Large Blastomeres Research Articles

Proteomics reveals the underlying mechanism by which the first uneven division affects embryonic development in pig

One of the most typical abnormal cleavage patterns during early embryonic development is uneven division, but the first uneven division of pig zygote is common. Uneven division results in different daughter cell sizes and an uneven distribution of organelles such as lipid droplet, mitochondria, but the developmental capacity of daughter cells and proteomic changes of daughter cells are still unclear. Therefore, the developmental ability and proteomic quantification were investigated on blastomeres from even division (ED) or uneven division (UD) embryos at 2-cell stage in the present study. Firstly, the developmental ability was affected by the blastomeric size, when compared with medium blastomeres (MBs), the large blastomeres (LBs) with the higher cleavage rate but the small blastomeres (SBs) with the lower rate was observed. Subsequently, proteomic analysis was performed on blastomeres of LBs, MBs and SBs, a total of 109 DEPs were detected, which were involved in protein metabolism and processing, energy metabolism and ribosome. In particular, DEPs in LBs vs. SBs were focused on RNA binding and actin cytoskeletal tissue. Two protein-dense networks associated with RNA binding and cytoskeleton were revealed by further protein-protein interaction (PPI) analysis of DEPs in LBs vs. SBs, that DDX1 related to RNA binding and ACTB related to cytoskeleton were confirmed in UD embryos. Therefore, a briefly information of DEPs in blastomeres of 2-cell stage pig embryos was described in the present study, and it further confirmed that the formation of uneven division of the first cell cycle of pig embryos might be controlled by the cytoskeleton; the developmental capacity of daughter cells might be affected by the energy metabolism, RNA binding and ribosome, and further account for the developmental potential of the whole embryo.

Meiotic and Early Zygotic Development in Crassostrea virginica Observed through Confocal Microscopy

Several studies have examined early development in oysters, but few have reported the normal (expected) behavior of meiosis and early mitosis or of the organization of the spindle by the centrosomes. To establish this baseline, normal development of fertilized eggs in Crassostrea virginica from meiosis until the four—cell stage using confocal microscopy was documented. To visualize the centrosomes and microtubules, eggs were stained with antibodies for γ- and a-tubulin. In addition to establishing normal development, two additional features of early development were documented. First, centrosome behavior was documented. The centrosome associated with the female pronucleus disappeared after polar body formation. The single centrosome associated with the male pronucleus duplicated by late anaphase of meiosis II to eventually form the spindle for mitosis I, thus the two centrosomes in the first cleavage were derived from the sperm. Second, linkage between the cell and centrosome cycles was established. In the first and second mitosis, centrosome duplication began at early anaphase. At late anaphase, the centrosomes duplicated and were observed as two distinct units at each pole. Asynchrony was also observed at the four—cell stage between the large and small blastomeres, with chromatin and centrosome duplication showing more advanced stages in the large blastomere. This documentation will serve as a baseline for future studies on chromosome loss in polyploid oysters.

Effects of laser-assisted hatching and exposure time to vitrification solution on mouse embryo development.

ObjectiveThis study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers.MethodsFirst, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed.ResultsThe hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p<0.05). In the control-8 group (22.1±4.6), the cell number of the inner cell mass was higher than in the LAH groups (p<0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group (92.8±8.9) than in the others (p<0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group (19.5±5.1, p<0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group (73.2±12.1) than in the other groups, but without statistical significance.ConclusionWhen LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

6 ANEUPLOIDY TOLERANCE IN RHESUS MACAQUE PRE-IMPLANTATION EMBRYOS VIA MICRONUCLEI FORMATION, CELLULAR FRAGMENTATION, AND BLASTOMERE EXCLUSION

