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At least 19 recordsLinked to original sources
PubMed1998

The genetic constitution of multinuclear blastomeres and their derivative daughter blastomeres.

The presence of multinuclear blastomeres (MNB) has been widely reported in in-vitro-cultured embryos. Multinucleation at the first mitotic division and affecting both blastomeres is considered abnormal and such embryos are not transferred. The objective of this study was to use fluorescent in-situ hybridization (FISH) and probes specific for chromosomes X, Y and 18 to examine the genetic constitution of embryos developing from the 2-cell stage in which both blastomeres were bi- or multinuclear. Initially, 2-cell embryos in which both blastomeres were bi- or multinuclear were cultured further. Of 101 embryos, 89 (88.1%) cleaved further and were analysed at the 3- to 8-cell stage on day 2 or 3. Among embryos analysed, 30.4% contained only mononuclear diploid blastomeres, 35.9% had a combination of mononuclear diploid and non-diploid blastomeres, and 33.7% had non-diploid blastomeres, indicative of chaotic division. Results obtained were similar with embryos derived from in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Also, no significant differences were found between 2-cell embryos with bi- or multinuclear blastomeres or between slowly or normally cleaved embryos. Twelve (11.9%) embryos arrested at the 2-cell stage on day 3; of these, one had diploid blastomeres and the others were abnormal and highly polyploid. Subsequently, 59 embryos were analysed at the 2-cell stage. Initial observations related to the high number of nuclei in metaphase at the moment of spreading, notably when multinuclear blastomeres were observed. Genetic analysis showed 44.7% of embryos to be susceptible to analysis; the genetic constitution corresponded in both blastomeres to a diploid status. A combined diploid blastomere and abnormal blastomere was found in 4.3% of embryos; both blastomeres were abnormal in 51%. These data show that the genetic constitution of bi- or multinuclear blastomeres, and the daughter cells developing from them, are not always abnormal.

Blastomeres↗
PubMed1988

Induction of cytoplasmic polarity in heterokaryons of mouse 4-cell-stage blastomeres fused with 8-cell- and 16-cell-stage blastomeres.

Cell surface and cytoplasmic polarity is exhibited by the blastomeres of mouse preimplantation embryos following compaction at the 8-cell stage of cleavage. It has been hypothesized that cytoplasmic polarity is initiated by plasma membrane functions of polar blastomeres that are absent from apolar blastomeres. To test this hypothesis the plasma membranes of "test" polar and apolar 8-cell- and 16-cell-stage blastomeres were inserted into the plasma membrane of "carrier" 4-cell-stage blastomeres by polyethylene glycol-mediated fusion of carrier-test blastomere pairs. After a 4-hr culture period each heterokaryon was scored for the distribution of two marker organelles--lipid droplets and nuclei--with respect to their proximity to the plasma membrane insert from the test blastomere. Plasma membrane inserts from polar test blastomeres were identified by labeling their apical domains with fluorescently tagged (succinylated) concanavalin A. The incidence of polar heterokaryons (those exhibiting a discrete fluorescently labeled area of plasma membrane corresponding to the apical domain inherited from the test blastomere) was 55/85 (69%) and 48/79 (61%) for 8-cell-stage and 16-cell-stage test blastomeres, respectively. In all polar heterokaryons, both nuclei were subjacent to the fluorescent label (apical domain of a polar plasma membrane insert), while the majority of lipid droplets resided in the hemisphere opposite the fluorescent label. In all 61 apolar heterokaryons examined (those lacking a discrete fluorescently labeled plasma membrane area) both nuclei were centrally located and lipid droplets were randomly distributed. These observations are consistent with the hypothesis that cytoplasmic polarity can be initiated by properties that distinguish the plasma membranes of polar blastomeres from those of apolar blastomeres.

Animals↗
PubMed2007

Noggin signaling from Xenopus animal blastomere lineages promotes a neural fate in neighboring vegetal blastomere lineages.

