77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

2019 ◽  
Vol 31 (1) ◽  
pp. 164
Author(s):  
A. E. Ynsaurralde Rivolta ◽  
M. Suvá ◽  
V. Alberio ◽  
C. Vazquez Echegaray ◽  
A. Guberman ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Monozygotic Twin ◽  
Cell Number ◽  
Control Group ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

High hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts

2011 ◽  
Vol 23 (6) ◽  
pp. 809 ◽  
Author(s):  
Luisa Bogliolo ◽  
Federica Ariu ◽  
Giovanni Leoni ◽  
Stefania Uccheddu ◽  
Daniela Bebbere
Keyword(s):  
Hydrostatic Pressure ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Lower Percentage ◽  
Control Group ◽  
Hatching Rate ◽  
Embryo Survival

Exposure to sub-lethal hydrostatic pressure (HP) treatment is emerging as an approach to improve the general resistance of gametes and embryos to in vitro conditions. The present study was aimed to evaluate the effect of HP treatment on in vitro-produced ovine blastocysts. Experiment 1 was aimed to define optimal treatment parameters: two different HP treatments were applied to blastocysts and embryo survival was evaluated. In Experiment 2, HP parameters (40 MPa, 70 min, 38°C) selected in Experiment 1 were used to treat blastocysts. Embryo quality was assessed and compared with untreated controls by counting total cell number, the inner cell mass (ICM) and trophectoderm (TE) cells and by evaluating nuclear picnosis. HP-treated blastocysts were processed for gene expression analysis (AQP3, ATP1A1, BAX, CDH1, HSP90β, NANOG, OCT4 and TP53) 1, 5 h after the end of HP exposure. Results showed that the hatching rate of embryos treated at 40 MPa was significantly higher than that of the 60 MPa-treated group (P < 0.01) and similar to untreated embryos. Blastocysts exposed at 40 MPa showed higher ICM (P < 0.05) and TE (P < 0.01) cell number and a lower percentage of picnotic nuclei (P < 0.05) compared with the control group. Significantly lower abundance for BAX (P < 0.01) and OCT4 (P < 0.05) transcripts were observed in HP embryos than in the control group. In conclusion, treatment with HP improved the quality of in vitro-produced ovine blastocysts by increasing their cell number and reducing the proportion of nuclear picnosis.

Abnormal development of pre-implantation mouse embryos grown in vitro with [3H]thymidine

Development ◽  
1973 ◽  
Vol 29 (3) ◽  
pp. 601-615
Author(s):  
M. H. L. Snow
Keyword(s):  
Specific Activity ◽  
Inner Cell Mass ◽  
Cell Damage ◽  
Cell Mass ◽  
Cell Number ◽  
Mouse Embryos ◽  
Abnormal Development ◽  
Tritium Concentration ◽  
Cell Stage

Mouse embryos were grown in vitro from the 2-cell stage to blastocysts in the presence of [3H]thymidine. Methyl-T-thymidine and thymidine-6-T(n) were used and both forms found to be lethal at concentrations above 0·1 μCi/ml. Both forms of [3H]Tdr at concentrations between 0·01 and 0·1 μCi/ml caused a highly significant (P &lt; 0·001) reduction in blastocyst cell number. The reduction in cell number, which was positively correlated with specific activity and tritium concentration, was associated with cell damage typical of radiation damage caused by tritium disintegration. Thymidine-6-T(n) also significantly reduced the number of 2-cell embryos forming blastocysts whereas methyl-T-Tdr did not. This difference in effect is assumed to be caused by contamination of one form of [3H]Tdr with a by-product of the tritiation process. A study of the cleavage stages showed that almost all the reduction in cell numbers could be accounted for by selective cell death occurring at the 16-cell stage. Cells which survive that stage cleave at a normal rate. The cells that are most susceptible to [3H]Tdr damage were found to normally contribute to the inner cell mass. The [3H]Tdr-resistant cells form the trophoblast. It is possible to grow blastocysts in [3H]Tdr such that they contain no inner cell mass but are composed entirely of trophoblast. Comparatively short (12 h) incubation with [3H]Tdr at any stage prior to the 16-cell stage will cause this damage. Possible reasons for this differential effect are discussed, and also compared with damage caused by X-irradiation.

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
C. Pontes Godoi ◽  
P. D. Moço ◽  
B. Cazari ◽  
P. T. Mihara ◽  
P. V. Silva ◽  
...  
Keyword(s):  
Cell Mass ◽  
Farm Animals ◽  
Control Group ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Chi Square Test ◽  
Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

scholarly journals Development of Preimplantation Mouse Embryos in vivo and in vitro

1982 ◽  
Vol 35 (2) ◽  
pp. 187 ◽  
Author(s):  
GM Harlow ◽  
P Quinn
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cell Stage ◽  
Preimplantation Mouse ◽  
Development In Vitro

