Daughter Blastomeres Research Articles

P-146 Larger time intervals between pronuclear fading and the first mitosis are strongly associated with trichotomous mitoses and cleavage arrest in human embryos

Abstract Study question Does the duration of the interval between pronuclear fading and first cell division affect preimplantation embryo developmental competence? Summary answer Larger pronuclear-fading to first-division intervals (t2-tPNf) are associated with greater risk of direct unequal cleavage (DUC) and lower blastulation rate, but not of blastocyst aneuploidy. What is known already During the last decade, time lapse technology (TLT) has made possible detailed and dynamic observation of in vitro embryo development, offering a powerful non-invasive tool for improving embryo selection. Recent evidence suggests that the phase between pronuclear fading and the first cleavage is a perilous bridge between the zygote and the cleaving embryo. Following pronuclear breakdown, delayed progression through mitosis caused by incomplete DNA replication may result in chromosome lagging, whole and segmental segregation errors, and breakage. In this analysis, we specifically tested the hypothesis that delays in a short final phase of fertilization have an impact on embryo development. Study design, size, duration Retrospective study of 1316 zygotes cultured until day 5 using TLT and derived from first ICSI cycles (111 no PGT-A and 94 PGT-A) performed between 2018 and 2022. Testicular sperm or cryopreserved gametes cycles were excluded. DUC was defined as the cleavage of one zygote or blastomere into three daughter blastomeres or cell cycle interval shorter than five hours at the first (DUC1), second (DUC2) or third (DUC3) cell division cycle. Participants/materials, setting, methods t2-tPNf intervals were clustered into quartiles (Q1, <2hpi; Q2, 2–2.5hpi; Q3, 2.5–3hpi and Q4,  > 3hpi), which were compared based on embryo development and live birth by linear and logistic regression. The Q1 cluster was set as control, while all other quartiles groups were considered as dummy variables. Multivariate analysis was conducted using the generalized estimating equations, to control for variable numbers of zygotes from each patient. The primary outcome was DUC rate. Main results and the role of chance Overall, rates of DUC1, DUC2 and DUC 3 rates were 10.3%, 8% and 2.9%, respectively. After adjusting for major confounders (maternal age, paternal age, maternal body mass index, cause of infertility, anti-Müllerian hormone, follicle stimulating hormone) we observed a higher DUC 1 and DUC 2 rate with increasing times of t2-tPNf. We observed abnormal distribution of DUC1 and DUC2 rate between quartiles groups. Specifically, 89% of DUC1 was shown among Q3 (21%) and Q4 (78%) and 61% of DUC2 among Q3 (22%) and Q4 (39%). The percentage of DUC1 was higher for Q3 (multivariate-OR = 3.29, 95%CI 0.99-10.85, adjusted-p = 0.01) and Q4 (multivariate-OR = 22.79, 95%CI 7.80-66.62, adjusted-p<0.01]. The percentage of DUC2 was higher only in Q4 (multivariate-OR = 2.58, 95%CI 1.13-5-57, adjusted-p = 0.01). Moreover, we observed a reduced blastulation rate in Q3 (multivariate-OR = 0.68, 95%CI 0.49-0.95, adjusted-p = 0.01) and Q4 (multivariate-OR = 0.27, 95%CI 0.19-0.38, adjusted-p<0.01). No significant differences were observed in DUC3 rate, top quality blastocyst, euploidy rate (PGT-A cycles analysis) and live birth rate. Limitations, reasons for caution The retrospective single-centre design and the limited sample size are limitations of the study Wider implications of the findings The study is consistent with the emerging view that the final phases of fertilization - although short - are crucial for development. Delays in progression towards mitosis probably express chromosome integrity loss and perturbances of chromosome segregation. In most embryos, such perturbances appear to cause trichotomous mitoses and cleavage arrest. Trial registration number Not applicable

Miro1 depletion disrupts spatial distribution of mitochondria and leads to oocyte maturation defects.