In Xenopus, localized factors begin to regionalize embryonic fates prior to the inductive interactions that occur during gastrulation. We previously reported that an animal-to-vegetal signal that occurs prior to gastrulation promotes primary spinal neuron fate in vegetal equatorial (C-tier) blastomere lineages. Herein we demonstrate that maternal mRNA encoding noggin is enriched in animal tiers and at low concentrations in the C-tier, suggesting that the neural fates of C-tier blastomeres may be responsive to early signaling from their neighboring cells. In support of this hypothesis, experimental alteration of the levels of Noggin from animal equatorial (B-tier) or BMP4 from vegetal (D-tier) blastomeres significantly affects the numbers of primary spinal neurons derived from their neighboring C-tier blastomeres. These effects are duplicated in blastomere explants isolated at cleavage stages and cultured in the absence of gastrulation interactions. Co-culture with animal blastomeres enhanced the expression of zygotic neural markers in C-tier blastomere explants, whereas co-culture with vegetal blastomeres repressed them. The expression of these markers in C-tier explants was promoted when Noggin was transiently added to the culture during cleavage/morula stages, and repressed with the transient addition of BMP4. Reduction of Noggin translation in B-tier blastomeres by antisense morpholino oligonucleotides significantly reduced the efficacy of neural marker induction in C-tier explants. These experiments indicate that early anti-BMP signaling from the animal hemisphere recruits vegetal equatorial cells into the neural precursor pool prior to interactions that occur during gastrulation.

Animals↗
PubMed1984

An improved method for destroying mouse blastomeres electrically inside the zona pellucida and the in vitro development of the surviving blastomeres.

An improved method for isolating mouse blastomeres by electrically destroying the other blastomeres inside the zona pellucida is described. Instruments required are two micromanipulators connected to an inverted microscope and an electronic stimulator equipped with an isolator. The target blastomere in which a glass microelectrode is inserted disintegrates completely within a few seconds after a direct current is applied. Complete destruction of a blastomere requires more than 60 microA delivered at 8 V. Destruction of blastomeres occurred mainly as a result of the medium penetrating the blastomere due to the change of membrane potential in both the zona and the blastomere. Most one-half, two-quarter, and three-quarter embryos obtained in this way developed in vitro into normal blastocysts (76.4--86.2%). By contrast, there was a marked decrease (35.3%) of developmental potential in one-quarter embryos. The results indicate that the present method is an improvement over previous methods of obtaining mammalian blastomeres inside the zona pellucida.

Animals↗
PubMed2001

Mouse singletons and twins developed from isolated diploid blastomeres supported with tetraploid blastomeres.

The aim of this study was to obtain mice, hopefully identical multiplets, from single diploid blastomeres isolated at the 4-cell stage, or from pairs of sister blastomeres isolated at the 8-cell stage. To this end isolated blastomeres were aggregated with one or two tetraploid carrier embryos produced by electrofusion of 2-cell embryos. Diploid embryos were albino and homozygous for the "a" allele of glucose-phosphate isomerase (GPI-1a1a) and tetraploid embryos were pigmented and GPI-1b1b. The aggregates were cultured in vitro up to the blastocyst stage. Each quartet (occasionally triplet or doublet) of chimaeric blastocysts was transplanted to the oviduct of a separate pseudopregnant recipient. Altogether 62 blastocysts were transplanted to 17 recipients. Eight full-term foetuses (two singletons and three pairs of twins) were rescued by Caesarian section on day 19, 20 or 21 of pregnancy. Three young (one singleton and twins) were successfully reared by foster mothers and proved to be normal and fertile females. All foetuses and animals were albino. In five individuals only the 1-A form of GPI (characteristic for 2n blastomere) was found. In one adult female traces of the 1-B form of GPI (characteristic for 4n carrier blastomeres) were detected in the heart and the lungs while 4 other organs contained only the 1-A form. These observations strongly suggest that the majority of foetuses/animals produced according to our experimental system are 'pure' diploids rather than 2n/4n chimaeras, and that the described method can be used in future to produce twins, triplets and quadruplets in the mouse. Our study confirms earlier work by Kelly (1975, 1977) that 'quarter' blastomeres of the mouse are still totipotent.

Animals↗
PubMed1996

Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos.

