Development of cytochalasin B-induced tetraploid and diploid/tetraploid mosaic mouse embryos

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 47-64 ◽  
Author(s):  
A. K. Tarkowski ◽  
A. Witkowska ◽  
J. Opas
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cytochalasin B ◽  
Primitive Streak ◽  
Cell Number ◽  
Mouse Embryos ◽  
Proliferation Rate ◽  
Foetal Membranes ◽  
Diploid Cells

By subjecting F1 (CBA × C57/BL) × A eggs at the time of 2nd cleavage to 10 μg/ml of cytochalasin B (CB), tetraploidy was produced in 52 % of 2-cell eggs and 35 % of 3-cell eggs. 2n/4n mosaic embryos were produced from 2-, 3- and 4-cell eggs and amounted to 20 % of all treated eggs. 80 % of tetraploid embryos developed in vitro into regular blastocysts with half the cell number of control diploids. The effectiveness of CB in producing tetraploid embryos is limited by the asynchrony of 2nd cleavage, both between eggs and between sister blastomeres. Two-cell presumed tetraploids were transplanted to recipients and examined between the 6th and 11th day of pregnancy. Up to 6½ days development is normal and most embryos form egg-cylinders. At 7½ days the embryonic part of the cylinders is underdeveloped and in later development fails to form an embryo. Development of foetal membranes is much less affected and in the most successfully developing egg-cylinders their formation can be fully accomplished. Failure of embryonic development appears to be due to subnormal activity of the primitive streak, resulting in shortage of mesoderm. Postimplantation development of 2n/4n mosaics was normal. While in embryos tetraploid cells were either absent or in very low proportion (below 4 %), their contribution to the foetal membranes amounted in some cases to up to 50 %. Elimination of tetraploid cells from mosaic embryos suggests that they have a lower proliferation rate than diploid cells.

scholarly journals Postimplantation development of CB-induced triploid mouse embryos

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 81-89
Author(s):  
Anna Niemierko
Keyword(s):  
Embryo Development ◽  
Cytochalasin B ◽  
Polar Body ◽  
Mouse Embryos ◽  
Foetal Membranes ◽  
Polar Body Extrusion

By subjecting A strain eggs at the time of fertilization and polar body extrusion to 5 µg/ml cytochalasin B, digynic triploidy was produced in 80% fertilized eggs. Triplonucleate eggs were transplanted to recipients and examined between 9–1lth day of pregnancy. Development of triploid mouse embryos up to day 7 is normal and most embryos form early egg cylinder. At day 8 the embryonic part of the cylinders is under-developed and later development fails to form an embryo. Development of foetal membranes is much less affected, CB-induced triploids survive to 10th- day of pregnancy.

Factors affecting the time of formation of the mouse blastocoele

Development ◽  
1977 ◽  
Vol 41 (1) ◽  
pp. 79-92
Author(s):  
Rosita Smith ◽  
Anne McLaren
Keyword(s):  
Cytochalasin B ◽  
Critical Factor ◽  
Cell Number ◽  
Experimental Manipulation ◽  
Factors Affecting ◽  
Elapsed Time ◽  
Developmental Age ◽  
Culture Formation

In normal mouse embryos developing in vivo, the first appearance of the blastocyst cavity was found to be associated more closely with developmental age, judged by cell number, than with chronological age, i.e. elapsed time since ovulation. When development was slowed by in vitro culture, formation of the blastocoele was delayed. However, cell number itself was not a critical factor, since the number of cells per embryo could be doubled or tripled or halved by experimental manipulation without substantially affecting the timing of blastocoele formation. Experiments in which one cell division was suppressed with cytochalasin-B, leading to tetraploidy, showed that the number of cell divisions since fertilization was also not critical. A possible role is suggested either for nucleocytoplasmic ratio, or for the number of nuclear or chromosomal divisions or DNA replications since fertilization, all of which increase during cleavage.

