The utilization of an inhibitor of spermidine and spermine synthesis as a tool for the study of the determination of cavitation in the preimplantation mouse embryo

Development ◽  
1979 ◽  
Vol 53 (1) ◽  
pp. 145-162
Author(s):  
H. Alexandre
Keyword(s):  
Biological Clock ◽  
Duration Of Treatment ◽  
Experimental Conditions ◽  
Cell Stage ◽  
Cytoplasmic Factor ◽  
Average Cell ◽  
Length Of Treatment ◽  
Preimplantation Mouse ◽  
The One

The inhibition of spermidine and spermine synthesis by methylglyoxal-Bis(guanylhydrazone) (MeGAG) at concentrations of 5, 10 and 20 µM, induces a reversible metabolic quiescence of mouse embryos, cultured in vitro from the 2-cell stage, at an average of 10·2, 8·5 and 6·9 cell stages respectively. In contrast, the inhibition of putrescine synthesis by α-methylornithine (α-MeOrn) at concentrations up to 10 mM fails to inhibit blastocyst formation, as shown previously. Complete reversibility of this induced arrest of development is observed for treatments up to 31 h with MeGAG at 10 µM. In agreement with the biological clock theory of Smith & MacLaren's hypothesis, the delay in cavitation is proportional to the length of treatment. However, the average cell numbers of the ‘delayed nascent blastocysts’ of all treated embryos (21·8–24·2) are consistently lower than that of control embryos (33·6) irrespective of the duration of treatment. It seems therefore that under some experimental conditions, DNA and chromosome replication on the one hand and cytoplasmic maturation on the other may be desynchronized. This suggests a role for a cytoplasmic factor in the induction of cavitation.

The effect of 5-bromodeoxyuridine on cell division and differentiation of preimplantation mouse embryos

Development ◽  
1976 ◽  
Vol 35 (1) ◽  
pp. 169-178
Author(s):  
D. R. Pollard ◽  
M. M. Baran ◽  
R. B. Bachvarova
Keyword(s):  
Cell Division ◽  
Cell Number ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Average Cell ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Excess Thymidine ◽  
System Cell

Mouse embryos exposed to concentrations of 5-bromodeoxyuridine (BUdR) ranging from 0·01 to 1·0 μg/ml in vitro for two days from the 8-cell stage exhibit a concentration-dependent decrease in the frequency of normal blastocysts and a decrease in average cell number per embryo. A 20-h exposure was adequate to achieve the full BUdR response. Both effects were eliminated in the presence of excess thymidine. Autoradiographs demonstrated that BUdR[3H] was incorporated into DNA during the first and second day of culture. Thus, BUdR appears to act through incorporation into DNA; and, in this system, cell division is at least as sensitive to BUdR as is differentiation.

scholarly journals Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 301-311 ◽  
Author(s):  
Paolo Rinaudo ◽  
Richard M Schultz
Keyword(s):  
Gene Expression ◽  
Preimplantation Embryos ◽  
Expression Analysis Systematic Explorer ◽  
Cell Stage ◽  
Global Pattern ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Global Patterns ◽  
The One

Culture of preimplantation embryos affects gene expression. The magnitude of the effect on the global pattern of gene expression, however, is not known. We compared global patterns of gene expression in blastocysts cultured from the one-cell stage in either Whitten’s medium or KSOM + amino acids (KSOM/AA) with that of blastocysts that developed in vivo, using the Affymetrix MOE430A chip. The analysis revealed that expression of 114 genes was affected after culture in Whitten’s medium, whereas only 29 genes were mis-expressed after culture in KSOM/AA. Expression Analysis Systematic Explorer was used to identify biological and molecular processes that are perturbed after culture and indicated that genes involved in protein synthesis, cell proliferation and transporter function were down-regulated after culture in Whitten’s medium. A common set of genes involved in transporter function was also down-regulated after culture in KSOM/AA. These results provide insights as to why embryos develop better in KSOM/AA than in Whitten’s medium, and highlight the power of microarray analysis to assess global patterns of gene expression.

Mouse one-cell embryos undergoing a radiation-induced G2 arrest may re-enter S-phase in the absence of cytokinesis

2002 ◽  
Vol 80 (7) ◽  
pp. 618-624 ◽  
Author(s):  
P Jacquet ◽  
J Buset ◽  
J Vankerkom ◽  
S Baatout ◽  
L de Saint-Georges ◽  
...  
Keyword(s):  
Calyculin A ◽  
Embryonic Cells ◽  
G2 Arrest ◽  
Cell Stage ◽  
Chromosome Fragments ◽  
X Irradiation ◽  
Radiation Induced ◽  
Mouse Zygotes ◽  
The One

