Viability and growth of mouse embryos after in vitro culture and fusion

Development ◽  
1970 ◽  
Vol 23 (3) ◽  
pp. 693-704
Author(s):  
Patricia Bowman ◽  
Anne McLaren
Keyword(s):  
In Vitro Culture ◽  
Success Rate ◽  
Zona Pellucida ◽  
Growth Regulation ◽  
Excess Mortality ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Foster Mothers ◽  
Mouse Eggs

About 80 % of 8-cell mouse eggs developed to the blastocyst stage in culture, whether the zona pellucida was left intact, or removed with pronase (pre-incubated and dialysed) and the eggs then cultured singly or as fused pairs. When pronase was used without prior incubation and dialysis, the success rate was reduced to 50 %. After transfer to uterine foster-mothers, 20–30 % of apparently normal blastocysts cultured with or without the zona, singly or fused, developed into live foetuses, compared with over 50 % of control blastocysts taken directly from the uterus. Some of the excess mortality of cultured embryos took place before implantation and some soon after. The foetuses derived from cultured blastocysts averaged 0·1 g lighter than those derived from control uterine blastocysts similarly transferred. No differences in the weights of the placentae were observed. Foetal and placental weights were unaffected by whether the eggs had been cultured singly or fused, implying that growth regulation of fused embryos is complete by the 17th day of gestation. The longer the eggs were maintained in culture, the lower was their viability after transfer, and the lighter were the foetuses derived from them.

Normal adenylate ribonucleotide content in mouse embryos homozygous for the t12 mutation

Development ◽  
1983 ◽  
Vol 78 (1) ◽  
pp. 43-51
Author(s):  
Horst Spielmann ◽  
Robert P. Erickson
Keyword(s):  
In Vitro Culture ◽  
Normal Values ◽  
Firefly Luciferase ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Luciferase Assay ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse

The recently improved firefly luciferase assay was used to determine ATP, ADP or AMP in single preimplantation mouse embryos from crosses yielding lethal t12/t12 embryos. Normal values of the three adenylate ribonucleotides were found in freshly collected 2-cell and 4-cell embryos and during in vitro culture to the blastocyst stage. A decrease in adenylate ribonucleotide content was seen in putative t12/t12 embryos only when they were degenerating.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation

1995 ◽  
Vol 29 (3) ◽  
pp. 320-326 ◽  
Author(s):  
Th. Rülicke ◽  
P. Autenried
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Freeze Thaw ◽  
Foster Mothers ◽  
Vital Stains ◽  
Partial Damage

Approximately 18% of cryopreserved 2-cell mouse embryos of 26 different batches showed various degrees of morphological damage after the freeze-thaw process. Normal and damaged morphology were assessed by light microscopy and the ability of an embryo to develop in vitro to a blastocyst, or to develop to term, after transfer to foster mothers. Using vital stains such as Fluorescein-diacetate (FDA) and 4',6-Diamidino-2-Phenylindole (DAPI) it was found that in approximately 82% of the cases, both of the 2 blastomeres of the cryopreserved embryos survived the freeze-thaw process; in 10% only one cell survived the process; and in 8% none survived. Normally, only intact 2-cell embryos are considered for transfer. Here it was shown that over 60% of the partially damaged embryos developed in vitro to the blastocyst stage and, of those, 26% developed to term after transfer to suitable foster mothers. Although the inner cell mass (ICM) appeared to remain smaller during culture after the transfer of partially damaged 2-cell stage embryos, no difference during gestation period was found compared with intact embryos.

The role of the zona pellucida in the development of mouse eggs in vivo

Development ◽  
1970 ◽  
Vol 23 (3) ◽  
pp. 539-547
Author(s):  
Jacek A. Modliński
Keyword(s):  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Development In Vitro ◽  
Single Blastomeres ◽  
Mammalian Eggs ◽  
Mouse Eggs

