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1
Toralová, Tereza, Veronika Benešová, Kateřina Vodičková Kepková, Petr Vodička, Andrej Šušor, and Jiří Kaňka. "Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage." REPRODUCTION 144, no. 3 (September 2012): 349–59. http://dx.doi.org/10.1530/rep-12-0033.
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This study was conducted to investigate the effect of silencing nucleophosmin in the development of in vitro-produced bovine embryos. Nucleophosmin is an abundant multifunctional nucleolar phosphoprotein that participates, for example, in ribosome biogenesis or centrosome duplication control. We showed that although the transcription of embryonic nucleophosmin started already at late eight-cell stage, maternal protein was stored throughout the whole preimplantation development and was sufficient for the progression to the blastocyst stage. At the beginning of embryogenesis, translation occurs on maternally derived ribosomes, the functionally active nucleoli emerge during the fourth cell cycle in bovines. We found that nucleophosmin localisation reflected the nucleolar formation during bovine preimplantation development. The protein was detectable from the beginning of embryonic development. Before embryonic genome activation, it was dispersed throughout the nucleoplasm. The typical nucleolar localisation emerged with the formation of active nucleoli. At the blastocyst stage, nucleophosmin tended to localise especially to the trophectoderm. To see for how long is maternal nucleophosmin preserved, we silenced the nucleophosmin mRNA using RNA interference approach. Although a large portion of nucleophosmin was degraded in embryos with silenced nucleophosmin mRNA, an amount sufficient for normal development was preserved and we detected only a temporal delay in nucleophosmin relocalisation to nucleoli. Moreover, we observed no defects in nuclear shape or cytoskeleton previously found in somatic cells and only a non-significant decrease in embryonic developmental competence. Thus, our results show that the preserved amount of maternal nucleophosmin is sufficient for preimplantation development of bovine embryo.
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Fu, Yao, Jia-Jun Xu, Xu-Lei Sun, Hao Jiang, Dong-Xu Han, Chang Liu, Yan Gao, Bao Yuan, and Jia-Bao Zhang. "Function of JARID2 in bovines during early embryonic development." PeerJ 5 (December 21, 2017): e4189. http://dx.doi.org/10.7717/peerj.4189.
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Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC) differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV), MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05), and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc) after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the efficiency ofin vitroembryo culture as well as a theoretical basis for further studying the regulatory mechanisms involved in early embryonic development.
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Perera, Chalani Dilshani, Muhammad Idrees, Abdul Majid Khan, Zaheer Haider, Safeer Ullah, Ji-Su Kang, Seo-Hyun Lee, Seon-Min Kang, and Il-Keun Kong. "PDGFRβ Activation Induced the Bovine Embryonic Genome Activation via Enhanced NFYA Nuclear Localization." International Journal of Molecular Sciences 24, no. 23 (December 1, 2023): 17047. http://dx.doi.org/10.3390/ijms242317047.
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Embryonic genome activation (EGA) is a critical step during embryonic development. Several transcription factors have been identified that play major roles in initiating EGA; however, this gradual and complex mechanism still needs to be explored. In this study, we investigated the role of nuclear transcription factor Y subunit A (NFYA) in bovine EGA and bovine embryonic development and its relationship with the platelet-derived growth factor receptor-β (PDGFRβ) by using a potent selective activator (PDGF-BB) and inhibitor (CP-673451) of PDGF receptors. Activation and inhibition of PDGFRβ using PDGF-BB and CP-673451 revealed that NFYA expression is significantly (p < 0.05) affected by the PDGFRβ. In addition, PDGFRβ mRNA expression was significantly increased (p < 0.05) in the activator group and significantly decreased (p < 0.05) in the inhibitor group when compared with PDGFRα. Downregulation of NFYA following PDGFRβ inhibition was associated with the expression of critical EGA-related genes, bovine embryo development rate, and implantation potential. Moreover, ROS and mitochondrial apoptosis levels and expression of pluripotency-related markers necessary for inner cell mass development were also significantly (p < 0.05) affected by the downregulation of NFYA while interrupting trophoblast cell (CDX2) differentiation. In conclusion, the PDGFRβ-NFYA axis is critical for bovine embryonic genome activation and embryonic development.
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Degrelle, Séverine A., Kim-Anh Lê Cao, Yvan Heyman, Robin E. Everts, Evelyne Campion, Christophe Richard, Céline Ducroix-Crépy, et al. "A small set of extra-embryonic genes defines a new landmark for bovine embryo staging." REPRODUCTION 141, no. 1 (January 2011): 79–89. http://dx.doi.org/10.1530/rep-10-0174.
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Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as ‘patterning’ the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.
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Stice, S. L. "Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer." Biology of Reproduction 54, no. 1 (January 1, 1996): 100–110. http://dx.doi.org/10.1095/biolreprod54.1.100.
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Asl, M. Pashai, K. Khodadadi, M. K. Holland, N. M. Richings, and P. J. Verma. "430. Investigation of pluripotency in derived embryonic stem cell (ESC) lines." Reproduction, Fertility and Development 20, no. 9 (2008): 110. http://dx.doi.org/10.1071/srb08abs430.
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To produce autologous ESCs for a bovine model of cell therapy, we activated oocytes by calcium ionophore (CI) and 6 dimethylaminopurine (6 DMAP) and isolated ESCs from the resulting parthenotes. Parthenote ESC lines (pbESC) would also provide a valuable tool for epigenetic studies on ESCs. Five pbESC like-cell lines were expanded for 12 passages over 120 days and differentiated to form embryoid bodies by suspension culture. The pbESC lines demonstrated typical ESC morphology and expressed ESC markers including alkaline phosphate and stage-specific embryonic antigen, SSEA1 and SSAE4 asssessed by histochemical and immuno-fluorescence staining, respectively. In addition, gene expression of Oct4, Rex1, SSEA1 and ALP was confirmed using RT–PCR. These cells had a normal karyotype. The cells formed EBs and showed expression of the markers of three embryonic germ layers. In summary, we show that ESCs can be derived from bovine parthenogenetic blastocysts and that these cells express pluripotent markers and have ability to form EBs and differentiate into cells indicative of the three embryonic germ layers. Additional work will focus on imprinted gene expression and will provide further evidence of the parthenogenetic origin of the pbESC lines.
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Saadeldin, Islam M., Ahmed Abdelfattah-Hassan, and Ayman Abdel-Aziz Swelum. "Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells." BioMed Research International 2017 (2017): 1–6. http://dx.doi.org/10.1155/2017/1061589.
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Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs) compared to the conventional mouse embryonic fibroblasts (MEFs) were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2), cytokeratin-8 (KRT8), and interferon tau (IFNT). The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.
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Wu, Shanshan, Xiaoyu Zhao, Meiling Wu, Lei Yang, Xuefei Liu, Danyi Li, Han Xu, et al. "Low Expression of Mitofusin 1 Gene Leads to Mitochondrial Dysfunction and Embryonic Genome Activation Failure in Ovine-Bovine Inter-Species Cloned Embryos." International Journal of Molecular Sciences 23, no. 17 (September 4, 2022): 10145. http://dx.doi.org/10.3390/ijms231710145.
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Inter-species somatic cell nuclear transfer (iSCNT) is significant in the study of biological problems such as embryonic genome activation and the mitochondrial function of embryos. Here, we used iSCNT as a model to determine whether abnormal embryo genome activation was caused by mitochondrial dysfunction. First, we found the ovine-bovine iSCNT embryos were developmentally blocked at the 8-cell stage. The reactive oxygen species level, mitochondrial membrane potential, and ATP level in ovine-bovine cloned embryos were significantly different from both bovine-bovine and IVF 8-cell stage embryos. RNA sequencing and q-PCR analysis revealed that mitochondrial transport, mitochondrial translational initiation, mitochondrial large ribosomal subunit, and mitochondrial outer membrane genes were abnormally expressed in the ovine-bovine embryos, and the mitochondrial outer membrane and mitochondrial ribosome large subunit genes, mitochondrial fusion gene 1, and ATPase Na+/K+ transporting subunit beta 3 gene were expressed at lower levels in the ovine-bovine cloned embryos. Furthermore, we found that overexpression and knockdown of Mfn1 significantly affected mitochondrial fusion and subsequent biological functions such as production of ATP, mitochondrial membrane potential, reactive oxygen species and gene expressions in cloned embryos. These findings enhance our understanding of the mechanism by which the Mfn1 gene regulates embryonic development and embryonic genome activation events.
