Blastomere Isolation Research Articles

Chromosomal information derived from single blastomeres isolated from cleavage-stage embryos and cultured in vitro

Objective To evaluate the potential of proliferation of single blastomeres isolated from human cleavage-stage embryos for use in preimplantation genetic diagnosis of chromosomal abnormalities. Design A laboratory study of chromosomal content of blastomeres isolated from embryos of patients from an in vitro fertilization program. Setting University hospital laboratory. Patient(s) Couples undergoing IVF or ICSI. Intervention(s) Blastomeres were isolated from normally fertilized cleavage-stage human embryos, cultured in vitro or fixed immediately, and analyzed by fluorescence in situ hybridization (FISH) probes. Main outcome measure(s) Chromosomal information yielded by blastomeres cultured in vitro compared with those obtained from blastomeres that were processed for chromosomal analysis directly after isolation. Result(s) The percentage of cultured blastomeres that produced FISH results was significantly lower than the percentage of blastomeres processed for FISH directly after isolation (72% vs. 90%). Lack of FISH results from cultured cells, which in most cases was related to nuclear anomalies, was significantly more frequent among nondivided than divided blastomeres (39% vs. 21%). Both cultured and noncultured cells showed diploid, aneuploid and polyploid chromosome complements on FISH. Compared with directly processed cells, cultured cells yielded a higher proportion of polyploid patterns (22.9% vs. 6.1%). Of the cultured blastomeres that divided, 18% produced progeny with mosaicism. Conclusion(s) Although blastomere culture may increase the number of cells available for chromosomal analysis, the high frequency of nuclear defects and the occurrence of polyploidy and mosaicism among cultured cells discourage the use of blastomere isolation and proliferation strategy for use in preimplantation genetic diagnosis.

Binary specification of nerve cord and notochord cell fates in ascidian embryos.

In the ascidian embryo, the nerve cord and notochord of the tail of tadpole larvae originate from the precursor blastomeres for both tissues in the 32-cell-stage embryo. Each fate is separated into two daughter blastomeres at the next cleavage. We have examined mechanisms that are responsible for nerve cord and notochord specification through experiments involving blastomere isolation, cell dissociation, and treatment with basic fibroblast growth factor (bFGF) and inhibitors for the mitogen-activated protein kinase (MAPK) cascade. It has been shown that inductive cell interaction at the 32-cell stage is required for notochord formation. Our results show that the nerve cord fate is determined autonomously without any cell interaction. Presumptive notochord blastomeres also assume a nerve cord fate when they are isolated before induction is completed. By contrast, not only presumptive notochord blastomeres but also presumptive nerve cord blastomeres forsake their default nerve cord fate and choose the notochord fate when they are treated with bFGF. When the FGF-Ras-MAPK signaling cascade is inhibited, both blastomeres choose the default nerve cord pathway, supporting the results of blastomere isolation. Thus, binary choice of alternative fates and asymmetric division are involved in this nerve cord/notochord fate determination system, mediated by FGF signaling.

Mesoderm formation by isolated and cultivated 8-cell stage blastomeres of the teleost, Leucopsarion ptersii (shiro-uo).

Isolation of cleavage-stage blastomeres and the study of their developmental potential has been used extensively for analyzing the mechanisms of embryogenesis in vertebrates, including amphibians and echinoderms. We devised a method to isolate 8-cell stage blastomeres in the teleost, shiro-uo, by utilizing its unique cleavage pattern of the horizontal 3rd cleavage plane. Removal of all the upper blastomeres at the 8-cell stage allowed almost normal embryogenesis from the remaining lower blastomeres and yolk cell mass. Isolated upper or lower blastomeres formed vesicles and spherical bodies, which later showed morphological changes during cultivation. Mesoderm formation was detected not only in the cultivated lower blastomeres or whole blastomeres but also in the upper blastomeres isolated from the yolk cell mass at the 8-cell stage, although at a lower frequency than the lower blastomeres. These results indicated the presence of very early signaling for mesoderm induction, which is independent from the currently postulated signals from the yolk syncytial layer at later stages. This also indicated non-equivalence or differentiation of the blastomeres from the very early cleavage stage in teleost embryos.

Ectopic mesodermal expression promoted by the eight-cell stage Bufo arenarum subequatorial cytoplasm.