Up to the present time the function and significance of the zona pellucida in the development of mammalian eggs has not been fully explained. Zona-free mouse eggs will develop in vitro from the 2-cell stage, or later, up to the blastocyst stage (Tarkowski, 1961; Mintz, 1962; Gwatkin, 1963). Single blastomeres isolated at the 2-cell (Mulnard, 1965), 4- and 8-cell stage (Tarkowski & Wróblewska, 1967) will also develop in vitro up to the blastocyst stage. Similar experiments on development in vitro of 1- and 2-cell rabbit eggs (Edwards, 1964) showed that in this species also cleavage can occur when the zona pellucida is absent, although the blastomeres exhibit a tendency to fall away from each other. Tarkowski's observations (unpublished) would appear to show, however, that naked 1-, 2- and 4-cell mouse eggs do not develop when transferred to the oviduct. A few hours after transplanting the naked eggs none could be recovered by flushing the oviduct, whereas eggs surrounded by zonae which were transplanted simultaneously were recovered.

In vivo survival of transferred sheep embryos following puncture of the zona pellucida and in vitro culture

1994 ◽  
Vol 35 (1-2) ◽  
pp. 81-89 ◽  
Author(s):  
P.A. Pugh ◽  
J.G. Thompson ◽  
K. Logan ◽  
H.R. Tervit
Keyword(s):  
In Vitro Culture ◽  
Zona Pellucida

Development of parthenogenetic and fertilized mouse embryos in the uterus and in extra-uterine sites

Development ◽  
1975 ◽  
Vol 34 (2) ◽  
pp. 387-405
Author(s):  
S. A. Iles ◽  
M. W. McBurney ◽  
S. R. Bramwell ◽  
Z. A. Deussen ◽  
C. F. Graham
Keyword(s):  
Reproductive Tract ◽  
Neural Tissue ◽  
Cell Types ◽  
Mouse Embryos ◽  
Female Reproductive Tract ◽  
Embryonic Cells ◽  
Haploid Embryos ◽  
Keratinized Epithelium ◽  
Mouse Eggs

Mouse eggs were activated with hyaluronidase in vitro and subsequently transferred to the oviduct. In the female reproductive tract they formed morulae and blastocysts which died soon after implantation. Haploid blastocysts were transferred beneath the kidney capsule and here some formed disorganized egg-cylinder structures in a week. Morulae and blastocysts from haploid and diploid parthenogenones were also transferred beneath the testis capsule. Two to four months later the growths which had formed were sectioned. They contained neural tissue, pigment, keratinized epithelium, glandular epithelium, ciliated epithelium, cartilage, bone, muscle, adipose tissue, and haemopoietic tissue. The range of cell types was similar to that produced by fertilized control blastocysts except that the parthenogenones did not form identifiable yolk-sac carcinoma or embryonal carcinomacells. The growths from haploid and diploid parthenogenones in the testis were stained with Feulgen and their DNA content measured. Growths from diploid embryos contained the normal diploid amount of DNA while growths from haploid embryos contained less than this amount. Cell cultures were prepared from the growths. The cells which were investigated contained no Y chromosome, suggesting that they were derived from the embryonic cells rather than the cells of the male host. These cells contained a near diploid chromosome number, although some of them were originally derived from haploid embryos.

Lethality of a tritiated amino acid in early mouse embryos

Development ◽  
1985 ◽  
Vol 88 (1) ◽  
pp. 209-217
Author(s):  
Janet L. Wiebold ◽  
Gary B. Anderson
Keyword(s):  
Specific Activity ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Radiation Induced ◽  
Specific Activities ◽  
Tritiated Amino Acids ◽  
Early Mouse

2- to 4-cell and morula- to blastocyst-stage mouse embryos were cultured for 1 h in tritiated leucine at two specific activities and their subsequent development followed in vitro and in vivo (after transfer to recipients), respectively. 2- to 4-cell embryos that incorporated an average of 42 d.p.m. per embryo were impaired in their ability to develop to the morula and blastocyst stage. Recipients receiving morulae and blastocysts that had incorporated an average of 384 d.p.m. per embryo failed to produce young. Reduction of the specific activity improved the viability of embryos both in vitro and in vivo but development was still less than that of unlabelled embryos. Protein degradation curves were different for both 2- to 4-cell and morulato blastocyst-stage embryos labelled at the two different specific activities. Most studies using tritiated amino acids have employed higher specific activities than those used here and they may have to be reevaluated due to the possibility of radiation-induced artifacts.

