Reassortment of cells according to position in mouse morulae

Sixteen-cell mouse morulae were disaggregated and blastomeres originally occupying outer or inner positions were separated. Outer, inner, or unsorted populations of blastomeres were labeled with either trinitrobenzene sulphonic acid (TNP) or fluorescein isothiocyanate (FITC) and individual blastomeres aggregated to unlabelled partially decompact eight- to ten-cell morulae. After up to 6 h in culture, the positions of the labelled blastomeres within the aggregates were examined. The combined results demonstrated that between 86 and 92% of outer cells remained on the surface of the aggregate and flattened into extensive polygonal shapes, whereas 76-77% of the inner cells had become engulfed by the host morula cells and retained their initial spherical shape. Using unsorted cells, 33-37% were internalised, which is compatible with the most recent estimates of the presence of six to eight inner cells at the 16-cell stage. The possibility that differential adhesiveness of the outer and inner cells is involved in the allocation of cells to the trophectoderm and inner cell mass of the blastocyst is discussed.     

Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos

We have examined the synthesis and distribution of the cell adhesion molecule uvomorulin in mouse preimplantation embryos. Uvomorulin can already be detected on the cell surface of unfertilized and fertilized eggs but is not synthesized in these cells. Uvomorulin synthesis starts in late two-cell embryos and seems not to be correlated with the onset of compaction. The first signs of compaction are accompanied by a redistribution of uvomorulin on the surface of blastomeres. During compaction uvomorulin is progressively removed from the apical membrane domains of peripheral blastomeres. In compact morulae uvomorulin is no longer present on the outer surface of the embryo but is localized predominantly in membrane domains involved in cell-cell contacts of adjacent outer blastomeres. On inner blastomeres of compact morulae uvomorulin remains evenly distributed. This uvomorulin distribution once established during compaction is maintained and also found in the blastocyst: on trophectodermal cells uvomorulin localization is very similar to that in adult intestinal epithelial cells while uvomorulin remains evenly distributed on the surface of inner cell mass cells. The possible role of the redistribution of uvomorulin for the generation of trophectoderm and inner cell mass in early mouse embryos is discussed.     

Delay of polarization event increases the number of Cdx2-positive blastomeres in mouse embryo

During preimplantation mouse embryo development expression of Cdx2 is induced in outer cells, which are the trophectoderm (TE) precursors. The mechanism of Cdx2 upregulation in these cells remains unclear. However, it has been suggested that the cell position and polarization may play a crucial role in this process. In order to elucidate the role of these two parameters in the formation of TE we analyzed the expression pattern of Cdx2 in the embryos in which either the position of cells and the time of polarization or only the position of cells was experimentally disrupted. Such embryos developed from the blastomeres that were isolated from 8-cell embryos either before or after the compaction, i.e. before or after the cell polarization took place. We found that in the embryos developed from polar blastomeres originated from the 8-cell compacted embryo, the experimentally imposed outer position was not sufficient to induce the Cdx2 in these blastomeres which in the intact embryo would form the inner cells. However, when the polarization at the 8-cell stage was disrupted, the embryos developed from such an unpolarized blastomeres showed the increased number of cells expressing Cdx2. We found that in such experimentally obtained embryos the polarization was delayed until the 16-cell stage. These results suggest that the main factor responsible for upregulation of Cdx2 expression in outer blastomeres, i.e. TE precursors, is their polarity.     

Blastomeres of the mouse embryo lose totipotency after the fifth cleavage division: expression of Cdx2 and Oct4 and developmental potential of inner and outer blastomeres of 16- and 32-cell embryos

Sixteen inner or outer blastomeres from 16-cell embryos and 32 inner or outer blastomeres from 32-cell embryos (nascent blastocysts) were reaggregated and cultured in vitro. In 24 h old blastocysts developed from blastomeres derived from 16-cell embryos the expression of Cdx2 protein was upregulated in outer cells (new trophectoderm) of the inner cells-derived aggregates and downregulated in inner cells (new inner cell mass) of the external cells-derived aggregates. After transfer to pseudopregnant recipients blastocysts originating from both inner and outer blastomeres of 16-cell embryo developed into normal, fertile mice, but the implantation rate of embryos formed from inner cell aggregates was lower. The aggregates of external blastomeres derived from 32 cell embryo usually formed trophoblastic vesicles accompanied by vacuolated cells. In contrast, the aggregates of inner blastomeres quickly compacted but cavitation was delayed. Although in the latter embryos the Cdx2 protein appeared in the new trophectoderm within 24 h of in vitro culture, these embryos formed only very small outgrowths of Troma1-positive giant trophoblastic cells and none of these embryos was able to implant in recipient females. In separate experiment we have produced normal and fertile mice from 16- and 32-cell embryos that were first disaggregated, and then the sister outer and inner blastomeres were reaggregated at random. In blastocysts developed from aggregates, within 24 h of in vitro culture, the majority of inner and outer blastomeres located themselves in their original position (internally and externally), which implies that in these embryos development was regulated mainly by cell sorting.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.     

Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning

To establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25–34), intermediate (40–55), and high (60–81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50–83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40–55) or high (60–81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40–55 cells) and 41% versus 25% (60–81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning. © 1995 wiley-Liss, Inc.     

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.     

Coordinate expression of X and Y haptens during murine embryogenesis

The X hapten (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) may play an important role in the adhesion of blastomeres during compaction. Therefore, we have investigated more thoroughly developmental changes in the fucosylation of lactoseries carbohydrate chains and the enzymatic basis of these fucosylation changes using well-characterized monoclonal antibodies. The Y hapten (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and polymeric X haptens were detected by fluorescence-activated flow cytometry on murine embryonal carcinoma cells. In paraffin sections of postimplantation mouse embryos, the Y hapten was detected in the embryonic ectoderm and visceral endoderm on Days 5.5-7.5; this pattern of antigen expression is identical to that previously reported for the X hapten (SSEA-1). Thus, the Gal:alpha 1----2 (H) and GlcNAc:alpha 1----3 (X) fucosyltransferases appear to be co-regulated during embryogenesis. Reciprocal changes in X and Y hapten expression were observed, however, during preimplantation development. Unlike the X hapten, the Y hapten is expressed maximally on 16-cell morulae and 32- to 64-cell blastocysts. Eight-cell embryos cultured to the blastocyst stage in vitro did not acquire the Y hapten, however, suggesting a role for the uterine environment in carbohydrate antigen expression. Homogenates of F9 embryonal carcinoma cells were found to possess a potent GlcNAc:alpha 1----3 fucosyltransferase activity, as well as a weaker Gal:alpha 1----2 fucosyltransferase activity, using paragloboside as a substrate. The results suggest that embryonic cell surface carbohydrate phenotypes represent a balance in the competition between glycosyltransferases for available substrates. Rapid changes in carbohydrate expression during development may reflect intermediate states of cellular commitment and determination that are critical for lineage formation and morphogenesis.     

Properties of polar and apolar cells from the 16-cell mouse morula

Summary The surface properties of newly formed, isolated 1/16 mouse blastomeres have been analyzed over the 10–12 h period prior to their division to 2/32 cells. Two populations of cells are formed at the 8- to 16-cell transition and their surface phenotypes vary with their relative position within the morula. Outer cells are polar, relatively non-adhesive and relatively large; inner cells are apolar, adhesive and smaller. The surface phenotypes of both inner and outer 1/16 cells are stable during culture for 11 h in isolation. However, the surface phenotypes can be induced to change by culture in combination with a second 1/16 cell, in a manner that is dependent upon the identity of the second cell. Two aggregated polar cells never flatten completely against each other, and both cells retain a clearly defined polar phenotype for 11–12 h. In aggregates of two apolar cells, cell outlines are lost as a result of intercellular flattening and microvilli are displaced away from areas of cell contact. However, if the two apolar cells are subsequently separated an even distribution of microvilli is restored. In most aggregates of an apolar and a polar cell, the polar cell envelops the apolar cell completely. These results are discussed in the context of the normal fate and potential of each cell type within the morula.     

Ultrastructural characteristics of fresh and frozen-thawed ovine embryos using two cryoprotectants

Cryopreservation of sheep embryos with ethylene glycol as a protectant appears to be more effective than glycerol, particularly at the morula stage, as has been demonstrated on the basis of in vitro and in vivo development rates after thawing. In this study we compare the ultrastructure of fresh morulae, thawed morulae, and blastocysts cryopreserved with either ethylene glycol or glycerol at the electron microscopic level, to look for cellular damage that could be responsible for proven differences in embryo survival after transfer. Embryos cryopreserved with glycerol showed unequal degrees of conservation even among blastomeres within a single embryo. In morulae, inner blastomeres were completely damaged, whereas external ones appeared to be intact. Both morulae and blastocysts cryopreserved with ethylene glycol showed a higher uniformity in blastomere conservation than embryos with glycerol. The most remarkable features in this experimental group were the presence of desmosomes following tight junctions between blastomeres and the presence of many microvilli on the outer surface of external blastomeres. These characteristics are similar in fresh embryos of the control group. Our results show that ethylene glycol protects membrane and cytoplasmic structures of embryonic cells from cryoinjury much better than glycerol. In vivo survival of embryos confirmed the ultrastructural observations. A limited permeability of glycerol would explain the observed ultrastructural differences in blastomere integrity, which depends on blastomere location and the differences between morulae and blastocysts. We conclude that the low reproductive yield after cryopreservation using glycerol can be attributed to the lack of protection of inner cells.     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Binucleate cells in mouse morulae

Summary Mouse morulae from two strains were examined in whole mounts after dissociation of embryos into single cells and were analysed in serial sections by light and electron microscopy. One or two binucleate cells per embryo were discovered in a statistically significant number of morulae. The frequency of morulae with binucleate cell(s) was higher in older morulae than in younger ones. Binucleate cells were always the outer cells of the embryo. Their ultrastructure did not differ from the ultrastructure of mononucleate cells. It is suggested that cell binuclearity at the morula stage is a possible way to polyploidization of nuclei, resulting in the formation of primary trophoblast giant cells.     

Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection

The mouse inner cell mass is established by cells that are allocated to internal positions after the 8-cell stage. We analyzed the timing of this allocation by microinjecting two cell lineage markers, horseradish peroxidase and rhodamine-conjugated dextran, into mouse blastomeres at the 8- to 32-cell stage. Prospective analysis was performed by coinjection of peroxidase and dextran, followed by 12-22 hr of culture and staining for peroxidase activity; retrospective analysis was performed by injection of peroxidase alone and localization of sister cells without further culture. Both approaches indicated that cells are allocated to internal positions during the fourth and fifth cleavage divisions, but not the sixth cleavage division, of the mouse embryo. Thus, outer cells can have inner descendants until the late morula/early blastocyst (32-cell) stage, but cells remaining outside after the fifth cleavage division are restricted to a trophectoderm fate. This information about cell lineage indicates that the previously observed totipotency of the cleaving mammalian embryo's cells is a regulative attribute that is used in normal development.     

The pattern of cell division in the early development of the sea urchin,Paracentrotus lividus

In the sea urchin Paracentrotus lividus, the first three cleavages are synchronous in all blastomeres. Upon the segregation of the micromeres at the fourth cleavage, a vegetal-animal gradient of cell division begins; i.e., the closer the cells are to the micromeres, the earlier they enter mitosis. The phase difference between mitotic cells along the vegetal-animal axis increases concurrently with the increase in cell number. At the blastula stage, mitoses appear organized in clusters. From the mesenchyme-blastula stage onward, the clusters become smaller and most of the mitotic cells are solitary. The size of the clusters increases upon treatment with colchicine, thus suggesting the existence of pacemaker units of mitotic activity. We confirm that the division of the micromeres is out of phase with respect to the other blastomeres. Of the eight cells originating from the first division of the micromeres, only the four outer ones continue to cleave; the four inner ones appear to have lost the ability to divide. The overall mitotic activity is high during cleavage and suddenly drops to very low levels around hatching. An interpretation of these results is presented in terms of a nonlinear oscillators theory.     

Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction

Cell lines expressing varying levels of ganglioside GM3 at the cell surface show different degrees of adhesion and spreading on solid phase coated with such glycosphingolipids (GSLs) as Gg3 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer), LacCer (Gal beta 1----4Glc beta 1----1Cer), or Gb4 (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer) (where Cer is ceramide), which may have structures complementary to GM3, but not on solid phase coated with various other GSLs. The degree of cell adhesion and spreading on Gg3 was correlated with the degree of cell-surface GM3 expression, as defined by reactivity with anti-GM3 monoclonal antibody (mAb) DH2. Only cells with high GM3 expression adhered on solid phase coated with LacCer or Gb4. Adhesion of GM3-expressing cells on Gg3-, LacCer-, and Gb4-coated solid phase is based on interaction of GM3 with Gg3 and, to a lesser extent, with LacCer and Gb4, as demonstrated by: (i) the interaction of the GM3 liposome with solid phase coated with Gg3, LacCer, and Gb4, respectively; (ii) the abolition of cell adhesion on each GSL-coated solid phase by treatment of cells with mAb DH2 or sialidase; and (iii) the inhibition of cell adhesion by treatment of GSL-coated solid phase with mAb specific to each GSL. Sialosyllactosyl-lysyllysine conjugate was bound to Gg3 adsorbed on a C18 silica gel column in the presence of bivalent cation, suggesting that the carbohydrate moiety of GM3 is involved in GM3-Gg3 interaction. Not only the adhesion and spreading of GM3-expressing cells, but also their cell motility was greatly enhanced on Gg3-coated solid phase, as determined by Transwell assay and phagokinetic track assay on a gold sol-coated surface. Spreading and motility of GM3-expressing cells on Gg3-coated solid phase were both inhibited by treatment of cells with mAb DH2 or sialidase. These results provide evidence that not only cell adhesion, but also spreading and motility in these cell lines are controlled by complementary GSL-GSL interaction.     

Role of cell adhesion in the specification of pigment cell lineage in embryos of the sea urchin, Hemicentrotus pulcherrimus

To clarify the role of cell adhesion in the specification of pigment cell lineage in sea urchin embryos, cell contacts were inhibited by Ca²⁺-free artificial seawater (ASW) treatment, and the number of differentiated pigment cells was examined by the method devised for the present study. Obtained results showed that inhibition of cell contacts during mid-to-late blastula stage greatly affects the number of pigment cells. Treatment with Ca²⁺-free ASW during 7.5–10.5h of development drastically decreased the number of pigment cells, indicating that cell adhesion during this period is indispensable for the specification of pigment cell lineage. On the other hand, the number of pigment cells were increased by the treatment during 9.5–12.5 h of development. It was suggested that this increase was caused by excess divisions of the precursor cells, that is, the division schedule of the precursor cells was altered by inhibition of cell contacts at this period. Interestingly, the number of pigment cells was a multiple of four in a majority of embryos in which pigment cells were drastically decreased in number. These findings suggest that the founder blastomeres of the pigment cell lineage are specified during 7–10 h of development, and that these blastomeres divide twice before they differentiate into pigment cells.