PCC (premature chromosome condensation) can be used for visualizing and scoring damage induced by radiation in the chromatin of cells undergoing a G1 or G2 arrest. A method involving the fusion of irradiated single embryonic cells with single MI oocytes was used to induce PCC in mouse zygotes of the BALB/c strain, which suffer a drastic G2 arrest after X-irradiation (dose used 2.5 Gy). Other G2-arrested embryos were exposed in vitro to the phosphatase inhibitor calyculin A. Both methods furnished excellent chromosome preparations of the G2-arrested embryos. The mean number of chromosome fragments did not change significantly during G2 arrest, suggesting that zygotes of this strain are unable to repair DNA damage leading to such aberrations. Forty to fifty percent of the irradiated embryos were unable to cleave after G2 arrest and remained blocked at the one-cell stage for a few days before dying. PCC preparations obtained from such embryos suggested that about 30% of them had undergone a late mitosis not followed by cytokinesis and had entered a new DNA synthesis. These results are discussed in the light of recent observations in irradiated human cells deficient in the p53/14-3-3sigma pathway.Key words: PCC, embryo, oocyte, calyculin A, G2 arrest, cytokinesis.

98 SUCCESSFUL PREGNANCIES FOLLOWING TRANSFER OF VITRIFIED PORCINE EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 157 ◽  
Author(s):  
K. Hiruma ◽  
H. Ueda ◽  
H. Saito ◽  
C. Tanaka ◽  
N. Maeda ◽  
...  
Keyword(s):  
Calf Serum ◽  
Developmental Competence ◽  
Penicillin G ◽  
Cell Stage ◽  
Epidermal Growth ◽  
Potassium Penicillin ◽  
The One ◽  
Parthenogenetic Embryos

To date only in vivo-produced embryos have successfully produced live piglets after cryopreservation. In this study, we aimed to produce piglets from vitrified embryos derived from in vitro matured (IVM) oocytes. Cumulus-oocyte complexes collected from ovaries obtained at a local slaughterhouse were matured for 44 to 45 h in NCSU23 MEDIUM supplemented with 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 �g/mL potassium penicillin G, 50 �g/mL streptomycin sulfate, and 10 IU/mL eCG/ hCG. These IVM oocytes were either activated for parthenogenesis or in vitro-fertilized (IVF). For IVF, oocytes were incubated with 5 � 106/mL of cryopreserved epididymal sperm in PGM-tac medium (Yoshioka et al. 2003 Biol. Reprod. 69, 2092-2099) for 20 h. Embryos were treated for removal of cytoplasmic lipid droplets (delipation; Nagashima et al. 1995 Nature 374, 416) at the 4- to 8-cell stages, around 50 to 54 h after activation or insemination. After culture in NCSU23 for 15 h, they were vitrified by the minimum volume cooling (MVC) method. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO), and 20% (v/v) calf serum for 4 min, followed by exposure to vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% calf serum. Embryos were then loaded onto a Cryotop (Kitazato Supply Co., Tokyo, Japan) and immediately plunged into liquid nitrogen. Vitrified embryos were examined for viability in vitro and in vivo after warming. Their in vitro developmental competence was compared to that of corresponding control (nonvitrified) embryos. Vitrified 4- to 8-cell stage embryos, both parthenogenetic and IVF, showed developmental competence into blastocysts comparable to that of control embryos (parthenogenetic: 46.8%, 36/77 vs. 51.7%, 31/60; IVF: 40.0%, 30/75 vs. 44.3%, 35/79). Of four surrogate gilts that received a total of 251 vitrified parthenogenetic embryos, three became pregnant and had 20 fetuses (8.0%, 22 to 23 days old). Three surrogates gilts that received 267 vitrified IVF embryos all became pregnant. Of those, the one that received 47 embryos was confirmed to have eight fetuses (17.0%, 22 days old) by autopsy. The other two were examined by ultrasonography at 56 and 95 days of gestation and found to be pregnant. These results suggest that porcine embryos derived from IVM oocytes have a potential to develop into live offspring after delipation and MVC vitrification. This study was supported by PROBRAIN.

scholarly journals Successful production of offspring after superovulation and in vitro culture of embryos from domestic ferrets (Mustela putorius furos)

Reproduction ◽  
2001 ◽  
pp. 611-618 ◽  
Author(s):  
ZY Li ◽  
QS Jiang ◽  
YL Zhang ◽  
XM Liu ◽  
JF Engelhardt
Keyword(s):  
Genetic Manipulation ◽  
Blastocyst Stage ◽  
Chorionic Gonadotrophin ◽  
Mustela Putorius ◽  
Cell Stage ◽  
Fetal Bovine ◽  
The One ◽  
Genetic Modelling ◽  
Successful Production