Localization of cytoskeletal proteins in preimplantation mouse embryos

Development ◽  
1980 ◽  
Vol 55 (1) ◽  
pp. 211-225
Author(s):  
E. Lehtonen ◽  
R. A. Badley
Keyword(s):  
Blastocyst Stage ◽  
Cytoskeletal Proteins ◽  
Mouse Embryos ◽  
Trophectoderm Cells ◽  
Apparent Concentration ◽  
Preimplantation Mouse ◽  
Early Mouse ◽  
Filament Protein

The immunofluorescence technique was used to detect the presence and distribution of actin, alpha-actinin, tubulin and 10 nm filament protein in early mouse embryos. Actin and alpha-actinin stainings showed a distinct concentration to a peripheral layer in the cleavage-stage blastomeres and in trophectoderm cells. Dots of fluorescence appeared in this cortical staining pattern. The distribution of tubulin staining in the blastomere cytoplasm was relatively even with apparent concentration at the perinuclear region and frequently at wide intercellular contact areas. 10 nm filament protein was distributed evenly in the blastomere cytoplasm without cortical concentration of the label. At the blastocyst stage, the trophectoderm cells in blastocyst outgrowths in vitro developed well organized cytoskeletons including both microfilament, microtubule and 10 nm filament elements. Comparable structures were not observed in blastocysts in vivo, or in late hatched blastocysts cultured in suspension. The morphogenetic significance of the observations is discussed.

185 EMBRYONIC LOSS IN PIGS ASSOCIATED WITH OVIDUCT TRANSPLANTATION OF EARLY-STAGE EMBRYOS WITH DAMAGES IN THE ZONA PELLUCIDA

2006 ◽  
Vol 18 (2) ◽  
pp. 200
Author(s):  
S. Ueno ◽  
M. Kurome ◽  
R. Tomii ◽  
K. Hiruma ◽  
N. Maeda ◽  
...  
Keyword(s):  
Immune Response ◽  
Zona Pellucida ◽  
Nuclear Transfer ◽  
Early Stage ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Embryo Recovery ◽  
Embryonic Loss ◽  
The Impact

It is assumed that if porcine early-stage embryos with damages in their zonae pellucidae are transplanted to the recipient's oviduct, they may suffer from mechanical and immunological stresses by oviduct contraction and the recipient's immune response. This study aimed to examine the impact of zona pellucida damages, which might arise during nuclear transfer and intra cytoplasmic sperm injection (ICSI), on the development and survival of transplanted embryos. Cumulus-oocyte complexes were collected from ovaries obtained at a local slaughterhouse and matured in vitro in NCSU23 to prepare MII-stage oocytes. The zonae pellucidae of these oocytes were either penetrated with 8- to 10-�m square-ended microinjection pipettes or incised with 35- to 40-�m beveled enucleation pipettes. Intact oocytes were used as controls. The oocytes were electroactivated to induce parthenogenesis and transplanted to the oviducts of estrus-synchronized recipient gilts (estrus-synchronized with 1000 IU eCG and 1500 IU hCG). After 5 to 7 days, the recipient uteri were flushed with PBS supplemented with 1% fetal bovine serum (FBS) to collect embryos, and their development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-penetrated, 129 zona-incised, and 57 intact embryos were transplanted to four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (59.0 to 81.8%). However, the zona-penetrated and zona-incised embryos showed inconsistent development and survival compared with controls; the development and survival rate were 92.6% (25/27) to 96.7% (29/30) and 77.8% (21/27) to 96.7% (29/30) in control embryos, respectively, whereas those of zona-penetrated embryos were 57.1% (28/49) to 95.7% (22/23) and 8.2% (4/49) to 78.3% (18/30), and those of zona-incised embryos were 47.6% (30/63) to 92.3% (36/39) and 23.8% (15/63) to 92.3% (22/23), respectively. Large foci of cells that appeared to be macrophage giant cells were observed at the surface or inside of the degenerated zona-damaged embryos. These results indicate that the recipient's immune response may impair development after transplantation of the embryo to the oviduct, when there is damage in the zona pellucida. This may be one of the factors attributable to the reduced efficiency of live progeny production by ICSI and nuclear transfer. This work was supported by PROBRAIN.

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