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(A) Development of chimeras aggregated with phytohemagglutinin-L and (B) by the sandwich technique at 0, 16, and 38 hours after aggregation.
Source publication
Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggreg...
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Context 1
... of chimeras aggregated with PHA or by the sandwich technique and the respective integration of eGFP-positive donor blastomeres to ICM, TE, or both line- ages are shown in Table 1 and Figure 2, respectively. Developmental kinetics of chimeras are shown in Figure 3. Table 1 Development of chimeras and donor blastomere integration. ...
Context 2
... rate when using PHA. The development of chimeras was observed using time-lapse microscopy, and we noticed that chimeras aggregated with PHA tightly compacted to one single morula before developing the blastocele cavity while compaction before blastocyst development was not visible that clearly in chimeras produced by the sandwich tech- nique (See Fig. 3). Additionally, integration of the donor blastomeres differed between aggregation techniques with PHA chimeras showing a higher contribution of eGFP-positive blastomeres to the ICM than sandwich chi- meras (40% vs. 16%, P < 0.05). We hypothesize that fixing the donor blastomeres in between the host embryos di- rects them to the inside ...
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Many crustacean groups show stereotyped cleavage patterns during early ontogeny. However, these patterns differ between the various major crustacean taxa, and a general mode is difficult to extract. Previous studies suggested that also copepods undergo an early cleavage with a more or less stereotyped pattern of blastomere divisions and fates. Yet,...
Citations
... Blastomere aggregation is essential to produce chimeric animals using early-stage embryos (2-cell stage to morula) [11,28]. In several mammals, embryo aggregation techniques are utilized to increase production efficiency by correcting developmental problems within in vitro embryos. ...
The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode’s solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%–38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 μm vs. 230–234 μm). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%–6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.
... Before fixation, the zona pellucida (ZP) was removed enzymatically using pronase (Merck) 40 or mechanically for in vitro produced or flushed embryos, respectively. Embryos from in vitro culture were fixed in a solution containing 2% paraformaldehyde (PFA, Merck) for 20 min at 37°C 41 and flushed embryos were fixed in 4% PFA overnight at 4°C. ...
... At day 4, the ZP was removed enzymatically and each one morula from OCT4 2bKOeGFP and NT Ctrl FSC were aggregated to a chimera using phytohemagglutinin (Merck) and cultured as described previously. 40 Chimera formation was confirmed by time-lapse imaging (Primo Vision, Vitrolife) and by detection of both eGFP-positive and -negative cells in the developed blastocysts. Chimeric blastocysts were fixed and stained as described above. ...
The mammalian blastocyst undergoes two lineage segregations, that is, formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) as the remaining pluripotent lineage. To clarify the expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9, and 12 blastocysts completely produced in vivo by staining for OCT4, NANOG, SOX2 (EPI), and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4‐deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4‐deficient cells. Our data show that OCT4 is required cell autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.
... To overcome these problems, we demonstrated that developmental competence increased in porcine IVP embryos by embryo aggregation via reducing the stress conditions and improving mitochondrial function. Embryo aggregation is widely used in the production of transgenic and chimeric animals in various mammals, including mice (Yamaguchi et al., 2017), bovine (Simmet et al., 2015) and monkeys (Tachibana et al., 2012), to improve the production efficiency by compensating for the developmental deficiency of IVP embryos. In particular, pigs have been reported to respond positively to embryo aggregation, with increases in blastocyst formation rate, total cell number, ICM/TE ratio, and cellular survival (Lee et al., 2007;Terashita et al., 2011). ...
Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.
... Phytohemagglutinin (PHA), an N-acetylgalactosamine/ galactose sugar-specific lectin, has a wide variety of biological activities and is widely used in animal research involving mouse [12], bovine [13,14], goat [15], porcine [6,16] and human [17,18]. The biological activities of PHA include strong agglutination of blood cells, mitogenesis, and mediation of cell recognition in both vertebrates and invertebrates [19,20]. ...
... As lectin, it specifically binds to carbohydrate. Isolated from red kidney bean (Phaseolus vulgaris), PHA has been used to facilitate the aggregation of blastomeres [5,14], the binding of donor cells to oocytes in handmade cloning [21,22], and to improve the efficiency of porcine parthenotes [16] and the nuclear transfer of somatic cells for oocytes, due to its capacity to induce closer contacts between adjacent cell membranes [6,15]. In this study, we assessed the effects of the phytohemagglutinin (PHA) treatment on the developmental competence of parthenotes and the somatic cell nuclear-transferred (NT) porcine embryos. ...
This study evaluated the effects of the phytohemagglutinin (PHA) treatment on the developmental competence of parthenotes and somatic cell nucleartransferred (NT) porcine embryos. In the first part of the experiment, the effects of PHA treatment of different durations (1 mg/ml; 0, 5, or 10 min) on parthenogenetically produced porcine embryos was examined. Although, PHA treatment did not affect the percentage of embryos that cleaved and developed into blastocysts, and the total number of cells per blastocyst, mitochondrial activity was higher (P<0.05) in PHA-treated oocytes. In the second part of the experiment, porcine NT embryos were treated in PHA for 5 min before electrofusion. The cleavage and blastocyst rates of NT embryos treated with PHA were higher (P<0.05). The expression of mitochondrial-related genes in NT embryos was demonstrated for the first time. The expression of ATPase6 and ND1 was higher (P<0.05) in PHA-treated embryos than in the other groups. In conclusion, our experiments clearly demonstrate that pre-fusion treatment in PHA (1 mg/ml; 5 min) improved the development of porcine NT embryos in vitro. The results suggest that PHA enhances embryonic mitochondrial activity, and other energy-related targetgene expressions.
