos arrested at earlier cleavages. The cells that expressed muscle actin in 8- to 64-cell cleavage-arrested embryos belonged to the primary muscle lineage; secondary muscle cell precursors did not express muscle actin. Zygotic muscle actin mRNA appeared to accumulate with myoplasmic pigment granules in the perinuclear region of cleavage-arrested embryos, suggesting that the myoplasm may have a role in the organization of muscle cells. In contrast to muscle actin, AChE was detected in a small proportion of embryos treated with cytochalasin as early as the 1- or 2-cell stage, and most embryos treated with cytochalasin at later cleavages expressed this enzyme in some of their cells. Most primary muscle lineage cells expressed both muscle actin mRNA and AChE, however, some cells expressed only muscle actin mRNA or AChE. The results suggest that at least three cleavages are required for muscle actin expression and that muscle actin and AChE expression can be uncoupled in cleavage-arrested embryos.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.     

Actin filament distribution in blocked and developing pig embryos

Actin filaments play an important role in cell division. The present study was designed to examine the relationship between actin filament distribution and pig embryo development. When in vivo matured and fertilised pig oocytes were cultured in TCM 199 or NCSU 23, in various proportions, 45-65% of inseminated oocytes developed to the 2- to 4-cell stages but blastocyst development was observed only in NCSU 23 (34%) or NCSU 23 containing 10% TCM 199 (7%). Supplementation of NCSU 23 medium with 20% or more TCM 199 resulted in no blastocyst formation. Examination of actin filaments indicated that microfilaments were distributed in the cortex, at the junction of blastomeres and in the perinuclear area in the embryos cultured in NCSU 23, but perinuclear actin filaments were not observed in embryos cultured in TCM 199. When 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23 medium at 36 h after in vivo fertilisation, 57% of the cleaved embryos developed to blastocysts, which was no different from the proportion obtained after culture in NCSU 23 alone (56%). In addition, when 2- to 4-cell stage embryos obtained from TCM 199 were transferred to NCSU 23, most embryos showed perinuclear actin filaments within 6h. The results indicate that the composition of the culture medium plays an important role in the polymerisation of actin filaments, which in turn influences embryo development. It is possible that pig embryo development was blocked by some components in TCM 199 which prevented actin filament polymerisation.     

Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose

The failure of hamster 2-cell embryos to develop in vitro (2-cell block) was examined with experiments in which concentrations of glucose and phosphate in the culture medium were varied. Embryos were cultured in a protein-free modified Tyrode's solution that normally contains 5.0 mM glucose and 0.35 mM sodium dihydrogen phosphate. In the presence of 0.35 mM phosphate but without glucose, 23% of 2-cell embryos reached the 4-cell stage or further after culture for 1 day and 27% after 2 days. Glucose inhibited embryo development even at 0.1 mM (4% development to greater than or equal to 4-cells after culture for 2 days); there was no dose-related inhibition above this glucose concentration. In a second experiment, phosphate levels were varied in the absence of glucose. Phosphate was highly inhibitory to development, with 97% of 2-cell embryos reaching the 4-cell stage or further after culture for 1 day in the absence of phosphate compared to 9-21% in the presence of 0.1-1.05 mM phosphate. After culture for 2 days, 26% of embryos reached the 8-cell stage or further when phosphate was absent compared to 0% development to 8-cells with 0.1 mM phosphate or higher. In a factorial experiment, phosphate blocked development when glucose was present or absent, whereas glucose did not block embryo development in the absence of phosphate. However, 2-deoxyglucose (a non-metabolizable analogue of glucose) inhibited embryo development in the absence of phosphate. These data show that the in vitro block to development of hamster 2-cell embryos is caused at least in part by glucose and/or phosphate. Deletion of these compounds from the culture medium eliminates the 2-cell block to development in virtually all embryos, and approximately 25-75% of embryos develop to the 8-cell or morula stages in vitro. The observations provide a possible explanation for the 2-cell and 4-cell blocks that occur in conventional culture media: stimulation of glycolysis by glucose and/or phosphate may result in inefficient adenosine triphosphate (ATP) production. The data indicate marked dissimilarities in the regulation of in vitro development of early cleavage stage hamster embryos compared with embryos of inbred mice, since the latter have an inactive glycolytic pathway prior to the 8-cell stage of development and will grow from 1-cell to blastocyst with both phosphate and glucose in the culture medium.     