In an effort to expand the use of ferrets as models for genetic disease, several experimental parameters that are required for successful genetic manipulation in this species were investigated. Optimum superovulation (19.3 +/- 0.6 oocytes and embryos per female) was achieved after injections of 100 iu equine chorionic gonadotrophin (eCG) and 150 iu human chorionic gonadotrophin (hCG). The ovulation rate achieved by the treatment was more than double that induced by mating. Mating with a male immediately after hCG treatment did not significantly alter the number of oocytes ovulated or the number of embryos present, indicating that mating is not required for superovulation in ferrets. Of embryos harvested at the one-cell stage, 64.5% and 47.1% developed into blastocysts when cultured in vitro in CZB or TCM-199 plus 10% fetal bovine serum (FBS) media, respectively. In contrast, only 17.1% of embryos cultured in vitro in NCSU-23 developed to the blastocyst stage. Both freshly retrieved and in vitro cultured embryos from cinnamon-coloured parents produced live young when transferred at the eight-cell stage into albino, pseudo-pregnant recipients. The percentage of kits delivered relative to embryos transferred was 61% for freshly retrieved embryos and 32% for embryos cultured in vitro. These results demonstrate successful embryo transfer in ferrets and provide a basis for further study of genetic modelling approaches in this species after embryo manipulation.

Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues

1994 ◽  
Vol 14 (7) ◽  
pp. 4694-4703
Author(s):  
E M Thompson ◽  
E Christians ◽  
M G Stinnakre ◽  
J P Renard
Keyword(s):  
Transgene Expression ◽  
Developmental Stages ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Cell Stage ◽  
Position Effects ◽  
Adult Mice ◽  
Preimplantation Mouse ◽  
Transgenic Lines

Eukaryotic interphase chromatin is thought to be organized into topologically discrete, independent domains acting as units upon which differential patterns of gene expression are established. Sequences which attach chromatin to in vitro preparations of a nucleoprotein matrix (scaffold attachment regions [SARs]) may act as domain boundaries, but their role remains poorly defined compared with those of other elements such as locus control regions. We have produced mice homozygous for a transgene which is transcribed as early as the activation of the embryonic genome at the two-cell stage and which is expressed ubiquitously in a number of differentiated tissues. Transgenic lines were generated in the presence or absence of flanking SAR sequences, creating an original model which enabled us to examine the effects of these elements at different developmental stages. In the preimplantation mouse embryo, flanking SARs stimulated transgene expression in a copy-dependent manner. In contrast, in the differentiated tissues of newborn and adult mice, no significant SAR-dependent increase in transgene expression was found, correlation with copy number was lost, and position effects were observed. These results suggest a limited capacity of SARs to act as insulating elements but are consistent with a proposed model of SAR-mediated chromatin opening and closing.

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.

scholarly journals The Influence of Light on Apoptosis in Preimplantation Mouse Embryos In Vitro

2019 ◽  
Author(s):  
Sepideh Khalili-Savadkouhi ◽  
Abbasali Karimpour Malekshah ◽  
Mehri Mirhoseini ◽  
Mahmood Moosazadeh ◽  
Maryam Shahidi
Keyword(s):  
Embryonic Development ◽  
Developmental Stages ◽  
Harmful Effect ◽  
Cell Number ◽  
Fluorescent Light ◽  
Cell Stage ◽  
Significant Difference ◽  
Preimplantation Mouse ◽  
Mammalian Embryos

Background: In vitro culture of mammalian embryos can slow or stop growth completely. This may be due to the medium used, pH, temperature, or light. There is considerable concern about the harmful effect of light in the laboratory environment. Cell number and apoptosis are useful parameters that indicate embryonic development and health. In this study, we assessed these two factors in the blastocyst. Materials and methods: A total of 128 embryos were extracted from NMRI mice at the 2-cell stage and were divided into 4 groups. The embryos were exposed to light for 0, 5, 15, and 30 min, and then cultured for 96 h. The degree of embryonic development were recorded every 24 h. Furthermore, several morphologically normal blastocysts were evaluated using the TUNEL assay. Results: There was no significant difference in developmental stages between the experimental and control groups. An evaluation of the percentage of blastomeres and apoptotic cells revealed significant differences among the four groups. The maximum number of apoptotic blastomeres was observed in the group exposed to light for 30 minutes. Conclusion: Up to thirty minutes of white fluorescent light can induce apoptosis in blastomeres, but it does not prevent embryo development.

