Removal of Cytoplasmic Lipid Enhances the Tolerance of Porcine Embryos to Chilling

Nagashima, Hiroshi; Kashiwazaki, Naomi; Ashman, Rodney J.; Grupen, Christopher G.; Seamark, R.F.; Nottle, Mark B.

doi:10.1095/biolreprod51.4.618

Cited by 195 publications

(149 citation statements)

References 23 publications

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“…This indicates that the method used for micromanipulation was not detrimental for subsequent in vitro development. In our protocol, we estimate that approximately 90% of the dense lipid fraction after centrifugation was removed from the zygote, which is very similar to the observations on porcine embryos by Nagashima et al (25,26,27) in which fully delipidated one cell eggs had the same developmental potential than intact controls. So, delipidated zygotes are able to develop in the absence of lipids normally found in one cell stage.…”

Section: Discussionsupporting

confidence: 86%

“…Similarly, Nagashima et at. (26,27) demonstrated that 1-to 4-cell stage centrifuged embryos were not as tolerant as delipidated embryos, but they had significantly higher survival rates than control embryos. In this work, only a part of the lipid droplets polarized by centrifugation could redistribute during in vitro development, producing blastocysts darker than delipidated embryos but clearer than the control ones, and retaining the rest of the droplets outside the cytoplasm.…”

Section: Discussionmentioning

confidence: 98%

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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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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 98%

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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“…Nagashima et al [16,17] demonstrated that cryotolerance of 1-8 cell stage porcine embryos can be increased by removing cytoplasmic lipid droplets from centrifuged embryos. Moreover, post-thaw survival of bovine blastocysts was improved after removal of lipid droplets at an earlier stage [18][19][20].…”

Section: Cytoplasmic Lipids Responsible For Cryoinjurymentioning

confidence: 99%

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GLTip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GLTip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination.

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“…Many studies have shown that in porcine and bovine embryos, increased lipid accumulation aggravated vitrification injury with respect to embryonic development and apoptosis [8][9][10][11][12][13], suggesting that high lipid content in embryos aggravated vitrification injury on morphologic parameters. The discordant results between mice and domestic animal species may be explained by the markedly higher lipid content in embryos from the latter, as the total lipid content per oocyte was 4 ng in mice [48], in contrast to 161 ng in pigs and 63 ng in cattle [49,50].…”

Section: The Effect Of Obesity On Vitrificationmentioning

confidence: 99%

“…Many studies have shown that increased lipid accumulation aggravates vitrification injury in porcine and bovine embryos and that delipidation by micromanipulation or the use of chemicals can significantly reduce embryonic sensitivity to chilling or cryopreservation [8][9][10][11][12][13]. However, the lipid contents of bovine and porcine oocytes are very high (63 and 161 ng/ oocyte, respectively), and we cannot adequately evaluate the effects of lipid content in other species [14,15].…”

Section: Introductionmentioning

confidence: 99%

Maternal obesity in mice not only affects fresh embryo quality but also aggravates injury due to vitrification

Huang

Yang

et al. 2016

J Assist Reprod Genet

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Purpose The aims of the present study are to identify the mechanism(s) whereby obesity impairs fresh embryos and to clarify the effects of vitrification on lipid droplet content within embryos from maternally obese mice. Methods The diet-induced obesity mouse model was established, and the zygotes were captured and cultured to day 3. The eight-cell embryos were selected and divided into fresh and vitrified groups. The blastocysts derived from fresh embryos were used as a control. The expression profiles of endoplasmic reticulum (ER) stress genes (Atf4, Grp78, and Hsp70) and other genes (MnSOD, p53, Gadd45g, caspase-3, IGF-II, ZO-1, and E-cadherin) on day-3 fresh and postwarming eight-cell embryos from obese and control groups were determined. For day-5 fresh blastocysts and blastocysts previously vitrified on day 3, the expression profiles for all of the above genes were also determined. Results For the fresh group, obesity significantly upregulated Hsp70, p53, IGF-II, and ZO-1 expression in embryos on day 3 and notably upregulated Atf4, MnSOD, Gadd45g, caspase-3, ZO-1, and E-cadherin expression in blastocysts on day 5. For vitrified ones, obesity significantly upregulated Atf4, MnSOD, and Gadd45g expression in embryos on day 3 and notably upregulated Hsp70 expression and downregulated MnSOD in day 5 blastocysts previously vitrified on day 3. Conclusions Obesity impairs fresh embryos and aggravates embryonic vitrification injury at a molecular level.

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Removal of Cytoplasmic Lipid Enhances the Tolerance of Porcine Embryos to Chilling

Cited by 195 publications

References 23 publications

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

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

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Maternal obesity in mice not only affects fresh embryo quality but also aggravates injury due to vitrification

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