The early asymmetric cleavages of Caenorhabditis elegans embryos produce blastomeres with distinct developmental potentials. Here, we show that the caudal-like homeodomain protein PAL-1 is required to specify the somatic identity of one posterior blastomere in the 4 cell embryo. We find that pal-1 activity is sequentially restricted to this blastomere. First, at the 4 cell stage, it is translated only in the two posterior blastomeres. Then, its function is restricted to one of these blastomeres. This second targeting step is dependent on the activities of the posteriorly localized SKN-1 and asymmetrically segregated PIE-1 proteins. We propose that the segregation of PIE-1, combined with the temporal decay of SKN-1, targets pal-1 activity to this posterior lineage, thus coupling the regulation of this conserved posterior patterning gene to asymmetric cell cleavages.

Animals↗
PubMed2005

Identical triplets and twins developed from isolated blastomeres of 8- and 16-cell mouse embryos supported with tetraploid blastomeres.

We studied the developmental potential of single blastomeres from early cleavage mouse embryos. Eight- and sixteen-cell diploid mouse embryos were disaggregated and single blastomeres from eight-cell embryos or pairs of sister blastomeres from sixteen-cell embryos were aggregated with 4, 5 or 6 tetraploid blastomeres from 4-cell embryos. Each diploid donor embryo gave eight sister aggregates, which later were manipulated together as one group (set). The aggregates were cultured in vitro until the blastocyst stage, when they were transferred (in sets) to the oviducts of pseudopregnant recipients. Eighteen live foetuses or pups were obtained from the transfer (11.0% of transferred blastocysts) and out of those, eleven developed into fertile adults (one triplet, one pair of twins and four singletons). In all surviving adults, pups and living foetuses, only diploid cells were detected in their organs and tissues as shown by analysis of coat pigmentation and distribution of glucose phosphate isomerase isoforms. In order to explain the observed high rate of mortality of transferred blastocysts, in an accompanying experiment, the diploid and tetraploid blastomeres were labelled with different fluorochromes and then aggregated. These experiments showed the diploid cells to be present not only in the inner cell mass (ICM) but also in the trophectoderm. The low number of diploid cells and the predominance of tetraploid cells in the ICM of chimaeric blastocysts might have been responsible for high postimplantation mortality of our experimental embryos.

Animals↗
PubMed1976

The effect of short-term labelling in (3H) thymidine on the viability of mouse blastomeres: alone and in combination with unlabelled blastomeres.

A method of labelling 8-cell-stage mouse blastomeres with [3H]thymidine is described, which allows them to be followed to the late blastocyst stage and is compatible with normal postimplantation development. However, the [3H]thymidine does affect the postimplantation vigour of the cells when placed in competition with unlabelled cells in blastomere aggregates. This suggests that caution should be used in interpreting results using [3H]thymidine as a label for early mammalian cells.

Animals↗
PubMed1990

Induced neural-type differentiation in the cleavage-arrested blastomere isolated from early ascidian embryos.

1. Isolated blastomeres and pairs of blastomeres from 8-cell embryos of Halocynthia roretzi and Halocynthia aurantium were cleavage-arrested with cytochalasin B and cultured. Their differentiation was examined in terms of membrane excitability, immunoreactivity to an epidermis-specific monoclonal antibody (2C5), and the presence of acetylcholinesterase. 2. The blastomeres that showed epidermal-type differentiation had Ca2(+)-dependent action potentials and membrane currents, and immunoreactivity to 2C5. The blastomeres that showed neural-type differentiation had Na(+)-, Ca2(+)- and TEA-sensitive delayed K+ channels, and lacked immunoreactivity to 2C5. 3. Cleavage-arrested anterior-animal blastomeres, a4-2, when cultured in isolation from an 8-cell embryo, differentiated exclusively into epidermal-type cells. However, when cultured in contact with anterior-vegetal blastomeres, A4-1, they mostly showed neural-type differentiation (seventeen out of twenty-four cells in H. roretzi). 4. Reduction of the cytochalasin B concentration enhanced neural-type development of a4-2 blastomeres in contact with A4-1 blastomeres in H. aurantium, possibly by tightening the physical contact between the blastomeres. 5. When a cleavage-arrested and isolated a4-2 blastomere was treated with 2% pronase at 10 degrees C for 15 min at the time when sister control embryos reached the 32-cell stage, the blastomere underwent neural-type differentiation in a manner identical to that of a4-2 blastomeres contacted by A4-1 cells. 6. The period during which neural-type differentiation of a4-2 blastomeres could be induced by treatment with pronase was from the 8-cell to the 110-cell stage. At the late gastrula stage neural-type differentiation of a4-2 blastomeres was not induced by pronase. The effective period for neural-type differentiation of a4-2 blastomeres in contact with A4-1 cells was between the 64-cell stage and late gastrula stage. Competence of the a4-2 blastomere to undergo neural-type differentiation decreased during gastrula stages, while the inducing ability of the A4-1 blastomere lasted longer. 7. In a few cases the posterior-animal blastomere, b4-2, could also be induced to undergo neural-type differentiation after contact with A4-1 cells or after pronase treatment. 8. The appearance of Na+ spikes in a4-2 blastomeres in contact with A4-1 cells was considered a manifestation of neural induction, similar in principle to the induction of ectoderm by the chorda-mesoderm in higher vertebrates.

