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1
Evsikov, A. V., W. N. de Vries, A. E. Peaston, E. E. Radford, K. S. Fancher, F. H. Chen, J. A. Blake, et al. "Systems biology of the 2-cell mouse embryo." Cytogenetic and Genome Research 105, no. 2-4 (2004): 240–50. http://dx.doi.org/10.1159/000078195.
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FUNAHASHI, Hiroaki, and Koji NIWA. "Electric fusion of 2-cell rat embryo balastomeres." Japanese journal of animal reproduction 36, no. 2 (1990): 114–19. http://dx.doi.org/10.1262/jrd1977.36.114.
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Zhang, M., L. Sui, Y. Li, Z. Chen, Y. Zhang, T. Liu, J. Xu, X. Zhang, and Y. Zhang. "96 EFFECT OF TWO DIFFERENT EMBRYO TRANSPORTERS ON DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS." Reproduction, Fertility and Development 26, no. 1 (2014): 162. http://dx.doi.org/10.1071/rdv26n1ab96.
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In the present study, we investigated two embryo transport methods, including a commercial cell transporter and a self-made, simple embryo transporter, for the pre-implantation development of porcine parthenogenetic embryos. The cleaved embryos were randomly distributed between the two types of embryo transport methods and were conserved in vitro for 2, 3, and 4 h. Embryo development efficiency testing and blastocyst differential staining were utilized to assess embryo developmental quality. There were no significant differences in embryo early development efficiency between the commercial cell transporter group, self-made embryo transporter group, and control group. The blastocyst hatch rate (7.75 ± 2.96%) in the self-made simple embryo transport method maintained for 3 h was significantly higher compared to the other groups (P < 0.05). The results (Table 1) showed that blastocyst differential staining showed that the ratio of inner cell mass (ICM) to total cells in both the 2-h-transport group and 3-h-transport group from the self-made simple embryo transport method and the 4-h-transport group from the commercial cell transporter were significantly higher than other groups (P < 0.05).The self-made simple embryo transporter and commercial cell transporter are both effective for transport and conservation of embryos for 3 h. Table 1.Effect of different modes of transport and transit time on embryo development1
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Li, Y., M. L. Day, and C. O'Neill. "235.PAF induced changes in intracellular Ca2+ and membrane potential in the 2-cell mouse embryo." Reproduction, Fertility and Development 16, no. 9 (2004): 235. http://dx.doi.org/10.1071/srb04abs235.
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Platelet-activating factor (PAF) is an autocrine survival factor for the preimplantation embryo. PAF induces a transient increase in intracellular Ca2+ ([Ca2+]i) in 2-cell embryos that is caused by the interdependent influx of external calcium and release of calcium from internal stores. A membrane current with L-type calcium channel properties is activated during PAF-induced calcium signalling. Since the L-type channel in many cell types is primarily voltage-gated we were interested to learn whether this was also the case in the 2-cell embryo. The present study investigated the relationship between the PAF-induced Ca2+ transient and changes in membrane potential (Em) in the 2-cell embryo. The perforated whole-cell patch-clamp technique was used to detect changes in Em and standard calcium imaging techniques were used to measure changes in [Ca2+]i in 2-cell embryos from QS mice. Embryos were first loaded with Fluo-3 and then pretreated with PAF:acetylhydrolase to degrade the embryo derived PAF before patch clamping. Whole-cell perforated patch-clamping was performed by inclusion of 240mg/ml Nystatin in the pipette solution. Changes in Em and [Ca2+]i were recorded simultaneously after treatment of the embryo with PAF. In 2-cell embryos PAF induced a change in Em, consisting of an initial small depolarisation of 2.4 � 0.2 mV (42 � 4 sec after addition of PAF) followed by one or more transient hyperpolarisations of -8 � 1 mV (100 � 9 sec after addition of PAF). Transient increases in [Ca2+]i paralleled the membrane hyperpolaristions and were initiated at 84 � 8 sec after addition of PAF. These responses to PAF were seen in 58% of 2-cell embryos (n = 52). It is not yet clear whether these changes in Em account for the activation of calcium influx through the L-type channel. The results show for the first time that the 2-cell embryo is an electrically active organism.
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Genet, Marion, and Maria-Elena Torres-Padilla. "The molecular and cellular features of 2-cell-like cells: a reference guide." Development 147, no. 16 (August 15, 2020): dev189688. http://dx.doi.org/10.1242/dev.189688.
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ABSTRACTCurrently, two main cell culture models predominate pluripotent stem cell research: embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Thanks to their ability to contribute to and form all tissues within the body, ESCs and iPSCs have proven invaluable in understanding pluripotent states, early embryonic development and cell differentiation, as well as in devising strategies for regenerative medicine. Comparatively little is known about totipotency – a cellular state with greater developmental potential. In mice, only the zygote and the blastomeres of the 2-cell-stage embryo are truly totipotent, as they alone can develop to form the embryo and all of its supportive extra-embryonic tissues. However, the discovery of a rare subpopulation of cells in murine ESC cultures, possessing features of 2-cell embryo blastomeres and expanded cell fate potential, has provided a biochemically tractable model to enable the in vitro study of totipotency. Here, we summarize current known features of these 2-cell-like cells (2CLCs) in an effort to provide a reference for the community, and to clarify what we know about their identity so far.
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Pratt, H. P., and A. L. Muggleton-Harris. "Cycling cytoplasmic factors that promote mitosis in the cultured 2-cell mouse embryo." Development 104, no. 1 (September 1, 1988): 115–20. http://dx.doi.org/10.1242/dev.104.1.115.
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A cytoplasmic component(s), previously shown to rescue the ‘blocked’ 2-cell mouse embryo in vitro, has been demonstrated to peak in activity during the transition between G2 and M phase and decline thereafter. The possible significance of this component(s) in the regulation of cleavage of the cultured mouse embryo is discussed.
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Sun, Jian Hong, Yong Zhang, Bao Ying Yin, Ji Xia Li, Gen Sheng Liu, Wei Xu, and Shuang Tang. "Differential expression of Axin1, Cdc25c and Cdkn2d mRNA in 2-cell stage mouse blastomeres." Zygote 20, no. 3 (July 12, 2011): 305–10. http://dx.doi.org/10.1017/s0967199411000347.
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SummaryThere is increasing evidence to show that 2-cell stage mouse blastomeres have differing developmental properties. Additionally, it has been suggested that such a difference might be due to their distribution of mRNA and/or protein asymmetry. However, to date, the exact genes that are involved in the orientation and order of blastomere division are not known. In this study, some differentially expressed transcripts were identified. Axin1, cell division cycle 25 homolog C (Cdc25c) and cyclin-dependent inhibitor 2D (Cdkn2d) were selected for validation by real-time polymerase chain reaction (PCR) based on published data. Our real-time PCR results demonstrated that Axin1, Cdc25c and Cdkn2d genes had different levels of expression among blastomeres of the mouse 2-cell embryo i.e. the level of Axin1 mRNA was significantly higher in one blastomere when compared with the other blastomeres of the 2-cell embryo (p < 0.05). The variation in Cdc25c (p < 0.05) and Cdkn2d (p < 0.01) mRNA expression followed a similar trend to that of Axin1. In addition, the highest levels of expression of these three genes were detected in the same blastomere in the 2-cell embryo. We confirmed that there was an asymmetrical distribution pattern for Axin1, Cdc25c and Cdkn2d transcripts in 2-cell embryos. In conclusion, this study demonstrated clearly that there is embryonic asymmetry at the 2-cell stage and that these differentially expressed genes may result in differentiation in expression in embryo development.
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Wurlina, Wurlina. "PENGARUH ANTIMITOSIS EKSTRAK Achyranthes Aspera Linn PADA PEMBELAHAN SEL EMBRIO (CLEAVAGE)." Berkala Penelitian Hayati 11, no. 2 (June 30, 2006): 161–65. http://dx.doi.org/10.23869/bphjbr.11.2.200610.
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To study the antimitotic effect of an extract of the leaf Achyranthes asapera Linn on embryonal cleavage rats (Rattus norwegicus). Rats ova were fertilized in vitro, and thes suitable zygote and embryo were selected to use as samples, which were divided into 5 groups. Each group comprised 80 ova/embryo. Those groups were: group 1. ova for fertilization, group 2. zygote, group 3. 2 cell embryo, group 4. 4 cell embryo and group 5. 8 cell embryo. Each group was divided into 4 treatmens, each of which consisted of 20 ova/ embryos. The treatments were as follows: control treatment receiving TCM 199 media, and treatment 1, 2 and 3 receiving Achyranthes aspera Linn alkaloid of 20, 30, and 40 ppm respectively. Observation to embryonal cleavage and development was out 12 and 24 hours after treatment. Concluded from the results that the administration of Achyranthes aspera Linn as much as 30 ppm in vitro could 1) inhibits fertilization zygote and 2, 4, and 8 cell embryonal cleavage, growth and development 2) induced zygote and embryonal membrane recruitment and blastomere degeneration and 3) inhibits zygote and embryonal mitotic cleavage at metaphase stage.