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Speckhart, Savannah L., Mary A. Oliver, and Alan D. Ealy. "Developmental Hurdles That Can Compromise Pregnancy during the First Month of Gestation in Cattle." Animals 13, no. 11 (May 25, 2023): 1760. http://dx.doi.org/10.3390/ani13111760.
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Several key developmental events are associated with early embryonic pregnancy losses in beef and dairy cows. These developmental problems are observed at a greater frequency in pregnancies generated from in-vitro-produced bovine embryos. This review describes critical problems that arise during oocyte maturation, fertilization, early embryonic development, compaction and blastulation, embryonic cell lineage specification, elongation, gastrulation, and placentation. Additionally, discussed are potential remediation strategies, but unfortunately, corrective actions are not available for several of the problems being discussed. Further research is needed to produce bovine embryos that have a greater likelihood of surviving to term.
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Yang, Cai-Xia, Zichuan Liu, Renaud Fleurot, Pierre Adenot, Véronique Duranthon, Xavier Vignon, Qi Zhou, Jean-Paul Renard, and Nathalie Beaujean. "Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development." REPRODUCTION 145, no. 2 (February 2013): 149–59. http://dx.doi.org/10.1530/rep-11-0421.
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To investigate the embryonic genome organization upon fertilization and somatic cell nuclear transfer (SCNT), we tracked HP1β and CENP, two well-characterized protein markers of pericentric and centromeric compartments respectively, in four types of embryos produced by rabbit in vivo fertilization, rabbit parthenogenesis, rabbit-to-rabbit, and bovine-to-rabbit SCNT. In the interphase nuclei of rabbit cultured fibroblasts, centromeres and associated pericentric heterochromatin are usually isolated. Clustering into higher-order chromatin structures, such as the chromocenters seen in mouse and bovine somatic cells, could not be observed in rabbit fibroblasts. After fertilization, centromeres and associated pericentric heterochromatin are quite dispersed in rabbit embryos. The somatic-like organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major embryonic genome activation in this species. In SCNT embryos, pericentric heterochromatin distribution typical for rabbit and bovine somatic cells was incompletely reverted into the 1-cell embryonic form with remnants of heterochromatin clusters in 100% of bovine-to-rabbit embryos. Subsequently, the donor cell nuclear organization was rapidly re-established by the 4-cell stage. Remarkably, the incomplete remodeling of bovine-to-rabbit 1-cell embryos was associated with delayed transcriptional activation compared with rabbit-to-rabbit embryos. Together, the results confirm that pericentric heterochromatin spatio-temporal reorganization is an important step of embryonic genome reprogramming. It also appears that genome reorganization in SCNT embryos is mainly dependent on the nuclear characteristics of the donor cells, not on the recipient cytoplasm.
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Kwon, Dae Kee, So Gun Hong, Jung Eun Park, Hee Jung Park, Jung Taek Kang, Ok Jae Koo, and Byeong Chun Lee. "Establishment of Bovine Embryonic Stem-Like Cells from Cloned Bovine Blastocyst." Biology of Reproduction 78, Suppl_1 (May 1, 2008): 229. http://dx.doi.org/10.1093/biolreprod/78.s1.229b.
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Arias, M. E., R. Sánchez, J. Risopatrón, L. Pérez, and R. Felmer. "Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection." Reproduction, Fertility and Development 26, no. 6 (2014): 847. http://dx.doi.org/10.1071/rd13009.
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The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10–50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.
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KANEDA, Masahiro, Masashi TAKAHASHI, Ken-ichi YAMANAKA, Koji SAITO, Masanori TANIGUCHI, Satoshi AKAGI, Shinya WATANABE, and Takashi NAGAI. "Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts." Journal of Reproduction and Development 63, no. 4 (2017): 365–75. http://dx.doi.org/10.1262/jrd.2017-040.
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ITOH, Takako, Yoshito AOYAGI, Masato KONISHI, Hatsue ITAKURA, Toshiro TAKEDOMI, Shigeto YAZAWA, and Kayoko AKANE. "Nuclear Transfer of Bovine Embryonic Disc Cells." Journal of Reproduction and Development 44, no. 6 (1998): j27—j32. http://dx.doi.org/10.1262/jrd.98-446j27.
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Mengeling, William L., and Martin J. Maaten. "Preparation of bovine embryonic spleen cell cultures." Journal of Tissue Culture Methods 11, no. 3 (September 1988): 135–38. http://dx.doi.org/10.1007/bf01404266.
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Itoh, T., Y. Aoyagi, M. Konishi, H. Itakura, T. Takedomi, S. Yazawa, and K. Akane. "Nuclear transfer of bovine embryonic disc cells." Theriogenology 49, no. 1 (January 1998): 322. http://dx.doi.org/10.1016/s0093-691x(98)90675-6.
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Gómez, E., JN Caamaño, A. Rodríguez, C. De Frutos, N. Facal, and C. Díez. "Bovine Early Embryonic Development and Vitamin A." Reproduction in Domestic Animals 41, s2 (October 2006): 63–71. http://dx.doi.org/10.1111/j.1439-0531.2006.00770.x.
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Plante, C., D. Bousquet, P. Guay, A. K. Goff, and W. A. King. "Luteotrophic factor secreted by bovine embryonic tissue." Theriogenology 23, no. 1 (January 1985): 217. http://dx.doi.org/10.1016/0093-691x(85)90123-2.
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Bogliotti, Yanina S., Nhi Chung, Erika E. Paulson, James Chitwood, Michelle Halstead, Colin Kern, Richard M. Schultz, and Pablo J. Ross. "Transcript profiling of bovine embryos implicates specific transcription factors in the maternal-to-embryo transition." Biology of Reproduction 102, no. 3 (November 11, 2019): 671–79. http://dx.doi.org/10.1093/biolre/ioz209.
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Abstract Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.
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Vallée, Maud, Isabelle Dufort, Stéphanie Desrosiers, Aurélie Labbe, Catherine Gravel, Isabelle Gilbert, Claude Robert, and Marc-André Sirard. "Revealing the bovine embryo transcript profiles during early in vivo embryonic development." REPRODUCTION 138, no. 1 (July 2009): 95–105. http://dx.doi.org/10.1530/rep-08-0533.
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Gene expression profiling is proving to be a powerful approach for the identification of molecular mechanisms underlying complex cellular functions such as the dynamic early embryonic development. The objective of this study was to perform a transcript abundance profiling analysis of bovine early embryonic development in vivo using a bovine developmental array. The molecular description of the first week of life at the mRNA level is particularly challenging when considering the important fluctuations in RNA content that occur between developmental stages. Accounting for the different intrinsic RNA content between developmental stages was achieved by restricting the reaction time during the global amplification steps and by using spiked controls and reference samples. Analysis based on intensity values revealed that most of the transcripts on the array were present at some point during in vivo bovine early embryonic development, while the varying number of genes detected in each developmental stage confirmed the dynamic profile of gene expression occurring during embryonic development. Pair-wise comparison of gene expression showed a marked difference between oocytes and blastocysts profiles, and principal component analysis revealed that the majority of the transcripts could be regrouped into three main clusters representing distinct RNA abundance profiles. Overall, these data provide a detailed temporal profile of the abundance of mRNAs revealing the richness of signaling processes in early mammalian development. Results presented here provide better knowledge of bovine in vivo embryonic development and contribute to the progression of our current knowledge regarding the first week of life in mammals.
