4n Embryos Research Articles

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

Production and development of porcine tetraploid parthenogenetic embryos.

The aim of this study was to produce porcine tetraploid (4N) parthenogenetic embryos using various methods and evaluate their developmental potential. In method 1 (M1), porcine 4N parthenogenetic embryos were obtained by inhibiting extrusion of both first (PB1) and second (PB2) polar bodies; in methods 2 (M2) and 3 (M3), 4N parthenogenetic embryos were obtained by electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 or PB1 extrusion, respectively. We found no differences in the rates of cleavage or blastocyst formation or the proportion of 4N embryos among M1, M2, and M3 groups. The different methods also did not influence apoptosis rates (number of TUNEL-positive cells/number of total cells) or expression levels of apoptosis-related BAX and BCL2L1 genes. However, total cell and EdU (5-ethynyl-2’-deoxyuridine)-positive cell numbers in 4N parthenogenetic blastocysts derived from M1 were higher (p < 0.05) than those for M2 and M3 groups. Our results suggest that, although porcine 4N parthenogenetic embryos could be produced by a variety of methods, inhibition of PB1 and PB2 extrusion (M1) is superior to electrofusion of 2-cell stage diploid parthenogenetic embryos derived from inhibition of PB2 (M2) or PB1 (M3) extrusion.

Screening of biotechnical parameters for production of bovine inter-subspecies embryonic chimeras by the aggregation of tetraploid Bos indicus and diploid crossbred Bos taurus embryos

The aggregation of a tetraploid zebu embryo (Bos indicus, a thermotolerant breed) with a diploid taurine embryo (Bos taurus, a thermosensitive breed) should create a complete taurine fetus, whose extra-embryonic components, e.g., the chorion, is derived mainly from the zebu embryo. These zebu-derived extra-embryonic components may interact positively with the taurine embryo/fetus during pregnancy in a tropical environment. We tested different parameters for the production of tetraploid Nelore (Bos indicus) embryos to be combined via aggregation with crossbred Bos taurus (diploid) embryos in order to produce viable chimeric blastocysts. Bovine (Bos indicus or crossbred Bos taurus) embryos were produced in vitro according to standard procedures. Two-cell Bos indicus embryos were submitted to electrofusion with varying numbers of pulses (1 or 2), voltages (0.4, 0.5, 0.75, 1.0, 1.4 and 5.0kV/cm) and time (20, 25, 50 and 60μs) to produce tetraploid embryos. Electrofused embryos were cultured with crossbred non-fused embryos to form chimeras that developed until the blastocyst stage. The best fusion parameter was 0.75kV/cm for 60μs. Four chimeric blastocysts (tetraploid Nelore with diploid crossbred Holstein) were formed after 31 attempts in 4 replicates (13%). We established an optimal procedure for the production of tetraploid Bos indicus (4n) embryos and embryonic chimeras by aggregation of crossbred Bos taurus (2n) with Bos indicus (4n) embryos. This technique would be valid in applied research, by producing exclusively taurine calves, but with placental elements from the Bos indicus breed, following transfer of these chimeras into recipient cows.

Generation and Developmental Characteristics of Porcine Tetraploid Embryos and Tetraploid/diploid Chimeric Embryos

The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9kV/cm for 30μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P<0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P>0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P<0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