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Forlani, S., C. Bonnerot, S. Capgras, and J. F. Nicolas. "Relief of a repressed gene expression state in the mouse 1-cell embryo requires DNA replication." Development 125, no. 16 (August 15, 1998): 3153–66. http://dx.doi.org/10.1242/dev.125.16.3153.
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In the mouse, transcriptional permissiveness is established in the fertilized egg prior to the activation of zygotic genes at the 2-cell stage. Therefore, gene inactivity initiated at the end of gametogenesis results from a complex process, involving more than an inhibition of the basal transcriptional apparatus. We have examined the ability of the first intron (I1) of the human hypoxanthine phosphoribosyl transferase gene, which functions as an enhancer in embryonic stem cells, to activate a reporter gene when placed proximally to or at a distance from the HSV-tk promoter, or when integrated into the mouse genome as part of a stable transgene. In microinjected embryos, I1 functions as an enhancer sequence; however, its competence for long-range activation appears only after the late 1-cell stage and depends on the first DNA replication. Moreover, activation of microinjected transgenes from proximal enhancers occurs in the late 2-cell embryo and in the male pronucleus of 1-cell embryos blocked for DNA replication; whereas, for integrated transgenes, proximal enhancer activity is subject to position effects in the 2-cell embryo and first occurs at the 2- or 4-cell stage, but only after completion of DNA replication. Therefore, the absence of long-range activation and a non-permissive genomic state (the relief of which both depend on DNA replication), together with an inactive transcriptional apparatus, appear to converge to prevent any gene activity in the 1-cell embryo. We propose that the embryo exploits the process of DNA replication to relieve the transcriptionally repressive state that was initially established to fulfil two purposes: (1) to arrest maternal gene expression in the maturing oocyte and (2) to protect the unicellular egg and 1-cell embryo from premature differentiation. Reactivation of gene expression by DNA replication would therefore serve to coordinate cell proliferation and differentiation in the preimplantation embryo.
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Chen, C. H., T. A. Lin, H. Y. Su, Y. S. Sung, L. J. Sung, S. C. Wu, W. T. K. Cheng, et al. "157 AGGREGATION AND DEVELOPMENT OF RABBIT CLONED EMBRYOS WITH ZYGOTIC, TETRAPLOID, AND PARTHENOGENETIC EMBRYO IN VITRO." Reproduction, Fertility and Development 22, no. 1 (2010): 237. http://dx.doi.org/10.1071/rdv22n1ab157.
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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 < 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 < 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 < 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.
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Cowan, Patrick, Victoria Allyn, Emma Krier, Hamilton Lee, Erik Strait, Allison Joy Updike, Airam Mex Puc, and Mark Larman. "MOUSE EMBRYO ASSAY (MEA): SHOWING THAT THE 1-CELL EMBRYO IS A MORE SENSITIVE STARTING POINT THAN THE 2-CELL." Fertility and Sterility 122, no. 4 (October 2024): e338. http://dx.doi.org/10.1016/j.fertnstert.2024.08.066.
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Rodriguez, M. D., A. Gambini, A. Sestelo, O. Briski, R. Fernandez-Martin, and D. F. Salamone. "81 Generation of presumptive domestic cat tetraploid embryos and its application for asynchronic complementation with diploid blastomeres." Reproduction, Fertility and Development 31, no. 1 (2019): 166. http://dx.doi.org/10.1071/rdv31n1ab81.
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Tetraploid complementation has been extensively used to verify the pluripotency of stem cells and also for improving placenta formation when tetraploid embryos are aggregated synchronously or asynchronously with diploid (2n) embryos. Generation of tetraploid embryos can be achieved by the electric fusion of a 2-cell embryo. However, the optimal electric intensity pulse to generate tetraploid embryos has not been studied in the feline. The aims of this study were to (1) evaluate the optimal fusion conditions to achieve the highest fusion rate without affecting embryo developmental competence, (2) compare the in vitro development of synchronic and asynchronic aggregated domestic cat IVF embryos, and (3) assess pre-implantation development of embryos generated by asynchronic complementation of presumptive 1-cell tetraploid embryos with diploid blastomeres. Domestic cat cumulus-oocyte complexes were matured in vitro on 21% O2 in air at 38.5°C for 22h. The IVF embryos were generated by co-incubation of in vitro-matured oocytes with 2×106 motile spermatozoa mL−1 on 21% O2 in air at 38.5°C for 18 to 20h. After 24h of IVF, 2-cell embryos were selected. For Experiment 1, membrane fusion of 2-cell IVF embryos (n=164) was performed with two 30-ms DC pulses at different electric field (0.8, 2, 4, and 8 kV/cm) in fusion media (Mannitol, MgSO4, CaCl2, and polyvinyl alcohol). Presumptive fused embryos and nonfused were cultured in vitro in 50-µL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C (Pope et al. 2006 Methods in Molecular Biology 254, 227-244). Cleavage was determined 24h after pulse. For Experiment 2, zona pellucida-free IVF embryos (n=110) were synchronically (two 4-cell embryos) or asynchronically (one 4-cell embryo and one 2-cell embryo) aggregated in 1 microwell. For Experiment 3, 1-cell presumptive tetraploid embryo (2-cell fused embryo) was asynchronically complemented with a 4-cell embryo (n=38). For all experiments, blastocyst stage was evaluated at Day 8, and embryos presenting more than one structure per microwell were considered non-aggregated. Data were analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), and differences were considered significant at P<0.05. The highest fusion rates (30 and 46%) with the best developmental competence (31 and 46%) were observed with 4 and 8 Kv/cm electric pulses, respectively. Electric fusion did not affect the embryo developmental competence. We observed that synchronic and asynchronic complementation reached similar blastocysts rates (54 and 65%, respectively), indicating that both techniques are suitable for tetraploid embryo complementation. Finally, when presumptive tetraploid embryos were asynchronically complemented with diploid blastomeres, the high blastocyst rate (90%) was obtained from embryos that form only one structure (aggregated embryos). Further experiments will be performed to track the distribution of cells using mitotrackers after complementation using tetraploid IVF and diploid somatic cell nuclear transfer embryos.
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Wooldridge, Lydia K., Madison E. Nardi, and Alan D. Ealy. "Zinc supplementation during in vitro embryo culture increases inner cell mass and total cell numbers in bovine blastocysts1." Journal of Animal Science 97, no. 12 (November 12, 2019): 4946–50. http://dx.doi.org/10.1093/jas/skz351.
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Abstract Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P < 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P < 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.
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Ding, Nai-Zheng, Cheng-Qiang He, and Zeng-Ming Yang. "Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR." Zygote 10, no. 3 (August 2002): 239–43. http://dx.doi.org/10.1017/s0967199402002319.
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Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 ± 0.0282 in the oocyte, 0.0102 ± 0.0036 in the zygote, 0.0007 ± 0.0003 in the 2-cell embryo, 0.0031 ± 0.0017 in the 4-cell embryo, 0.0084 ± 0.0024 in the 8-cell embryo, 0.0537 ± 0.0121 in the morula and 0.0392 ± 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.
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Gardner, DK, L. Selwood, and M. Lane. "Nutrient uptake and culture of Sminthopsis macroura (stripe-faced dunnart) embryos." Reproduction, Fertility and Development 8, no. 4 (1996): 685. http://dx.doi.org/10.1071/rd9960685.
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Glucose and pyruvate uptake by individual embryos were measured in a marsupial species (stripe-faced dunnart) and a eutherian species (mouse). At each stage of development, nutrient uptake by the dunnart embryo was around an order of magnitude greater than that of the mouse embryo. The pattern of glucose uptake by the dunnart embryo was not like that for any eutherian embryo, all of which have a low glucose uptake before the blastocyst stage. Rather, in the dunnart embryo there was a significant increase in glucose uptake after the third cleavage division, increasing from 13.6 pmol embryo h-1 at the 4-cell stage to 34.9 pmol embryo h-1 by the 8-cell stage. This increase in glucose uptake before blastocyst formation may be attributed to an increased energy demand associated with the movement of cells within the dunnart embryo. Using a new culture system, it was possible to culture 66% of dunnart embryos at the 2-4-cell stage and 80% of those at the 8-16-cell stage to the unilaminar blastocyst stage. Embryos cultured from the 2-cell to the 4-cell stage were retarded by around 12 h when they reached the blastocyst stage. Developmental retardation was also reflected in the pattern of nutrient uptake, which lagged behind that of embryos developed in vivo. The present study has shown that it is possible to culture the early marsupial embryo to the blastocyst stage in a serum-free culture system, while concomitantly quantifying embryonic nutrient requirements. Such an approach is essential for species where there is a paucity of material for study.