Action Potentials↗
PubMed1996

Neuronal expression in cleavage-arrested ascidian blastomeres requires gap junctional uncoupling from neighbouring cells.

1. When anterior-animal (a4-2) blastomeres isolated from 8-cell ascidian embryos were cultured under cleavage-arrested conditions in contact with anterior-vegetal (A4-1) blastomeres (a-A blastomere pairs), the a4-2 blastomeres differentiated into neuronal cells that expressed Na+ and delayed K+ channels at a time when normal sister embryos became tadpole larvae (after 40 developmental hours at 9 degrees C). When a4-2 blastomeres were cultured in contact with posterior-animal (b4-2) blastomeres (a-b blastomere pairs), the a4-2 blastomeres differentiated into epidermal cells expressing Ca2+ channels and tunic on their exterior surface. In these blastomere pairs, we analysed changes in gap junctional communication during neural and epidermal differentiation by using both dye transfer and double voltage clamp. 2. In both types of blastomere pairs, gap junctional communication was detectable at 5 h by double voltage clamp and at 7 h by dye transfer. Gap junctional communication in both types gradually increased until 25 h (equivalent to the neurula stage). However, during 25-35 h (late neurula or tailbud) in the a-A pair it decreased and finally disappeared, while it increased steeply in the a-A pair. When blastomere pairs were treated with a transcription inhibitor, actinomycin D, gap junctional communication also appeared at around 7 h but remained at a plateau level, showing neither a steep increase in a-b pairs nor a disappearance in a-A pairs. 3. In blastomere triplets in which an epidermally committed a4-2 was in contact with both blastomeres of an a-A pair, the epidermally committed a4-2 blastomere did, but the neurally committed a4-2 blastomere did not, communicate through gap junctions with the A4-1 blastomere, indicating that gap junctional communication is restricted when a4-2 blastomeres are neurally committed. 4. When a kinase inhibitor, K252a (0.5-1.0 microM), was applied at 20 h (prior to the disappearance of gap junctional communication), gap junctional communication was maintained in the a-A pair for more than 40 h. The persistence of gap junctional communication delayed the expression of Na+ and K+ channels in the a4-2 blastomere. However, channel expression followed an almost normal time sequence in single neuronally committed a4-2 blastomeres separated from A-a pairs and treated with K252a. 5. We conclude that the persistence of gap junctions causes a delay in expression of neuronal characteristics, and suggest that one of the functional roles of embryonic gap junctions is to time the expression of neuron-specific ion channels and other markers in preneuronal cells.

Animals↗
PubMed2004

Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos.

BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients referred for ICSI treatment were included. Sequences of digital images were taken by focusing at 5- micro m intervals through the embryo. Blastomere sizes and number of nuclear structures were evaluated based on these sequences. The degree of embryonic fragmentation was evaluated by normal morphological assessment prior to transfer and correlated to the blastomere sizes. RESULTS: As a result of normal cell cleavage, mean blastomere size decreased significantly from a volume of 0.28 x 10(6) microm(3) at the 2-cell stage to 0.15 x 10(6) microm(3) at the 4-cell stage (P < 0.001). Mean blastomere size decreased significantly (P < 0.001) with increasing degree of embryonic fragmentation, where highly fragmented embryos showed a 43-67% reduction in blastomere volume compared with embryos with no fragmentation. Multinucleated blastomeres were significantly larger than non-multinucleated blastomeres (P < 0.001). On average, multinucleated blastomeres were 51.5, 67.8 and 73.1% larger than their non-multinucleated sibling blastomeres at the 2-, 3- and 4-cell stage, respectively. Furthermore, the average volume of non-multinucleated blastomeres originating from multinucleated embryos was significantly smaller than the average volume of the blastomeres from mononucleated embryos (P < 0.001). CONCLUSIONS: The results of this study show that the average blastomere size is significantly affected by degree of fragmentation and multinuclearity, and that computer-assisted, multilevel analysis of blastomere size may function as a biomarker for embryo quality.

Adult↗
PubMed1995

An analysis of multinucleated blastomere formation in human embryos.

Human embryos were disaggregated into component blastomeres 42-72 h after insemination. The blastomeres were scored for the number of nuclei present and blastomeres of known nuclear morphology were returned to individual culture drops for 16-20 h, after which they were scored for cleavage and nuclear morphology. In all, 48% of mononucleated blastomeres cleaved during this period, but only 76% of these produced two mononucleated daughter blastomeres; in the remainder, one or more of the blastomeres was abnormally nucleated. During overnight culture, 30% of multinucleated blastomeres and 30% of anucleate blastomeres cleaved, the majority producing abnormally nucleated daughter blastomeres. The majority of blastomeres which showed no sign of cleavage after overnight culture retained the same nuclear morphology as when originally disaggregated. However, a small number of mononucleated blastomeres contained two nuclei after culture, indicating that karyokinesis may have taken place in the absence of cytokinesis. Overall, approximately 30% of blastomeres with more than one nucleus seemed to arise by this mechanism, the remainder probably arising by errors of chromosome segregation and/or packaging at mitosis. In addition, 25/111 mononucleated daughter cells arose either after abnormal division of mononucleated parent cells or after division of multinucleated cells, suggesting that approximately 23% of newly formed mononucleated cells might be chromosomally abnormal. The results of DNA quantitation indicated that very few (12/131, 9.2%) blastomeres (whether uni- or multinucleated) had a DNA content outside the 2-4C range. The embryos used for these studies had been cultured in one of three commonly used in-vitro fertilization (IVF) media: modified T6, Earle's balanced salts or Universal IVF medium (a commercial medium from Medi-Cult). A retrospective analysis was carried out of the number of embryos containing multinucleated blastomeres at disaggregation and of the total proportion of isolated blastomeres which were multinucleated in three groups of embryos, each of which had been cultured in one of the IVF media. Both these parameters were found to vary between cohorts of embryos cultured in the different media. The mechanism(s) by which culture medium composition might affect multinucleation of human blastomeres is discussed, as is the significance of these data for reliable preimplantation diagnosis of genetic status.

Blastomeres↗
PubMed1993

Neural differentiation in cleavage-arrested ascidian blastomeres induced by a proteolytic enzyme.

1. As previously reported, ectodermal a4-2 blastomeres isolated from 8-cell embryos of the ascidian, Halocynthia roretzi or aurantium, and cultured under conditions of cleavage arrest always differentiated into an epidermal phenotype, showing long-lasting Ca(2+)-dependent action potentials and/or tunic on the cell surface. a4-2 blastomeres contacted by a chordamesodermal blastomere, A4-1, differentiated into a neural phenotype, characterized by fast Na(+)-dependent spikes. Differentiation to a similar neural phenotype occurred when isolated a4-2 blastomeres from H. aurantium embryos were treated with > 0.003% subtilisin for 60 min at the 32-cell stage of the control embryo. Comparisons between induction by cell contact and induction by proteolytic enzymes were made and showed them to be similar in several respects. 2. When the serine protease, subtilisin, was used as the neural inducer, neural competence of a4-2 blastomeres, measured as the percentage frequency of the induction of Na+ spikes, increased after the 32-cell stage and decreased during the gastrula stage. The time course of the neural competence was the same as that for contact with the A4-1 blastomere. 3. The neural competence of four different ectodermal blastomeres isolated from the 16-cell embryo was also examined using subtilisin as a neural inducer, and by contact with the A4-1 blastomere from the 8-cell embryo. The competence was higher in anterior blastomeres than in posterior blastomeres for both types of induction. This regional difference in neural competence along the antero-posterior axis paralleled that expected from neural cell lineage during normal development, i.e. blastomeres with more cells of neural lineage among their derivatives showed higher competence. 4. Streptomyces subtilisin inhibitor, SSI (0.1%), a specific protease inhibitor for subtilisin-type serine proteases, significantly suppressed (50%) neural induction of the ectodermal blastomere, a4-2, by contact with the chordamesodermal blastomere, A4-1. 5. Monensin, brefeldin A and bafilomycin A1, all of which affect secretory processes, suppressed the neural inducing ability of the chordamesodermal blastomere, A4-1. 6. These results permit the hypothesis that a protease secreted from the chordamesoderm-generating blastomere induces the ectodermal blastomere to differentiate into neural cell type.