Characteristic of structural and functional organization of two-cell mouse embryos exposed to inhibitors of cell proliferation

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.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos

In early cleavage stage hamster embryos, the inability to regulate intracellular pH (pHi) properly is associated with reduced developmental competence in vitro. The disruption of mitochondrial organization is also correlated with reduced development in vitro. To determine the relationship between pHi and the disruption of cytoplasmic organization, we examined the effects of altering pHi on hamster embryo development, mitochondrial distribution, and cytoskeletal organization. The weak base trimethylamine was used to increase pHi and was found to reduce embryo development and disrupt the perinuclear organization of mitochondria. The weak acid 5,5-dimethyl-2,4-oxazolinedione was used to decrease pH(i) and was also found to reduce development and disrupt the perinuclear organization of mitochondria. With either treatment, the microfilament organization was perturbed, but the microtubule cytoskeleton was not. However, the temporal progression of the disruption of mitochondrial distribution was more rapid in alkalinized embryos than acidified embryos, as revealed by two-photon imaging of living embryos. Additionally, the disruption of the microfilament network by the two treatments was not identical. The cytoplasmic disruptions observed were not due to acute toxicity of the compounds because embryos recovered developmentally when the treatment compounds were removed. These observations link ionic homeostasis, structural integrity and developmental competence in preimplantation hamster embryos.     

Phosphatidylcholine-specific phospholipase C and RhoA are involved in the thyrotropin-induced activation of phospholipase D in FRTL-5 thyroid cells

Liquid nitrogen (LN2) infusions are currently used in a slow controlled-rate freezing during cryopreservation. The effects of two different LN2 infusion frequencies (conventional, slow 50 infusions/min and high 120 infusions/min) were studied with frozen-thawed two-cell mouse embryos and their subsequent development to blastocysts. The embryos that were subjected to the high frequency LN2 infusion (HFLI) showed a significantly higher survival rate over the low frequency LN2 infusion (LFLI) (50.7 vs. 34.6%, P < 0.05). The blastocyst formation was also higher in HFLI (76.7%) than LFLI (44.0%, P < 0.05) with respective to the number of cells in a blastocyst of 71.6 8.0 (n = 20) and 62.5 +/- 4.7 (n = 20) (P < 0.05). The relative amount of H2O2 in an embryo that was assessed by a fluorescence intensity of 2',7'-dichlorofluorecein (DCF) showed a difference between the procedures with 16.6 +/- 1.6 (n = 21) and 23.4 +/- 1.8 (n = 24) for HFLI and LFLI, respectively (P < 0.05). Mitochondrial staining by Rhodamine 123 showed that the number and distribution of viable mitochondria were similar in both procedures, but fewer mitochondria were observed with a marked aggregation in the arrested embryos, indicating a mitochondrial disintegration. The mitochondrial membrane potential was visualized by a membrane potential-sensitive fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). There was a decrease in the number of mitochondria that had a high membrane potential, and they showed a peripheral redistribution along the cell membrane in LFLI. A fluorescent staining of the actin filaments revealed a discontinuity that was noticeably at the peripheral "actin band" in LFLI. The DNA fragmentation was assessed by the dUTP nick end-labeling (TUNEL). The results showed a higher DNA fragmentation of blastocyst nuclei in LFLI compared to HFLI (65.6 vs. 36.0%, P < 0.05). Based on these observations, it was concluded that HFLI was better than LFLI in the case of freezing the mouse 2-cell embryos for preserving cytoskeletons and mitochondrial integrities. This could subsequently lead to a higher survival and developmental rate of the cryopreserved mouse embryos.     

Development of cellular polarity of hamster embryos during compaction.

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.     

Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis.