Mesodermal determinants were investigated by cytoplasmic transfer and blastomere isolation in the eight-cell stage of Bufo arenarum. Their existence was confirmed by assaying the subequatorial cytoplasm's ability to respecify the developmental potency of animal quartets. The gray subequatorial cytoplasm, but not animal cytoplasm, is able to divert the ectodermal fate of animal quartets to several mesodermal components. The source of the transplanted cytoplasm was important in determining the category of the resulting structures. Ventral subequatorial cytoplasm from ventrovegetal blastomeres generated ventral derivatives, namely erythrocytes and mesenchyma. Dorsal subequatorial cytoplasm from dorsovegetal blastomeres produced dorsolateral derivatives, such as notochord, muscle, nephric tubules, and coelomic epithelium, including mesenchyma. On the other hand, transfer of vegetal pole cytoplasm to animal quartets resulted in the formation of groups of endoderm-like cells dispersed among epidermal cells. However, the presence of such cells did not cause any mesodermal induction. The present findings suggest the existence of cytoplasmic information responsible for mesodermal specification. The alternative hypothesis that animal blastomeres become mesoderm due to vegetal induction is questioned.

Influence of the timing of blastomere isolation after the removal of nocodazole in bovine nuclear transfer

This study was conducted to investigate the influence of the timing of blastomere isolation after the removal of nocodazole on the subsequent division of blastomeres and developmental ability of reconstituted bovine embryos. The division rate of isolated blastomeres was examined at 3, 5 and 24 h of culture after nocodazole removal. Furthermore, isolated blastomeres and those of whole embryos were used as donors in nuclear transfer to determine the development of reconstituted embryos. The division rate of isolated blastomeres at 3 h was significantly lower than the presumptive division rate of blastomeres from whole embryos (P<0.05). When these blastomeres were used as donor nuclei, the dividing blastomeres yielded a significantly higher development rate than blastomeres from whole embryos (P<0.05). These results confirm that the timing of blastomere isolation influences the subsequent division of blastomeres and the developmental ability of the reconstituted embryos.

Experimental Manipulation of Parental Investment in Echinoid Echinoderms

SYNOPSIS. In free spawning species, maternal investment is limited to the contents of the egg. Striking correlations between egg size and larval characteristics, such as development rate, larval size, body form, feeding capability, nutritional requirements, and size at metamorphosis, have been observed in many taxa of marine invertebrates, in spite of tremendous morphological and ecological diversity. Analysis of these relationships has historically been based on comparative observations or quantitative modeling. Advances in our understanding of life history ecology in marine organisms require combining evolutionary theory with functional analyses of larvae as pelagic organisms. I believe that development will prove to be an important integrative link between these fields. In taxa with regulative development {e.g., echinoid echinoderms), it is possible to experimentally manipulate the amount of material that is available for larval morphogenesis. This provides a powerful tool for elucidating the developmental consequences of changes in maternal investment. Here, I will examine the rationale and methodology underlying this experimental approach and review the conclusions and some outstanding questions concerning the influence of maternal investment on the morphology, function, growth, and development of larvae. The four main effects of an experimental reduction of egg size (blastomere isolation) are: 1) smaller larval size, 2) simpler larval form, and 3) slower development in the early-stage larvae, but 4) regulation of size, shape, and development rate in late-stage larvae.

Onset of competence to respond to activin A in isolated eight-cell stage Xenopus animal blastomeres.

Single animal hemisphere blastomeres isolated from the eight-cell stage Xenopus embryos differentiate into mesoderm when treated with activin A, whereas when cultured without activin they form atypical epidermis. The mesoderm tissue induced by activin is different between dorsal and ventral blastomeres. In the present study, the duration and timing of activin treatment was varied, in order to identify the critical stage when animal blastomeres acquire competence to respond to activin A. It was shown that the critical time was 45 min after blastomere isolation, which corresponds approximately to NF stage 6 (32-cell stage) of normal development. The competence gradually increased during the morula stages.

Chapter 13 Blastomere Culture and Analysis

This chapter discusses embryo devitellinization and blastomere isolation. A method of permeabilization and blastomere isolation and assay methods for assessing the degree of development and its relative normality after experimental manipulation are minimal requirements for a satisfactory Caenorhabditis elegans (C. elegans) embryonic culture system. In the procedure described in this chapter, embryos are stripped of the eggshell and vitelline membranes by chitinasing the eggshell, and mechanically stripping off the vitelline membrane. Batches of up to about 200 embryos can be prepared at the dissecting microscope. Blastomeres may be isolated manually following this denuding. The procedure takes from 15 to 40 minutes, depending on the number of worms cut. The criteria that indicates, both a good preparation and a good batch of culture medium includes: (1) embryos show very little lysis, initially or after overnight culture; (2) embryos continue to divide on schedule; (3) intact embryos after overnight incubation have more than 300 cells, and ideally more than 500; and (4) gut granules are visible under polarized light in more than 90% of these embryos.

Establishment of gut fate in the E lineage of C. elegans: the roles of lineage-dependent mechanisms and cell interactions.