Animals↗
PubMed1993

Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage.

The nuclei of disaggregated blastomeres from two hundred preimplantation human embryos were examined between days 2 and 4 after insemination in vitro by vital labelling with a polynucleotide-specific fluorochrome. Although the majority of blastomeres had a single nucleus, binucleate blastomeres containing two nuclei of equal size were common and other blastomeres had fragmented nuclei or were anucleate. Seventeen per cent of normally fertilized embryos at two- to four-cell stage had at least one binucleate blastomere, and this increased to 65% at the nine- to 16-cell stage when individual embryos had between one and six binucleate blastomeres. The proportion of binucleate blastomeres in normally fertilized embryos increased from 5 to 10% over this period, whereas in abnormally fertilized, polyspermic or parthenogenetic, embryos the proportion was significantly higher during early cleavage stages but decreased at the nine- to 16-cell stage when the majority of these embryos arrest (25 and 6%, respectively). The incidence of anucleate blastomeres in normally fertilized embryos was also high, especially in those of poor morphology. In contrast, blastomeres with fragmented nuclei were relatively uncommon and the incidence was variable among classes and stages of development. Estimates of the volume of binucleate blastomeres based on measurement of their diameters and comparison with mononucleate blastomeres at various cleavage stages indicated that these blastomeres arise from a failure of cytokinesis between the second and fourth cleavage divisions. On this basis, assignment of binucleate blastomeres to particular cleavage stages in normally fertilized day 4 embryos suggests that at least some of these blastomeres arising during early cleavage persist without further cell division for up to 48 h. At the cellular level, therefore, blastomeres with either binucleate or abnormal nuclei contribute to cleavage stage arrest in vitro.

Blastomeres↗
PubMed1990

Differentiation of membrane excitability in isolated cleavage-arrested blastomeres from early ascidian embryos.

1. Differentiation of excitable cells was studied electrophysiologically and histochemically in cleavage-arrested blastomeres isolated from early ascidian embryos. Blastomeres were isolated at the 4- or 8-cell stage, and cultured in sea water containing cytochalasin B until the time of hatching of control larvae. Electrical responses, immunoreactivity to epidermis-specific monoclonal antibody (2C5) and activity of muscle-specific acetylcholinesterase were examined. 2. All cleavage-arrested blastomeres isolated from an 8-cell embryo differentiated to elicit either muscular- or epidermal-type action potentials, but no neural-type action potentials were observed in these blastomeres. The anterior-animal and the posterior-animal blastomeres developed only epidermal-type action potentials, which involved expression of Ca2+ channels and immunoreactivity to 2C5. One-third of anterior-vegetal blastomeres developed epidermal-type action potentials which are mediated by Ca2+ channels though the immunoreactivity to 2C5 was absent. A majority of remaining blastomeres showed action potentials composed of Ca2+ currents and TEA-sensitive delayed K+ currents (type I response), and a few of them had fast transient K+ currents (A-currents) in addition (type II response). One-third of posterior-vegetal blastomeres developed epidermal-type action potentials without expression of the immunoreactivity to 2C5. The remainder differentiated into muscular-type cells, which expressed Ca2+ currents, TEA-sensitive and TEA-insensitive delayed K+ currents, and showed acetylcholinesterase activity. 3. Cleavage-arrested blastomeres isolated from a 4-cell embryo also differentiated into epidermal- or muscular-type cells, but not neural-type cells. The anterior blastomere, which is the parent cell of anterior-animal and anterior-vegetal blastomeres of an 8-cell embryo, developed epidermal-type, type I or type II responses, as was the case in the anterior-vegetal blastomere isolated from an 8-cell embryo. The posterior blastomere, which was the parent cell of posterior-animal and posterior-vegetal blastomeres of an 8-cell embryo, differentiated into either epidermal-type or muscular-type cells in terms of both membrane excitability and immunochemical reactivity. 4. Cleavage-arrested 1-cell embryos differentiated exclusively into epidermal-type cells in terms of membrane excitability and 2C5 immunoreactivity, even when the cytochalasin B concentration was decreased below 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed2006