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Macklin, Ruth. "Ethics, politics, and human embryo stem cell research." Women's Health Issues 10, no. 3 (May 2000): 111–15. http://dx.doi.org/10.1016/s1049-3867(00)00040-2.
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Croteau, S., and Y. Menezo. "Methylation in fertilised and parthenogenetic preimplantation mouse embryos." Zygote 2, no. 1 (February 1994): 47–52. http://dx.doi.org/10.1017/s0967199400001751.
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SummaryDNA methylation is one of the proposed biochemical mechanisms involved in cell differentiation and in genomic imprinting, and DNA methyltransferase (DMT) is a key enzyme in the embryo since mutation of its gene is lethal early in development. In order to verify that non-viability of uniparental embryos was not due to a defect in the regulation of DMT activity, we compared the metabolism of methylation in parthenogenetic embryos (maternal genome) and in fertilised embryos (maternal and paternal genomes). As regards total methylation, estimated by a measure of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) formation, no significant difference was found between the two kinds of embryos during preimplantation development. Mean values were 4.5 ± 0.6 fmol (SAM +SAH)/h per 2-cell embryo and 0.40 ± 0.05 fmol SAH/h per 2-cell embryo, i.e. a SAH/(SAM + SAH) ratio of 9%; there was no detectable SAH formation in blastocysts. The same observation can be made for DMT activity, with mean values of: 7.8 fmol/h per oocyte, 8.5 fmol/h per 2-cell embryo, 6.1 fmol/h per 4-cell embryo, 4.1 fmol/h per morula, and no detectable activity in blastocysts. Total methylation as well as DNA methylation is characterised by a progressive drop in activity during preimplantation development.
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Li, Y., M. L. Day, and C. O.'Neill. "257. Activation of a calcium-activated chloride channel by paf is required for normal preimplantation mouse embryo development in vitro." Reproduction, Fertility and Development 20, no. 9 (2008): 57. http://dx.doi.org/10.1071/srb08abs257.
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Platelet activating factor (paf) is an autocrine survival factor for preimplantation embryo. Binding of paf to its receptor activates PI3kinase, causing an IP3-dependent release of Ca2+ from intracellular stores as well as activation of Ca2+ influx via a dihydropyridine-sensitive Ca2+ channel. These actions result in the generation of a defined intracellular calcium ([Ca2+]i) transient in the 2-cell embryo[1]. By using combined whole-cell patch-clamp and real-time [Ca2+]i analyses, we have shown that paf also induces a concomitant hyperpolarisation of the membrane potential in 2-cell embryos, accompanied by an increased net outward ion current. Both the membrane hyperpolarisation and outward current were dependent upon the occurrence of the paf-induced [Ca2+]i transient[2]. The aim of this study was to investigate the characteristics of the paf-induced outward current in 2-cell embryos and to assess whether it has a role in normal mouse preimplantation development. We show that: (1) removal of extracellular anions or treatment with niflumic acid (NFA, 100 μM, a Ca2+-activated Cl- channel blocker) prevented activation of the outward current by paf but had no effect on the paf-induced [Ca2+]i transient; and (2) The culture of embryos with NFA (100 μM) from the 1-cell to late 2-cell stage significantly reduced their development to the blastocyst stage (P < 0.001), but treatment with NFA from the late 2-cell stage had no effect on development. The results show that paf induces an increase in [Ca2+]i which in turn activates a Ca2+-activated Cl- channel. The activity of this NFA-sensitive channel during the zygote to 2-cell stage is required for normal embryo development. (1) C. O’Neill (2008) The potential roles of embryotrophic ligands in preimplantation embryo development. Hum Reprod Update 14:275–288. (2) Y. Li, M.L. Day & C. O’Neill (2007) Autocrine activation of ions currents in the two-cell mouse embryo. Exp Cell Res. 313:2785–2794.
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Seshagiri, Polani B., and Barry D. Bavister. "Assessment of hamster blastocysts derived from eight-cell embryos cultured in hamster embryo culture medium-2 (HECM-2): Cell numbers and viability following embryo transfer." Journal of In Vitro Fertilization and Embryo Transfer 7, no. 5 (October 1990): 229–35. http://dx.doi.org/10.1007/bf01129524.
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Smolyaninova, Yevgeniya, Victor Shigimaga, Lyudmila Popivnenko, and Igor Kovalenko. "Cell-to-Cell Adhesion and Electrical Parameters of Murine Embryo Membranes After Cryopreservation by Vitrification." Problems of Cryobiology and Cryomedicine 32, no. 4 (December 30, 2022): 256–66. http://dx.doi.org/10.15407/cryo32.04.256.
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In this work, we have studied the impact of various stages of cryopreservation by vitrification in ethylene glycolsucrose medium on plasma membranes adhesive properties of 8-cell murine embryos and their specific electrical conductivity. Embryos were divided into the following experimental groups: the control, group 1 (incubation in vitrification medium) and group 2 (complete cycle of cryopreservation). The embryo exposure to vitrification medium did not affect their ability to cell-to-cell adhesion. After a complete cycle of cryopreservation, no embryo adhesion was observed. Electrical conductivity of embryo membranes was determined using the pulsed conductometry. After incubation in vitrification medium, their resistance to electric pulse was shown to decrease, that was manifested in the phenomenon of irreversible electric breakdown. The average values of electrical conductivity varied within the following ranges: ((12.1 ± 1.5)...(55.5 ± 2.6)), ((28.7 ± 5.7)...(44.± 8.9)), ((31.0 ± 9.3)...(87.9 ± 26.1)) μS/cm in the control, groups 1 and 2, respectively. These findings may be explained by appearance of first structural disorders in lipid bilayer of embryo membranes even at the stage of their incubation in vitrification medium. The lack of adhesive properties of blastomeres after vitrification-warming testified to a damage to the membrane adhesion proteins.
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KWON, Ivo. "EU Policy and Legislation on Stem Cell Research." Korean Journal of Medical Ethics 7, no. 2 (December 2004): 247–57. http://dx.doi.org/10.35301/ksme.2004.7.2.247.
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EU policy on the research of the human embryo and stem cell is based on the 1997 Convention - Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine : Convention on Human Rights and Biomedicine. The purpose of this convention is to protect the dignity and identity of all human beings without discrimination, respect for their integrity and other rights and fundamental freedoms with regard to the application of biology and medicine. Applying this convention's view to the human embryo and stem cell research, 1) the human embryo research can only be permitted only if it protect the embryo for the purpose of the health and medicine. 2) Making human embryo is prohibited by any method-IVF or SCNT technique. 3) Other stem cell resources(adult stem cell, cord blood stem cell, etc) can be used under the condition of full informed consent without any financial interest of the donor. 4) In all cases, the privacy and human right as well as health of all related persons should be guaranteed. As a result, the research on the human embryo and stem cell has not been done actively except in few country. But now most member states permit stem cell research using spare embryo, but prohibit therapeutic cloning by SCNT. However EU itself has failed to agree with to fund the scientific research on stem cell using spare embryo. It is hardly to say the decision on stem cell research in the future by EU, but EU will continue to stress on the basic human right, social justice and human freedom in the field of biotechnology and its applications.
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Hutter, H., and R. Schnabel. "Establishment of left-right asymmetry in the Caenorhabditis elegans embryo: a multistep process involving a series of inductive events." Development 121, no. 10 (October 1, 1995): 3417–24. http://dx.doi.org/10.1242/dev.121.10.3417.
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Bilateral pairs of blastomeres derived from the founder cell AB, the anterior blastomere of the 2-cell stage, in the Caenorhabditis elegans embryo are initially equivalent in their developmental potential. Recently, we showed that an induction at the 12-cell stage by a blastomere called MS is necessary to establish the differences between left and right pairs of blastomeres in the anterior part of the embryo. Further analysis of the process of creating left-right asymmetry reveals that the induction at the 12-cell stage is only the first of a series of inductions establishing the left-right asymmetry of the embryo. We describe here two further inductions that create additional asymmetries in the posterior part of the embryo. One induction occurs at the 24-cell stage among AB descendants themselves. This induction is restricted to the left side of the embryo as a consequence of the fate changes induced by MS at the 12-cell stage. The second induction requires again blastomeres of the MS lineage and also occurs around the 24-cell stage. Together these inductions establish the fate differences observed in the development of left-right pairs of blastomeres in the embryo.
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MING, ZHANG, and CHEN SUNHONG. "ESTABLISHMENT OF COLOSSOMA BRACHYPOMUM EMBRYO CELL LINE." In Vitro Cellular & Developmental Biology - Animal 36, no. 10 (2000): 617. http://dx.doi.org/10.1290/1071-2690(2000)036<0617:eocbec>2.0.co;2.