61 DIPLOID (BOS TAURUS) AND TETRAPLOID (BOS INDICUS) EMBRYO AGGREGATION TO PRODUCE BOVINE EMBRYONIC CHIMERAS

The formation of tetraploid embryos (4n) by electrofusion and their subsequent chimerism with a diploid embryo (2n) must result in a chimeric conceptus, whose inner cell mass (ICM) is entirely 2n. Hence, the aggregation of a zebu embryo (4n, thermotolerant) with a taurine embryo (2n, thermosensitive) would result in an exclusively taurine ICM, but the trophectoderm (future extraembryonic components) would be mostly from the zebu embryo, which could interact with the taurine embryo/fetus in a different way (than it occurs in whole Bos taurus conceptus) during pregnancy in a tropical environment. The purpose of this study was to standardize the production of 4n Nelore embryos (Bos indicus) and the production of embryonic chimeras by aggregation of 1/2 B. taurus (2n) with B. indicus (4n) embryos. Oocytes from Nelore cows from the abattoir were matured, fertilized with semen from Nelore and Holstein bulls and cultured in SOF. Two-cell stage Nelore embryos (30 h post-insemination), with a well-defined inter-blastomeric axis, were selected for the electrofusion procedure (ECM 830-BTX, Harvard Apparatus) to produce 4n embryos. For this procedure, some parameters were tested according to the number of pulses (1 or 2), voltage (40, 50, 75, 100, 140 and 500 V) and duration of electroshock (20, 25, 50 and 60 μs). Nelore 4n embryos produced after electrofusion and 2n taurine embryos, both at 8 to 16-cell stages (72 h post-insemination) were subjected to protease treatment to remove the zona pellucida and subsequently treated with the agglutinant agent phytohemagglutinin. Bos indicus (4n) and B. taurus (2n) embryos were added in pairs (4n+2n) into individual wells (well of the well, WOW) for culture until the blastocyst stage to validate the chimeric embryos formation. Among the tested parameters, the best fusion results (92%) and rates of cleavage after fusion (66%) were obtained with a single pulse of 75 V for 60 μs. The production rate of expanded 4n blastocysts was 31.5% using these parameters. After 4 replicates (still in the final stage of standardization), 4 blastocyst chimeras (4n+2n) were obtained from 31 attempts (13%). The production of bovine embryonic chimeras [Bos indicus (4n) + Bos taurus (2n)], with a non-random pattern of distribution of their cell aggregates, will enable the validation of this technique in applied research, by producing exclusively taurine calves, but with placental elements from the B. indicus breed, following transfer of these chimeras into recipient cows. Financial support received from FAPESP, Brazil.

Defects in Trophoblast Cell Lineage Account for the Impaired In Vivo Development of Cloned Embryos Generated by Somatic Nuclear Transfer

The low success rate of somatic nuclear transfer (NT) is hypothesized to be mainly due to functional defects in the trophoblast cell lineage rather than the inner cell mass (ICM); this hypothesis, however, remains to be tested directly. Here we separated the ICMs from cloned blastocysts and aggregated the cloned ICM with two fertilization-derived (FD) tetraploid (4N) embryos. We found that the full-term development of cloned ICMs was dramatically improved after the trophoblast cells in the cloned blastocysts were replaced by cells from tetraploid embryos, thus providing direct evidence that defects in trophoblast cell lineage underlie the low success rate of somatic NT.

Acceptance of Embryonic Stem Cells by a Wide Developmental Range of Mouse Tetraploid Embryos1

Tetraploid (4N) complementation assay is regard as the most stringent characterization test for the pluripotency of embryonic stem (ES) cells. The technology can generate mice fully derived from the injected ES cell (ES-4N) with 4N placentas. However, it remains a very inefficient procedure owing to a lack of information on the optimal conditions for ES incorporation into the 4N embryos. In the present study, we injected ES cells from embryos of natural fertilization (fES) and somatic cell nuclear transfer (ntES) into 4N embryos at various stages of development to determine the optimal stage of ES cells integration by comparing the efficiency of full-term ES-4N mouse generation. Our results demonstrate that fES/ntES cells can be incorporated into 4N embryos at 2-cell, 4-cell and blastocyst stages and full-term mice can be generated. Interestingly, ntES cells injected into the 4-cell group resulted in the lowest efficiency (5.6%) compared to the 2-cell (13.8%, P > 0.05) and blastocyst (16.7%, P < 0.05) stages. Because 4N embryos start to form compacted morulae at the 4-cell stage, we investigated whether the lower efficiency at this stage was due to early compaction by injecting ntES cells into artificially de-compacted embryos treated with calcium free medium. Although the treatment changed the embryonic morphology, it did not increase the efficiency of ES-4N mice generation. Immunochemistry of the cytoskeleton displayed microtubule and microfilament polarization at the late 4-cell stage in 4N embryos, which suggests that de-compaction treatment cannot reverse the polarization process. Taken together, we show here that a wide developmental range of 4N embryos can be used for 4N complementation and embryo polarization and compaction may restrict incorporation of ES cells into 4N embryos.