Mitochondria are dynamic organelles that undergo regulated microtubule- and actin-mediated trafficking to meet local energy and metabolic needs. Mitochondrial trafficking may be particularly critical in large cells such as eggs and early embryos where spindle formation and polar body extrusion occur in specific regions of the cytoplasm. To investigate the role of mitochondrial distribution in oocytes we have targeted the mitochondrial membrane protein, MIRO1, which couples mitochondria to the motor protein-TRAK complex. Oocyte-specific deletion of MIRO1 leads to the formation of large aggregates of mitochondria in perinuclear and cortical compartments. Mitochondria remain capable of long-range trafficking during maturation, indicating redundancy in the mechanisms coupling mitochondria to motor proteins. Polar body extrusion in the absence of MIRO1 was reduced by approximately 20%. In MIRO1-deleted zygotes, mitochondria showed increased accumulation around the pronuclei but this did not affect mitochondrial distribution to daughter blastomeres. In vitro development of parthenogenetic embryos was also reduced, although no differences were found in the fertility of oocyte-specific Miro1 KO mice. These findings demonstrate MIRO1 acts as a mitochondrial adaptor, setting mitochondrial distribution in oocytes and early embryos, and disrupting this process compromises in vitro oocyte maturation and embryo development.

O-100 Incidence, morphology, ploidy and developmental arrest in direct cleavage embryos: what are they teaching us? Morphokinetic analysis of 15.081 embryos

Abstract Study question What is the impact of direct cleavage (DC) occurrence during embryo/blastocyst development regarding morphology grade, embryo arrest and ploidy status? Summary answer Blastocyst formation and morphology grade are significantly reduced in DC when compared to non-DC embryos; however, there is no difference in blastocyst ploidy status. What is known already Chromosome instability leading to aneuploidy during early cleavage is well known in humans. DC is defined as the abrupt cleavage of one into three or more daughter blastomeres as the result from an irregular mitosis. Three or more mitosis poles can impair early embryo development; however, how some embryos with these irregular divisions might be able to develop into expanded blastocysts is still unknown. Time-lapse system provides an uninterrupted evaluation of embryo morphological and dynamic parameters, allowing our understanding in such events and if those embryos may be associated with a self-correction mechanism to reach blastocyst stage. Study design, size, duration Retrospective cohort study from 15.081 embryos, 3.962 cycles and 3.106 patients that were undergoing in vitro fertilization (IVF) treatment according to medical referral in a single private ART center from December 2018 to November 2021. Only two-pronuclear (2PN) embryos cultured in time-lapse incubators (EmbryoScope, Vitrolife, Sweden) were included in the study. Direct cleavage occurance was annotated when a single blastomere directly cleaved into three or more daughter blastomeres. Participants/materials, setting, methods DC embryos were also classified in DC 1-3: abnormal cleavage occurred in zygote (1-cell) resulting in 3–4 blastomeres and in DC 2+: abnormal cleavage occurred at the 2-cell stage resulting in 5 or 6 blastomeres. Embryo arrest, blastocyst formation rate and morphology grade (Gardner and Schoolcraft, 1999) were annotated. Blastocyst biopsy reports for ploidy status were also considered for analysis. Mann-Whitney, chi-squared and Fisher tests were applied for statistical analysis. p < 0,05 was considered significant. Main results and the role of chance There were 1168 DC embryos (7,7%) from 896 cycles/841 patients; 502 DC1-3 (3,3%) from 391 cycles/366 patients and 666 DC2+ embryos (4,4%) from 475 patients/505 cycles. In DC group, around 21% of patients have the incidence of more than 1 DC embryo per cycle. Maternal age was lower in DC groups: 38,0±3,61 (DC1-3) and 38,60±3,21 (DC2+) years old versus not-DC (39,02±3,01, p < 0,0001). Embryo arrest was higher (DC1-3:85,5% and DC2+:56,8% versus not-DC:35,4%, p < 0,0001) and blastocyst formation rate was lower (14,5% and 43,2% versus 64,6%, p < 0,0001) in both DC groups and also between DC groups (p < 0,0001). There were 4124 biopsied not-DC blastocysts, 24 in DC1-3 and 154 in DC2+. There was no difference in ploidy between not-DC (57,9%, 40,2%, 1,9%, aneuploid, euploid and mosaic respectively) and DC groups (54,2%, 41,7%, 4,2% and 61%, 37% and 1,9% respectively, p = 0.86) and neither between DC-groups (p = 0.69). Inner cell mass morphology grades were distinct in all groups (not-DC A:45,8%, B:45,2% C:9,0%, DC1-3 A:35,6%, B:33,9%, C:30,5% and DC2+ A:23,1%, B:57,0% C:19,9%, p < 0,0001) and between DC-groups (p < 0,0001). Trophoectoderm morphology grades were also higher for not-DC group (not-DC A:30,5%, B:48,9% C:20,6%, DC1-3 A:15,3%, B:47,5%, C:37,3% and DC2+ A:13,6%, B:44,4% C:42,0%, p < 0,0001), however there was no difference between DC-groups (p = 0.80). Limitations, reasons for caution Limited data are available on the implantation potential of embryos with DC, as they are not prioritize for transfer, although our study did not demonstrated a higher aneyploidy rate. Patient parameters, with the exception of maternal age, were not considered in this study. Wider implications of the findings Although the morphology grades are lower and embryo arrest is higher in DC embryos, probably due to unequal cleavage, if they reach blastocyst stage, the ploidy status of these embryos are similar to not-DC embryos, speculative for a self-correction mechanism. Future steps should focus on their performance in pregnancy/live-birth. Trial registration number Not Applicable