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David, G., X. M. Bai, B. Van der Schueren, P. Marynen, J. J. Cassiman, and H. Van den Berghe. "Spatial and temporal changes in the expression of fibroglycan (syndecan-2) during mouse embryonic development." Development 119, no. 3 (November 1, 1993): 841–54. http://dx.doi.org/10.1242/dev.119.3.841.
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Fibroglycan (syndecan-2) is a member of a family of cell surface heparan sulfate proteoglycans that interact with adhesion molecules, growth factors and a variety of other effector systems that support the shaping, maintenance and repair of an organism. To investigate this apparent redundancy of proteoglycans at the cell surface, we have studied the expression of fibroglycan in the mouse embryo and compared this expression with that of syndecan-1. The characterisation of mouse embryo cDNA clones that crosshybridized to human fibroglycan-cDNA predicted that murine and human fibroglycan were highly similar in structure. Consistently, the analysis of transfectant cells, murine cell lines and embryo extracts indicated that the murine proteoglycan reacted specifically with monoclonal antibody 10H4 developed against the human protein. Fibroglycan, as detected by monoclonal antibody 10H4 in sections of embryonic tissues, occurred exclusively on mesenchymal cells that represented the putative precursors of the hard and connective tissue cells. No fibroglycan was detected in epithelia or in muscle cells. Areas where fibroglycan was particularly abundant were sites of high morphogenetic activity where intense cell-cell and cell-matrix interactions are known to occur (e.g. the epithelial-mesenchymal interfaces, the prechondrogenic and preosteogenic mesenchymal condensations). The expression of fibroglycan was weak in the early embryo, culminated during the morphogenetic phase and at the moment of cell lineage differentiation, and persisted in the perichondrium, periosteum and connective tissue cells. Syndecan-1, in contrast, was primarily detected in epithelia, and transiently in some mesenchymal cells, with mesenchymal localisations that did not or only partially overlap with those of fibroglycan. In situ hybridization analyses confirmed these expression patterns at the transcriptional level, identifying mesenchymal cells as the major source of fibroglycan production. These data indicate that the expression of fibroglycan occurs along unique and developmentally regulated patterns, and suggest that fibroglycan and syndecan-1 may have distinctive functions during tissue morphogenesis and differentiation.
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Goutte, C., W. Hepler, K. M. Mickey, and J. R. Priess. "aph-2 encodes a novel extracellular protein required for GLP-1-mediated signaling." Development 127, no. 11 (June 1, 2000): 2481–92. http://dx.doi.org/10.1242/dev.127.11.2481.
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In animal development, numerous cell-cell interactions are mediated by the GLP-1/LIN-12/NOTCH family of transmembrane receptors. These proteins function in a signaling pathway that appears to be conserved from nematodes to humans. We show here that the aph-2 gene is a new component of the GLP-1 signaling pathway in the early Caenorhabditis elegans embryo, and that proteins with sequence similarity to the APH-2 protein are found in Drosophila and vertebrates. During the GLP-1-mediated cell interactions in the C. elegans embryo, APH-2 is associated with the cell surfaces of both the signaling, and the responding, blastomeres. Analysis of chimeric embryos that are composed of aph-2(+) and aph-2(−) blastomeres suggests that aph-2(+) function may be provided by either the signaling or responding blastomere.
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Caudle, J., C. K. Hamilton, F. A. Ashkar, and W. A. King. "90 TRANSCRIPTION OF USP9Y AND ZFY IN DEVELOPING AND ARRESTED BOVINE EMBRYOS IN VITRO." Reproduction, Fertility and Development 25, no. 1 (2013): 193. http://dx.doi.org/10.1071/rdv25n1ab90.
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Sexual dimorphisms such as differences in growth rate and metabolism have been observed in the early embryo, suggesting that sex chromosome-linked gene expression may play an active role in early embryo development. Furthermore, in vitro sex ratios are often skewed toward males, indicating that Y-linked genes may benefit development. While little attention has been paid to the Y chromosome, expression of some Y-linked genes such as SRY and ZFY has been identified in the early embryo, and only a few studies have systematically examined early stages. Identification of transcripts of Y-linked genes in the early embryo may provide insights into male development and provide markers of embryonic genome activation in male embryos. The objectives of this study were i) to examine the timing of transcription of 2 Y chromosome-linked genes involved with sperm production and male development, ubiquitin-specific peptidase 9 (USP9Y) and zinc finger protein (ZFY), in in vitro-produced bovine embryos from the 2-cell stage to the blastocyst stage and ii) to determine if USP9Y and ZFY transcripts are present in in vitro-produced embryos arrested at the 2- to 8-cell stages. To examine the chronology of transcription of these genes, pools of 30 embryos for each developmental stage, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst, were produced by bovine standard in vitro embryo production (Ashkar et al. 2010 Hum. Reprod. 252, 334–344) using semen from a single bull. Pools of 30 were used to balance sex ratios and to account for naturally arresting embryos. Embryos for each developmental stage were harvested and snap frozen. Total RNA was extracted from each pool, reverse transcribed to cDNA and by using PCR, and transcripts of USP9Y and ZFY were detected as positive or negative. In addition pools of 30 embryos arrested at the 2- to 8-cell stage harvested 7 days after IVF were processed and analysed in the same way to determine if transcripts from the Y chromosomes are present in developmentally arrested embryos. Transcripts of USP9Y and ZFY were detected in the pooled embryos from the 8-cell stage through to the blastocyst stage, but none were detected in the 2-cell or 4-cell pools. Transcripts of ZFY were detected in the arrested 2- to 8-cell embryo pool, but transcripts of USP9Y were not detected. Given that these Y genes begin expression at the 8-cell stage, coincident with embryonic genome activation, it was concluded that these genes may be important for early male embryo development. Furthermore, the results suggest that arrested embryos that have stopped cleaving before the major activation of the embryonic genome are still capable of transcribing at least some of these genes. The absence of USP9Y transcripts in the arrested embryos suggests that it may be important for early male embryo development. Funding was provided by NSERC, the CRC program, and the OVC scholarship program.
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Hyatt, G. A., and D. C. Beebe. "Regulation of lens cell growth and polarity by an embryo-specific growth factor and by inhibitors of lens cell proliferation and differentiation." Development 117, no. 2 (February 1, 1993): 701–9. http://dx.doi.org/10.1242/dev.117.2.701.
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We used a double-label method, which monitors the rate at which cells enter S-phase of the cell cycle, to identify factors that control the growth of chicken embryo lens epithelial cells in vivo. With this assay, we identified a mitogen for lens epithelial cells in the anterior segment of the embryonic eye. When the anterior chamber was opened briefly, by tearing the cornea or displacing the lens, the growth-promoting activity was lost. None of the purified growth factors tested replaced this growth activity, including EGF, bFGF, PDGF, IGF-1, IGF-2, TGF beta and mixtures of these factors. However, chicken embryo serum or plasma did cause chicken embryo lens epithelial cells to progress through the cell cycle. The activity in serum was destroyed by heat and protease treatment. It was most active in serum from 10-day embryos, decreased with subsequent development and was undetectable from 2 days after hatching through adulthood. When embryo serum or plasma was mixed with vitreous humor or IGF-1, agents that induce lens fiber cell formation, cell elongation was prevented. In contrast to the mitogenic activity in serum, this inhibitor of differentiation was insensitive to trypsin treatment. We also identified an activity in vitreous humor that inhibited the growth-promoting agent in embryo serum. Plasma proteins readily enter the anterior chamber of the eye of chicken embryos.(ABSTRACT TRUNCATED AT 250 WORDS)
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Voronov, D. A., and Y. V. Panchin. "Cell lineage in marine nematode Enoplus brevis." Development 125, no. 1 (January 1, 1998): 143–50. http://dx.doi.org/10.1242/dev.125.1.143.
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Early cleavages of the marine nematode Enoplus brevis are symmetrical and occur in synchrony. At the 2- to 16-cell stages, blastomeres are indistinguishable. The progeny of blastomeres was investigated by intracellular injections of fluorescent dyes and horse radish peroxidase. One blastomere of the 2-cell embryo gives rise to a compact group of cells occupying about half of an embryo. The border between labeled and unlabeled cells differs in each embryo dividing it to anterior-posterior, left-right or intermediate parts. At the 8-cell stage, one blastomere gives rise to only endoderm, whereas the other blastomeres produce progeny that form multiple cell types, including nerve, muscle and hypoderm cells, in various proportions. Thus the fates of the blastomeres of early E. brevis embryos, with the exception of the endoderm precursor, are not determined. The process of gastrulation in E. brevis is very similar to that in Caenorhabditis elegans and other nematodes. At the beginning of gastrulation, the 2-celled endoderm precursor lies on the surface of embryo and then sinks inwards. After labeling of cells on the ventral side (near endoderm precursor) at the beginning of gastrulation, their progeny differentiate predominantly into body muscles or pharyngeal cells of the first stage larva. Cells that are located more laterally give rise mainly to neurons. The dorsal blastomeres differentiated principally into hypoderm cells. Our study suggests that a precise cell lineage is not a necessary attribute of nematode development.