INDUCTION OF 2N GAMETES AND 4N EMBRYO IN LILIUM (LILIUM × FORMOLONGI HORT.) BY NITROUS OXIDE GAS TREATMENT

ISHS XXIII International EUCARPIA Symposium, Section Ornamentals, Colourful Breeding and Genetics - Part II INDUCTION OF 2N GAMETES AND 4N EMBRYO IN LILIUM (LILIUM × FORMOLONGI HORT.) BY NITROUS OXIDE GAS TREATMENT

157 AGGREGATION AND DEVELOPMENT OF RABBIT CLONED EMBRYOS WITH ZYGOTIC, TETRAPLOID, AND PARTHENOGENETIC EMBRYO IN VITRO

The rabbit compared to other domestic animals, such as cattle and sheep, was a relatively more difficult species to clone. One of the major reasons may be attributed to the low cell number in cloned embryo before implantation. This study was designed to aggregate 2 nuclear transfer (NT) embryos and one with a different origin, determine their developmental potential in vitro, and finally examine the cell number of aggregated embryos. NT was performed with our standard procedure using in vivo derived oocytes and donor cells from adult skin fibroblasts. Zygotes (ZY) were collected from does at 18 h post-hCG and mating. Parthenogenetic (PA) embryos were generated from oocytes with activation protocol, whereas tetraploid embryos (4N) were prepared by fusing fertilized embryos at 2-celled stage into 1-celled stage by electrical pulse. All of the embryos were cultured for 20-24 h into 4-/8-celled stage prior to aggregation. Zona pellucida was then removed and 2 NT embryos were aggregated with 1 embryo originated from ZY, PA, and 4N groups in a depressed droplet containing culture medium. Aggregated embryos were cultured for another 48 h (total 3 days, initiation of activation = Day 0) before being fixed for cell counting. Both single embryo from NT, ZY, PA, and 4N and 3 embryo aggregates (3X) from the same category were used as controls for NT aggregation. The results of 3-day embryo culture in vitro showed that the development of aggregated embryos to blastocyst (BL) stage was 2 NT + ZY, 68.6% (n = 35); 2 NT + 4N, 91.7% (n = 36); and 2 NT+PA, 37.5% (n = 24), whereas 3X aggregates developed to BL at a rate of ZY, 100% (n = 24); 4N, 100% (n = 19); and PA, 100% (n = 14). The BL rates of single embryo control developed into early BL were ZY, 100% (n = 34) and 4N, 93% (n = 36); however, NT and PA developed slower, only 45.4% NT (n = 187) and 79.2% PA (n = 72) to compacted morula/early BL stage. Cell counting data (Table 1) showed that there was no difference in cell number per embryo between NT and PA, whereas ZY and 4N possessed significantly higher cell number than NT and PA (128-162 v. 52-61, P &lt; 0.05) for single embryo category. In the 3X aggregation group, significantly higher cell number per aggregated embryo was found in ZY and 4N compared to that in PA (549-564 v. 196, P &lt; 0.05). More importantly, there was significantly higher cell number found in 2 NT + 4N embryo than that in single 4N (293 v. 162, P &lt; 0.05). This result demonstrated that 2 cloned embryos had propagated and incorporated into 1 tetraploid embryo during pre-implantational development. The next step is to study how NT embryos successfully interact within the aggregated embryo during further development and differentiation, in order to increase the birth rate of clones. Table 1.The cell number of aggragated rabbit embryos between NT and other embryos of different origin after 3 days of culture in vitro Supported by NIH 5R44HL091605-03.