Asymmetric Contribution of Blastomere Lineages of First Division of the Zygote to Entire Human Body Using Post-Zygotic Variants.

In humans, after fertilization, the zygote divides into two 2n diploid daughter blastomeres. During this division, DNA is replicated, and the remaining mutually exclusive genetic mutations in the genome of each cell are called post-zygotic variants. Using these somatic mutations, developmental lineages can be reconstructed. How these two blastomeres are contributing to the entire body is not yet identified. This study aims to evaluate the cellular contribution of two blastomeres of 2-cell embryos to the entire body in humans using post-zygotic variants based on whole genome sequencing. Tissues from different anatomical areas were obtained from five donated cadavers for use in single-cell clonal expansion and bulk target sequencing. After conducting whole genome sequencing, computational analysis was applied to find the early embryonic mutations of each clone. We developed our in-house bioinformatics pipeline, and filtered variants using strict criteria, composed of mapping quality, base quality scores, depth, soft-clipped reads, and manual inspection, resulting in the construction of embryological phylogenetic cellular trees. Using our in-house pipeline for variant filtering, we could extract accurate true positive variants, and construct the embryological phylogenetic trees for each cadaver. We found that two daughter blastomeres, L1 and L2 (lineage 1 and 2, respectively), derived from the zygote, distribute unequally to the whole body at the clonal level. From bulk target sequencing data, we validated asymmetric contribution by means of the variant allele frequency of L1 and L2. The asymmetric contribution of L1 and L2 varied from person to person. We confirmed that there is asymmetric contribution of two daughter blastomeres from the first division of the zygote across the whole human body.

Germplasm and the origin of the primordial germ cells in the oriental river prawn Macrobrachium nipponense.

Germplasm is a special cytoplasmic component containing special RNAs and proteins, and is located in specific regions of oocytes and embryos. Only the blastomeres inheriting the germplasm can develop into primordial germ cells (PGCs). By using Vasa mRNA as a germline marker, we previously demonstrated that germline specification followed the preformation mode in the prawn Macrobrachium nipponense. In this study, we raised the Vasa antibody to identify germplasm in the oocyte and trace the origin and migration of PGCs. In previtellogenic oocytes, Vasa protein was detected in the perinuclear region, in which electron-dense granules associated with numerous mitochondria were mostly visualized under a transmission electron microscope. In mature oocytes, immunosignal was localized to a large granule under the plasma membrane. During early embryogenesis, the granule was inherited by a single blastomere from 1-cell to 16-cell stages, and thereafter was segregated into two daughter blastomeres at the 32-cell stage. In gastrula, the Vasa-positive cells were large with typical PGC characteristics, containing a big round nucleus and a prominent nucleolus. The immunosignal was localized to the perinuclear region again. In the zoea stage, the Vasa-positive cells migrated toward the genital ridge and clustered in the dorsomedial region close to the yolk portion. Accordingly, we concluded that the prawn PGCs could be specified from the 16-cell stage by inheriting the germplasm. To our knowledge, this is the first report on the identification of the prawn germplasm and PGCs. The continuous expression of Vasa protein throughout oogenesis and embryogenesis suggests that Vasa protein could be an important factor in germplasm that functions in early germ cell specification.