P–060 Dose- dependent mitigation of lead acetate toxicity in males on embryo development in female mice

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
P Dolati ◽  
M J Zamiri ◽  
A Akhlaghi ◽  
Z Jahromi
Keyword(s):  
Adverse Effects ◽  
Embryonic Development ◽  
Embryo Development ◽  
Lead Acetate ◽  
Endocrine System ◽  
Cell Number ◽  
The Other ◽  
Control Group ◽  
Male Mice ◽  
Female Mice

Abstract Study question Does quercetin (75 or 100 mg/kg BW/day) co-administration with lead acetate to male mice affects embryonic development in female mice? Summary answer The low-dose quercetin (75 mg/kg BW/day) ameliorated the adverse effects of lead acetate on mouse embryogenesis. What is known already Lead causes male infertility by impacting on endocrine system and spermatogenesis, and may exert undesirable effects on the offspring. The currently approved treatment for lead poisoning is the use of chelating agents, which form an insoluble complex with lead and shield it from biological targets; thus, reducing its toxicity. One of the main mechanisms of lead-induced toxicity is oxidative stress, and it has been reported that natural antioxidants can reduce the heavy metals toxicity. The aim of the present study was to examine the protective effects of quercetin on the toxicity induced by lead acetate on the embryogenesis in mice. Study design, size, duration Sexually mature (eight-week-old) NMRI male mice (n = 24) were randomly divided into four groups (n = 6 per group) receiving (i) distilled water (control group); (ii) lead acetate (150 mg/kg BW/day) dissolved in deionized water (LA); (iii) lead acetate (150 mg/kg BW/day) + quercetin (75 mg/kg BW/day) (LQ75); (IV) lead acetate (150 mg/kg BW/day) + quercetin (100 mg/kg BW/day) (LQ100). Treatments were applied daily as oral gavages for one cycle of the seminiferous epithelium (35 days). Participants/materials, setting, methods At the end of treatment administration, the males were joined with super-ovulated females, and the retrieved zygotes were cultured for evaluation of the embryo development (at 2-cell, 4-cell, 8-cell, and blastocyst stages), and blastocyst cell number using differential staining (propidium iodide and bisbenzimide). After incubation of capacitated sperm with oocytes, an ultraviolet light microscope was used following 3 min incubation with 25 µg⁄mL bisbenzamide solution for fertilization assessment. Main results and the role of chance Lead acetate (LA) treatment of male mice decreased the 2-cell stage compared with the control group (P > 0.05). There was no difference between control and LQ75, and between LA and LQ100. The other stages of embryonic development were not significantly affected by the treatment. Overall, early embryonic development in the control and LQ75 mice were better than LQ100 and LA mice. The number of cells in the trophectoderm and inner-cell mass were not affected by treatments. However, the total blastocyst cell number in the control was higher than in the other groups; there was no significant difference between LQ100, LQ75 and LA groups. Fertilization rate was not affected by the treatments (P < 0.05). Quercetin acts as a potent antioxidant at low doses, but at high doses exerts a pro-oxidant action. According to previous reports, higher concentrations of quercetin increased apoptosis and necrosis while decreasing the activities of the antioxidant enzymes. Also, it has been suggested that quercetin might disrupt the endocrine system and interfere with Sertoli cell function and sperm motility. Limitations, reasons for caution A limitation of this study is narrow dose selection; more studies are needed to determine the effective dose of quercetin in ameliorating the lead toxicity. There are also side effects of lead-quercetin chelates such as metal redistribution, essential metal loss, accumulation and persistency in intracellular sites, and peroxidation. Wider implications of the findings: Lead administration adversely impacted on the embryogenesis; on the other hand, paternal quercetin co-administration somewhat ameliorated the adverse effects of lead on mice embryogenesis. Trial registration number Not applicable

Effects of changing culture medium on preimplantation embryo development in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Haixia Wang ◽  
Wenbin Cao ◽  
Huizhong Hu ◽  
Chenglong Zhou ◽  
Ziyi Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Apoptotic Index ◽  
Total Cell ◽  
Toxic Substances ◽  
Total Cell Number ◽  
Preimplantation Embryo Development

Summary Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

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.

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.

Embryo development and sex ratio of in vitro-produced porcine embryos are affected by the energy substrate and hyaluronic acid added to the culture medium

2014 ◽  
Vol 26 (4) ◽  
pp. 570 ◽  
Author(s):  
Eva Torner ◽  
Eva Bussalleu ◽  
M. Dolors Briz ◽  
Marc Yeste ◽  
Sergi Bonet
Keyword(s):  
Hyaluronic Acid ◽  
Sex Ratio ◽  
Embryo Development ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Porcine Embryo ◽  
Preferential Loss