A primary contributor to in vitro fertilization (IVF) failure is the presence of unbalanced chromosomes in pre-implantation embryos. Previous array-based and next-generation sequencing (NGS) studies determined that ~50 to 80% of human embryos are aneuploid at the cleavage stage. During early mitotic divisions, many human embryos also sequester mis-segregated chromosomes into micronuclei and concurrently undergo cellular fragmentation. We hypothesised that cellular fragmentation represents a response to mis-segregated chromosomes that are encapsulated into micronuclei. Here, we utilised the rhesus macaque pre-implantation embryo as a model to study human embryonic aneuploidy using a combination of EevaTM time-lapse imaging for evaluating cell divisions, single-cell/-fragment DNA-Sequencing (DNA-Seq), and confocal microscopy of nuclear structures. Results from our time-lapse image analysis demonstrated that there are considerable differences in the timing of the first and third mitotic divisions between rhesus blastocysts and those that arrested before this stage in development (P &lt; 0.01; ANOVA). By examining the chromosome content of each blastomere from cleavage stage embryos via DNA-Seq, we determined that rhesus embryos have an aneuploidy frequency up to ~62% (N = 26) with several embryos exhibiting chromosomal mosaicism between blastomeres (N = 6). Certain blastomeres also exhibited reciprocal whole chromosomal gains or losses, indicating that these embryos had undergone mitotic non-disjunction early in development. In addition, findings of reciprocal sub-chromosomal deletions/duplications among blastomeres suggest that chromosomal breakage had occurred in some embryos as well. Embryo immunostaining for the nuclear envelope protein, LAMIN-B1, demonstrated that fragmented cleavage-stage rhesus embryos often contain micronuclei and that cellular fragments can enclose DNA. Our DNA-Seq analysis confirmed that cellular fragments might encapsulate whole and/or partial chromosomes lost from blastomeres. When embryos were immunostained with gamma-H2AX, a marker of chromatin fragility, we observed distinct foci solely in micronuclei and DNA-containing cellular fragments. This suggests that micronuclei may be ejected from blastomeres through the process of cellular fragmentation and, once sequestered, these mis-segregated chromosomes become highly unstable and undergo DNA degradation. Finally, we also observed that ~10% of embryos prevented cellular fragments or large blastomeres from incorporating into the inner cell mass or trophectoderm at the blastocyst stage (n = 5). Upon confocal imaging, multiple nuclei and intense gamma-H2AX foci were found in a large unincorporated blastomere in one of the blastocysts. Altogether, our findings demonstrate that the rhesus embryo responds to segregation errors by eliminating chromosome-containing micronuclei via cellular fragmentation and/or selecting against aneuploid blastomeres that fail to divide during pre-implantation development with significant implications for human IVF.

Development of large and small blastomeres from 2‐cell embryos produced in vitro in pigs

In the present study, we examined the development to blastocysts of large and small blastomeres from unevenly cleaved 2-cell embryos (uneven 2-cell embryos) in pigs. Proportion of blastocysts derived from large blastomeres (52.8 ± 6.4%) was significantly higher (P<0.05) compared with small ones (32.1 ± 4.6%). However, there were no differences in total cell number, inner cell mass (ICM) cell number and ICM/total cells ratio between them. Of 53 sister blastomere pairs in the same embryos examined there were 12 pairs (22.6%) in which both blastomeres developed to blastocysts, 16 pairs (30.2%) in which only large blastomeres developed to blastocysts, and five pairs (9.4%) in which only small blastomeres developed to blastocysts. Relative total amount of active mitochondria in small blastomeres were lower (P<0.05) than that of large blastomeres and blastomeres from evenly cleaved 2-cell embryos. However, there was no difference in relative density of active mitochondria in these three types of blastomeres. In conclusion, blastocysts derived from small and large blastomeres in uneven 2-cell embryos had comparable quality in terms of cell number, ICM number, ICM/total cell ratio and distribution of active mitochondria. The results suggest that these blastomeres may contribute multiple offspring production in pigs.

Nuclear Transplantation of Mouse Inner Cell Mass and Trophectoderm Cells into Enucleated Two-Cell Embryos.

. Trophectoderm (TE) and inner cell mass (ICM) cells from blastocytsts were compared for their ability to produce chimeric mice or conceptuses (pluripotency) using nuclear transplantation in which a single cell was transplanted into an enucleated blastomere of a 2-cell embryo. The developmental ability of the embryos reconstituted with TE and ICM cells was dependent on when the recipient 2-cell embryos were recovered after hCG administration. When 2-cell embryos obtained 38-42 and 43-46 h post hCG injection were used, cell division of the reconstituted embryos rarely progressed to the 4-cell stage. However, when embryos recovered 48-51 h post hCG were used, the proportion of cleaved embryos increased significantly, developing to blastocysts with several large blastomeres. When reconstituted embryos were treated with nocodazole after nuclear transplantation, the percentage of cleaved embryos significantly increased, with some developing to blastocysts. Such blastocysts were transferred to recipient females, and no donor nuclei were detected in the conceptuses at midgestation. The in vitro developmental ability of the chimeric 2-cell embryos reconstituted with TE and ICM cells from blastocysts was quite limited and no chimeric conceptuses were obtained.