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Choi, W. J., S. J. Lee, W. W. Lee, S. J. Kim, I. M. Saadeldin, J. K. Cho, B. C. Lee, and G. Jang. "52 IMPLANTATION OF TRANSGENIC BOVINE CLONED EMBRYOS DERIVED FROM TRANSFECTED CELLS BY PiggyBac TRANSPOSITION." Reproduction, Fertility and Development 25, no. 1 (2013): 173. http://dx.doi.org/10.1071/rdv25n1ab52.
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A gene-delivery system, PiggyBac (PB) transposition, has been applied to transgene expression in mammalian cells or animals. In this study, to produce transgenic cattle, we used PB in bovine fibroblasts and then the transfected cells were microinjected into enucleated bovine oocytes to produce embryos and offspring. For this study, 2 different fluorescence genes (GFP, transcribed by constitutive promoter and RFP, transcribed by tetracycline-dependent promoter), which were flanked by PB sequences, were transfected into the bovine fetal fibroblasts by the FuGENE transfection protocol. The developmental rate of blastocysts among the cleaved embryos derived from GFP cells and doxycycline-induced RFP cells was developed at 23.1% (31/134) and 40.9% (442/1082), respectively. After transferring the GFP- or RFP-expressing blastocysts into recipient cows, pregnancies were detected by ultrasonography from both recipients of GFP or RFP. To know gene expression in fetal stage, embryonic sacs were collected surgically. The primary cells were successfully isolated from both embryonic sacs. Every cell from the GFP embryonic sac expressed GFP. When the cells from the RFP embryonic sac were treated with doxycycline, RFP was homogenously expressed. In conclusion, this study demonstrated that PB transposition could be applied to deliver genes into bovine somatic cells. Furthermore, transgenic embryos from transfected cells using the PB system were developed into blastocysts, implanted, and were able to form embryonic sacs. The PB system will be a useful method to produce transgenic cattle. This study was financially supported by IPET (grant no. 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 Program for Veterinary Science.
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Yang, Qi-En, Manabu Ozawa, Kun Zhang, Sally E. Johnson, and Alan D. Ealy. "The requirement for protein kinase C delta (PRKCD) during preimplantation bovine embryo development." Reproduction, Fertility and Development 28, no. 4 (2016): 482. http://dx.doi.org/10.1071/rd14160.
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Protein kinase C (PKC) delta (PRKCD) is a member of the novel PKC subfamily that regulates gene expression in bovine trophoblast cells. Additional functions for PRKCD in early embryonic development in cattle have not been fully explored. The objectives of this study were to describe the expression profile of PRKCD mRNA in bovine embryos and to examine its biological roles during bovine embryo development. Both PRKCD mRNA and protein are present throughout early embryo development and increases in mRNA abundance are evident at morula and blastocyst stages. Phosphorylation patterns are consistent with detection of enzymatically active PRKCD in bovine embryos. Exposure to a pharmacological inhibitor (rottlerin) during early embryonic development prevented development beyond the eight- to 16-cell stage. Treatment at or after the 16-cell stage reduced blastocyst development rates, total blastomere numbers and inner cell mass-to-trophoblast cell ratio. Exposure to the inhibitor also decreased basal interferon tau (IFNT) transcript abundance and abolished fibroblast growth factor-2 induction of IFNT expression. Furthermore, trophoblast adhesion and proliferation was compromised in hatched blastocysts. These observations provide novel insights into PRKCD mRNA expression profiles in bovine embryos and provide evidence for PRKCD-dependent regulation of embryonic development, gene expression and post-hatching events.
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Bowen, R. A., R. P. Elsden, and G. E. Seidel. "Infection of early bovine embryos with bovine herpesvirus-1." American Journal of Veterinary Research 46, no. 5 (May 1, 1985): 1095–97. https://doi.org/10.2460/ajvr.1985.46.05.1095.
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SUMMARY Recently hatched bovine embryos were exposed in vitro to 1 of 4 strains of bovine herpesvirus-1 to determine whether the viruses would replicate in these embryos and, if so, what pathologic consequences would ensue. Exposure to each of the viruses resulted in embryonic infection and death, and replication of the agents was demonstrated by electron microscopy and titration of progeny virus. There were no dramatic differences between virus strains in pathogenicity or in the ultrastructural pathologic findings of infection.
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Torres, Ana Catarina, Dorota Boruszewska, Mariana Batista, Ilona Kowalczyk-Zieba, Patricia Diniz, Emilia Sinderewicz, Jean Sebastian Saulnier-Blache, Izabela Woclawek-Potocka, and Luis Lopes-da-Costa. "Lysophosphatidic Acid Signaling in Late Cleavage and Blastocyst Stage Bovine Embryos." Mediators of Inflammation 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/678968.
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Lysophosphatidic acid (LPA) is a known cell signaling lipid mediator in reproductive tissues. In the cow, LPA is involved in luteal and early pregnancy maintenance. Here, we evaluated the presence and role of LPA in bovine early embryonic development. In relevant aspects, bovine embryos reflect more closely the scenario occurring in human embryos than the mouse model. Transcription of mRNA and protein expression of enzymes involved in LPA synthesis (ATX andcPLA2) and of LPA receptors (LPAR1–4) were detected in Days 5 and 8in vitroproduced embryos. Embryonic LPA production into culture medium was also detected at both stages of development. Supplementation of culture medium with LPA (10−5 M) between Days 2 and 8 had no effect on embryo yield and quality and on blastocyst relative mRNA abundance of genes involved in prostaglandin synthesis (PTGS2,PGES, andPGFS) and steroidogenesis (3βHSD). However, LPA treatment affected transcription levels of embryo quality markers, decreasingBAX(apoptotic) and increasingBCL2(antiapoptotic) andIGF2R(growth marker) gene transcription levels. Blastocyst transcription ofOCT4(pluripotency marker) was not affected by LPA stimulation. In conclusion, LPA is an early bovine embryonic autocrine/paracrine signaling mediator, and LPA action may be relevant in early embryo-maternal interactions leading to embryonic survival.
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Memili, Erdogan, and Neal L. First. "Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species." Zygote 8, no. 1 (February 2000): 87–96. http://dx.doi.org/10.1017/s0967199400000861.
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Early embryonic development is largely dependent on maternal RNAs and proteins synthesised during oogenesis. Zygotic transcription is an essential event that occurs at a species-specific time after fertilisation. In the absence of zygotic transcription the embryo dies since it can no longer support requirements for successful embryo development. Molecular genetics of gene expression during early embryogenesis, especially in the bovine species, remain one of the unsolved questions in modern biology. Earlier studies suggested that embryonic transcription in cattle begins at the late 4-cell or 8-cell stage. However, more recent studies suggest that bovine zygotes and 2-cell embryos are both transcriptionally and translationally active. Moreover, changes in chromatin structure due to acetylation of core histones and DNA replication play important roles in the regulation of zygotic/embryonic gene expression. This review will summarise results of recent studies about the timing and mechanisms of zygotic/embryonic gene expression in cattle. In addition, terminology in the literature regarding gene expression during early embryogenesis will be clarified. These terminologies include: ‘zygotic/embryonic gene expression’, ‘maternal to embryonic transition in control of development (MET)’ and ‘zygotic/embryonic genome activation (ZEGA)’.
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DiGiacomo, Ronald F., Barbara J. Deeb, Scott J. Brodie, Thomas E. Zimmerman, Eugene R. Veltkamp, and Clarence E. Chrisp. "Toxin production by Pasteurella multocida isolated from rabbits with atrophic rhinitis." American Journal of Veterinary Research 54, no. 8 (August 1, 1993): 1280–86. http://dx.doi.org/10.2460/ajvr.1993.54.08.1280.