Culture of hamster embryos with 0.35 mM inorganic phosphate results in developmental arrest at the 2-cell stage. These arrested 2-cell embryos were found to have significantly elevated levels of both intracellular pH and intracellular free calcium. Culture of 2-cell embryos with both glucose and phosphate did not further alter intracellular ionic homeostasis. Developmental arrest of 2-cell embryos was dependent on the concentration of phosphate used. Culture with 1.25 microM phosphate did not alter development, while concentrations of 2.5 microM and 5.0 microM resulted in a percentage of embryos arresting development at the 2-cell stage. Analysis of intracellular levels of pH and calcium after culture with different phosphate concentrations revealed a significant negative correlation between intracellular calcium levels and development beyond the 2-cell stage. There was no correlation between the increase in intracellular pH and embryo development in the presence of phosphate. The increase in intracellular calcium levels after culture with phosphate appears to be derived from intracellular pools, as preventing the influx of extracellular calcium did not alter development beyond the 2-cell stage. Therefore, it is apparent that a disruption in ionic homeostasis is associated with developmental arrest of hamster embryos cultured with phosphate.     

Patterns of organelle distribution in mouse embryos during preimplantation development

We have evaluated the distribution of mitochondria and acidic organelles using, respectively, the specific vital fluorescent dyes rhodamine 123 and acridine orange during preimplantation embryonic development in the mouse. Under conditions used to visualize organelles in living embryos, staining with either dye was found to have no effect on either the rate or extent of in vitro development of five- to eight-cell embryos up to the blastocyst stage. Mitochondria were randomly distributed throughout the cytoplasm and located around nuclei in blastomeres of uncompacted embryos. During compaction, mitochondria initially reorganized to the blastomere cortex; however, these organelles were later confined to the perinuclear region in the trophectoderm (TE) of expanded blastocysts. Acidic organelles were randomly distributed in the cytoplasm of uncompacted embryos, but following compaction, they were concentrated in cortical and perinuclear locations. Moreover, in TE cells of expanded blastocysts, acidic organelles were found exclusively in a tight perinuclear pattern. Microtubules and microfilaments in TE cells were localized in fixed embryos stained with antitubulin antibodies and rhodamine phalloidin, respectively; these structures were found primarily in the cortical cytoplasm at areas of cell-cell contact and secondarily in a perinuclear location. Thus mitochondria and acidic organelles undergo stage-specific redistributions from a diffuse or cortical pattern at the eight-cell stage to a tight perinuclear localization in the TE. We conclude that the polarized distributions of some organelles and cytoskeletal proteins during compaction may not be reliable permanent markers of the mature TE.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

Presence and distribution of E-cadherin in the 4-cell golden hamster embryo. Effect of maternal age and parity

Maternal age dependency of gestation time in hamster and in other mammals is a well demonstrated fact. We have recently shown that adult nulliparous and multiparous hamster females show significant asynchrony and retard on early embryo development (from two blastomeres to morula stages) when compared with nulliparous young females. The number of cell-cell adhesions between blastomeres in early embryo development has been reported to be a good indication of the ability of embryos to cleave and develop. In this work we studied, by indirect immunofluorescence, the presence and distribution of E-cadherin in 4-cell embryos obtained from nulliparous young (NYF), nulliparous adult (NAF) and multiparous adult (MAF) hamster females. Distribution and intensity of fluorescence was observed and registered using confocal microscopy. Staining intensities for E-cadherin were quantified by computed densitometry in the free membrane regions, in the cytoplasm region and in the cell-cell adhesion zones of each embryo. E-Cadherin in all the studied zones was significantly higher (p<0.01) in NYF. Cadherin concentration in the intercellular membranes was always statistically higher (p<0.05) than in the free membrane regions. An appreciable concentration of E-cadherin was found in the cytoplasm of the 4-cell embryos obtained from the three groups of females, but was significantly higher in NYF. No statistical differences were observed in any of the parameters studied between NAF and MAF. Our results seem to indicate that changes in the reproductive behavior related to age and/or multiparity may be correlated with changes in the processes related to intercellular adhesions during early cleavage.     

Microcompartmentation of glycolytic enzymes in cultured cells.