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Pozzatti, R., M. McCormick, M. A. Thompson, and G. Khoury. "The E1a gene of adenovirus type 2 reduces the metastatic potential of ras-transformed rat embryo cells." Molecular and Cellular Biology 8, no. 7 (July 1988): 2984–88. http://dx.doi.org/10.1128/mcb.8.7.2984-2988.1988.
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We have previously demonstrated that second-passage rat embryo cells transformed by the ras oncogene alone are both tumorigenic and highly metastatic when injected into nude mice. In contrast, rat embryo cells cotransformed with the ras oncogene and the adenovirus type 2 (Ad2) E1a gene are tumorigenic but either fail to metastasize or exhibit a very low metastatic potential. In this report, we demonstrate that transfection of the Ad2 E1a gene into four independent ras-transformed rat embryo cell lines results in a dramatic reduction in metastatic potential relative to that of the parental cell line. Transfection of cDNAs for the 12S and 13S E1a transcripts showed that the 12S cDNA was highly effective in reducing the metastatic potential of ras-transformed cell lines, while the 13S cDNA showed an effect in only one of the two cell lines tested. This effect is specific to the Ad2 E1a gene, since ras-transformed cell lines expressing the Ad12 E1a gene maintained their high metastatic potential. We hypothesize that the Ad2 E1a gene may regulate the expression of one or more cellular genes that contribute to the metastatic phenotype.
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Pozzatti, R., M. McCormick, M. A. Thompson, and G. Khoury. "The E1a gene of adenovirus type 2 reduces the metastatic potential of ras-transformed rat embryo cells." Molecular and Cellular Biology 8, no. 7 (July 1988): 2984–88. http://dx.doi.org/10.1128/mcb.8.7.2984.
Full textAbstract:
We have previously demonstrated that second-passage rat embryo cells transformed by the ras oncogene alone are both tumorigenic and highly metastatic when injected into nude mice. In contrast, rat embryo cells cotransformed with the ras oncogene and the adenovirus type 2 (Ad2) E1a gene are tumorigenic but either fail to metastasize or exhibit a very low metastatic potential. In this report, we demonstrate that transfection of the Ad2 E1a gene into four independent ras-transformed rat embryo cell lines results in a dramatic reduction in metastatic potential relative to that of the parental cell line. Transfection of cDNAs for the 12S and 13S E1a transcripts showed that the 12S cDNA was highly effective in reducing the metastatic potential of ras-transformed cell lines, while the 13S cDNA showed an effect in only one of the two cell lines tested. This effect is specific to the Ad2 E1a gene, since ras-transformed cell lines expressing the Ad12 E1a gene maintained their high metastatic potential. We hypothesize that the Ad2 E1a gene may regulate the expression of one or more cellular genes that contribute to the metastatic phenotype.
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Kang, Hye Na, and Jong-Sik Hah. "The Effects of Vero Cell-Conditioned Medium on Mouse Late 2-Cell Embryo Development." Ewha Medical Journal 19, no. 4 (1996): 551. http://dx.doi.org/10.12771/emj.1996.19.4.551.
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Hobbs, JG, and PL Kaye. "Glycine uptake in pre-implantation mouse embryos: kinetics and the effects of external." Reproduction, Fertility and Development 2, no. 6 (1990): 651. http://dx.doi.org/10.1071/rd9900651.
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The kinetic parameters (with standard errors) describing glycine uptake by mouse 2-cell embryos and blastocysts were determined by non-linear regression. Uptake at both stages was best described by a combination of a non-saturable component and a single saturable uptake system. During development, the rate constants for both components increased, as would be expected from the known increases in surface area, from 9.4 +/- 3.7 pL per 10 min per embryo and 128 +/- 12 fmol per 10 min per embryo in 2-cell embryos to 38.9 +/- 2.1 pL per 10 min per embryo and 258 +/- 15 fmol per 10 min per embryo in blastocysts. In contrast to earlier reports, there was no change in Km, which was 88 +/- 13 microM in 2-cell embryos and 115 +/- 10 microM in blastocysts. Reducing the external [Na+] from 230 mM increased Km for both stages. This effect on Km appeared to be related to [Na+]-2 or [Na+]-3. Vmax was increased in embryos of both stages by increasing [Na+] from 60 to 100 mM. However, whilst further increases to 400 mM were without major effect on uptake by 2-cell embryos, they inhibited uptake by blastocysts. This may result from osmotic effects on trophectodermal transport in the blastocysts. These results suggest that during development to blastocysts, the gly-system that operates in 2-cell embryos may be modified to a less restricted system with a similar Km and a complex dependence on [Na+].
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Vigneault, Christian, Serge McGraw, and Marc-Andre Sirard. "Spatiotemporal expression of transcriptional regulators in concert with the maternal-to-embryonic transition during bovine in vitro embryogenesis." REPRODUCTION 137, no. 1 (January 2009): 13–21. http://dx.doi.org/10.1530/rep-08-0077.
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Cleavage-stage bovine embryos are transcriptionally quiescent until they reach the 8- to 16-cell stage, and thus rely on the reserves provided by the stored maternal mRNAs and proteins found in the oocytes to achieve their first cell divisions. The objective of this study was to characterize the expression and localization of the transcriptional and translational regulators, Y box binding protein 2 (YBX2), TATA box-binding protein (TBP), and activating transcription factor 2 (ATF2), during bovine early embryo development. Germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, as well as 2-, 4-, 8-, 16-cell-stage embryos, morula, and blastocysts, producedin vitrowere analyzed for temporal and spatial protein expression. Using Q-PCR,ATF2mRNA expression was shown to remain constant from the GV-stage oocyte to the four-cell embryo, and then decreased through to the blastocyst stage. By contrast, the protein levels of ATF2 remained constant throughout embryo development and were found in both the cytoplasm and the nucleus. Both TBP and YBX2 showed opposite protein expression patterns, as YBX2 protein levels decreased throughout development, while TBP levels increased through to the blastocyst stage. Immunolocalization studies revealed that TBP protein was localized in the nucleus of 8- to 16-cell-stage embryos, whereas the translational regulator YBX2 was exclusively cytoplasmic and disappeared from the 16-cell stage onward. This study shows that YBX2, TBP, and ATF2 are differentially regulated through embryo development, and provides insight into the molecular events occurring during the activation of the bovine genome during embryo developmentin vitro.
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Schultz, GA, A. Hogan, AJ Watson, RM Smith, and S. Heyner. "Insulin, insulin-like growth factors and glucose transporters: temporal patterns of gene expression in early murine and bovine embryos." Reproduction, Fertility and Development 4, no. 4 (1992): 361. http://dx.doi.org/10.1071/rd9920361.
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mRNA phenotyping by the reverse transcription-polymerase chain reaction (RT-PCR) method was used to compare the patterns of expression of insulin and insulin-like growth factor (IGF) ligand and receptor genes in preimplantation bovine embryos with those established previously for preimplantation murine embryos. In the early bovine embryo, transcripts for IGF-I, IGF-II and mRNAs encoding receptors for insulin, IGF-I and IGF-II were all detectable at all embryo stages from the 1-cell zygote to the blastocyst. In the mouse, IGF-II ligand and receptor mRNAs were not expressed until the 2-cell stage, and the insulin and IGF-I receptor mRNAs were not detectable until the 8-cell stage. Since transcriptional activation of the embryonic genome occurs at the 8- to 16-cell stage in the bovine embryo and at the 2-cell stage in the murine embryo, it is suggested that these transcripts are products of both the maternal and embryonic genomes in the bovine embryo whereas in the mouse they are present only after activation of the embryonic genome. Transcripts for insulin were not detected in preimplantation embryos of either species. Colloidal-gold immunocytochemistry with antibodies directed against the insulin receptor, IGF-I receptor and IGF-I ligand has confirmed the presence of these molecules in bovine blastocysts. RT-PCR and indirect immunofluorescence procedures demonstrated that the glucose transporter (GLUT) isoform 1 is present in murine embryos from the oocyte to blastocyst stage whereas GLUT 2 expression begins at the 8-cell stage.
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Sepulveda-Rincon, L. P., N. Islam, P. Marsters, B. K. Campbell, N. Beaujean, and W. E. Maalouf. "Embryo cell allocation patterns are not altered by biopsy but can be linked with further development." Reproduction 154, no. 6 (December 2017): 807–14. http://dx.doi.org/10.1530/rep-17-0514.