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Summary Naturally acquired turbinate atrophy in rabbits was associated with Pasteurella multocida infection. Several in vitro and in vivo studies were conducted to document toxin production from P multocida isolates and to determine the relation of toxin to atrophic rhinitis in rabbits. Ten isolates of P multocida serotype A:12 were obtained from adult New Zealand White rabbits with noninduced atrophic rhinitis. Specific-pathogen-free rabbits inoculated intranasally with isolates of P multocida developed rhinitis and turbinate atrophy. However, inoculation with filtrates of the same bacteria failed to induce turbinate atrophy. Cytotoxicity was observed in assays, using bovine embryonic turbinate cell cultures with extracts of P multocida, but not in agar overlay cytotoxicity assays, using bovine embryonic turbinate, bovine embryonic lung, or Vero cell cultures, or in a sandwich elisa, using monoclonal antibodies to purified P multocida toxin. Thus, turbinate atrophy was experimentally reproduced in rabbits with isolates of P multocida, but toxin was only detected in vitro by cell culture assay of P multocida extracts.
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Chang, Kai-Hsin, Angelique M. Nelson, Hua Cao, Linlin Wang, Betty Nakamoto, Carol B. Ware, and Thalia Papayannopoulou. "Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin." Blood 108, no. 5 (September 1, 2006): 1515–23. http://dx.doi.org/10.1182/blood-2005-11-011874.
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Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45+ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45- erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (ϵ) and fetal (γ) globins, with little or no adult globin (β). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms.
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Tripurani, S. K., K. B. Lee, G. W. Smith, and J. Yao. "6 CLONING AND EXPRESSION OF BOVINE FACTOR IN THE GERMLINE ALPHA (FIGLA) IN OOCYTES AND EARLY EMBRYOS: A POTENTIAL TARGET OF microRNA-212." Reproduction, Fertility and Development 23, no. 1 (2011): 109. http://dx.doi.org/10.1071/rdv23n1ab6.
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Factor In the GermLine Alpha (FIGLA), a basic helix-loop-helix transcription factor, was first identified in regulating coordinate expression of zona pellucida genes in mice. It plays a crucial role in the formation of primordial follicles and lack of FIGLA in mice alters the expression of many oocyte specific genes that are required for fertilization and early embryonic survival. The objective of this study was to characterise the expression and regulation of bovine FIGLA during early embryogenesis. The cloned bovine FIGLA cDNA is 660 bp in length, which encodes a protein of 165 amino acids. Expression of bovine FIGLA mRNA is restricted to ovarian tissue and can be detected in fetal ovaries harvested as early as 90 days of gestation when primordial follicles start to form. Expression analysis demonstrated that FIGLA mRNA is abundant in germinal vesicle and metaphase II stage oocytes, as well as in embryos from pronuclear to 8-cell stage, but barely detectable in embryos collected at morula and blastocyst stages, suggesting that FIGLA might be a maternal effect gene. Recent studies in zebrafish have highlighted the importance of non-coding small RNAs (microRNAs) as key regulatory molecules targeting maternal mRNAs for degradation during embryonic development. We hypothesised that FIGLA, as a maternal transcript, is regulated by microRNAs during early embryogenesis. Using microInspector, an algorithm for detection of possible interactions between microRNAs and target mRNA sequences, a microRNA binding site (miR-212) was identified in the 3′-UTR of the bovine FIGLA mRNA. Alignment of the 3′-UTR of FIGLA mRNAs from bovine, human and mouse shows complete conservation of the ‘seed’ region indicating that miR-212 might be a post-transcriptional regulator of FIGLA and the microRNA: mRNA interaction is evolutionary conserved. Expression analysis indicates that bovine miR-212 is expressed in oocytes and tends to increase at the 4-cell and 8-cell stage embryos followed by a decline at morula and blastocyst stages, indicating that miR-212 is presumably of maternal origin and potentially involved in maternal transcript degradation during the maternal-to-embryonic transition. To validate the role of miR-212 in silencing FIGLA, a luciferase reporter assay was performed using HeLa cells. The luciferase activity in cells expressing a luciferase construct containing the entire 3′ UTR of bovine FIGLA was suppressed by ∼40% in the presence of miR-212. We also investigated the stability of FIGLA mRNA in cells transfected with bovine FIGLA expression plasmid in the presence or absence of miR-212. Expression of bovine FIGLA mRNA was significantly reduced in the presence of mir-212 compared to control cells transfected with FIGLA construct alone. In summary, our data establish miR-212 as a potential post-transcriptional regulator of FIGLA during the maternal-to-embryonic transition in bovine embryos. Future studies aim to determine if miR-212 mimic can inhibit endogenous FIGLA expression in bovine embryos and its effect on subsequent embryonic development.
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Bauersachs, Stefan, Pascal Mermillod, and Carmen Almiñana. "The Oviductal Extracellular Vesicles’ RNA Cargo Regulates the Bovine Embryonic Transcriptome." International Journal of Molecular Sciences 21, no. 4 (February 14, 2020): 1303. http://dx.doi.org/10.3390/ijms21041303.
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Oviductal extracellular vesicles (oEVs) are emerging as key players in the gamete/embryo–oviduct interactions that contribute to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To determine whether these effects are associated with changes of embryonic gene expression, the transcriptomes of embryos supplemented with bovine fresh (FeEVs) or frozen (FoEVs) oEVs during in vitro culture compared to controls without oEVs were analyzed by low-input RNA sequencing. Analysis of RNA-seq data revealed 221 differentially expressed genes (DEGs) between FoEV treatment and control, 67 DEGs for FeEV and FoEV treatments, and minor differences between FeEV treatment and control (28 DEGs). An integrative analysis of mRNAs and miRNAs contained in oEVs obtained in a previous study with embryonic mRNA alterations pointed to direct effects of oEV cargo on embryos (1) by increasing the concentration of delivered transcripts; (2) by translating delivered mRNAs to proteins that regulate embryonic gene expression; and (3) by oEV-derived miRNAs which downregulate embryonic mRNAs or modify gene expression in other ways. Our study provided the first high-throughput analysis of the embryonic transcriptome regulated by oEVs, increasing our knowledge on the impact of oEVs on the embryo and revealing the oEV RNA components that potentially regulate embryonic development.
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Datta, Tirtha K., Sandeep K. Rajput, Gabbine Wee, KyungBon Lee, Joseph K. Folger, and George W. Smith. "Requirement of the transcription factor USF1 in bovine oocyte and early embryonic development." REPRODUCTION 149, no. 2 (February 2015): 203–12. http://dx.doi.org/10.1530/rep-14-0445.
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Upstream stimulating factor 1 (USF1) is a basic helix–loop–helix transcription factor that specifically binds to E-box DNA motifs, knowncis-elements of key oocyte expressed genes essential for oocyte and early embryonic development. However, the functional and regulatory role of USF1 in bovine oocyte and embryo development is not understood. In this study, we demonstrated thatUSF1mRNA is maternal in origin and expressed in a stage specific manner during the course of oocyte maturation and preimplantation embryonic development. Immunocytochemical analysis showed detectable USF1 protein during oocyte maturation and early embryonic development with increased abundance at 8–16-cell stage of embryo development, suggesting a potential role in embryonic genome activation. Knockdown ofUSF1in germinal vesicle stage oocytes did not affect meiotic maturation or cumulus expansion, but caused significant changes in mRNA abundance for genes associated with oocyte developmental competence. Furthermore, siRNA-mediated depletion ofUSF1in presumptive zygote stage embryos demonstrated thatUSF1is required for early embryonic development to the blastocyst stage. A similar (USF2) yet unique (TWIST2) expression pattern during oocyte and early embryonic development for related E-box binding transcription factors known to cooperatively bind USF1 implies a potential link to USF1 action. This study demonstrates that USF1 is a maternally derived transcription factor required for bovine early embryonic development, which also functions in regulation ofJY1, GDF9, andFSTgenes associated with oocyte competence.
