Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo

Iwasaki, Setsuo; Yoshiba, Nagako; Ushijima, Hitoshi; Watanabe, Shinji; Nakahara, Tsutomu

doi:10.1530/jrf.0.0900279

Cited by 198 publications

(108 citation statements)

References 15 publications

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“…The number of cells in day 7 blastocysts was determined by either: (1) counting the Giemsa stained nuclei of fixed embryos spread onto glass slides; or (2) by combining the differential cell counts of the trophectoderm and ICM using procedures as previously described (Iwasaki et al 1990). In vivo embryos were obtained as described above using a total of 20 animals in two separate experiments.…”

Section: Cell Counting Of Blastocyst Embryosmentioning

confidence: 99%

Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Smith¹,

Berg²,

Beaumont³

et al. 2007

Reproduction

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During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4, Otx2, Ifitm3, GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (b-actin, b-tubulin and GAPDH) in 21 NT blastocysts with that in genetically half-identical in vitro produced (IVP, nZ19) and in vivo (nZ15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts. Ifitm3 expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NT and IVP embryos increased between two-and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP or in vivo embryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer. Oct4 levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed that in vivo embryos resembled each other much more than did NT and IVP blastocysts. Furthermore, in vivo embryos, consisting of 1.5-fold more cells, generally contained two-to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivo versus in vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

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Section: Cell Counting Of Blastocyst Embryosmentioning

confidence: 99%

Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Smith¹,

Berg²,

Beaumont³

et al. 2007

Reproduction

View full text Add to dashboard Cite

show abstract

“…Finally, from these results it appears that the viability of in vitro-derived embryos is still lower than for in viw embryos. Morphological, cellular and physiological differ-' ences have been described (Iwasaki et al, 1990; Van Soom and De Kruif, 1992; Leibo and Loskutoff, 1993;Leibo, 1993, 1994) which affect the viability and sensitivity to cooling and freezing. For example, in vitro embryos have a more buoyant density (< 1.2) than in vivo ones (> 1.3).…”

Section: Statisticsmentioning

confidence: 99%

Survival and viability of fresh and frozen-thawed in vitro bovine blastocysts

Massip¹,

Mermillod²,

Langendonckt³

et al. 1995

Reprod. Nutr. Dev.

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“…Iwasaki et al (1990) found that IVP embryos had a lower ratio of ICM to TE cells, and a less compact ICM compared with both embryos produced entirely in vivo and embryos matured and fertilised in vivo but cultured in vitro. A minimum number of ICM cells is required to obtain a pregnancy and excessive allocation of cells to the TE may lead to pregnancy abnormalities (Van Soom et al 1996;Leese et al 1998).…”

Section: Discussionmentioning

confidence: 90%

127 Development and Quality of Bovine Morulae Cultured in Serum-Free Medium With Retinoic Receptor Specific Agonists

Gómez¹,

Caamaño²,

Muñoz³

et al. 2008

Reprod. Fertil. Dev.

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Abstract.Retinoids regulate development and differentiation of the bovine blastocyst in vitro, although the underlying mechanisms remain to be clarified. A challenge in reproductive biotechnology is the identification of pathways that regulate early embryonic development and their influence on blastocyst differentiation, apoptosis and survival to cryopreservation as traits of embryo quality. The present paper analyses the effects of short-term exposure (24 h) to retinoids on in vitroproduced bovine morulae. Immature cumulus oocyte complexes were in vitro matured and fertilised. Presumptive zygotes were subsequently cultured in modified synthetic oviduct fluid up to Day 6, in which morulae were randomly allocated to the different experimental groups. The treatments consisted of 0.1 µm LG100268 (LG; a retinoid X receptor agonist), 0.7 µm all-trans retinoic acid (ATRA; a retinoic acid receptor agonist) or no additives. Day 8 blastocyst development was increased in the ATRA-treated group compared with the LG and untreated embryos. In Day 7 embryos, the number of total cells and cells allocated to the trophectoderm were higher in the ATRA-treated group compared with untreated embryos. Apoptosis in the inner cell mass increased after LG treatment, whereas ATRA had no effect. After vitrification and warming, survival and hatching rates of Day 7 blastocysts did not change with retinoid treatment. Within the LGtreated and untreated blastocyst groups, survival and hatching rates were higher for Day 7 than Day 8 embryos; however, Day 8 blastocysts treated with ATRA showed improved hatching rates. In conclusion, treatment of morulae with ATRA in serum-free medium improves embryo development and quality without increasing the incidence of apoptosis and necrosis.

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Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo

Cited by 198 publications

References 15 publications

Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Survival and viability of fresh and frozen-thawed in vitro bovine blastocysts

127 Development and Quality of Bovine Morulae Cultured in Serum-Free Medium With Retinoic Receptor Specific Agonists

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