The gut of C. elegans derives from all the progeny of the E blastomere, a cell of the eight cell stage. Previous work has shown that gut specification requires an induction during the four cell stage (Goldstein, B. (1992) Nature 357, 255-257). Blastomere isolation and recombination experiments were done to determine which parts of the embryo can respond to gut induction. Normally only the posterior side of the EMS blastomere contacts the inducing cell, P2. When P2 was instead placed in a random position on an isolated EMS, gut consistently differentiated from the daughter of EMS contacting P2, indicating that any side of EMS can respond to gut induction. Additionally, moving P2 around to the opposite side of EMS in an otherwise intact embryo caused EMS's two daughter cells to switch lineage timings, and gut to differentiate from the descendents of what normally would be the MS blastomere. The other cells of the four cell stage, ABa, ABp, and P2, did not form gut when placed in contact with the inducer. To determine whether any other inductions are involved in gut specification, timed blastomere isolations were done at the two and eight cell stages. In the absence of cell contact at the two cell stage, segregation of gut fate proceeded normally at both the two and four cell stages. Gut fate also segregated properly in the absence of cell contact at the eight cell stage. A model is presented for the roles of lineage-dependent mechanisms and cell interactions in establishing gut fate in the E lineage.

Scanning electron microscope (SEM) study of mouse embryos obtained from isolated blastomeres

Preimplantation diagnosis and embryo sexing offer great possibilities in the prevention of human diseases and in the field of animal production. These techniques involve blastomere isolation. Isolated blastomeres can grow in culture and develop as whole embryos. In this paper we describe, at the scanning electron microscope level, the characteristics of the plasma membrane surface of isolated blastomeres obtained from mouse embryos at the two-cell stage and of embryos grown to the 2/4- and 4/8-cell stages and compare them to control embryos grown in vitro. According to our results the in vitro manipulation of these embryos does not affect the surface characteristics of the plasma membrane in the early cleavage stages.

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.

Isolation and culture of single 8-cell blastomeres in-rabbits with a novel approach

This study was undertaken to develop a new technique to produce identical offspring by aggregating a quarter or eighth embryo with a parthenogenetically activated egg in the mouse. One or two blastomeres from 8-cell embryos were aggregated with a parthenogenetic 4-cell egg from which one or two blastomeres had been removed. After micromanipulation and culture for 2 d in vitro, the morphologically normal blastocysts were transferred to the uterus of recipient females. The success rate in micromanipulation of eggs was 93 to 100%: aggregation of blastomeres occured about 60% of the time and the proportion of live young after transfer of aggregated eggs was 11 to 33% for the quarter and 2 to 24% for the eighth egg. The proportion of chimaeras as judged by coat color was 10% for the quarter and 20% for the eighth egg. However, GPI-1 analysis and progeny testing could not detect a parthenogenetic contribution in all offspring. The mean number of young obtained from one embryo was 1.7 for the quarter and 1.6 for the eighth embryo. The maximal number of young obtained from splitting one 8-cell embryo into quarters was three and into eighths was four. The mice of each set derived from a single embryo were of the same sex.Our study clearly demonstrates that the parthenogenone can assist development of the quarter and eighth mouse embryo to term. The proportion of chimaeras is low compared with that obtained when two fertilied eggs are combined.

Chromatin diminution and early cleavage in Parascaris univalens (Nematoda).

In Parascaris developmental commitment to the germ line and somatic lineages is indicated by the orientation of the mitotic spindle in blastomeres, the topology of cells in the embryo, and chromatin diminution in presomatic blastomeres. Using three different experimental techniques: transient pressure treatment, application of cytochalasin B, and isolation of blastomeres, we have succeeded in uncoupling several developmental processes during cleavage of P. univalens. The following results were obtained: (1) Following mitotic nondisjunction we observed identical behavior of all chromatids in each blastomere. Thus chromosome differentiation by differential replication does not occur. (2) Chromosome fragments obtained by pressure treatment of egg cells underwent chromatin diminution. Thus this process does not require an intact germ-line chromosome. However, chromosomes immobilized on a monopolar spindle did not undergo chromatin diminution. Thus diminution appears to require segregation of chromatids. (3) Blastomeres that completely lacked chromosomes as a result of mitotic nondisjunction underwent normal early cleavage divisions. (4) Pressure treatment or prolonged treatment with cytochalasin B caused egg cells or germ line blastomeres to lose their germ line quality, as deduced from the coincident occurrence of symmetrical (presomatic-like) cleavage and chromatin diminution. (5) Isolated blastomeres from 2-cell embryos, i.e. 1/2 blastomeres, usually cleaved according to their prospective fates in the whole embryo. However, in some partial embryos derived from such blastomeres, chromatin diminution was delayed for either one or two cleavage mitoses. An "activation" model as an alternative to a prelocalization model is presented, which can account for early blastomere topogenesis and chromatin diminution.