The microcompartmentation of aldolase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was investigated in four different cell types (3T3 cells, SV 40 transformed 3T3 cells, mouse fibroblasts, chick embryo cardiomyocytes) combining cell permeabilization and indirect immunofluorescence technique. Permeabilization of the cells prior to fixation released the soluble fractions, whilst the total amount of enzymes was preserved in nonpermeabilized cells. Both enzymes exist in a soluble as well as in a structure-bound form. The soluble fraction of aldolase and GAPDH is distributed homogeneously throughout the cytoplasm, excluding the nucleus and vesicles. The permeabilization-resistant form is associated with the actin cytoskeleton. A considerable amount of both enzymes is located in the perinuclear region and cannot be attributed to a definite structure. Comparing the staining patterns of aldolase and GAPDH in four different cell types we found that the distribution of the enzymes corresponds with diverse forms of actin cytoskeletal organization of these cells. The codistribution is maintained in cells treated with cytochalasin D.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

Spreading of porcine kidney epithelial cells under normal conditions and under the effect of inhibitors of energy metabolism. II. Behavior of cellular organelles

In the present work the behavior of mitochondria and lysosomes during cell spreading has been investigated in normal conditions and under ATP-synthesis inhibitors: sodium aside and N,N-dicyclohexylcarbodiimide (DCCD). In the control culture, microtubules run along the stable edge and perpendicular to the leading edge in most of spreading cells. As a whole, microtubules form a dense network in these cells. However, the radial cells contain bundles of microtubules, radiating from the perinuclear area or form circular arrays around the nucleus. The microtubule network is more dense under inhibitory treatment, than in control conditions. In the control culture the spherical cells display numerous small mitochondria (staining with Rhodamine 123). In the process of cell spreading some elongated mitochondria appear, most of them being localized in the perinuclear area. The mitochondria of cells with radial microtubule organization are directed towards the cell periphery, while in cells with circular bundles of microtubules the mitochondria are localized chaotically. Under DCCD treatment the mitochondria retain the staining for 2-3 h. In the spreading cells, round mitochondria may be distributed all over the cytoplasm. In the presence of sodium aside the mitochondria are not stained. However, by means of phase contrast microscopy some disoriented thread-shaped structures are observed, obviously corresponding to mitochondria. In the control conditions, lysosomes (stained with Acridine orange) in spreading cells are dispersed chaotically, all over the cytoplasm, or are localized in the perinuclear area. In the presence of sodium aside lysosomes are observed only in the perinuclear area. Under DCCD treatment lysosomes do not accumulate the dye. Thus, the cytoskeleton modification and changes in the properties of membrane organelles, induced by ATP-synthesis inhibitors, do not prevent attachment, spreading or cell polarization.     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

Localization of actin networks during early development of Tubifex embryos

In precleavage zygotes of Tubifex, actin filaments segregate to the animal and vegetal poles forming the polar actin filament networks (AFNs). In this study, the fate of the polar AFNs during early development of Tubifex embryos has been followed using rhodamine-phalloidin as a specific stain for F-actin. During the first two cleavages, which are unequal and meridional, the polar AFNs are retained at the regions of cells corresponding to the poles of the precleavage zygote; thereby, they are segregated to the CD-cell at the 2-cell stage then to the D-cell at the 4-cell stage. As the mitotic apparatus forms in the D-cell, however, the vegetal polar AFN translocates toward the animal pole of the cell where the mitotic apparatus is located and unites with the animal polar AFN there. This redistribution of the AFNs is impaired by colchicine treatment, suggesting the involvement of microtubules. Thereafter, the unified AFN is found to be associated with nuclear regions of the macromeres of the D-cell line, and finally partitioned to the teloblast precursors 2d and 4d and an endodermal cell 4D. Cytochalasin B experiments indicate that the AFNs play a cytoskeletal role in generating and maintaining the spatial organization of the cytoplasm which gives rise to the intracellular localization of the cytoplasm and the mitotic apparatus orientations. The developmental and cellular significance of the AFNs is discussed in relation to the localization of developmental potential and the regulation of the mitotic apparatus organization in the Tubifex embryo.     

Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band