Cellular evaluation of bovine nuclear transfer embryos developed in vitro

Heyman, Y.; Degrolard, J.; Adenot, Pierre; Chesné, P.; Fléchon, B.; Renard, J.P.; Fléchon, J.‐E.

doi:10.1051/rnd:19950611

Cited by 16 publications

(6 citation statements)

References 14 publications

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“…We have noted a greater incidence of aberrant mitochondrial morphologies in embryos cultured in WMϪ, regardless of stage. Cloned bovine embryos also display deficiencies in mitochondrial ultrastructure (19). We observed little difference in MitoTracker staining of mitochondria at the two-cell stage.…”

Section: Discussionmentioning

confidence: 53%

Role of glucose in cloned mouse embryo development

Han¹,

Vassena²,

Maggie³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

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Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early beneficial effect during the first cell cycle. This early beneficial effect of glucose is not displayed by parthenogenetic, fertilized, or tetraploid nuclear transfer control embryos, indicating that it is specific to diploid clones. Precocious localization of the glucose transporter SLC2A1 to the cell surface, as well as increased expression of glucose transporters and increased uptake of glucose at the one-and two-cell stages, is also seen in cloned embryos. To examine the role of glucose in early cloned embryo development, we examined glucose metabolism and associated metabolites, as well as mitochondrial ultrastructure, distribution, and number. Clones prepared with cumulus cell nuclei displayed significantly enhanced glucose metabolism at the two-cell stage relative to parthenogenetic controls. Despite the increase in metabolism, ATP content was reduced in clones relative to parthenotes and fertilized controls. Clones at both stages displayed elevated concentrations of glycogen compared with parthenogenetic controls. There was no difference in the number of mitochondria, but clone mitochondria displayed ultrastructural alterations. Interestingly, glucose availability positively affected mitochondrial structure and localization. We conclude that cloned embryos may be severely compromised in terms of ATP-dependent processes during the first two cell cycles and that glucose may exert its early beneficial effects via positive effects on the mitochondria. glucose metabolism; mitochondrial ultrastructure; somatic cell nuclear transfer; cumulus cell nuclei; pentose phosphate pathway THE SUCCESSFUL DEVELOPMENT OF CLONED EMBRYOS produced by the technique of somatic cell nuclear transfer (SCNT) requires that the donor cell genome be reprogrammed by the oocyte. Although the specific molecular mechanisms of reprogramming remain largely undefined, the process should, in principle, involve some degree of silencing of the donor cell gene expression program followed by activation of the embryonic gene expression program. We recently reported that although many genes apparently are reprogrammed successfully within the first two cell cycles, Ͼ2,000 mRNAs are differentially expressed between clones and control embryos at the two-cell stage, including 880 mRNAs that are overexpressed in clones in a transcription-dependent manner and another 302 transcribed mRNAs that are underexpressed. The affected mRNAs span a variety of functional categories, most notably transcription factors, oxidoreductase activity, and transporter functions (43). These observations revealed a substantial amount of difference in gene expression between clones and control embryos that may render the two very different phenotypically.Preimplantation-stage cloned mouse embryos display a number of characteristics very different from control embryos and, in partic...

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Section: Discussionmentioning

confidence: 53%

Role of glucose in cloned mouse embryo development

Han¹,

Vassena²,

Maggie³

et al. 2008

American Journal of Physiology-Endocrinology and Metabolism

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“…Representative samples of delipidated and control embryos were fixed at the end of Day 2 (four-cell stage) and by Day 7 (blastocysts) for ulmtstructure assessment by electron microscopy. Quality of Day 7 blastocysts derived from delipidated or control zygotes was assessed also by counting nuclei, by using propidium iodide staining and by confocal microscopy (15). To confirm the in vivo developmental competence of "'lipid free" blastocysts, embryo transfers were made to synchronous recipient heifers.…”

Section: Experimental Designmentioning

confidence: 99%

Delipidating in vitro-produced bovine zygotes: Effect on further development and consequences for freezability

Díez

Heyman

Bourhis³

et al. 2001

Theriogenology

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To study the effect of partial removal of intracytoplasmatic lipids from bovine zygotes on their in vitro and in vivo survival, presumptive zygotes were delipidated by micromanipulation and cocultured with Veto cells in B2+10% FCS. Blastocyst rates of delipidated (n=960), sham (centrifuged but not delipidated, n=830) and control embryos (n=950) were 42.1, 42.3 and 39.9% respectively (P > 0.05). Day 7 blastocysts derived from delipidated zygotes had a mean of 123.9 + 45.6 nuclei compared to 137.5 + 32.9 for control blastocysts (P > 0.05). The full-term development of delipidated blastocysts after single transfer to recipients was similar to that of control IVF blastocysts (41.2% vs 45.4% respectively). To assess the effect of delipidation on the embryo tolerance to freczing/thawing, delipidated (n=73), control (n=67) and shmn (n=50) Day 7 blastocysts were frozen in 1.36 M glycerol + 0.25 M sucrose in PBS. After thawing, embryos were cocultured for 72 h with Vero cells in B2+10% FCS. Survival rates at 24 h were not significantly different between groups. However, in the delipidated group, the survival rate after 48 h in culture was significantly higher than in the control group (56.2 vs 39.8, P < 0.02), resulting in a higher hatching rate after 3 days in culture (45.2 vs 22.4, P < 0.02). Pregnancy rates for delipidated and control frozen/thawed embryos were respectively 10.5 and 22.2% (P > 0.05). Electron microscopic observations showed much fewer lipid droplets (and smaller) in delipated blastocysts than in controls. Taken together, our data show that delipidation of one cell stage bovine embryos is compatible with their normal development to term and has a beneficial effect on their tolerance to freezing and thawing at the blastocyst stage. This procedure, however, alters the developmental potential of such blastocysts, suggesting that maternally inherited lipid stores interfere with metabolic recovery after thawing.

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“…Cell number evaluation was performed as previously described by Heyman et al (1995). Briefly, individual embryos were removed from co-culture on day 6-7 and fixed in a solution of 2.5% paraformaldehyde for 1 h at room temperature, treated with 0.5 M NaOH for 30 min to permeabilise them, and stained with propidium iodide for 15 min at 37 °C.…”

Section: Cell Number Evaluationmentioning

confidence: 99%

Comparative glucose and fructose incorporation and conversion by in vitro Produced bovine embryos

Guyader-Joly¹,

Khatchadourian²,

Ménézo³

1996

Zygote

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SummaryWe have investigated the quality of bovine IVM/IVF embryos co-cultured on Vero cells. Blastocyst cell numbers are very similar to those obtained in vivo, and higher than those obtained by co-culture with oviduct cells. The metabolism and conversion of fructose and glucose are not equivalent even though carbon dioxide production is similar and increasing from morula to blastocyst. Formation of free amino acids and incorporation into proteins are higher and faster for glucose than for fructose, but this conversion is rather stable with embryonic growth. Moreover, the by-products formed are not the same. Glucose at physiological concentrations (i.e.2 mM)seems to be a more appropriate fuel for the burst of embryonic development at the blastocyst stage in preparation for hatching.

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Cellular evaluation of bovine nuclear transfer embryos developed in vitro

Cited by 16 publications

References 14 publications

Role of glucose in cloned mouse embryo development

Role of glucose in cloned mouse embryo development

Delipidating in vitro-produced bovine zygotes: Effect on further development and consequences for freezability

Comparative glucose and fructose incorporation and conversion by in vitro Produced bovine embryos

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