... The technology and procedures for bovine chimera generation have been well established. Chimeric embryos and/or live-born bovine chimeric calves have been reported by microinjecting or aggregating embryonic cells of one breed/subspecies to the diploid (2N) host embryos of another breed/ subspecies [33][34][35][36] . Relatively high efficiency of chimeric embryo generation (12%-86%) has been reported. ...
Although the pursuit of bovine embryonic stem cells started more than 26 years ago for the purpose of gene-targeting, true pluripotent stem cells in this economically important species are still elusive. With the rapid advances in genome-editing and cloning using homologously recombined somatic cells, the need for pluripotent stem cells for precise genetic modification in any species became questionable. With the pig being the better model for human regenerative biology, the identification of the commonalities and uniqueness of the pluripotency circuitry across mammalian species may be the main objective for studying pluripotent stem cells in the bovine.
... Rabbit blastocysts were collected after 4 days of in vitro culture of zygotes flushed from oviducts of oestrus-induced rabbits (Brero et al. 2009). Pig and cattle blastocysts were produced in vitro as described previously and cultured until blastocyst stage for 6-7 days in pigs (Kurome et al. 2017) and 7-8 days in cattle (Simmet et al. 2015). ...
Immunosurgical isolation of the inner cell mass (ICM) from blastocysts is based on complement-mediated lysis of antibody-coated trophectoderm (TE) cells. Conventionally, anti-species antisera, containing antibodies against multiple undefined TE-cell epitopes, have been used as the antibody source. We previously generated α-1,3-galactosyltransferase deficient (GTKO) pigs to prevent hyperacute rejection of pig-to-primate xenotransplants. Since GTKO pigs lack galactosyl-α-1,3-galactose (αGal) but are exposed to this antigen (e.g. αGal on gut bacteria), they produce anti-αGal antibodies. In this study, we examined whether serum from GTKO pigs could be used as a novel antibody source for multi-species embryo immunosurgery. Mouse, rabbit, pig and cattle blastocysts were used for the experiment. Expression of αGal epitopes on the surface of TE cells was detected in blastocysts of all species tested. GTKO pig serum contained sufficient anti-αGal antibodies to induce complement-mediated lysis of TE cells in blastocysts from all species investigated. Intact ICMs could be successfully recovered and the majority showed the desired level of purity. Our study demonstrates that GTKO pig serum is a reliable and effective source of antibodies targeting the αGal epitopes of TE cells for multi-species embryo immunosurgery.
... Enzyme reaction was stopped by washing embryos in THP supplemented with 10% foetal calf serum (FCS) and the dissolved ZP was completely removed by gentle pipetting. Embryos were incubated in drops of PBS without Mg 2+ and Ca 2+ supplemented with 4 mg/ml polyvinylpyrrolidone under oil for 5-10 minutes and blastomeres were subsequently disaggregated by gentle pipetting 51 . Single cells were transferred individually to 0.5-µl drops of lysis buffer (Buffer A of Prelude Direct Lysis Module, NuGEN) under mineral oil, collected in a 384-well plate, and stored at −80 °C. ...
Embryonic development is initially controlled by maternal RNAs and proteins stored in the oocyte, until gene products gradually generated by the embryo itself take over. Major embryonic genome activation (EGA) in bovine embryos occurs at the eight- to 16-cell stage. Morphological observations, such as size of blastomeres and distribution of microvilli, suggested heterogeneity among individual cells already at this developmental stage. To address cell heterogeneity on the transcriptome level, we performed single-cell RNA sequencing of 161 blastomeres from 14 in vitro produced bovine embryos at Day 2 (n = 6) and Day 3 (n = 8) post fertilization. Complementary DNA libraries were prepared using the Single-Cell RNA-Barcoding and Sequencing protocol and sequenced. Non-supervised clustering of single-cell transcriptome profiles identified six clusters with specific sets of genes. Most embryos were comprised of cells from at least two different clusters. Sorting cells according to their transcriptome profiles resulted in a non-branched pseudo-time line, arguing against major lineage inclination events at this developmental stage. In summary, our study revealed heterogeneity of transcriptome profiles among single cells in bovine Day 2 and Day 3 embryos, suggesting asynchronous blastomere development during the phase of major EGA.
... In contrast to CRISPR-Cas9-mediated editing of zygotes, this approach guarantees consistent modification of all cells of the embryo and allows efficient screening for off-target effects. We used one cell line with a PGK-EGFP reporter construct, to enable later chimeric complementation studies of OCT4-deficient blastomeres (34). To exclude possible effects of the reporter construct, we also used a wild-type fibroblast cell line. ...
... SCNT and IVP procedures were performed as described previously (44). At 5 or 7 d after activation of fused complexes or IVF, respectively, the zona pellucida was removed, and embryos were fixed in 2% paraformaldehyde (34) or stored at −80°C until RNA extraction. ...
Significance
Studies of mammalian development are mostly performed in the mouse model, where reverse genetics is most advanced. Recent developments in gene editing, including CRISPR-Cas9, facilitated functional studies of specific genes in other mammalian species and revealed important differences (e.g., between human and murine development). In this study, we generated loss-of-function mutations of the OCT4 gene in bovine fibroblasts and produced embryos by nuclear transfer cloning. We demonstrated that, similar to human development but in contrast to mouse development, OCT4 is required for maintaining NANOG-positive epiblast cells in the inner cell mass of blastocysts. Our study outlines a general strategy for dissecting the roles of specific genes in preimplantation development in domestic species.
The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3–21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.
Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 μg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly ( p < 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.