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It has been suggested that first embryo cleavage can be related with the embryonic–abembryonic axis at blastocyst stage in mice. Thus, cells of the 2-cell embryo might be already biased to form the inner cell mass or trophectoderm. This study was conducted to observe the possible effects of embryo biopsy on cell allocation patterns during embryo preimplantation in two different mouse strains and the effects of these patterns on further development. First, one blastomere of the 2-cell embryo was injected with a lipophilic tracer and cell allocation patterns were observed at blastocyst stage. Blastocysts were classified into orthogonal, deviant or random pattern. For the first experiment, embryos were biopsied at 8-cell stage and total cell counts (TCC) were annotated. Furthermore, non-biopsied blastocysts were transferred into foster mothers. Then, pups and their organs were weighed two weeks after birth. Random pattern was significantly recurrent (≈60%), against orthogonal (<22%) and deviant (<22%) patterns among groups. These patterns were not affected by biopsy procedure. However, TCC on deviant embryos were reduced after biopsy. Moreover, no differences were found between patterns for implantation rates, litter size, live offspring and organ weights (lungs, liver, pancreas and spleen). However, deviant pups presented heavier hearts and orthogonal pups presented lighter kidneys among the group. In conclusion, these results suggest that single blastomere removal does not disturb cell allocation patterns during pre-implantation. Nonetheless, the results suggest that embryos following different cell allocation patterns present different coping mechanisms against in vitro manipulations and further development might be altered.
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Gebhardt, K. M., D. Feil, M. Lane, and D. L. Russell. "262. Human cumulus cell gene expression as a marker of clinical embryo grade." Reproduction, Fertility and Development 20, no. 9 (2008): 62. http://dx.doi.org/10.1071/srb08abs262.
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In Australia, Assisted Reproductive Technology (ART) accounts for ~3% of births. However, the success rate remains around 65% for women under 35 years of age, hence multiple embryo transfer is frequently preferred to improve the probabiity of achieving a term pregnancy. A biochemical marker for oocyte and embryo developmental potential would augment successful pregnancy outcomes following IVF/ICSI by optimising oocyte and embryo selection, therefore increasing the number of single embryo transfers (SET) performed in ART cycles. Changes in expression levels in human cumulus cells may reflect the quality of their enclosed oocyte. We investigated cumulus cell gene expression and subsequent embryo development to find a marker of embryo quality. Paired samples of cumulus cells were collected from oocytes that progressed to embryos of either high or low grade from eleven IVF/ICSI patients. Following cumulus oocyte complex retrieval cumulus cells were trimmed from the oocyte, and all oocytes and resulting embryos were cultured and tracked individually. Cumulus cell gene expression was assessed using a real-time RT–PCR assay, measuring expression of cyclooxygenase 2 (COX2; PTGS2), Pentraxin 3 (PTX3), Versican (VCAN), Tumour Necrosis Factor Alpha Induced protein 6 (TNAIFP6; TSG6), Lactate Dehydrogenase A (LDHA), Phosphofructokinase Platelet (PFKP), Gremlin (GREM1), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and 18S rRNA. Standard curves using plasmid subclones for each target were run to assess copy numbers of genes. Embryo morphology was assessed by an embryologist and correlated with relative gene expression. Cumulus cell gene expression was altered in cumulus cells from oocytes which subsequently developed into higher quality (Grade 1 and 2) embryos compared with cumulus cells from oocytes which developed into lower quality (Grade 3 and 4) embryos. This may lead to establishment of markers prognostic for developmental outcome, facillitating more reliable selection of higher quality embryos, increasing single embryo transfers and improving health outcomes from ART.
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Taylor, K. D., and L. Piko. "Patterns of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos." Development 101, no. 4 (December 1, 1987): 877–92. http://dx.doi.org/10.1242/dev.101.4.877.
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Considerable evidence indicates that the 2-cell stage is a critical period of mouse embryo development when a transition from maternal to zygotic genomic control takes place. The overall changes in the structure of the mRNA population as a result of this transition were explored using a random cDNA library of 69 clones derived from late 2-cell embryos. The prevalence of the cloned sequences was analysed by dot hybridization of the cDNA clones with labelled cDNA probes synthesized to poly(A)+ RNA from different stages of development from 1-cell through blastocyst. The number of copies of individual transcripts was quantitatively estimated by comparison to standard clones of known prevalence. About one half of the transcripts that gave a measurable reaction at the 2-cell and later stages were not represented detectably in egg RNA, suggesting that a large set of zygote-specific genes not included in the maternal gene set becomes transcriptionally active in the 2-cell embryo. Six of the cDNA clones represented B1 and B2 repeat sequences. As measured by hybridization with labelled cDNA, B1 and B2 transcripts were abundantly expressed throughout cleavage, being represented by about 10(5) to 10(6) copies per embryo. However, the developmental pattern of prevalence was different for the two transcripts suggesting that their expression is regulated independently. The results of this study corroborate previous evidence derived from protein synthetic patterns and in vitro translation experiments that a major qualitative shift in the mRNA population occurs in the 2-cell embryo.
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Rahbaran, Mohaddeseh, Ehsan Razeghian, Marwah Suliman Maashi, Abduladheem Turki Jalil, Gunawan Widjaja, Lakshmi Thangavelu, Mariya Yurievna Kuznetsova, et al. "Cloning and Embryo Splitting in Mammalians: Brief History, Methods, and Achievements." Stem Cells International 2021 (November 30, 2021): 1–11. http://dx.doi.org/10.1155/2021/2347506.
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Embryo splitting is one of the newest developed methods in reproductive biotechnology. In this method, after splitting embryos in 2-, 4-, and even 8-cell stages, every single blastomere can be developed separately, but the embryos are genetically identical. Embryo splitting, as an approach in reproductive cloning, is extensively employed in reproductive medicine studies, such as investigating human diseases, treating sterility, embryo donation, and gene therapy. In the present study, cloning in mammalians and cloning approaches are briefly reviewed. In addition, embryo splitting and the methods commonly used in embryo splitting and recent achievements in this field, as well as the applications of embryo splitting into livestock species, primate animals, and humans, are outlined. Finally, a perspective of embryo splitting is provided as the conclusion.
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Dale, B., L. Santella, and E. Tosti. "Gap-junctional permeability in early and cleavage-arrested ascidian embryos." Development 112, no. 1 (May 1, 1991): 153–60. http://dx.doi.org/10.1242/dev.112.1.153.
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Using the whole-cell voltage clamp technique, we have studied junctional conductance (Gj), and Lucifer Yellow (LY) coupling in 2-cell and 32-cell ascidian embryos. Gj ranges from 17.5 to 35.3 nS in the 2-cell embryo where there is no passage of LY, and from 3.5 to 12.2 nS in the later embryo where LY dye spread is extensive. In both cases, Gj is independent of the transjunctional potential (Vj). Manually apposed 2-cell or 32-cell embryos established a junctional conductance of up to 10 nS within 30 min of contact. Furthermore, since we did not observe any significant number of cytoplasmic bridges at the EM and Gj is sensitive to octanol, it is probable that blastomeres in the 2-cell and 32-cell embryos are in communication by gap junctions. In order to compare Gj in the two stages and to circumvent problems of cell size, movement and spatial location, we used cytochalasin B to arrest cleavage. Gj in cleavage-arrested 2-cell embryos ranged from 25.0 to 38.0 nS and remained constant over a period of 2.5 h. LY injected into a blastomere of these arrested embryos did not spread to the neighbour cell until they attained the developmental age of a 32- to 64-cell control embryo. Our experiments indicate a change in selectivity of gap junctions at the 32-cell stage that is not reflected by a macroscopic change in ionic permeability.
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Pereira, A. F., L. M. Melo, V. J. F. Freitas, and D. F. Salamone. "Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos." Zygote 23, no. 4 (April 15, 2014): 485–93. http://dx.doi.org/10.1017/s0967199414000100.
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SummaryIn vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.
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Sayed, Shabana, Marte Myhre Reigstad, Bjørn Molt Petersen, Arne Schwennicke, Jon Wegner Hausken, and Ritsa Storeng. "Nucleation status of Day 2 pre-implantation embryos, acquired by time-lapse imaging during IVF, is associated with live birth." PLOS ONE 17, no. 9 (September 22, 2022): e0274502. http://dx.doi.org/10.1371/journal.pone.0274502.