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Wang, Xue, Lili Guo, and Wenguang Zhang. "Extraction of Innate Immune Genes in Dairy Cattle and the Regulation of Their Expression in Early Embryos." Genes 15, no. 3 (March 18, 2024): 372. http://dx.doi.org/10.3390/genes15030372.
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As more and more of the available genomic data have been published, several databases have been developed for deciphering early mammalian embryogenesis; however, less research has been conducted on the regulation of the expression of natural immunity genes during early embryonic development in dairy cows. To this end, we explored the regulatory mechanism of innate immunity genes at the whole-genome level. Based on comparative genomics, 1473 innate immunity genes in cattle were obtained by collecting the latest reports on human innate immunity genes and updated bovine genome data for comparison, and a preliminary database of bovine innate immunity genes was constructed. In order to determine the regulatory mechanism of innate immune genes in dairy cattle early embryos, we conducted weighted co-expression network analysis of the innate immune genes at different developmental stages of dairy cattle early embryos. The results showed that specific module-related genes were significantly enriched in the MAPK signaling pathway. Protein–protein interaction (PPI) analysis showed gene interactions in each specific module, and 10 of the highest connectivity genes were chosen as potential hub genes. Finally, combined with the results for differential expressed genes (DEGs), ATF3, IL6, CD8A, CD69, CD86, HCK, ERBB3, LCK, ITGB2, LYN, and ERBB2 were identified as the key genes of innate immunity in dairy cattle early embryos. In conclusion, the bovine innate immunity gene set was determined and the co-expression network of innate immunity genes in the early embryonic stage of dairy cattle was constructed by comparing and analyzing the whole genome of bovines and humans. The findings in this study provide the basis for exploring the involvement and regulation of innate immune genes in the early embryonic development of dairy cattle.
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Avila-Alejandre, Alma X., Fulgencio Espejel, Esmeralda Paz-Lemus, Edith Cortés-Barberena, Fernando Díaz de León-Sánchez, Tzvetanka D. Dinkova, Estela Sánchez de Jiménez, and Laura J. Pérez-Flores. "Effect of insulin on the cell cycle of germinating maize seeds (Zea mays L.)." Seed Science Research 23, no. 1 (January 11, 2013): 3–14. http://dx.doi.org/10.1017/s0960258512000281.
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AbstractDuring seed germination, metabolism is reactivated, DNA is repaired and cell division is restarted in the meristems. The mechanisms that co-ordinate cell growth and division in maize embryonic axes during germination are not well understood. However, the presence of a factor similar to IGF (insulin-like growth factor) that accelerates germination has been reported. In the present work, the regulation of the cell-cycle restart by bovine insulin [which has been demonstrated to produce similar effects as insulin-like growth factor of maize (ZmIGF) in maize seeds] was studied in germinating embryonic axes. Our results showed that bovine insulin differentially stimulates growth, S6K phosphorylation, S6rp transcript accumulation on the polysomal fraction, as well as de novo DNA synthesis in the radicles and the coleoptiles of the embryonic axis. A stronger and earlier effect was observed in radicles compared to coleoptiles; therefore, the effect of insulin on the cell cycle of the root meristem was studied by flow cytometry. The G1–S transition was stimulated and cell proliferation was induced. Furthermore, it was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) that bovine insulin increased E2F and PCNA (proliferating cell nuclear antigen) transcription after 15 h of germination and PCNA de novo synthesis at 15 h of germination. These results show that bovine insulin preferentially stimulates growth in the radicles of germinating embryonic axes and suggest that its effect on the G1–S transition and the activation of cell proliferation is mediated by the induction of E2F and PCNA transcription.
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Zhang, K., and H. Wang. "61 Expression Profiles and Functional Roles of H3.3 and HIRA in Bovine Early Embryos." Reproduction, Fertility and Development 30, no. 1 (2018): 169. http://dx.doi.org/10.1071/rdv30n1ab61.
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Early embryo death is one major reason for poor reproductive efficiency in dairy cows. In particular, ~20 to 50% of high-producing cows are subject to pregnancy loss during the first week of gestation, indicating the importance of embryonic development from fertilization to the blastocyst stage. To highlight this importance, multiple critical molecular and developmental events, including zygote reprogramming, maternal RNA decay, and embryonic genome activation, occur during bovine pre-implantation development. However, the molecular mechanisms of these events have yet to be defined. H3.3 is a histone H3 variant that encoded by 2 genes, namely, H3F3A and H3F3B. It is generally believed that H3.3 is closely related to active transcribed genes. Of interest, H3.3 required for establishing proper chromatin structure during mouse oogenesis. Immediately following fertilization, H3.3 is incorporated to parental chromatins and essential for blastocyst formation in mice. HIRA is a chaperone for H3.3 deposition and indispensable for zygote development. Previously, our results showed that H3.3 is needed for bovine early embryonic development. Herein, experiments were designed to determine the mechanisms of functional requirement of H3.3 in bovine early embryos. Slaughterhouse-derived cumulus–oocyte complexes (COC) were matured in vitro and IVF was performed. To knock down genes of interest, small interfering (si)RNAs were delivered into zygotes via microinjection. The qPCR results showed that H3F3A mRNA level is stable, whereas H3F3B and HIRA mRNA are dynamic during early embryonic development (4 replicates). The mRNA abundance of H3F3B is significantly higher than that of H3F3A (4 replicates; P < 0.05), which is also found in mouse and human. Immunostaining results revealed a stage-specific pattern for the localization of H3.3 in bovine early embryos, and the H3.3 signal was not different between paternal and maternal pronuclei in zygotes, which was different from the pattern in mice. The siRNA-mediated silencing of H3.3 dramatically reduces the expression of CTGF (a putative trophectoderm marker) in bovine blastocysts (3 replicates; P < 0.05). Furthermore, we found that the signal intensity of dimethylation of histone H3 lysine 36 (H3K36me2) and linker histone H1 decreases in H3.3-ablated embryos, which is similar to CHD1 knockdown (3 replicates; P < 0.05). However, no difference was found for the intensity of trimethylation of histone H3 lysine 4, dimethylation of histone H3 lysine 9 (H3K9me2) and splicing factor 3 B1 (SF3B1). We also found that HIRA deletion does not affect bovine early embryonic development. Taken together, the results described herein suggest that H3.3 is required for proper epigenetic modifications and H1 deposition during bovine early embryonic development. This project was supported by National Natural Science Foundation of China grant (No. 31672416) and the Fundamental Research Funds for the Central Universities.
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Vigneault, Christian, Catherine Gravel, Maud Vallée, Serge McGraw, and Marc-André Sirard. "Unveiling the bovine embryo transcriptome during the maternal-to-embryonic transition." REPRODUCTION 137, no. 2 (February 2009): 245–57. http://dx.doi.org/10.1530/rep-08-0079.
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Bovine early embryos are transcriptionally inactive and subsist through the initial developmental stages by the consumption of the maternal supplies provided by the oocyte until its own genome activation. In bovine, the activation of transcription occurs during the 8- to 16-cell stages and is associated with a phase called the maternal-to-embryonic transition (MET) where maternal mRNA are replaced by embryonic ones. Although the importance of the MET is well accepted, since its inhibition blocks embryonic development, very little is known about the transcripts expressed at this crucial step in embryogenesis. In this study, we generated and characterized a cDNA library enriched in embryonic transcripts expressed at the MET in bovine. Suppression subtractive hybridization followed by microarray hybridization was used to isolate more than 300 different transcripts overexpressed in untreated late eight-cell embryos compared with those treated with the transcriptional inhibitor, α-amanitin. Validation by quantitative RT-PCR of 15 genes from this library revealed that they had remarkable consistency with the microarray data. The transcripts isolated in this cDNA library have an interesting composition in terms of molecular functions; the majority is involved in gene transcription, RNA processing, or protein biosynthesis, and some are potentially involved in the maintenance of pluripotency observed in embryos. This collection of genes associated with the MET is a novel and potent tool that will be helpful in the understanding of particular events such as the reprogramming of somatic cells by nuclear transfer or for the improvement of embryonic culture conditions.
