A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide‐specific fluorochromes

Handyside, Alan H.; Hunter, Susan

doi:10.1002/jez.1402310317

Cited by 165 publications

(76 citation statements)

References 7 publications

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“…In order to obtain a great developmental potential of the embryos, 120 h of in vitro culture was defined as the final time point. Blastocysts produced in CZB, KSOM and HCO 3 HTF media covered with either 0.5 mL or 1 mL of paraffin oil were stained to differentiate ICM and TE cells, using a modified method of Handyside and Hunter [20]. Briefly, the blastocysts were transferred from the above mentioned three culture media to acid Tyrode solution, under constant observation for 30-60 s, until the zonae pellucidae were completely dissolved.…”

Section: Cell Count and Cell Ratio Between Te And Icm In In Vitro Promentioning

confidence: 99%

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos

2004

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-This study was undertaken to investigate the effects of three media, volume and type of oil and frequency of observation on the in vitro development of mouse zygotes. B6CBF1 female mice (4 to 6 wk old) were superovulated using PMSG/hCG and mated with a proven fertile male of the same strain. Putative zygotes with polar bodies were collected from the oviducts of mated mice, 25-28 h after hCG injection, and were cultured in vitro. Embryo development was evaluated at either 96 h and 120 h or every 24 h for 120 h. The results obtained showed that the CZB medium was better than the KSOM and HCO 3 HTF media, and the use of 1 mL of paraffin oil was better than the use of 0.5 mL of paraffin oil. The effect of paraffin oil and mineral oil on embryo development was examined and the results indicated that the use of paraffin oil was better than the use of mineral oil. Repeated observations did not influence the proportion of embryos developing to blastocysts.in vitro development / media / type of oil / frequency of observation / mouse embryos

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Section: Cell Count and Cell Ratio Between Te And Icm In In Vitro Promentioning

confidence: 99%

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos

2004

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“…Differential cell counts of the ICM and trophecloderm of the blastocyst, or inner and outer cells of compact morulae, were made using a modification of the method of Handyside and Hunter (1984) which does not involve fixation. The procedure depends on the complement-mediated lysis of outside cells that have been exposed to anti-species antiserum while the inner cells are protected from antibody by their position inside the embryo (Solter and Knowles, 1975).…”

Section: Culture Media and Cell Countsmentioning

confidence: 99%

Mouse half embryos: Viability and allocation of cells in the blastocyst

Papaioannou

Ebert

1995

Developmental Dynamics

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We have investigated the developmental capacity of mouse embryos in which one blastomere was destroyed by lysis at the 2-cell stage. The allocation of cells to the trophectoderm and inner cell mass (ICM) was documented by differential cell counts on single embryos after 2 days under different culture conditions. Viability and further developmental potential were tested by embryo transfer to foster mothers. The conditions used were: (1) in vitro culture in modified BMOC-2 medium, (2) in vivo oviduct transfer to immature (prepuberal) females, and (3) in vivo oviduct transfer to pseudopregnant females. Half embryos almost always fared less well for all parameters of development than control embryos developing under the same conditions. Lower total cell numbers in half embryos were accounted for by decreases in both ICM and trophectoderm with a disproportionate decrease in ICM in smaller embryos. In both half and control embryos, the growth conditions affected the rate of morphological development, the total cell number, and embryo viability. Unlike the effect of halving embryos, the growth condition effects on total cell number can be accounted for primarily by differences in ICM cell number, with trophectoderm cell number remaining constant. These results provide new information on the ability of the mouse embryo to differentially regulate ICM and trophectoderm cell number under different conditions, and confirm our previous work showing the advantage of shortterm development in vivo over short-term in vitro culture (Papaioannou and Ebert [19861 J. Reprod. Fertil. 76603-608). They also clearly document that lower total cell number and a lower proportion of ICM is correlated with compromised development. 0 1995 Wiley-Liss, Inc.

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“…Total cell number and the proportion of TE and ICM cells were assessed in F2 rat blastocysts using a modification of a method described previously (nZ30-60 blastocysts/group from nZ6 females/group) (Handyside & Hunter 1984, Hardy et al 1989. Briefly, blastocysts were incubated in pronase (Sigma, 0.5% in GMOPS, 5 min at 37 8C) to remove the zona pellucida, then incubated in picrylsulfonic acid (Sigma, 10 min at 37 8C) and washed in GMOPS with 5 mg/ml HSA.…”

Section: Differential Nuclear Stainingmentioning

confidence: 99%

Low female birth weight and advanced maternal age programme alterations in next-generation blastocyst development

et al. 2015

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Low birth weight is associated with an increased risk for adult disease development with recent studies highlighting transmission to subsequent generations. However, the mechanisms and timing of programming of disease transmission to the next generation remain unknown. The aim of this study was to examine the effects of low birth weight and advanced maternal age on second-generation preimplantation blastocysts. Uteroplacental insufficiency or sham surgery was performed in late-gestation WKY pregnant rats, giving rise to first-generation (F1) restricted (born small) and control offspring respectively. F1 control and restricted females, at 4 or 12 months of age, were naturally mated with normal males. Second-generation (F2) blastocysts from restricted females displayed reduced expression of genes related to growth compared with F2 control (P!0.05). Following 24 h culture, F2 restricted blastocysts had accelerated development, with increased total cell number, a result of increased trophectoderm cells compared with control (P!0.05). There were alterations in carbohydrate and serine utilisation in F2 restricted blastocysts and F2 restricted outgrowths from 4-month-old females respectively (P!0.05). F2 blastocysts from aged restricted females were developmentally delayed at retrieval, with reduced total cell number attributable to reduced trophectoderm number with changes in carbohydrate utilisation (P!0.05). Advanced maternal age resulted in alterations in a number of amino acids in media obtained from F2 blastocyst outgrowths (P!0.05). These findings demonstrate that growth restriction and advanced maternal age can alter F2 preimplantation embryo physiology and the subsequent offspring growth.

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A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide‐specific fluorochromes

Cited by 165 publications

References 7 publications

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos

Mouse half embryos: Viability and allocation of cells in the blastocyst

Low female birth weight and advanced maternal age programme alterations in next-generation blastocyst development

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