Matching Mitochondria DNA Haplotypes Circumvent Tissue-Specific Segregation Bias

Segregation of different mtDNA genotypes is common in tissues of mitochondrial disease patients, tissues of heteroplasmic mice and human cells of treated with mitochondrial replacement, allowing for the expression of pathogenic phenotypes, suggesting that functional compatibility between “donor” and “recipient” mtDNA, as well as between the mtDNA and nuclear genome is of the utmost importance to future potential clinical applications, such as mitochondria replacement. Here, we hypothesis that shortened genetic distance (matching mtDNA haplotypes) between donor and recipient mtDNA has the potential to circumvent the segregation bias towards mutant mtDNA in tissues of mitochondria replacement offspring. Based on heteroplasmic mice with only three single nucleotide polymorphisms difference, we found that tissues within each adult and daughter blastomeres within each embryo of the 2-cell, 4-cell and 8-cell groups shared the same dispersion of mtDNA heteroplasmy: diverse heteroplasmy levels in each blastomere across the 2-cell to 8-cell embryos and tissues in each adult with low mean levels of heteroplasmy. However, a more uniform distribution was witnessed in the blastomeres of embryos at 2 to 8-cell stage and tissues in adults with high mean levels of heteroplasmy. CV values present negative correlation with the mean heteroplasmy of embryos at 2-cell to 8-cell stage and adults, respectively. There is no difference for both the heteroplasmy distribution (p > 0.05) and CV (p > 0.05) values among blastomeres of embryos at 2-, 4- to 8-cell stages and adult tissues, suggesting no selective replication of mtDNA is taking place during the progressive cleavage across developmental stages. The deviations of individual tissue in adult with low mean levels of heteroplasmy were caused by uneven inheritance of donor mitochondria in each blastomere of early embryos, rather than tissue segregation. Our finding indicated that by matching the haplotypes of the donor and recipient mtDNA, we could “fool” the nucleus into treating the donated mtDNA and the native pathogenic mtDNA the same, thereby eliminating any proliferative advantage and circumventing any segregation bias and preventing the occurrence of mitochondrial diseases.

Specification of Larval Axes of Partial Embryos in the Temnopleurid Temnopleurus toreumaticus and the Strongylocentroid Hemicentrotus pulcherrimus.

Many sea urchins, including the strongylocentroid Hemicentrotus pulcherrimus, produce an amniotic cavity on the left for adult rudiment formation at the late larval stage. In contrast, temnopleurids form a cell mass at the early larval stage instead of an amniotic cavity. Although the mechanisms establishing left-right polarity of the amniotic cavity involve cell-cell interactions and signaling pathways, corresponding pathways for the cell mass are unknown. We analyzed the effects of blastomere isolation on the specification of larval axes in the temnopleurid Temnopleurus toreumaticus and compared them to those in H. pulcherrimus. Blastomere isolation at the two- or four-cell stages in T. toreumaticus disturbed the location of the cell mass and adult rudiment in approximately 10-20% of specimens. In contrast, isolation at the two-cell stage in H. pulcherrimus caused the left-right polarity to become random. When blastomeres isolated at the two-cell stage were cultured as pairs, approximately 20% of pairs had atypical polarity in both species. Following isolation at the four-cell stage, 71.4% of quartets produced larvae with atypical polarity in T. toreumaticus. Thus, cell-cell interaction between two daughter blastomeres after the second cleavage may be involved in the mechanism determining left-right polarity. Dye injection into a blastomere and subsequent observations indicated that the location of the boundary of the first cleavage showed similar patterns in both species. These observations suggest that species-specific mechanisms establish the larval axes and blastomeres at the two- and four-cell stages redistribute their cytoplasm, forming gradients that establish left-right polarity.