Mass Isolation of Muscle Lineage Blastomeres from Ascidian Embryos: (ascidian embryos/muscle lineage cells/mass isolation).

The aim of this investigation was to establish an experimental system for studying the causal relationship between DNA replication and tissue-specific enzyme development in ascidian embryos. Blastomeres were dissociated from 44∽64-ceIl Halocynthia roretzi embryos and fractionated by centrifugation through a discontinuous Percoll density gradient. When cells harvested from the fraction at the bottom of the tube were division-arrested with cytochalasin B (an inhibitor of cytokinesis) soon after their isolation, more than 70% of them developed histochemically-detectable muscle-specific acetylcholinesterase (AChE) activity, suggesting that they were almost all blastomeres of muscle lineage. When these cells were arrested with aphidicolin (an inhibitor of DNA replication) and cytochalasin B immediately after their isolation, however, none of them showed AChE activity. When they were allowed to divide once and then arrested with the inhibitors, nearly 40 % of them developed AChE activity, and when they were allowed to divide twice before arrest, about 70% of them showed AChE activity.

Restriction of developmental potential and trochoblast ciliation in Patella embryos

At the 64-cell-stage embryos of Patella develop a prototroch consisting of four groups of four cilia-bearing cells. Ciliogenesis of isolated blastomeres and trochoblasts was studied, as well as the effect on it of cleavage arrest caused by cytochalasin B treatment. Isolation of blastomeres or trochoblast cells has no influence on ciliogenesis; neither has arrest of cleavage in whole embryos after the third cleavage. However, cleavage arrest before third cleavage completely prevents ciliogenesis. Thus, third cleavage is decisive for the expression of the developmental potential of the primary trochoblasts. Impairment of DNA synthesis by aphidicolin in the S-phase preceding third cleavage also prevents ciliogenesis. It is concluded that a determinant for ciliogenesis as well as certain nuclear factors must be segregated into the micromeres at third cleavage for ciliogenesis to occur in the prototroch cells.

The second polar lobe of theSabellaria cementarium embryo plays an inhibitory role in apical tuft formation.

The embryo ofSabellaria cementarium (Polychaeta) forms a polar lobe at each of the first two cleavage divisions which becomes absorbed into one of the blastomeres at the end of the division. Lobe removal experiments show that the polar lobe preceding first cleavage is necessary for the development of the apical tuft and the posttrochal region of the trochophore larva. The polar lobe preceding second cleavage is smaller than the first polar lobe and is necessary only for post-trochal region development. In blastomere isolation experiments, isolates containing the C but not the D blastomere form apical tufts. Isolates containing the D but not the C blastomere do not form apical tufts. When the polar lobe preceding second cleavage is removed and the C and D blastomeres are separated and raised in isolation, each can form an apical tuft. When the second cleavage is equalized with sodium dodecyl sulfate (SDS) such that both the C and the D blastomeres receive second polar lobe material, no apical tuft is formed. These results suggest that apical tuft determinants are distributed to both the C and D blastomeres at second cleavage but that the second polar lobe contains an inhibitor for apical tuft formation which is shunted to the D blastomere after the completion of second cleavage.

Fusion of dissociated fish embryonic cells

The induction of a frequent fusion in dissociated fish embryonic cells is reported. Fusion was induced during mechanical isolation of blastomeres in normal saline solution or Ca2+-free saline solution by quickly bringing about physical contact between cells within about 1-5 min of dissociation. Fused blastomeres were obtained in about 18% yield from early morula cells in about 68% yield from early blastula cells and in about 5% yield from early gastrula cells.

Modifications in Development of the Sand Dollar by NaCNS

Experiments conducted at the Hopkins Marine Station, Pacific Grove, California, during the summer of 1938 on the unfertilized and fertilized eggs of Dendraster excentricus have given certain interesting modifications in the early developmental patterns of this form.Unfertilized eggs treated for 12 hours with a solution of NaCNS (20 cc .54 M NaCNS plus 100 cc Ca-free sea water) showed a retardation in cleavage rate when returned to normal sea water and fertilized. The developing larvae ranged from forms showing considerable inhibition (increased mesenchyme, shortened anal arms, undifferentiated oral lobe) to larvae (approximately 35%) which consisted entirely of ectoderm and ectodermal structures. These ectodermized forms were modifications in the same direction as the ‘animalized’ forms produced in the sea urchin by Lindahl1 and Horstadius2 with NaCNS treatment and experimental isolation of animal blastomeres. Neither mesenchyme, pigment nor gut was present. With higher concentrations or exposure periods ...