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The primary purpose of this time-lapse data analysis was to identify the association between the nucleation status of a Day 2 preimplantation embryo and live births following in vitro fertilization (IVF). The retrospective data analysis was based on 2769 transferred embryos from 1966 treatment cycles and utilised only Known Implantation Data (KID) for live births. Nucleation errors (NE) such as micronucleation, binucleation, multinucleation and minor error groups, were annotated in the time-lapse images which were taken every 15 minutes for a minimum of 44 hours post insemination. Further, factors that may impact NE and the relationship of early morphological attributes and morphokinetic variables with NE occurrence were explored. The frequency of NE among the transferred embryos was 23.8%. The reversibility of NE evidenced by their presence at the two-cell stage, but absence at the four-cell stage was 89.6%. Embryos exhibiting nucleation errors at the two-cell stage had significantly lower live birth rates compared to embryos with no nucleation errors, constituting a significant predictor. A Generalized Additive Mixed Model was used to control for confounders and for controlling clustering effects from dual embryo transfers. Increased incidences of NE were observed with increasing age, with delayed occurrence of cell divisions and in oocytes inseminated with surgically retrieved spermatozoa. NE assessment and their impact on live birth provides valuable markers for early preimplantation embryo selection. In addition, the high incidence of reversibility of NE and their possible impact on live birth suggest that incorporating two-cell nuclear status annotations in embryo selection, alongside morphology and morphokinetics, is of value.
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Wiekowski, M., M. Miranda, J. Y. Nothias, and M. L. DePamphilis. "Changes in histone synthesis and modification at the beginning of mouse development correlate with the establishment of chromatin mediated repression of transcription." Journal of Cell Science 110, no. 10 (May 15, 1997): 1147–58. http://dx.doi.org/10.1242/jcs.110.10.1147.
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The transition from a late 1-cell mouse embryo to a 4-cell embryo, the period when zygotic gene expression begins, is accompanied by an increasing ability to repress the activities of promoters and replication origins. Since this repression can be relieved by either butyrate or enhancers, it appears to be mediated through chromatin structure. Here we identify changes in the synthesis and modification of chromatin bound histones that are consistent with this hypothesis. Oocytes, which can repress promoter activity, synthesized a full complement of histones, and histone synthesis up to the early 2-cell stage originated from mRNA inherited from the oocyte. However, while histones H3 and H4 continued to be synthesized in early 1-cell embryos, synthesis of histones H2A, H2B and H1 (proteins required for chromatin condensation) was delayed until the late 1-cell stage, reaching their maximum rate in early 2-cell embryos. Moreover, histone H4 in both 1-cell and 2-cell embryos was predominantly diacetylated (a modification that facilitates transcription). Deacetylation towards the unacetylated and monoacetylated H4 population in fibroblasts began at the late 2-cell to 4-cell stage. Arresting development at the beginning of S-phase in 1-cell embryos prevented both the appearance of chromatin-mediated repression of transcription in paternal pronuclei and synthesis of new histones. These changes correlated with the establishment of chromatin-mediated repression during formation of a 2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates and core histones are deacetylated.
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Menezo, Yves, Jamal Hamidi, Ch Khatchadourian, and C. Nardon. "The Murine Prepuberal Oviduct Supports Early Embryo Development In Vitro. (mouse embryo coculture/2 cell-block/immature oviduct/protein secretion/early embryo signal)." Development, Growth and Differentiation 31, no. 6 (December 1989): 557–61. http://dx.doi.org/10.1111/j.1440-169x.1989.00557.x.
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Lee, Choon-Soo, Hyun-Jai Cho, Jin-Woo Lee, Hyun Ju Son, Jaewon Lee, Minjun Kang, and Hyo-Soo Kim. "The G Protein-Coupled Receptor Latrophilin-2, A Marker for Heart Development, Induces Myocardial Repair After Infarction." Stem Cells Translational Medicine 11, no. 3 (March 1, 2022): 332–42. http://dx.doi.org/10.1093/stcltm/szab015.
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Abstract Discovering cell–surface markers based on a comprehensive understanding of development is utilized to isolate a particular cell type with high purity for therapeutic purposes. Given that latrophilin-2 (Lphn2) substantially contributes to cardiac differentiation, we examined whether Lphn2 regulates functional significance in heart development and repair. We performed whole-mount immunostaining followed by clearing technique of embryo, RNA sequencing related to Lphn2-knockout (KO) embryo, and in vivo functional analyses of Lphn2+ cells using echocardiography. After immunostaining the cleared embryo sample, Lphn2 was exclusively observed in cardiac cells expressing α-sarcomeric actinin at embryonic days E9.5 and E10.5. Homozygous Lphn2-KO mice were embryonically lethal and showed underdevelopment of the ventricular myocardium. However, Lphn2 was not required to develop vessels, including endothelial cells and smooth muscle cells. For the purpose of cardiac regeneration, we transplanted pluripotent stem cell (PSC)–derived Lphn2+ cells into the infarcted heart. PSC–derived Lphn2+ cells differentiated into cardiomyocytes and regenerated the myocardium when transplanted into the infarcted heart, unlike Lphn2− cells. Transplanted Lphn2+ cells improved left-ventricle systolic function and reduced infarct size. We demonstrated that Lphn2 exhibits potential as a cardiomyogenic marker to facilitate targeted stem cell therapy for heart repair in clinical practice.
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Hamdi, M., B. Rodríguez-Alonso, A. Almansa-Ordonez, A. Gutierrez-Adán, P. Lonergan, and D. Rizos. "116 In Vitro Transcriptomic Response of Bovine Oviduct Epithelial Cells to Direct or Indirect Embryo Contact." Reproduction, Fertility and Development 30, no. 1 (2018): 197. http://dx.doi.org/10.1071/rdv30n1ab116.
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We observed that in vitro transcriptomic response of bovine oviduct epithelial cells (BOEC) to the early embryo could be the result of a contact-dependent signalling effect or interactions with embryo secretions. In order to determine this, BOEC were co-cultured directly with embryos or indirectly with embryo-conditioned media (CM); BOEC from the isthmus of oviducts at early luteal phase were cultured with TCM-199+10% fetal calf serum (FCS) in 4-well plates in 5% CO2 in air at 38.5°C for 6 days until confluence. In vitro 2- and 8-cell embryos as well as their CM were produced in parallel. A day before co-culture, BOEC medium was replaced with SOF+10% FCS. Groups for 2- and 8-cell embryos were established: BOEC in direct contact with embryos; BOEC in the same well as embryos but not in indirect contact; BOEC with embryo CM; and BOEC without embryos, as a control. Polyester mesh was used to maintain embryos position on top of the cells. After 48 h of co-culture, BOEC were recovered for gene expression analysis (4 replicates). The relative abundance of candidate genes previously shown to be affected by the presence of embryo in vivo (Maillo et al. 2015 Biol Reprod. 92, 144) [SMAD6 (BMP signalling pathway); ROCK1, ROCK2 (cytokinesis); SOCS3 (inflammatory response); PRELP (extracellular matrix)] or in vitro (Schmaltz-Panneau et al. 2014 Anim. Reprod. Sci. 149, 103-106) [GPX4, NFE2L2 (oxidative stress); SCN9A (sodium ion binding); EPSTI1 (tissue remodelling); IGFBP3 (insulin-like growth factor binding); TDGF1 (BMP signalling pathway); AGR3 (regulation of ciliary beating)] was assessed by RT-qPCR. H2A.Z and ACTG1 were used as housekeeping genes. Statistical analysis was assessed by ANOVA. The BOEC responded to the presence of 2-cell embryos only when in direct contact by significantly decreasing abundance of NFE2L2. Both direct and indirect embryo contact or culture with CM significantly decreased GPX4, ROCK2, and SCN9A transcripts compared with control. The presence of 2-cell embryos irrespective of being in direct or indirect contact reduced the expression of SMAD6 compared with the control and CM groups. In the case of CM, expression of IGFBP3 was enhanced compared with the control but was similar to the presence of the 2-cell embryos. In the presence of 8-cell embryos, direct contact with BOEC significantly down-regulated the expression for GPX4 and SOCS3, whereas expression of SCN9A was up-regulated. The opposite was observed when compared with control. The presence of 8-cell embryos down-regulated the expression of SMAD6 and ROCK2 compared with the CM group, whereas direct or indirect contact with BOEC or culture with CM down-regulated the expression of PRELP compared to control. In conclusion, these results provide evidence for a differential affect on the transcriptome of BOEC in vitro depending on embryo stage. These changes may be related either with direct embryo contact or embryo secretions released into the media. Research supported by Spanish MINECO-AGL2015-70140-R; AGL2015-66145-R; OECD-Co-operative Programme TAD/CRP JA00092482.
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Fleming, T. P., D. R. Garrod, and A. J. Elsmore. "Desmosome biogenesis in the mouse preimplantation embryo." Development 112, no. 2 (June 1, 1991): 527–39. http://dx.doi.org/10.1242/dev.112.2.527.