Developmental pattern of hexaploid mouse embryos produced by blastomere fusion of diploid and tetraploid embryos at the 2-cell stage

Polyploid mouse embryos are important models for understanding the mechanisms of cleavage and preimplantation development in mammals. In this study, hexaploid (6n) mouse embryos were produced by the electrofusion of blastomeres from diploid (2n) and tetraploid (4n) embryos at the 2-cell stage. Furthermore, the developmental pattern of hexaploid embryos was evaluated by blastocyst rate, cell number, karyotype analysis, cytoskeleton staining and Oct-4 immunofluorescence. The results showed that 72.7% of the hexaploid embryos were able to develop to the blastocyst stage, which is a lower number than that found with normal diploid embryos (98.0%, p < 0.05). The cell number in hexaploid blastocyst was 12.3 +/- 2.0, which was less than that found in diploid or tetraploid blastocysts (41.2 +/- 7.2; 18.4 +/- 3.5). Karyotype analysis confirmed that the number of chromosomes in hexaploid embryos was 120. beta-Tubulin and Oct-4 immunofluorescence indicated that the hexaploid blastocysts were nearly lacking inner cell mass (ICM), but some blastomeres did show Oct-4-positive expression.

Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages

We previously showed that increasing the cell number of host tetraploid (4n) embryos by aggregating multiple 4n embryos at two to four-cell stages can improve the birthrate of mice from embryonic stem cells (ES mice). In the present study, we assessed whether in vitro aged blastocysts (e.g., E4.5 or E5.5), where their cell number also increased with development, can be used as hosts for generating ES mice. As expected, the cell number of in vitro aged 4n blastocysts increased with development, i.e., 26.5+/-2.4, 49.6+/-8.4, and 84.9+/-20.9 cells for E3.5, E4.5, and E5.5 respectively. Three independent ES cell lines were injected into 4n aged blastocysts, and their developmental ability was compared with that of E3.5 4n blastocysts commonly used for this procedure. We found that the birthrate of ES mice derived from E4.5 blastocysts were comparable with those of mice generated from E3.5 blastocysts. On the other hand, the birthrates decreased when E5.5 blastocysts were used. These results suggest that not only the cell number but also developmental age is important for producing ES mice. We also discuss a comparison of the present findings with those of our previous study, where ES mice were generated using an aggregation method employing the same ES cell lines.

Increasing the Cell Number of Host Tetraploid Embryos Can Improve the Production of Mice Derived from Embryonic Stem Cells1

Tetraploid (4n) embryo complementation assay has shown that embryonic stem (ES) cells alone are capable of supporting embryonic development (ES mouse), allowing the recovery of mouse lines directly from cultured ES cell lines. Although the advantages of this technique are well recognized, it remains inefficient for generating ES mice. In the present study, we investigated the effects of cell number of host 4n embryos on the production of ES mice. Four independent ES cell lines (two general ES cell lines and two nuclear transfer-derived ES cell lines) were used, and each cell line was aggregated with single (1X) to triple (3X) host 4n embryos. We found that birth rate of ES mice using 1X 4n embryos was quite low (0-2%) regardless of cell line, whereas except for one cell line, approximately 6-14% of embryos developed to full term in the case of 3X 4n embryos. Contamination of host 4n cells in ES mice was quite rare, being comparable to that generated using general methods even if they were delivered from 3X 4n host embryos. These results demonstrate that the use of 3X 4n embryos is effective for generating ES mice. Our technique described here will be applicable to any ES cell line, including general ES cell lines used for gene targeting.

Developmental potential and behavior of tetraploid cells in the mouse embryo

Tetraploid (4n) mouse embryos die at variable developmental stages. By examining 4n embryos from F2 hybrid and outbred mice, we show that 4n developmental potential is influenced by genetic background. The imprinted inactivation of an X chromosome-linked eGFP transgene in extraembryonic tissues occurred correctly in 4n embryos. A decrease of the cleavage rate in 4n preimplantation embryos compared to diploid (2n) embryos was revealed by real-time imaging, using a histone H2b:eGFP reporter. It has previously been known that mouse chimeras produced by the combination of diploid (2n) embryos with embryonic stem (ES) cells result in mixtures of the two components in epiblast-derived tissues. In contrast, the use of 4n host embryos with ES cells restricts 4n cells from the embryonic regions of chimeras, resulting in mice that are believed to be completely ES-derived. Using H2b:eGFP transgenic mice and ES cells, the behavior of 4n cells was determined at single cell resolution in 4n:2n injection and aggregation chimeras. We found a significant contribution of 4n cells to the embryonic ectoderm at gastrulation in every chimera analyzed. We show that the transition of the embryonic regions from a chimeric tissue to a predominantly 2n tissue occurs after gastrulation and that tetraploid cells may persist to midgestation. These findings suggest that the results of previously published tetraploid complementation assays may be influenced by the presence of tetraploid cells in the otherwise diploid embryonic regions.