[HLA-A site genotyping on single blastomeres is studied by nest-PCR-SSP method].

OBJECTIVE: To assess the accuracy and reliability of the nest-PCR-sequence specific primer(SSP) method in HLA-A site genotyping of single blastomeres retrieved from human pre-implantation embryos. METHODS: By nest PCR on HLA-A exon 2, the success rate of first-round amplification was estimated for single blastomeres. Based on the first-round amplification, the HLA-A genotype of every single blastomeres was analyzed by commercially available PCR-SSP kits. RESULTS: The amplification of HLA-A exon 2 were performed to 120 blasotmeres retrieved from in vitro fertilization(IVF) surplus embryos donated by 10 couples. The average success rate of family 1-5 and 6-10 was 78.2%(43/55) and 93.8%(61/65), respectively. And 86.7%(104/120) in total. Eighty blastomeres were further tested by nest-PCR-SSP, among which 11 blastomeres failed to HLA-A exon 2 amplification and then failed to genotyping while the other 69 blastomeres succeed in HLA-A exon 2 amplification and succeed in genotyping. Except for 6 blastomeres that were uncertain for allele lost because of parents' homozygosity, the left 63 blastomeres had accurate HLA genotyping. Among these 63 blastomeres, 59 blastomeres had genotypes confirmed from their parents(93.6%), 3 blastomeres lost one of parents' alleles(4.8%), and only one blastomere had two more than parents' alleles(1.6%). CONCLUSION: The above research results indicated that based on the successful first round amplification of single blastomeres, nest-PCR-SSP strategy offers a convenient and reliable option for HLA genotyping on single blastomeres, which is a key process in pre-selecting HLA-identical sibling for allogeneic cord blood cell transplantation.

Base Sequence↗
PubMed1990

Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres.

A detailed fate map of all the progeny derived from each of the blastomeres of the 4- and 8-cell stage South African clawed frog (Xenopus laevis) embryo is presented. Each "identified" blastomere that results from stereotypic cleavages has a characteristic set of progeny that distinguishes it from the other blastomeres of the embryo. The 4-cell dorsal (D) blastomere is the major progenitor of the stomodeum, cement gland, retina, notochord, head somite, pharynx and liver. The 4-cell ventral (V) blastomere is the major progenitor of the trunk and fin epidermis, ventral somite, nephrotome, lateral plate mesoderm and proctodeum. The other organs are derived from both blastomeres. At the next cell division, the animal hemisphere daughters of both blastomeres (D1 and V1, respectively) become the major progenitors for head ectodermal and mesodermal structures, and the vegetal hemisphere daughters become the major progenitors for trunk mesodermal (D2) or trunk endodermal (V2) structures. Semiquantitative lineage diagrams, using data from this and from previous studies demonstrate that as cleavage proceeds from the 2- to the 32-cell stage, the progenitors for particular organs or for specific regions of organs segregate into defined regions of the blastula. To determine whether this segregation is related to the position of the blastomere or to its geneological lineage, we compared the fates of radial 8-cell blastomeres to those of stereotypic 8-cell blastomeres. Radial blastomeres have fates nearly equivalent to the sum of the two 16-cell blastomeres that occupy the same position in the embryo, demonstrating that fate depends upon blastomere position rather than lineage.

Animals↗
PubMed2001

How many blastomeres of the 4-cell embryo contribute cells to the mouse body?

The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body.

Animals↗