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Berg, D. K., S. E. Beaumont, and P. L. Pfeffer. "168 miRNA LEVELS DURING BOVINE PREIMPLANTATION EMBRYONIC DEVELOPMENT." Reproduction, Fertility and Development 20, no. 1 (2008): 164. http://dx.doi.org/10.1071/rdv20n1ab168.
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MicroRNAs (miRNAs) are a class of naturally occurring non-coding RNAs that play a role in gene regulation. They are highly conserved, single-stranded RNAs, 22 nucleotides in length, that are cleaved from larger inactive hairpin precursor transcripts, and use the RNA interference-related pathways to repress their mRNA targets. They play diverse regulatory roles in cellular proliferation, morphogenesis, apoptosis, and differentiation. Maternal miRNAs are crucial for early mammalian development (Murchison et al. 2007 Genes Dev. 21, 682–693; Tang et al. 2007 Genes Dev. 21, 655–648), while sperm-borne miRNAs do not contribute significantly to miRNAs in the zygote (Amanai et al. 2006 Biol. Reprod. 75, 877–884). Our objective was to identify miRNAs that are expressed during bovine in vitro oocyte maturation (MII) and blastocyst stages as well as during parthenogenic development. MII oocytes (n = 1680) were generated from abattoir-derived oocytes and matured in vitro for 24 h. Cumulus cells were removed and the first polar body was visually assessed before the oocytes were frozen in liquid N2. Parthenogenic blastocysts (n = 575) were produced using ionomycin/6DMAP activation, and IVF blastocysts (n = 1150) were produced using standard in vitro fertilization followed by in vitro culture in synthetic oviduct fluid (Thompson et al. 2000 J. Reprod. Fertil. 118, 47–55). Blastocysts (grades 1 and 2) were selected on Day 7 post-activation/insemination and frozen in liquid N2. RNA was isolated using the mirVana miRNA isolation kit (Ambion, Scoresby, Victoria, Australia). miRNAs were quantified using the TaqMan� MicroRNA Human Panel-Early Access Kit (Applied Biosystems, Scoresby, Victoria, Australia) following the manufacturer's protocol. Absolute copy numbers per embryo were estimated. Of the 157 miRNAs in the panel, 102, 136, and 118 were detected above background in oocytes, IVF, and parthenogenic blastocysts, respectively. Only 28 miRNAs were present at over 100 copies in MII oocytes, with maximum levels reaching 1300 copies. Levels were generally much higher at blastocyst stages, with 21 miRNAs present at more than 10 000 copies. miR-16 was one of the most abundant miRNAs in all samples tested. Copy numbers per blastomere cell were 5-fold higher in IVF blastocysts compared to parthegenotic blastocysts for miR-19a, 21, and 30b. The low copy numbers of mature miRNAs before embryonic genome activation may have implications for somatic cell nuclear transfer experiments in that exogenously added miRNAs from the donor cell could impact on the embryonic gene expression profiles.
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Kalenberg, Christina Alexandra, and Michael Hubert Stoffel. "The embryonic development of the bovine stomach revisited." Anatomia, Histologia, Embryologia 49, no. 2 (March 2020): 270–80. http://dx.doi.org/10.1111/ahe.12525.
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Strelchenko, N., S. Saito, and H. Niemann. "Towards the establishment of bovine embryonic stem cells." Theriogenology 35, no. 1 (January 1991): 274. http://dx.doi.org/10.1016/0093-691x(91)90250-h.
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Barnes, F. L., and N. L. First. "Embryonic transcription in in vitro cultured bovine embryos." Molecular Reproduction and Development 29, no. 2 (June 1991): 117–23. http://dx.doi.org/10.1002/mrd.1080290205.
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Johnson, F. Brent, Laura B. Fenn, Thomas J. Owens, Laura J. Faucheux, and Shawn D. Blackburn. "Attachment of bovine parvovirus to sialic acids on bovine cell membranes." Journal of General Virology 85, no. 8 (August 1, 2004): 2199–207. http://dx.doi.org/10.1099/vir.0.79899-0.
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Although it has previously been shown that bovine parvovirus (BPV) attaches to the sialated glycoprotein glycophorin A on erythrocytes, the nature of virus-binding moieties on mammalian nucleated cells is less clear. Buffalo lung fibroblasts (Bu), primary bovine embryonic kidney cells, Madin–Darby bovine kidney cells and bovine embryonic trachea (EBTr) cells were assessed for molecules capable of binding BPV. Competition studies were carried out on both erythrocyte and nucleated cell targets using a variety of sialated compounds and sialic acid-negative compounds. Glycophorin A was found to inhibit BPV binding, while mucin exhibited low-level inhibition. These two sialated compounds also blocked attachment of BPV-modified microsphere carriers to the Bu cell membrane. Influenza A virus was used as a sialic acid competitor and interfered with BPV attachment to erythrocytes and replication in Bu cells. Significantly, the enzyme sialidase removed BPV-binding sites from Bu and EBTr cells. The binding sites could be reconstituted on sialidase-treated cells by the enzymes α-2,3-O-sialyltransferase and α-2,3-N-sialyltransferase. These results indicated that BPV can attach to sialic acid on cell membranes and that the sialylglycoproteins available for virus attachment appear to contain both N- and O-linked carbohydrate moieties, but that not all members of the sialic acid family can bind BPV. Moreover, there may be other moieties that can bind BPV, which may act as either primary or secondary receptors.
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Poppicht, F., H. Stinshoff, and C. Wrenzycki. "70 PROTEIN EXPRESSION OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT." Reproduction, Fertility and Development 26, no. 1 (2014): 149. http://dx.doi.org/10.1071/rdv26n1ab70.
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Insulin-like growth factor 1 (IGF1) is essential for regulating physiological processes such as growth and development of fetal and placental tissues (Bauer et al. 1998, Fowden 2003). During early embryonic development, IGF1 plays an important role, as it leads to a reduction of apoptosis and decreases early embryonic mortality (Block et al. 2007). The signal transduction of IGF1 is carried out by its specific binding to the membrane-embedded insulin-like growth factor 1 receptor (IGF1R). The expression of IGF1R is a potential quality marker of in vitro produced embryos (Liu et al. 1997, Yaseen et al. 2001). Thus far, analysis of the relative amount of specific transcripts is the method of choice to study bovine pre-implantation embryos, as information on protein expression is scarce. Therefore, it is of great interest to analyse protein expression and to determine if and to which extent these results differ from results obtained in previous mRNA expression analyses. In the present study, a total of 4800 cumulus-oocyte-complexes were deployed in 60 in vitro produced runs. The cleavage rates averaged 57.4 ± 7.3% and blastocyst rates were 27.6 ± 7.5% at Day 8 of culture. Embryos at the blastocyst stage were frozen and stored at –80°C for further experiments. The protein expression of the IGF1R during early embryonic development was investigated by Western blot analysis testing 8 different antibodies. Seven of these antibodies were commercially available and mainly not tested in the bovine species. Only 1 of these antibodies resulted in a weak signal for the IGF1R protein in bovine blastocysts. Therefore, a specific peptide antibody against 2 peptide sequences of the α unit of the bovine IGF1R was produced. The analysis of the IGF1R protein with this antibody resulted in the determination of a signal in a pool of 100 blastocysts, which was weaker than in the positive control (20 μg of bovine liver protein extract). The detection of the IGF1R protein localization was possible in all different stages of embryonic development from the zygote to the expanded blastocyst using immunfluorescence staining with the specific peptide antibody. The IGF1R protein was mainly expressed in the plasma membrane of single blastomeres and also weakly in the cytoplasm. As the early bovine embryo expresses IGF1R throughout all stages, the main function of IGF1 in embryonic development needs to be further elucidated. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).