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.

Dynamics of Germ Plasm Localization and Its Inhibition by Ultraviolet Irradiation in Early Cleavage Xenopus Embryos

In the spawned Xenopus egg, germ plasm is found as cytoplasmic islands spread over a wide cortical region of the vegetal pole. However, by the blastula stage, the same material is found concentrated into a few large blastomeres at the floor of the blastocoel. Components of the germ plasm can be specifically labeled with a fluorescent dye, DiOC6(3), permitting the dynamic movements of germ plasm localization to be followed in live embryos via time-lapse confocal scanning optical microscopy. During the first cell cycle, the small islands initially appear to be fixed to the vegetal yolk mass and to move with it during the cortical rotation. After rotation, the islands appear to be released from the vegetal yolk mass and to begin fusing with one another. During early cleavages, the germ plasm aggregates into large islands at the vegetal pole in a movement dependent on microtubules. Two distinct actions can be discerned: (1) a continuous process of local fusing and (2) periodic surface contraction waves (SCWs) that gather the islands toward the vegetal pole. These SCWs are inhibited by ultraviolet irradiation of the vegetal pole. Near the vegetal pole, germ plasm patches ingress into the embryo's interior along the cleavage furrow in periodic movements contemporaneous with the SCWs.

Centrifugal elutriation of large fragile cells: Isolation of RNA from fixed embryonic blastomeres

In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96,94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed nonuniformly among these cells.

Transcription in leech: mRNA synthesis is required for early cleavages in Helobdella embryos

Zygotic transcription was analyzed in embryos of the glossiphoniid leech Helobdella triserialis by autoradiographic detection of tritiated uridine incorporated in the presence of absence of low concentrations of α-amanitin. RNA synthesis was first detected after the second cleavage and α-amanitin-sensitive RNA synthesis was first detected during the divisions yielding the embryonic stem cells, or teloblasts. RNA synthesis increased as development progressed, and the bulk of α-amanitin-sensitive RNA synthesis was found in two classes of cells, the blast cells, which are the progeny of the teloblasts, and the micromere-derived cells. The time during which zygotic gene products are required was determined by observing the developmental consequences of α-amanitin exposure. Zygotes microinjected with α-amanitin underwent the first several cleavages with normal timing and symmetry, but underwent aberrant cleavages and produced supernumerary large blastomeres during the time that the control embryos generated teloblasts. Once the teloblasts were formed, the microinjection of α-amanitin did not affect the production of blast cells by the teloblasts, but it did block the divisions and movements of the blast cells and the micromere-derived cells. These data suggest that zygotic transcription is activated during the early cleavages of Helobdella embryos and that newly synthesized transcripts are required for the generation of teloblasts. Thus, there is an early, critical period of messenger RNA synthesis essential for teloblast production that is distinct from the later phase of messenger RNA synthesis required for cell divisions and cell movements during gastrulation.

Rare maternal mRNAs code for regulatory proteins that control lineage-specific gene expression in the sea urchin embryo.

The prevalence of mRNAs coding for the sea urchin embryo regulatory factors P3A1 and P3A2 was measured by single-strand probe excess solution hybridization. P3A1 and P3A2 are not homologous proteins, though they both bind specifically to a particular cis-regulatory sequence. Interaction at this target site is known to be required for lineage-specific expression of an aboral ectoderm-specific gene and probably for several other genes as well. Genome blot hybridizations show that both factors are encoded by single-copy genes. Maternal mRNAs for both factors are present at less than 10(3) molecules per egg, which places them in the rare mRNA class. During development to the mesenchyme blastula stage, the amount of P3A1 mRNA (per embryo) increases severalfold while that of P3A2 remains approximately constant. Specification of the aboral ectoderm founder cells and of their initial patterns of gene expression must occur during early to mid-cleavage stage. Therefore, the regulatory proteins needed for this process must be produced by this stage. We show that the quantities of the P3A proteins that can be synthesized from the numbers of mRNA molecules present in the large blastomeres of the early embryo are sufficient to be functional, because these proteins will be accumulated in the nuclei. Thus maternal P3A1 or P3A2 proteins asre not required, nor were these detected in earlier studies. Furthermore, differential spatial (as well as temporal) distribution of both of these newly synthesized factor species could result from the unequal cleavage pattern utilized in the sea urchin egg.