In the present study, the effects of replacing glucose with pyruvate–lactate and supplementing these in vitro culture (IVC) media with hyaluronic acid (HA) on porcine embryo development and sex ratio were examined. The in vitro-produced (IVP) porcine embryos were cultured in NCSU-23 medium with 0.0, 0.5 or 1.0 mg mL–1 HA, and with either 5.55 mM glucose (IVC-Glu) or pyruvate (0.17 mM)–lactate (2.73 mM) from 0 to 48 h post insemination (h.p.i.) and then with glucose from 48 to 168 h.p.i. (IVC-PL). Those embryos cultured with IVC-PL had significantly higher blastocyst rates (23.7 ± 1.5%) than those cultured with IVC-Glu (14.27 ± 2.75%). At 1.0 mg mL–1, HA tended to skew the sex ratio of blastocysts towards males in those embryos cultured in IVC-PL, and led to a significant decrease in the blastocyst rate compared with embryos cultured in the presence of 0.5 and 0.0 mg mL–1 HA and IVC-Glu (4.28 ± 0.28% vs 11.01 ± 1.42% and 10.14 ± 2.77%, respectively) and IVC-PL (14.37 ± 1.35% vs 20.96 ± 2.85% and 22.99 ± 1.39%, respectively). In contrast, there were no significant differences in the total cell number per blastocyst or in apoptosis rates. In conclusion, pyruvate and lactate were the preferred energy substrates in the early stages of IVP porcine embryos. Moreover, 1.0 mg mL–1 HA significantly decreased the percentage of blastocyst rates in both the IVC-Glu and IVC-PL groups, but only by a preferential loss of female embryos for those cultured in IVC-PL.

159 THE EFFECT OF DIFFERENT ZWITTERIONIC BUFFERS AND PBS ON IN VITRO DEVELOPMENT AND MORPHOLOGY OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
J. De la Fuente ◽  
A. Gutiérrez-Adán ◽  
P. Beltrán Breña ◽  
S. S. Pérez-Garnelo ◽  
A. T. Palasz
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Apoptotic Cells ◽  
Bovine Embryos ◽  
Chi Square ◽  
Oh Groups ◽  
In Vitro Development ◽  
Chi Square Analysis ◽  
Vitro Development

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.

22 EFFECT OF OOCYTE AGE ON THE DEVELOPMENT OF MOUSE PARTHENOGENETIC EMBRYOS AND EFFECT OF OXYGEN CONCENTRATION ON EMBRYO DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 119
Author(s):  
H. Bagis ◽  
S. Arat ◽  
H. Odaman ◽  
A. Tas
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Group 4 ◽  
Mouse Embryo Development ◽  
O2 Concentration ◽  
Group 3 ◽  
Development In Vitro

The objective of this study was to investigate the effects of two parameters on mouse embryo development in vitro. These parameters were the effect of oocyte age on activation and the effect of O2 concentration in culture. In the first experiment, oocytes were recovered from superovutated mice at 15 h (group 1) or 20 h (group 2) after human chorionic gonadotropin (HCG) injection. All oocytes were activated for 6 h with 10 mM Sr2+ in Ca2+ free medium in the presence of 5 �g/mL of cytochalasin B. After activation, embryos were cultured in KSOM.aa medium for 4.5-5.5 days. Zygotes from naturally bred mice were used as control. Differences in blastocyst formation rate and blastocyst cell number among treatments were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, blastocyst formation rate in the first group was higher than in the second group (62.6% vs. 47.1%; P < 0.05). In addition, blastocyst cell number was also higher in the first group than in the second one (69.4 � 3.2 vs. 52.4 � 2.2; P < 0.05). However, both values were higher in control group (80%, 76.2 � 1.2; P < 0.05) than in the experimental groups. These results showed that young oocytes were activated more effectively than aged oocytes. In the second experiment, mouse zygotes were cultured in a humidified atmosphere of 5% CO2 in air (group 3) or 5% CO2, 5% O2, and 90% N2 (group 4). Blastocyst formation rate and blastocyst cell number of zygotes cultured in low O2 concentration (group 4) for 4.5 days were higher than for group 3 (76.3% vs. 56.4 and 69.0 � 3.4 vs. 52.8 � 2.3; P < 0.05). There was a significant difference in blastocyt formation rate of embryos for 5.5 days between the two groups (25.8% for group 4 vs. 14.4% for group 3; P < 0.05). This suggests that the embryos developed more slowly in high O2 concentration. These results showed that low O2 concentration provided a more suitable environment for mouse embryo development in vitro. The same experiment was repeated with parthenogenetic embryos recently in our laboratory. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

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