Latent effects on in vitro development following cytochalasin B treatment of 8-cell mouse embryos

Development ◽  
1979 ◽  
Vol 51 (1) ◽  
pp. 97-108
Author(s):  
N. H. Granholm ◽  
G. M. Brenner ◽  
J. T. Rector
Keyword(s):  
Cytochalasin B ◽  
Observation Interval ◽  
Calf Serum ◽  
Essential Amino Acids ◽  
Mouse Embryos ◽  
High Incidence ◽  
Latent Effects ◽  
Vitro Development ◽  
Plastic Substratum

Eight-cell mouse embryos when treated with 4·0 μg/ml cytochalasin B (CB) in vitro undergo a reversible developmental arrest. Upon rinsing of embryos and subsequent culture in control medium, normal morphogenetic processes such as compaction of 8-cell embryos, cavitation, and post-blastocyst attachment and outgrowth are restored. However, the effects of CB on mouse embryos are not completely reversible; latent post-blastocyst defects become increasingly more prevalent as CB treatment duration increases. The present study was conducted to quantitatively determine latent effects of CB on post-blastocyst embryos by comparing their ability to attach and to sustain the growth and differentiation of ICM and trophoblast tissues. Groups of 8-cell embryos were cultured in Brinster's BMOC-3 medium containing 40 μg/ml cytochalasin B for 6, 12, 18, and 24h. Following treatment, embryos were rinsed and cultured until 190 h post coitun (h.p.c.) in Eagle's MEM/10% fetal calf serum modified to contain optimal levels of essential amino acids. Blastocysts generally attached to the surface of the plastic substratum by 120 h.p.c. At selected time periods after attachment (130, 160, and 190 h.p.c), embryos were scored for outgrowth size, ICM size, extent of peripheral hyaloplasmic fan, and number of trophoblast nuclei per outgrowth. Analyses of variance (ANOVAs) were conducted for each of the four parameters listed above. Rates of attachment were analyzed by χ2 test. Results show that the treatments affect (P < 0·01) embryo attachment, number of trophoblast nuclei per outgrowth, hyaloplasmic fan production, and ICM growth in a durationdependent manner. Interestingly, since treatment effects on outgrowth areas are nonsignificant apparently CB does not significantly change total outgrowth area. But CB tieatment does cause abnormal fan production and decreased trophoblast nuclei numbers. However, trophoblast cells are apparently more resistant than ICM to CB as is evident by the high incidence of tiophoblast outgrowths devoid of ICM. CB (4·0 μg/ml) treatments at 8-cell stages for relatively short durations (6 and 12 h) induce latent effects on post-blastocyst embryos. Finally, there exists a definite 4·0 μg/ml CB duration response over the 68–190 h.p.c. observation interval.

Effect of docosahexaenoyl dopamine on the in vitro development of early mouse embryos

2012 ◽  
Vol 442 (1) ◽  
pp. 38-41 ◽  
Author(s):  
N. Yu. Sakharova ◽  
L. N. Markova ◽  
A. A. Smirnov ◽  
E. F. Vikhlyantseva ◽  
L. A. Fialkovskaya ◽  
...  
Keyword(s):  
Mouse Embryos ◽  
In Vitro Development ◽  
Early Mouse ◽  
Vitro Development

In vitro development of inner cell masses isolated from t0/t0 and tw5/tw5 mouse embryos

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 419-428
Author(s):  
Brigid Hogan ◽  
Martha Spiegelman ◽  
Dorothea Bennett
Keyword(s):  
Outer Layer ◽  
Inner Core ◽  
Mouse Embryos ◽  
Advanced Stage ◽  
In Vitro Development ◽  
Vitro Development

Inner cell masses (ICMs) isolated immunosurgically from mouse blastocysts segregating the homozygous lethal mutants t0/t0 and tw5/tw5 were cultured in vitro. Presumed t0/t0 ICMs fail to grow after three days in culture (equivalent gestational day 7·5) when they consist of an outer layer of endoderm cells surrounding about 30 epiblast cells. Presumed homozygous tw5/tw5 ICMs develop to a more advanced stage in culture and on the seventh day (equivalent gestational day 11·5) consist of an inner core of disorganized ectoderm cells with a small proamniotic cavity, surrounded by multiple layers of endoderm cells.