Cleavage pattern predicts developmental potential of day 3 human embryos produced by IVF

Time-lapse technique provides opportunities to observe the dynamic process of human early development. Previous studies have suggested several abnormal division patterns were associated with decreased developmental potential, but no systematic results are currently available. In this study, seven abnormal division patterns were observed during early cleavage, and these had different effects on the further development potential of daughter blastomeres. According to the severity and occurrence of abnormal division patterns during the initial three cleavages, an embryo hierarchical classification model was developed and day 3 embryos were classified into six grades (from A to F). The good-quality blastocyst formation rate for these grades decreased from 70.8–3.8% (P < 0.001). In a prospective observational study, 139 IVF cycles were recruited to assess the efficiency of this classification model. In the embryos that had confirmed implantation results, the implantation rate decreased from 67.0% (Grade A) to 0% (Grade D;P < 0.001). These results indicated that cleavage patterns can predict the developmental potential of day 3 human embryos.

How to be at the right place at the right time: the importance of spindle positioning in embryos.

Spindle positioning is an imperative cellular process that regulates a number of different developmental events throughout embryogenesis. The spindle must be properly positioned in embryos not only for the segregation of chromosomes, but also to segregate developmental determinants into different daughter blastomeres. In this review, the role of spindle positioning is explored in several different developmental model systems, which have revealed the diversity of factors that regulate spindle positioning. The C. elegans embryo, the Drosophila neuroblast, and ascidian embryos have all been utilized for the study of polarity-dependent spindle positioning, and exploration of the proteins that are required for asymmetric cell division. Work in the sea urchin embryo has examined the influence of cell shape and factors that affect secondary furrow formation. The issue of size scaling in extremely large cells, as well as the requirement for spindle positioning in developmental fate decisions in vertebrates, has been addressed by work in the Xenopus embryo. Further work in mouse oocytes has examined the roles of actin and myosin in spindle positioning. The data generated from these model organisms have made unique contributions to our knowledge of spindle positioning. Future work will address how all of these different factors work together to regulate the position of the spindle.

Chromosomal stability of second polar bodies in mouse embryos

Incorporation of a second polar body (PB2) into one of the blastomeres has been considered as a causal mechanism underlying diploid/triploid mixoploidy in humans. Using a mouse model, we examined whether PB2s can participate in the formation of mixoploidy. Uptake of BrdU was examined to determine DNA synthesis in PB2s up to 28 h after fertilization. PB2s from embryos at 4-6 (1-cell), 24 (2-cell), 48 (4-cell), and 72 h (morula) were fused with MII oocytes to induce premature chromosome condensation. Caspase and TUNEL assays were used to detect apoptotic PB2s at 24, 48, and 72 h. PB2s were fused with one of the blastomeres of the 2-cell embryos to produce mixoploid embryos. DNA synthesis in the PB2s continued until 22 h after fertilization. At 4-6 h, nearly all of the PB2s showed G1-type chromosomes and there was no significant increase in chromosome damage. At 24, 48, and 72 h, S-type chromatin predominated. Few PB2s showed apoptotic response until 72 h. Regardless of the fusion with the PB2, more than 90 % of the embryos developed to 4-cell stage, and over 80 % of the resultant 4-cell embryos had daughter blastomeres with a morphologically normal nucleus. Some of the daughter blastomeres displayed triploidy. The PB2 is viable for at least 72 h after fertilization, with slow progression through the cell cycle. Once the PB2 has been incorporated into a blastomere, the cell cycle of the PB2 might be synchronized with that of the host resulting in diploid/triploid mixoploidy.

Rapid Mitochondrial DNA Segregation in Primate Preimplantation Embryos Precedes Somatic and Germline Bottleneck

The timing and mechanisms of mitochondrial DNA (mtDNA) segregation and transmission in mammals are poorly understood. Genetic bottleneck in female germ cells has been proposed as the main phenomenon responsible for rapid intergenerational segregation of heteroplasmic mtDNA. We demonstrate here that mtDNA segregation occurs during primate preimplantation embryogenesis resulting in partitioning of mtDNA variants between daughter blastomeres. A substantial shift toward homoplasmy occurred in fetuses and embryonic stem cells (ESCs) derived from these heteroplasmic embryos. We also observed a wide range of heteroplasmic mtDNA variants distributed in individual oocytes recovered from these fetuses. Thus, we present here evidence for a previously unknown mtDNA segregation and bottleneck during preimplantation embryo development, suggesting that return to the homoplasmic condition can occur during development of an individual organism from the zygote to birth, without a passage through the germline.