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Memili, E., E. Behboodi, H. M. Meade, and Y. Echelard. "60EPIGENETIC CHARACTERISTICS OF BOVINE AND CAPRINE EMBRYOS AND DONOR CELLS USED FOR NUCLEAR TRANSFER." Reproduction, Fertility and Development 16, no. 2 (2004): 152. http://dx.doi.org/10.1071/rdv16n1ab60.
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The molecular aspects of epigenetic events taking place in nuclear transfer (NT)-derived embryos are not well defined, but DNA methylation is known to be involved. One leading hypothesis is that the significant losses that occur during both pre- and post-implantation development are in great part due to improper epigenetic reprogramming. Aims of this study were to perform comparative quantitative analyses of the overall DNA methylation of bovine (both IVF- and NT-derived) and caprine IVF-derived preimplantation embryos as well as of donor cells used for NT. Caprine IVF was performed according to Blash S et al. (2001 Theriogenology 54, 899–905). Bovine and caprine 8- to 16-cell embryos were harvested on Day 4 post-insemination (dpi). Caprine donor cells (two adult fibroblast cell lines, T75-514 and F638) at the G1 phase of the cell cycle were prepared as previously described (Memili E et al. 2003 Theriogenology 59, 274 abst). Bovine cumulus cells were serum starved for four days prior to use and NT were performed as previously described (Echelard Y et al. 2002 Theriogenology 57, 779 abst). Embryos and the donor cells were vacuum-fixed onto 10-μm filters, and DNA methylation was determined by immunoassaying with a well-defined anti-5-methyl-cytosine antibody (Dean W et al. 2001 Prod Natl Acad Sci 98(24), 13734–13738) and fluorescent labeled anti-mouse IgG as secondary antibody following the protocol of Shi and Haaf (2002Mol Reprod Dev 63, 329–334). Fluorescent imaging was performed by epifluorescence microscopy. The methylation-specific signal was recorded digitally with a high-resolution charge-coupled devise camera, followed by analysis with the MetaMorph™ imaging software (Universal Imaging Corporation, Downingtown, PA, USA). These experiments showed a high level of heterogeneity in the methylation levels of bovine and caprine donor cells. However, caprine 8- to 16-cell IVF embryos exhibited similar levels of DNA methylation to their bovine IVF counterparts. Conversely, when the DNA methylation level of 97 bovine IVF nuclei was compared to that of 55 bovine NT embryonic nuclei, significant differences were found. Levels of signal intensities per nucleus were almost 9-fold greater for NT embryos (133 v. 15), a significant difference (P<0.01). In conclusion, donor cell populations with heterogeneous DNA methylation and incomplete reprogramming of DNA methylation in NT embryos are likely to be the underlying reasons for low level of successful NT embryonic and fetal development. Thus, designing NT protocols supporting better reprogramming may result in improvement since, in order for a successful embryonic development after NT, DNA methylation needs to be reprogrammed from a somatic cell pattern to an early embryonic pattern. Furthermore, similar levels of DNA methylation between caprine and bovine IVF embryos may be an indication of a similar pattern of embryonic gene expression between these species.
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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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Rodríguez-Alvarez, Lleretny, José Cox, Felipe Navarrete, Cristián Valdés, Teresa Zamorano, Ralf Einspanier, and Fidel Ovidio Castro. "Elongation and gene expression in bovine cloned embryos transferred to temporary recipients." Zygote 17, no. 4 (June 8, 2009): 353–65. http://dx.doi.org/10.1017/s0967199409005486.
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SummaryElongated embryos provide a unique source of information about trophoblastic differentiation, gene expression and maternal-embryonic interactions; however they are difficult and costly to obtain, especially elongated cloned embryos. One alternative is their production in heterologous temporary recipients such as sheep and goats. We aimed to produce elongated bovine cloned embryos using heterologous transfer to temporary recipients. Day-7 cloned cattle blastocysts were transferred to the uteri of ewes and goats and recovered as elongated structures at day 17. We evaluated elongation, length, presence of embryonic disc and expression of several important genes for embryonic development. We also produced homologous (cloned cattle embryos transferred into cattle uteri). Cloned bovine blastocysts were able to proceed with preimplantation development through elongation with high efficiency despite the species to which they were transferred. In qualitative and quantitative RT-PCR experiments we found differences in the pattern of gene expression among embryos recovered from different species. Sox2, Nanog and FGF-4 were markedly deregulated. No previous reports about the expression pattern of the studied genes had been published for elongated bovine cloned embryos produced in intermediate recipients, furthermore, the pattern of expression of Nanog, Oct4, Eomes, Cdx2, IFN-tau, Dicer, FGF-4 and Sox2 shown here are novel for elongated cloned bovine embryos created by hand-made cloning. Our data confirmed that sheep and goats can be used as temporary recipients. This model could serve as a basis for further research on gene expression and cellular changes during bovine peri-implantation development.
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Morovic, Martin, Matej Murin, Frantisek Strejcek, Michal Benc, Dusan Paál, Olga Østrup, Heiner Niemann, Lazo Pendovski, and Jozef Laurincik. "The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos." Macedonian Veterinary Review 39, no. 2 (October 1, 2016): 209–17. http://dx.doi.org/10.1515/macvetrev-2016-0085.
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AbstractOne of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.
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Sirard, Marc-André. "How the environment affects early embryonic development." Reproduction, Fertility and Development 34, no. 2 (2022): 203. http://dx.doi.org/10.1071/rd21266.
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In the field of animal reproduction, the environment associated with gametes and embryos refers to the parents’ condition as well as conditions surrounding gametes and embryos in vivo or in vitro. This environment is now known to influence not only the functionality of the early embryo but potentially the future phenotype of the offspring. Using transcriptomic and epigenetic molecular analysis, and the bovine model, recent research has shown that both the female and the male metabolic status, for example age, can affect gene expression and gene programming in the embryo. Evidence demonstrates that milking cows, which are losing weight at the time of conception, generates compromised embryos and offspring with a unique metabolic signature. A similar phenomenon has been associated with different culture conditions and the IVF procedure. The general common consequence of these situations is an embryo behaving on ‘economy’ mode where translation, cell division and ATP production is reduced, potentially to adapt to the perceived future environment. Few epidemiological studies have been done in bovines to assess if these changes result in a different phenotype and more studies are required to associate specific molecular changes in embryos with visible consequences later in life.
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Goissis, M. D., P. J. Ross, and J. B. Cibelli. "148 EFFECTS OF Wnt3A SUPPLEMENTATION ON BOVINE BLASTOCYST CELL NUMBER AND ALLOCATION." Reproduction, Fertility and Development 22, no. 1 (2010): 232. http://dx.doi.org/10.1071/rdv22n1ab148.
Full textAbstract:
Derivation of true bovine embryonic stem cells (ESC), as defined by their capacity to form robust teratomas and/or contribute to the germ line in chimeras, has not been achieved despite several attempts. It is possible that failures to derive bonafide bovine ESC are due to the inability of bovine embryonic cells to adapt to in vitro culture conditions that favor ESC derivation. Wnt pathways are involved in pluripotency and self-renewal of mouse and human ESC. Wnt signaling is also required for implantation competence in mouse blastocysts. Given the shared developmental potential between inner cell mass (ICM) and ESC, we hypothesized that Wnt could act on the ICM of bovine embryos increasing its proliferation potential. The objective of this study was to evaluate the effect of post-embryonic genome activation Wnt3A supplementation on blastocyst formation and cell allocation to ICM and trophectoderm (TE). In vitro fertilized bovine embryos at Day 4 of culture in KSOM medium were divided into 3 treatments: Control, no co-culture; co-culture with regular mouse embryonic fibroblasts (MEF); and co-culture with mouse L fibroblasts overexpressing Wnt3A protein (L-Wnt3A, Willert et al. 2003 Nature 423, 448-452). Embryos were cultured until Day 8 when blastocyst and hatching rates were recorded. Then, embryos were submitted to differential staining of ICM and TE by brief exposure to 0.25% Triton X-100 in PBS and staining with bisbenzimide and propidium iodide. Six IVF replications were performed and a total of 39 embryos were counted: 11 for Control, 16 for MEF, and 12 for L-Wnt3A. Only intact embryos after processing were used for cell count. Statistical analysis was performed by ANOVA using PROC MIXED of SAS software (SAS Institute Inc., Cary, NC, USA) in which each IVF was considered as a block with Tukey’s adjustment for mean comparison of rates and Bonferroni adjustments for mean comparison of cell counts. Results for blastocyst rate, hatching rate, ICM, TE, and total cell number are presented in the table below. Different superscript letters within columns indicate significant statistical difference (P < 0.05). These results indicate that L-Wnt3A fibroblast co-culture exerts a positive effect on bovine embryo cell number, resulting in a larger number of ICM cells in bovine embryos, which could be beneficial for ESC derivation attempts. Table 1.Blastocyst and hatching rates, ICM, TE, and total cell number results
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Labrecque, Rémi, and Marc-André Sirard. "Gene expression analysis of bovine blastocysts produced by parthenogenic activation or fertilisation." Reproduction, Fertility and Development 23, no. 4 (2011): 591. http://dx.doi.org/10.1071/rd10243.