The culture conditions for the development in vitro of (C57BL/6 X CBA) F2 hybrid two-cell embryos to the blastocyst stage have been optimized. Commercially available pre-sterile disposable plastic culture dishes supported more reliable development than re-usable washed glass tubes. The presence of an oil layer reduced the variability in development. An average of 85 % of blastocysts developed from hybrid two-cell embryos cultured in drops of Whitten's medium under oil in plastic culture dishes in an atmosphere of 5% O2 : 5% CO2 : 90% N2 ? The time taken for the total cell number to double in embryos developing in vivo was 10 h, and in cultured embryos 17 h. Embryos cultured in vitro from the two-cell stage to blastocyst stage were retarded by 18-24 h in comparison with those remaining in vivo. Day-4 blastocysts in vivo contained 25-70 cells (mean 50) with 7-28 (mean 16) of these in the inner cell mass. Cultured blastocysts contained 19-73 cells (mean 44) with 8-34 (mean 19) of these in the inner cell mass. In the uterine environment, inner-cell-mass blastomeres divided at a faster rate than trophectoderm blastomeres and it is suggested that a long cell cycle is associated with terminal differentiation. Although cultured blastocysts and inner cell masses contained the same number of cells as blastocysts and inner cell masses in vivo, the rate of cell division in cultured inner cell masses was markedly reduced.

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 39-44 ◽  
Author(s):  
P. Chrenek ◽  
A.V. Makarevich
Keyword(s):  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Control Group ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Egfp Gene ◽  
Chimeric Blastocysts ◽  
Rabbit Embryos

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16 h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p<0.05), but significantly lower than in TR<>N chimeric blastocysts (p<0.001). Similarly, a higher proportion (p<0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage

2014 ◽  
Vol 26 (3) ◽  
pp. 395 ◽  
Author(s):  
Chawalit Siriboon ◽  
Ching-Fu Tu ◽  
Michel Kere ◽  
Ming-Sing Liu ◽  
Hui-Jung Chang ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Cell Stage ◽  
Surrogate Mothers ◽  
Cell Numbers ◽  
Pluripotency Gene ◽  
Apoptosis Gene

The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P < 0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P < 0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass : total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P < 0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P < 0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term.

scholarly journals Insulin increases the cell number of the inner cell mass and stimulates morphological development of mouse blastocysts in vitro

Development ◽  
1990 ◽  
Vol 110 (3) ◽  
pp. 963-967 ◽  
Author(s):  
M.B. Harvey ◽  
P.L. Kaye
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Insulin Receptors ◽  
Cell Number ◽  
Morphological Development ◽  
Cell Stage ◽  
Morphological Studies ◽  
Preimplantation Mouse ◽  
Stimulation Of

Previous studies showed that insulin promotes cell proliferation and morphological development of preimplantation mouse embryos. In this report, the receptor responsible for these actions and the cell populations that are affected were investigated. Insulin's 9% stimulation of blastocyst cell number was entirely due to a 23% increase in ICM cell number with an EC50 of 0.54 pM. This and the similar degrees of stimulation of immunosurgically isolated ICMs by both physiological and supraphysiological insulin concentrations suggest that insulin receptors are present on the ICM and respond to exogenous insulin transcytosed through the TE to promote expansion of the ICM cell numbers. In morphological studies, insulin increased the number of blastocysts and decreased the number of morulae by 10% after 54 h culture from 2-cell embryos with EC50s of about 0.95 pM. The equivalence of these EC50s suggests mediation of insulin's stimulation of blastocyst formation via insulin receptors which are functionally expressed around the time of compaction at the 8-cell stage. These results support our hypothesis that insulin has an important role in the regulation of growth during preimplantation development.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

The human blastocyst: cell number, death and allocation during late preimplantation development in vitro

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 597-604 ◽  
Author(s):  
K. Hardy ◽  
A.H. Handyside ◽  
R.M. Winston
Keyword(s):  
Cell Death ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cell Numbers ◽  
Human Blastocyst ◽  
Development In Vitro

The development of 181 surplus human embryos, including both normally and abnormally fertilized, was observed from day 2 to day 5, 6 or 7 in vitro. 63/149 (42%) normally fertilized embryos reached the blastocyst stage on day 5 or 6. Total, trophectoderm (TE) and inner cell mass (ICM) cell numbers were analyzed by differential labelling of the nuclei with polynucleotide-specific fluorochromes. The TE nuclei were labelled with one fluorochrome during immunosurgical lysis, before fixing the embryo and labelling both sets of nuclei with a second fluorochrome (Handyside and Hunter, 1984, 1986). Newly expanded normally fertilized blastocysts on day 5 had a total of 58.3 +/− 8.1 cells, which increased to 84.4 +/− 5.7 and 125.5 +/− 19 on days 6 and 7, respectively. The numbers of TE cells were similar on days 5 and 6 (37.9 +/− 6.0 and 40.3 +/− 5.0, respectively) and then doubled on day 7 (80.6 +/− 15.2). In contrast, ICM cell numbers doubled between days 5 and 6 (20.4 +/− 4.0 and 41.9 +/− 5.0, respectively) and remained virtually unchanged on day 7 (45.6 +/− 10.2). There was widespread cell death in both the TE and ICM as evidenced by fragmenting nuclei, which increased substantially by day 7. These results are compared with the numbers of cells in morphologically abnormal blastocysts and blastocysts derived from abnormally fertilized embryos. The nuclei of arrested embryos were also examined. The number of TE and ICM cells allocated in normally fertilized blastocysts appears to be similar to the numbers allocated in the mouse. Unlike the mouse, however, the proportion of ICM cells remains higher, despite cell death in both lineages.

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