Asymmetrical allocation of mitochondrial DNA to blastomeres during the first two cleavages in mouse embryos

A recent report showed higher oxygen consumption, adenosine triphosphate (ATP) production and mitochondrial localisation in trophectoderm cells than in the inner cell mass of mouse blastocysts. We hypothesised that this phenomenon was due to the asymmetrical distribution of mitochondria in the blastomeres during the earlier stages. Oocytes, 2-cell embryos and 4-cell embryos were analysed to determine the volume, ATP content and mitochondrial DNA (mtDNA) copy number in the whole egg and individual blastomeres. Significant differences were detected in the volumes of cytoplasm and ATP contents between blastomeres from the 2-cell and 4-cell embryos. Moreover, whilst remaining stable in whole embryos, mtDNA copy number differed between blastomeres, indicating that mitochondria in oocytes are unevenly delivered into the daughter blastomeres during the first two cleavages. Although their volume and ATP content were not correlated, there was a significant correlation between volume and mtDNA copy number in 2- and 4-cell blastomeres. These results indicate that the number of mitochondria delivered to blastomeres during early cleavage is not precisely equal, suggesting that the allocation of mitochondria into daughter blastomeres is affected by uneven cytoplasmic distribution during cytokinesis in the oocyte and mother blastomeres.

Initial observation of potential factors involved in the specification process of oral‐aboral axis in the sand dollar Scaphechinus mirabilis

To elucidate factors involved in the oral-aboral axis specification, several observations and experiments were undertaken using the sand dollar Scaphechinus mirabilis. Unlike in Strongylcentrotus purpuratus, localization of mitochondria was not detected in unfertilized eggs. After fertilization, however, the bulk of mitochondria became localized to the opposite side of sperm entry. The first cleavage divided this mitochondrial cluster into daughter blastomeres. On the other hand, a second cleavage produced daughter blastomeres containing quite different amounts of mitochondria. To know whether such mitochondrial localization affects the oral-aboral axis specification, 4-cell-stage embryos were separated along the second cleavage plane. Although both half embryos developed into morphologically normal plutei, some differences, such as the number of pigment cells, were noticed between the siblings. In contrast, cell tracing revealed that the first cleavage separated the oral from the aboral part in most cases, indicating that the unequal distribution of mitochondria is not critical for the oral-aboral axis specification. Further, stained and non-stained half embryo fragments were combined. Such combined embryos developed into normal plutei with a single oral-aboral axis. The plane dividing labeled and non-labeled parts were incident, oblique or perpendicular to the median plane of the combined embryo, and the appearance frequencies of those labeling patterns were similar to those obtained by cell tracing in intact embryos. Interestingly, the half fragments derived from embryos inseminated earlier showed a tendency to form the oral part. These suggest that several factors as well as the localized cytoplasmic components would be involved in the specification process of oral-aboral axis.

Analysis of Heterogeneous Mitochondria Distribution in Somatic Cell Nuclear Transfer Porcine Embryos

We previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Occurrence of maternal and paternal spindles in unfertilized human oocytes: possible relationship to nucleation defects after silent fertilization

Experience with conventional clinical IVF indicates that a first cleavage can occur in the absence of detectable pronuclear formation (so-called silent fertilization). In these instances, the first division is often asymmetrical and delayed when compared with normally fertilized siblings. In this study, DNA configurations and spindle organization were examined by fluorescence microscopy in metaphase II human oocytes that remained unfertilized after conventional IVF and were considered likely candidates for silent fertilization. The results show comparatively high frequencies of penetration in the absence of detectable pronuclear evolution, and that both a maternal meiotic and a sperm-derived mitotic-like spindle can coexist in the same oocyte. Patterns of cell division and blastomere nucleation in silent fertilizations suggest the possibility that this division may involve uniparental chromosomal segregation in which maternal and paternal DNA is differentially partitioned into daughter blastomeres. This pattern of inheritance may generate certain types of ploidy and nucleation defects detected at the 2- to 4-cell stage.

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.

Ultraviolet-irradiated spermatozoa activate oocytes but arrest preimplantation development after fertilization and nuclear transplantation in cattle.