Modification of cell division by phorbol ester in preimplantation mouse embryos.

2-cell mouse embryos were treated in vitro with a 2 h pulse of phorbol myristate acetate (PMA) at 32nd, 38th and 50th h after hCG, then chased in culture for up to 46 h. Embryos were fixed at various time intervals of chasing, then stained and inspected. Some embryos were carefully inspected with a video recording system, every 1.44s and the cell divisions (cytokinesis) as well as formation of large, single blastomeres, each from two smaller ones, were recorded. PMA pulse let to the suppression of cell divisions. The rate of the suppression was time dependent: with a delay of 0-1, 12 and 18 h between the PMA pulse and time of scheduled cell division about 99, 87 and 44% of 2-cell embryos remained at this stage of development, for at least 10 h, respectively, and 90, 58 and 12% of their blastomeres revealed binuclearity. Since we found that PMA-mediated formation of binuclearity was not the effect of cell fusions, it was assumed that the inhibition of cytokinesis preceded by karyokinesis was responsible for binuclearity. PMA effect on cell divisions was reversible. PMA-treated embryos revealed formation of large, single blastomeres, each from two smaller ones. If cell division appeared after PMA pulse, in about 52% of 3- to 6-cell embryos, the large blastomere formation was recorded in the course of the subsequent 38 h. Large blastomere formation was concluded to be the result of either cell fusion or reversion of incompleted cytokinesis brought about by PMA.

Formation of nucleus-like structure in the cytoplasm of λ-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis

The fate of bacteriophage λ-DNA was examined after injection into the fertilized eggs of Xenopus laevis. Injection of a large amount of λ-DNA (ca. 24 ng) into a fertilized Xenopus egg induced the formation around the injected DNA of a giant nucleus-like structure which was surrounded by an apparently normal bilayered nuclear membrane with nuclear pore complexes. Southern blot analysis revealed the persistence of injected λ-DNA until the blastula stage. The nucleus-like structure was partitioned into blastomeres during cleavage through a process of nuclear fission, and was maintained in a group of extraordinarily large blastomeres until the blastula stage.

Determination of excitability types in blastomeres of the cleavage-arrested but differentiated embryos of an ascidian.

Cleavage of the embryo of Halocynthia roretzi was arrested with cytochalasin B at 1- to 32-cell stages and the embryo was cultured in sea water containing cytochalsin B until a developmental time equivalent to the hatching of the control larva. Membrane properties of the blastomeres were studied with constant-current and voltage-clamp techniques. Four types of membrane response - neural, epidermal, muscular and non-excitable - were identified on the basis of the shapes and ionic dependence of action potentials in the blastomeres of 8- to 32-cell embryos. Only the epidermal type of response was found in the blastomeres of 1- to 4-cell embryos. The blastomeres with responses of neural type had Na, Ca, delayed K rectifier, anomalous K rectifier and Ca-induced K channels. Those of epidermal type had Ca, anomalous K rectifier and Ca-induced K channels. Those of muscular type had Ca, delayed K rectifier, anomalous K rectifier and possibly Ca-induced K channels. Those of non-excitable type had almost none or small amounts of outward- and inward-going rectifier channels. The characteristic responses of neural type were found in small blastomeres in the animal hemisphere, which included some presumptive neural regions. The responses of muscular type were found in large blastomeres of the vegetal hemisphere, which included some presumptive regions for muscle. Those of epidermal type were found in the blastomeres of the animal hemisphere which did not differentiate into the neural type. Those of non-excitable type were found in some blastomeres of the vegetal hemisphere. Blasomeres of 1- to 32-cell cleavage-arrested embryos, which were presumed to possess more than one possible developmental fate, did not develop mosaic membrane properties but differentiated into one of the four types, with a probability dependent upon a gradient of ooplasmic segregation at the time of arrest.

MALATE DEHYDROGENASE: MULTIPLE FORMS IN SEPARATED BLASTOMERES OF SEA URCHIN EMBRYOS.

Sea urchin embryos at the 64-cell stage were dissociated by treatment with trypsin and separated by centrifugation on a sucrose gradient. The large blastomeres have two and the small blastomeres have three bands of L-malate dehydrogenase activity, which are separated by disk microelectrophoresis on polyacrylamide gel, whereas unfertilized eggs have five.