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The molecular processes underlying the formation of the first desmosomes in the mouse early embryo have been examined by immunocytochemical and biochemical techniques using antibody probes recognising desmosomal proteins 1 and 2 (dp1 + 2, desmoplakins), dp3 (plakoglobin), desmosomal glycoprotein 1 (dg1, desmoglein) and dg2 + 3 (desmocollins). Immunofluorescence labelling of staged intact embryos and synchronised cell clusters indicates that dp1 + 2, dg1 and dg2 + 3 are first detectable on the lateral membrane contact sites between trophectoderm cells in early cavitating blastocysts, coincident with the onset of desmosome formation as seen in ultrastructural preparations. Membrane localisation of these antigens is predominantly punctate in appearance, occurs after division to the 32-cell stage and appears to be coincident with blastocoele formation since non-cavitated embryos/cell clusters of equivalent age/cell cycle are usually unlabelled. In contrast, dp3 is first detectable at the 32-cell stage at all internal membrane contact sites (including those with inner cell mass cells) in a continuous linear pattern, and appears in both cavitated and non-cavitated specimens. Subsequently during blastocyst expansion, dp3 localisation becomes punctate and restricted to trophectodermal membranes. Immunoprecipitation of desmosomal antigens following metabolic labelling indicates that synthesis of dp3 is underway from at least compaction in the 8-cell embryo, while dp1 + 2 synthesis is first evident in 16-cell morulae. Synthesis of dg1 and dg2 + 3 is not detectable until the early blastocyst stage. These results suggest that desmosome biogenesis in the preimplantation embryo might be regulated by transcription or translation of desmosomal glycoproteins and by maturational changes in the trophectoderm layer associated with blastocoele formation. The earlier expression and wider distribution of dp3 at cell contact areas may reflect non-desmosomal sites (eg, adherens junctions) for this protein and a possible role for dp3 in the development of intercellular junctions.
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Gurung, S., D. W. Greening, S. Catt, L. Salamonsen, and J. Evans. "Exosomes and soluble secretome from hormone-treated endometrial epithelial cells direct embryo implantation." Molecular Human Reproduction 26, no. 7 (May 13, 2020): 510–20. http://dx.doi.org/10.1093/molehr/gaaa034.
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Abstract A successful pregnancy requires a synchronous dialogue between endometrium and embryo within the endometrial milieu. The aim of this study was to assess the role in the implantation of mediators in the endometrial milieu. Total secretome (TS), soluble secretome (SS) and small extracellular vesicles (containing exosomes) were generated from hormonally primed human endometrial epithelial cell culture medium. Human trophectoderm stem cell-derived spheroids were cultured with TS, SS or exosomes (30 µg/ml) on hormonally primed epithelial cells, with exosomes significantly increasing cell adhesion and outgrowth. Furthermore, F1 mouse 2-cell embryos were cultured in groups for 48 h followed by culture with each secretome fraction (30 µg/ml) for 48 h. Blastocyst cell number and hatching were quantified. In addition, blastocysts were further cultured on a fibronectin matrix for 72 h or transferred to recipient mice (with corresponding secretomes) with embryo implantation assessed after 6 days. Exosomes significantly increased total cell number in mouse embryos and complete hatching from zona pellucida, with both exosomes and SS significantly enhancing mouse embryo outgrowth. Importantly, exosomes increased the embryo implantation rate in comparison to other secretome fractions (normalized based on treatment amount) from the endometrial epithelia. These data indicate that endometrial epithelial exosomes support embryo growth, development and implantation while the SS has selective involvement specifically on mouse embryo outgrowth. This finding provides new insights into the molecular differences of endometrial secretome components in implantation and early embryo development and may implicate endometrial exosomes in the pathophysiology of implantation failure in infertility.
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García-Herreros, Manuel, Constantine A. Simintiras, and Patrick Lonergan. "Temporally differential protein expression of glycolytic and glycogenic enzymes during in vitro preimplantation bovine embryo development." Reproduction, Fertility and Development 30, no. 9 (2018): 1245. http://dx.doi.org/10.1071/rd17429.
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Proteomic analyses are useful for understanding the metabolic pathways governing embryo development. This study investigated the presence of enzymes involved in glycolysis and glycogenesis in in vitro-produced bovine embryos at five developmental stages leading up to blastocyst formation. The enzymes examined were: (1) glycolytic: hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase mutase 1/2 (PKM-1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (2) glycogenic: glycogen synthase kinase-3 isoforms α/ β (GSK-3α/β). Glucose transporter-1 (GLUT-1) was also analysed. The developmental stages examined were: (1) 2–4-cell, (2) 5–8-cell, (3) 16-cell, (4) morula and (5) expanded blastocyst. The enzymes HK-I, PFK-1, PKM-1/2, GAPDH and GLUT-1 were differentially expressed throughout all stages (P < 0.05). GSK-3α and β were also differentially expressed from the 2–4-cell to the expanded blastocyst stage (P < 0.05) and GLUT-1 was identified throughout. The general trend was that the abundance of PFK1, GAPDH and PKM-1/2 decreased whereas HK-I, phospho-GSK3α (P-GSK3α) and P-GSK3β levels increased as the embryo advanced. In contrast, GLUT-1 expression peaked at the 16-cell stage. These data combined suggest that in vitro bovine embryo metabolism switches from being glycolytic-centric to glycogenic-centric around the 16-cell stage, the developmental window also characterised by embryonic genome activation.
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Utami, Edy Setiti Wida, Issirep Soemardi, Taryono Taryono, and Endang Semiarti. "EMBRIOGENESIS SOMATIK ANGGREK BULAN Phalaenopsis amabilis (L.) Bl: STRUKTUR DAN POLA PERKEMBANGAN." Berkala Penelitian Hayati 13, no. 1 (December 31, 2007): 33–38. http://dx.doi.org/10.23869/bphjbr.13.1.20075.
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Research of the structure and development pattern of somatic embryos from callus of leaf explants moon orchid Phalaenopsis amabilis (L) Bl had been done. One year old of plantlets were used as explants sources. Basal leaf of these explants were cultured in Somatic Embryo Induction Medium (SEIM) e.i.: NP(New Phalaenopsis) medium added with 2 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Then somatic embryos were transferred to EMM (Embryo Maturation Medium) e.i. NP medium added with 1 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Finally, mature somatic embryo were transferred to NP medium without plant growth regulator as Embryo Germination Medium (EGM). The origin of somatic embryos initially from single cell at the pheriphery of embryogenic callus. These cells then devided in mitotic repeatedly formed globular proembryo, elongation embryo, and completed embryo. The structure and development pattern of somatic embryos as the same as with zygotic embryo.
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Klein, C., S. Bauersachs, S. Ulbrich, H. Meyer, S. Schmidt, H. Reichenbach, M. Vermehren, H. Blum, F. Sinowatz, and E. Wolf. "2 IDENTIFICATION OF GENES INDUCED BY THE CONCEPTUS IN THE BOVINE ENDOMETRIUM DURING THE PRE-IMPLANTATION PERIOD." Reproduction, Fertility and Development 18, no. 2 (2006): 109. http://dx.doi.org/10.1071/rdv18n2ab2.
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Early embryonic development, implantation, and maintenance of a pregnancy are critically dependent on an intact embryo-maternal communication. So far, only few signals involved in this dialogue have been identified. In ruminants, interferon tau (IFN�) plays a key role in the process of maternal recognition of pregnancy by exhibiting antiluteolytic activity. Even though many experimental findings indicate a pivotal role of IFN� in the context of embryo-maternal communication in ruminants, a number of other systems may be involved. To identify genes induced in the bovine endometrium by the signaling of the embryo, a combination of subtracted cDNA libraries and cDNA array hybridization was applied. Monozygotic twin pairs (n = 5) were used as the biological model. Pregnancy was created in one twin by transferring two in vitro-produced embryos on Day 7 of the estrous cycle; the other twin received a sham embryo transfer. Pregnant and nonpregnant twins were slaughtered at Day 18; endometrial tissue samples were recovered and processed for transcriptome analysis as described (Bauersachs et al. 2005 J. Mol. Endocrinol. 34, 889-908). Screening of 4608 clones of two subtracted libraries revealed 90 different up-regulated genes and mRNAs, of which almost 50% are known to be stimulated by type I interferons. Among these interferon-stimulated genes, the ISG15 system is assumed to be of particular interest, and several components were studied in more detail using in situ hybridization. The pattern of mRNA expression suggests that modification of endometrial proteins through ISG15ylation plays a fundamental role during the pre-implantation period. A classification of the identified genes based on Gene Ontologies revealed the prevalence of genes involved in regulation of gene expression, cell communication, cell growth, cell differentiation, cell proliferation, and cell adhesion, and also the prevalence of genes with immune-related functions. These results underline the intense response of the endometrium to the presence of a conceptus, culminating in the preparation of the maternal environment for embryonic implantation. Further, for eleven selected genes the expression in the endometrium was quantified by the use of real-time RT-PCR. Overall, the results of quantitative RT-PCR and array hybridization correlated very well. To our knowledge this study provides the first holistic gene expression analysis of the bovine endometrium during the pre-implantation period. The results underline the importance of IFN� as an embryo-derived pregnancy recognition signal and depict the molecular mechanisms at the mRNA level underlying the intense embryo-maternal dialog taking place at Day 18 of gestation.