103 BIRTH OF CLONED PIGLETS DERIVED FROM AN OPTIMIZED IN VITRO BLASTOCYST PRODUCTION SYSTEM BY TRICHOSTATIN A TREATMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 168
Author(s):  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
K. Zhang ◽  
J. Li ◽  
P. M. Kragh ◽  
...  
Keyword(s):  
Culture Media ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Deacetylase Inhibitor ◽  
The One ◽  
Effective Group

The present study was designed to examine the effect of trichostatin A (TSA, a histone deacetylase inhibitor) treatment on in vitro developmental ability of pig cloned embryos and to evaluate the feasibility of producing piglets from these embryos. Cell lines were established from 40-day-old fetuses, and adult ear skin was used as nuclear donor. In vitro-matured oocytes from abattoir-derived sow ovaries were used as cytoplast recipients for micromanipulator-assisted somatic cell nuclear transfer (SCNT). Data were analyzed by using SPSS (11.0) with one-way ANOVA, and each experiment was replicated at least 3 times. In Experiment 1, immediately after simultaneous fusion and activation, the reconstructed couplets were randomly cultured in porcine zygote medium 3 (PZM3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) with 10 �g mL-1 cytochalasin B (CB), 10 �g mL-1 cycloheximide (CHX), and 0 nM, 5 nM, or 50 nM TSA for the first 4 h. Cloned embryos (fused reconstructed couplets) were moved to the same culture media but without CB and CHX and further cultured at 38.5�C, under 5% CO2, 5% O2, 90% N2 and 100% humidity. After incubation for a total of 8–14 h in 50 nM, 19–24 h in 50 nM or 5 nM, and 31–36 h in 50 nM TSA in PZM3 (0 nM TSA serves as control for each group), the embryos were further cultured in vitro without TSA in PZM3 for up to 168 h. Cleavage and blastocyst development rates, based on embryos cultured, were recorded at 48 and 168 h of IVC, respectively. Results showed that 50 nM TSA treatment for 19-24 h supported a higher blastocyst development rate than the control group [No. blastocysts/No. embryos cultured (mean � SEM): 107/258, 47.4 � 5.9% vs. 65/324, 20.0 � 2.3%, respectively; P < 0.05], whereas similar pre-implantation development was obtained between the other 3 test groups and the control. In Experiment 2, TSA-treated cloned embryos at the one-cell stage or blastocyst stage were transferred to recipients to examine the possibility of producing piglets. Ten cloned piglets (2 are healthy and 8 died shortly after birth) and one ongoing pregnancy were obtained from 3 recipients who received an average of 110 one-cell stage embryos, whereas 4 piglets originating from traditional cloning were produced from one recipient which received 28 traditional cloned blastocysts (produced from the effective group in Experiment 1) and 30 handmade but non-TSA-treated ones. Our data demonstrate that TSA treatment after SCNT in porcine can significantly improve the in vitro blastocyst production, and embryos treated with TSA could support full-term development and result in healthy offspring.

scholarly journals Developmental exposure window influences silver toxicity but does not affect the susceptibility to subsequent exposures in zebrafish embryos

2020 ◽  
Vol 154 (5) ◽  
pp. 579-595
Author(s):  
Paige C. Robinson ◽  
Hannah R. Littler ◽  
Anke Lange ◽  
Eduarda M. Santos
Keyword(s):  
Early Development ◽  
Toxic Metal ◽  
Positive Control ◽  
Zebrafish Embryos ◽  
Chemical Toxicity ◽  
Experimental Conditions ◽  
Cell Stage ◽  
Developmental Exposure ◽  
Methylation Inhibitor ◽  
The One

AbstractSilver is a non-essential, toxic metal widespread in freshwaters and capable of causing adverse effects to wildlife. Its toxic effects have been studied in detail but less is known about how sensitivity varies during development and whether pre-exposures affect tolerance upon re-exposure. We address these knowledge gaps using the zebrafish embryo (Danio rerio) model to investigate whether exposures encompassing stages of development prior to mid-blastula transition, when chorion hardening and epigenetic reprogramming occur, result in greater toxicity compared to those initiated after this period. We conducted exposures to silver initiated at 0.5 h post fertilisation (hpf) and 4 hpf to determine if toxicity differed. In parallel, we exposed embryos to the methylation inhibitor 5-azacytidine as a positive control. Toxicity increased when exposures started from 0.5 hpf compared to 4 hpf and LC50 were significantly lower by 1.2 and 7.6 times for silver and 5-azacyitidine, respectively. We then investigated whether pre-exposure to silver during early development (from 0.5 or 4 hpf) affected the outcome of subsequent exposures during the larvae stage, and found no alterations in toxicity compared to naïve larvae. Together, these data demonstrate that during early development zebrafish embryos are more sensitive to silver when experiments are initiated at the one-cell stage, but that pre-exposures do not influence the outcome of subsequent exposures, suggesting that no long-lasting memory capable of influencing future susceptibility was maintained under our experimental conditions. The finding that toxicity is greater for exposures initiated at the one-cell stage has implications for designing testing systems to assess chemical toxicity.

Sign in / Sign up

Export Citation Format

Share Document