Tetraploid development in the mouse.

Spontaneous duplication of the mammalian genome occurs in approximately 1% of fertilizations. Although one or more whole genome duplications are believed to have influenced vertebrate evolution, polyploidy of contemporary mammals is generally incompatible with normal development and function of all but a few tissues. The production of tetraploid (4n) embryos has become a common experimental manipulation in the mouse. Although development of tetraploid mice has generally not been observed beyond midgestation, tetraploid:diploid (4n:2n) chimeras are widely used as a method for rescuing extraembryonic defects. The tolerance of tissues to polyploidy appears to be dependent on genetic background. Indeed, the recent discovery of a naturally tetraploid rodent species suggests that, in rare genetic backgrounds, mammalian genome duplications may be compatible with the development of viable and fertile adults. Thus, the range of developmental potentials of tetraploid embryos remains in large part unexplored. Here, we review the biological consequences and experimental utility of tetraploid mammals, in particular the mouse.

Developmental potential of mouse tetraploid cells in diploid tetraploid chimeric embryos.

Mouse 2n (lacZ-) <--> 4n (lacZ+) aggregation chimeras were examined 5 or 10 days after uterine transfer to test the potential of 4n cells to contribute to embryonic tissues. Recovered embryos corresponded to embryonic day 7.5 approximately 8.0 and 12.5, respectively. Ten days after transfer, 4n cells were never detected, as reported earlier, in embryonic tissues of chimeras produced by the standard procedure in which one 2n embryo at the8-cell stage is aggregated with a4n embryo at the4-cell stage. However, beta-gal positive cells were present in embryonic tissues, though in a low number, in chimeras produced by a 2n and a 4n embryo at the 4-cell stage. Similar results were obtained when one 2n embryo atthe 8-cell stage was aggregated with two 4n embryos atthe 4-cell stage. beta-gal positive cells were found in the heart, liver, skin and intestinal epithelium. The majority of chimeras 5 days after uterine transfer retained beta-gal positive cells in embryonic tissues. The complete lack of 4n cell contribution to chimeras produced by the standard procedure is therefore attributed to the initial low proportion of 4n cells allocated to epiblast and their severe elimination from embryonic tissues.

Improved develpomental rate of DNA-injected bovine embryos co-cultured with mouse embryonic fibroblasts

The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P < 0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P > 0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P < 0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

The parental source of heteroploidy in chick embryos determined with chromosomally marked gametes.

Males homozygous for chromosomal translocations were mated to karyologically normal females. The resulting embryos, after incubation for 16-18 h, were prepared for cytogenetic analysis. Of the 62 embryos analysed, 54 (7.8%) were heteroploid or contained a major heteroploid cell line. Each of the 638 normal 2n embryos contained one marker and one normal chromosome, indicating that none arose from gynogenesis. Thirteen homogeneous haploid embryos were identified; 24 embryos were chimaeric 1n/2n and one was 1n/3n. All haploid cell lines contained a marker chromosome indicating androgenetic origins. The 9 homogeneous triploid (3n) embryos and the 3n cell line in a single 1n/3n embryo contained a single marker. All resulted from fertilization by single spermatozoon of eggs that were diploid as a result of suppression of the second meiotic division. The 3n lines of two 2n/3n embryos were derived from other mechanisms. A single homogeneous tetraploid (4n) embryo and the 4n cell lines of three 2n/4n mosaic embryos each contained two marker chromosomes and presumable resulted from failure of cytokinesis in an early cleavage division.