Artificial means of parthenogenetically activating mammalian oocytes are believed to lack an essential sperm epigenetic component required for normal development. The main goal of this study was to examine the potential of ultraviolet (UV)-irradiated sperm as a means of functionally eliminating the chromatin component of spermatozoa without affecting the ability to induce activation and support parthenogenetic development in cattle. Spermatozoa were stained with a DNA dye, exposed to various UV irradiation doses, and used to fertilize secondary oocytes. Although the percentage of pronuclei at 18 h postinsemination was similar using treated and control sperm, most oocytes fertilized by UV-irradiated sperm failed to develop beyond the 2-cell stage, suggesting that UV irradiation can functionally destroy the genomic component of spermatozoa with limited effects on the ability to induce oocyte activation. However, when oocytes activated with UV-irradiated sperm were used as hosts for nuclear transfer, developmental rates to cleavage and to blastocyst improved only marginally and remained lower than in the controls, indicating that UV-treated spermatozoa blocked development even in the presence of a diploid donor nucleus. Although DNA replication was not inhibited by UV irradiation treatment, abnormal chromatin morphology after cleavage suggests improper segregation of chromatin to daughter blastomeres during the first mitotic division. Together, these results indicate that although sperm exposed to UV can activate oocytes, a developmental block occurs at or soon after the first mitosis in parthenotes and oocytes reconstructed by nuclear transfer.

Suppression of Muscle Fate by Cellular Interaction Is Required for Mesenchyme Formation during Ascidian Embryogenesis

The tadpole larva of the ascidian Halocynthia roretzi has several hundred mesenchyme cells in its trunk. Mesenchyme cells are exclusively derived from the B8.5 and B7.7 blastomere pairs of the 110-cell embryo. It has been believed that specification of mesenchyme cells is an autonomous process. In the present study, we have demonstrated that presumptive-mesenchyme blastomeres isolated from early 32-cell embryos did not express mesenchyme-specific features, whereas those isolated after the late 64-cell stage developed mesenchyme markers autonomously. Results of experiments involving coisolation and recombination of blastomeres showed that cellular interaction with adjacent presumptive-endoderm blastomeres during the late 32- and early 64-cell stages is required for mesenchyme formation. When such interaction was absent, the presumptive-mesenchyme blastomeres developed into muscle cells. Therefore, a signal from endoderm precursor blastomeres promotes mesenchyme fate, suppressing the muscle fate that is specified by ooplasmic muscle determinants. In Halocynthia, the muscle actin gene was precociously activated in mesenchyme–muscle precursor blastomeres at the 32-cell stage, and the mesenchyme and muscle fates were separated into two daughter blastomeres at the next cleavage. In presumptive-mesenchyme blastomeres at the 64-cell stage, expression of the muscle actin gene was immediately down-regulated by the signal from the neighboring endoderm precursor blastomeres. Thus, mesenchyme formation involves a novel mechanism of fate specification in ascidians, where formation of mesenchyme cells requires cellular interaction that suppresses muscle fate in the mesenchyme precursor blastomeres.

Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis.

During the first few cleavages of the zebrafish embryo, daughter blastomeres are loosely associated immediately after furrow ingression, but then gradually cohere. Cohesion appears to be cadherin-dependent, as cadherin and beta-catenin are found at the membrane between cohering blastomeres, and blastomeres fail to cohere in calcium-free medium. Cadherin and beta-catenin are not initially found on the blastomere surface, but are deposited specifically at the furrow surface. An array of parallel microtubules is present on either side of the furrow tip during ingression, as seen by confocal microscopy of alpha-tubulin labelled embryos. Transient incubation of embryos in 1 microg/ml nocodazole at the start of furrowing, which causes a loss of the furrow array, inhibits the localization of beta-catenin to the furrow surface but does not prevent furrow ingression. During ingression, intracellular membranes are transported to the furrow, as shown by labelling with DiD or DiOC6(3). Concentration of these membranes near the furrow surface is microtubule-dependent. These findings suggest that microtubules are required for cohesion of blastomeres because they mediate trafficking of intracellular membranes to the furrow surface, where they are exocytosed and allow cohesion via cadherins.

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.