Full textAbstract:
The processes underlying the very first moments of embryonic development are still not well characterised in mammals. To better define the kinetics of events taking place following fertilisation, it would be best to have perfect synchronisation of sperm entry. With fertilisation occurring during a time interval of 6 to 12 h in the same group of fertilised oocytes, this causes a major variation in the time of activation of embryonic development. Bovine parthenogenesis could potentially result in better synchronisation and, if so, would offer a better model for studying developmental competence. In the present study, bovine oocytes were either parthenogenetically activated or fertilised and cultured in vitro for 7 days. Gene expression analysis for those two groups of embryos at early and expanded stages was performed with BlueChip, a customised 2000-cDNA array developed in our laboratory and enriched in clones from various stages of bovine embryo development. The microarray data analysis revealed that only a few genes were differentially expressed, showing the relative similarity between those two kinds of embryos. Nevertheless, the fact that we obtained a similar diversity of developmental stages with parthenotes suggests that synchronisation is more oocyte-specific than sperm entry-time related. We then analysed our data with Ingenuity pathway analysis. Networks of genes involved in blastocyst implantation but also previous stages of embryo development, like maternal-to-embryonic transition, were identified. This new information allows us to better understand the regulatory mechanisms of embryonic development associated with embryo status.
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Zhao, Lixia, Xuefei Gao, Yuxuan Zheng, Zixin Wang, Gaoping Zhao, Jie Ren, Jia Zhang, et al. "Establishment of bovine expanded potential stem cells." Proceedings of the National Academy of Sciences 118, no. 15 (April 8, 2021): e2018505118. http://dx.doi.org/10.1073/pnas.2018505118.
Full textAbstract:
Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.
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Zhang, D., and H. M. Zhou. "64 RECONSTRUCTION OF HETEROGENEOUS EMBRYOS BY HUMAN SOMATIC CELLS AND BOVINE ENUCLEATED OOCYTES AND ISOLATION OF PUTATIVE HUMAN EMBRYONIC STEM CELL CLONES." Reproduction, Fertility and Development 20, no. 1 (2008): 113. http://dx.doi.org/10.1071/rdv20n1ab64.
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This study was undertaken to reconstruct heterogeneous nuclear-transferred embryos by using human fetal skin fibroblast cells as nuclear donor cells and the enucleated bovine oocytes as recipient cytoplasts for the purpose of investigating the feasibility of enucleated bovine oocyte cytoplasm as a means of reprogramming human somatic cell nuclei in an attempt to generate an accessible, autologous, and potentially unlimited source of totipotent human embryonic stem cells for transplantation medicine. Bovine ovaries were recovered at a local abattoir and oocytes were in vitro-matured and employed as recipient cytoplasts. A human fibroblast cell line was derived from an aborted fetus at 4 months of age, serum-starved, and used as donor somatic cells. The cultured nuclear transfer embryos were visually assessed for the first completion of cleavage at 48 h of culture, and for subsequent developmental stages at 72 to 168 h. The fusion of fibroblast cells into recipient cytoplasm was induced by electroporation. The fused oocytes were activated by ionomycin with 2 m mL–1 6-DMAP. The activated reconstructed embryos were co-cultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% (v/v) fetal calf serum (FCS) for 168 h. Morulae and blastocysts were used for isolating embryonic stem cells. The results indicated that human fetal skin fibroblast cells could maintain normal morphology and characteristics in culture conditions. They proliferated constantly and presented a regular growth curve in culture. Of these cells, 83.3% retained normal numbers of chromosomes after over 20 culture passages. The skin fibroblast cells exhibited normal morphology and retained normal numbers of chromosomes (2n = 64) in serum starvation culture after undergoing freezing and thawing treatment. The first completed cleavage of xenonuclear transplantation human embryos occurred between 24 and 48 h after activation, and morula and blastocyst development was completed between 72 and 168 h. The cleavage rate and the percentage of blastocyst development of the reconstructed embryos were 80.0% and 7.5%, respectively. Putative embryonic stem cell clones were observed, with nest-like morphology, after 3–7 days of culture on a fibroblast cell layer. Identifications by alkaline phosphatase (AKP) showed that the clones presented a positive reaction, which demonstrated that the isolated stem cell clones were embryonic stem cells. This study demonstrated that xenonuclear-transferred human embryos can undergo embryonic division and subsequent development to morula and blastocyst stage, and that human fetal fibroblast nuclei can be reprogrammed in bovine enucleated oocytes. Xenonuclear-transferred human embryos can be an alternative for obtaining human embryonic stem cells.
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Kovpak, V. V., O. S. Kovpak, S. S. Derkach, O. A. Valchuk, Y. V. Zhuk, and Y. S. Masalovych. "Influence of calcium ionophore on the fertilization of bovine oocytes and their further embryonic development." Regulatory Mechanisms in Biosystems 14, no. 1 (February 7, 2023): 137–44. http://dx.doi.org/10.15421/022321.
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Intracytoplasmic spermatozoid injection (ICSI) is one of the commonest methods used in assisted reproductive technologies in human medicine. However, this procedure has low efficacy for bovines, mainly because of insufficient activation of oocytes after spermatozoid microinjection. One of the most effective methods of activating oocytes is considered to be the use of phosphorus calcium, though the optimal concentration of activator and its effect on pre-implant development of embyo are still open questions. An oocyte-cumulus complex of clinically healthy cows, retrieved from the ovaries during slaughter, matured over 22–24 h in in vitro conditions. Oocytes with visible polar body had been subjected to intracytoplasmic spermatozoid injection (ICSI), and were 15–30 min later activated in the environment with different concentrations of calcium ionophore for 15–20 min and then transferred for further cultivation in a culture medium with sodium pyruvate. The fertilization rate was identified on the second day at the 2–4th stages of cellular embryo, and the quality of obtained embyos was evaluated on day 8. Based on the statistical analysis of the data, we determined that the artificial activation of bovine oocytes using calcium ionphore after intracytoplasmic spermatozoid injection (ICSI) led to statistically significant improvement in conception and ratio of blastocytes obtained to oocytes injected. In the study, we confirmed that addition of 5, 10 and 50 µМ of the agent had the same efficacy on the activation of occytes of bovine cattle. However, it has to be noted that during further cultivation of the obtained zygotes up to the blastocyte stage (day 8), we saw no significant differences in quality of embryos obtained. Therefore, use of calcium ionophore for the activation of bovine oocytes after intracytoplasmic spermatozoid injection is effective, for it promotes increase in fertilization parameters and ratio of blastocytes obtained to oocytes injected, facilitating production of higher numbers of embyos suitable for transplantation or cooling. Our previous conclusions are valuable for increasing the efficacy of methods of intracytoplasmic injection of bovine spermatozoid and its further use for purposes of science and production.