Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts

Li, G. P.; White, Kenneth L.; Aston, Kenneth I.; Meerdo, Lora N.; Bunch, Tom D.

doi:10.1530/rep.1.00089

Cited by 25 publications

(10 citation statements)

References 35 publications

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“…Our research, as well as that of others, has demonstrated that chromatin remodeling and blastomere ploidy frequently deviate from normal after NT in various species, including cattle (Booth et al 2003, Bureau et al 2003, Li et al 2004a, 2004b, rabbits (Shi et al 2004) and pigs (Kim et al 2005). Several significant morphologic changes occur in the donor nucleus after NT into cytoplasts with high MPF activity.…”

Section: Discussionsupporting

confidence: 65%

“…Maturation of bovine oocytes was performed as described previously (Li et al 2004a(Li et al , 2004b. Briefly, cumulus oocyte complexes (COC) were aspirated from 3 -8 mm follicles with an 18 gauge needle from ovaries collected from a local abattoir.…”

Section: Oocyte Maturationmentioning

confidence: 99%

“…The exact mechanism(s) contributing to losses are still unclear. Epigenetic alterations (Wrenzycki et al 2001, Cezar et al 2003 and chromosomal abnormalities (Burgoyne et al 1991, Li et al 2004a, 2004b) probably contribute to developmental failure.…”

Section: Introductionmentioning

confidence: 99%

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Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Aston¹,

Li²,

Hicks³

et al. 2006

Reproduction

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This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stage in vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.

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Section: Discussionsupporting

confidence: 65%

Section: Oocyte Maturationmentioning

confidence: 99%

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Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Aston¹,

Li²,

Hicks³

et al. 2006

Reproduction

Self Cite

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“…Several studies have reported embryo co-culture with somatic cells (Desai and Goldfarb, 1998;Funston et al, 1997;Kobayashi et al, 1992), oviduct cells (Bavister, 1988;Lee et al, 2003;Lee et al, 2001;Liu et al, 1998;Mermillod et al, 1993;Van Langendonckt et al, 1996;Xu et al, 2001), and CM (Li et al, 2004a;Li et al, 2004b) to mimic the microenvironment conditions associated with the maternal tract. However, only a single report has studied the co-culture of different kinds of embryos together (Terashita et al, 2011) because of the difficulty in discrimination or separation of different kinds of embryos during the co-culture.…”

Section: Introductionmentioning

confidence: 99%

Improvement of Cloned Embryos Development by Co-Culturing with Parthenotes: A Possible Role of Exosomes/Microvesicles for Embryos Paracrine Communication

Saadeldin

Kim

Choi

et al. 2014

Cellular Reprogramming

139

140

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It is well known that embryos cultured in a group can create a microenvironment through secretion of autocrine and paracrine factors that can support and improve the embryos' development when compared to the embryos cultured individually. In this study, we used a co-culture system for paracrine communication between different kinds of embryos. The results showed that co-culture of porcine parthenogenetic (PA) embryos significantly improved the in vitro development of cloned (nuclear transfer, NT) embryos. To reveal the possible mechanism of communication between the two groups, we isolated exosomes/microvesicles (EXs/MVs) from the PA embryos conditioned medium (PA-CM) through differential centrifugation and identified them through transmission electron microscope and immunoflourescence against exosomal/membrane marker CD9. Furthermore, these EXs/MVs were found to contain mRNA of pluripotency genes (Oct4, Sox2, Klf4, c-Myc, and Nanog), and the PKH67-labeled EXs/MVs could be internalized by the NT embryos. The current study demonstrates that cloned embryos' developmental competence can be improved through co-culturing with PA embryos and revealed, for the first time, that in vitro-produced embryos can secrete EXs/MVs as a possible communication tool within their microenvironment. Moreover, it provides a new paradigm for embryo-to-embryo communication in vitro.

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“…Genetic damage may occur during in vitro culture of somatic cells. For example, a suboptimal in vitro culture system may contribute to an increase in the incidence of chromosomal abnormalities in somatic cells (Denning et al, 2001) and embryos, both IVF and NT derived (Lonergan et al, 2004;Li et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Chromosomal Aneuploidy in African Wildcat Somatic Cells and Cloned Embryos

Gómez

Pope

López

et al. 2006

Cloning and Stem Cells

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In the present study, we compared the incidence of aneuploidy in in vitro fertilized domestic cat embryos (DSH-IVF) with that of African Wildcat (AWC) cloned embryos reconstructed with AWC fibroblast donor cells from different passages (AWC-NT). Fibroblast cells were cultured to passages 1 (P1), 3 (P3), 4 (P4), and 9 (P9), after which cells at each passage were karyotyped and serum-starved before being frozen for nuclear transfer. AWC-NT embryos were produced by fusion of a single AWC somatic cell at P1, P3, P4, or P9 to enucleated domestic cat cytoplast derived from in vitro matured (IVU) oocytes. DSH-IVF embryos were produced after IVU oocytes were fertilized in vitro with domestic cat spermatozoa. To determine chromosome numbers, embryos (2-4-cell) or fibroblast cells were cultured in medium containing 0.28 microg/mL of Colcemid for 22-24 h or 15-24 h, respectively. Subsequently, embryos and cells were placed in hypotonic solution, fixed, and stained for analysis of chromosome spreads by bright field microscopy. Chromosomal abnormalities in AWC fibroblast cells increased progressively during culture in vitro: P1 (43%), P3 (46%), P4 (62%), and P9 (59%). In fibroblast cells, hypoploidy (94/202, 46%) was the major chromosomal abnormality, and it occurred more frequently than hyperploidy (14/202, 7%; p < 0.05). While the percentage of hyperploid cells remained stable during all passages, the proportion of hypoploidy in fibroblast cells increased significantly after P4. The overall incidence of chromosomal abnormalities in AWC-NT embryos at P1 (45%), P3 (60%), and P4 (50%) was similar to that of the fibroblast cells from which they were derived; however, the incidence was higher for embryos reconstructed with donor fibroblasts at P9 (89%). Hypoploidy was the most common chromosomal abnormality observed in either AWC-NT or DSH-IVF embryos. AWCNT embryos reconstructed with donor cells at early passages (P1, P3, and P4) had similar frequencies of chromosomal diploidy, as did DSH-IVF embryos. Accordingly, based on the present results, for NT we are currently using cat donor cells at early passages, when the percentage of cells with chromosomal abnormalities is low. It is recommended that the chromosomal stability of each cell line be analyzed before use as NT donor cells to reduce the incidence of chromosomal anomalies in reconstructed embryos and to possibly produce a subsequent increase in cloning efficiency.

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Conditioned medium increases the polyploid cell composition of bovine somatic cell nuclear-transferred blastocysts

Cited by 25 publications

References 35 publications

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Effect of the time interval between fusion and activation on nuclear state and development in vitro and in vivo of bovine somatic cell nuclear transfer embryos

Improvement of Cloned Embryos Development by Co-Culturing with Parthenotes: A Possible Role of Exosomes/Microvesicles for Embryos Paracrine Communication

Chromosomal Aneuploidy in African Wildcat Somatic Cells and Cloned Embryos

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