Preimplantation development of gynogenetic diploid mouse embryos

Development ◽  
1982 ◽  
Vol 69 (1) ◽  
pp. 215-222
Author(s):  
Ewa Borsuk
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Mouse Embryos ◽  
Fertilization In Vitro ◽  
Body Formation ◽  
Male Pronucleus ◽  
Step Procedure ◽  
Gynogenetic Diploid ◽  
Polar Body Formation

Diploid gynogenetic mouse embryos were produced in a three-step procedure: fertilization in vitro, suppression of the 2nd polar body formation by Cytochalasin B, and microsurgical removal of the male pronucleus. The operated eggs were transplanted to the oviduct of recipient females for 72 or 96 h. The overall recovery rate was 73%, but compacted morulae and blastocysts constituted only 28·6% of transplanted eggs. After 72 h blastocysts were rare (3·5%) but 24 h later their incidence increased to 21·2%. In eggs homozygous for T6 chromosome it was possible to prove karyologically that the male pronucleus was effectively removed and that the diploid genome was of purely maternal origin.

47 ACTIVATION WITH IONOMYCIN FOLLOWED BY DEHYDROLEUCODINE AND CYTOCHALASIN B OF CLONED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 181
Author(s):  
N. Canel ◽  
R. Bevacqua ◽  
D. Salamone
Keyword(s):  
Cytochalasin B ◽  
Combined Treatment ◽  
Cumulus Cells ◽  
Normal Embryo ◽  
Intracytoplasmic Injection ◽  
Glass Slides ◽  
Cloned Bovine ◽  
Pronuclear Formation ◽  
Vitro Development

A combined treatment of dehydroleucodine (DhL) and cytochalasin B (CB) was previously demonstrated to induce pronuclear formation of bovine oocytes (Canel and Salamone 2008 Reprod. Fertil. Dev. 21, 214-215). The aim of this study was to evaluate the potential of DhL combined with CB to induce diploid activation of parthenogenetic embryos and to employ this treatment to assist cloning by intracytoplasmic injection of whole cumulus cells. To do that, COCs were collected from cow ovaries obtained from a slaughterhouse and in vitro-matured in TCM-199, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were treated with 5 μM ionomycin (Io) for 4 min and randomly assigned to the following activation groups: a) DhL/CB (incubation with 1 μM DhL and 5 μg mL-1 CB, for 3 h); b) DhL/long CB (treatment DhL/CB for 3 h, followed by exposure to 5 μg mL-1 CB alone, for 3 additional hours); and c) DMAP (incubation with 2 mM 6-DMAP for 3 h). In experiment 1, activated oocytes underwent IVC for 48 h and cleaved embryos were treated with 1 μg mL-1 colchicine for 6 h, fixed on glass slides, and stained with 5% vol/vol Giemsa solution to assess chromosomal complements. In experiment 2, MII oocytes were mechanically enucleated and injected with whole cumulus cells obtained from IVM COCs. After 2 h, reconstructed eggs were treated with 5 μM Io for 4 min and randomly exposed to the activation treatments a, b, or c. Parthenogenetic control groups were also included. All embryos were cultured in SOF medium and rates of cleavage, morulae, and blastocysts were evaluated on Days 2, 5, and 8 (Table 1). Results showed that DhL/long CB diploidy rates were significantly higher than those of DhL/CB and DMAP (63.8, 40. and 31.6%, respectively; Fisher’s test, P < 0.05). Both DhL treatments induced polyploidy rates lower than DMAP (5.2, 10.6, and 31.6%, respectively; P < 0.05). Finally, Io followed by DhL/CB or DhL/long CB was able to induce cloned blastocyst rates not statistically different from Io plus DMAP (P > 0.05), but presumably with a higher degree of normal embryo ploidy. Table 1.In vitro development of bovine cloned embryos activated with DhL and CB

Oesophagostomum radiatum: inhibition of in vitro development by newborn calf serum

1990 ◽  
Vol 64 (1) ◽  
pp. 9-14
Author(s):  
I. J. East ◽  
C. J. Fitzgerald
Keyword(s):  
Inhibitory Action ◽  
Natural Infection ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Newborn Calf Serum ◽  
In Vitro Development ◽  
Specific Antibodies ◽  
Serum Inhibition ◽  
Vitro Development

ABSTRACTOesophagostomum radiatum developed to fourth stage larvae after 14 days in in vitro culture. However, development was totally inhibited if the standard 50% foetal calf serum in the medium was replaced by newborn calf serum. Inhibition did not occur with serum from cattle immune to O. radiatum through natural infection or experimental vaccination irrespective of the titre of specific antibodies to O. radiatum in each serum. The inhibitory action of NCS could be abolished by heat treatment at 56°C for 1 h but not by dialysis or repeated freeze-thawing. The inhibition was not consistent with observed differences in the activity of 19 enzymes in the various sera or the absence of various thiol-containing stimulants of worm development.

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