Effect of RU486 on different stages of mouse preimplantation embryos in vitro

1990 ◽  
Vol 68 (11) ◽  
pp. 1457-1460 ◽  
Author(s):  
Subhash C. Juneja ◽  
Melvin G. Dodson
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
In Vitro Development ◽  
Stage Embryo ◽  
Morula Stage ◽  
Vitro Development

17β-Hydroxy-11β-(4-dimethylaminophenyl)-17α-(1-propynyl)estra-4,9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 °C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 μg/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 μg/mL culture medium (p < 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 μg/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 μg/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.Key words: RU486, mouse, preimplantation embryos, embryo culture, postcoital contraceptive.

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

Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium

2012 ◽  
Vol 24 (3) ◽  
pp. 443 ◽  
Author(s):  
Tomomi Mito ◽  
Koji Yoshioka ◽  
Shoko Yamashita ◽  
Chie Suzuki ◽  
Michiko Noguchi ◽  
...  
Keyword(s):  
Culture Medium ◽  
Survival Rates ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Atp Content ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Defined Culture ◽  
Vitro Development

In the present study, the effects of glucose and/or glycine on the in vitro development of Day 5 (Day 0 = IVF) porcine blastocysts were determined. The addition of 2.5–10 mM glucose to the chemically defined culture medium porcine zygote medium (PZM)-5 significantly increased blastocyst survival rates compared with those of blastocysts cultured in the absence of glucose. The addition of 5 and 10 mM glycine to PZM-5 containing 5 mM glucose significantly enhanced the development to hatching and the number of hatched blastocysts compared with no addition of glycine. However, the addition of glycine to PZM-5 with no glucose did not improve blastocyst development. The ATP content of Day 6 blastocysts cultured with glucose was significantly higher than that of blastocysts cultured in the absence of glucose, regardless of glycine supplementation. The diameter and total cell numbers were significantly greater, and the apoptotic index was significantly lower, in Day 6 blastocysts cultured with both glucose and glycine. These results indicate that glucose is an important energy source for the porcine blastocyst and that glucose and glycine act synergistically to enhance development to the hatching and hatched blastocyst stage in vitro.

scholarly journals The role of Kifc1, Kchn5 and miRNA-302 on in vitro development in 8-cell, morula and blastocyst stage of mouse embryos

2019 ◽  
Vol 18 (3) ◽  
pp. 71-79
Author(s):  
Konstantinos Ntzeros ◽  
Despoina Mavrogianni ◽  
Athina Koutsi ◽  
Antonia Kandaraki ◽  
Anastasios Papadelas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Embryo Development ◽  
Expression Profile ◽  
Cell Cycle Control ◽  
Blastocyst Stage ◽  
Circadian Oscillators ◽  
Mouse Embryo Development ◽  
Morula Stage ◽  
Vitro Development

Introduction: Embryo development is characterized by lack of cell cycle check-points and overexpression of core circadian oscillators. On previous report we have identified several genes over-and under-detected at human embryo blastomeres. In this study, we investigated the expression profile of Kcnh5, KIFC1 and miRNA-302 genes at three pre-implantation stages of mouse embryo development. Material and methods: Total RNA was extracted from mouse embyos at 8-cell, morula and blastocyst stage. The expression profile of Kcnh5, KIFC1 and miRNA-302 was assessed by RT-PCR and the results were normalized with G6pdh expression levels. Results: Kcnh5 showed absence of expression at all stages, indicating novel mechanisms of cell cycle control during blastomeres divisions. KIFC1 showed positive expression at all stages, with decreasing levels as the embryogenesis progresses. This finding indicates that KIFC1 may have more important role at early events. miRNA-302 showed increased levels of expression at all stages, with morula having the highest levels. Therefore, miRNA-302 might play an important role at the events that happen during morula stage such as compaction. Conclusions: Cell cycle control of blastomeres at early embryogenesis might be based on different mechanisms compared to somatic cells and more research is needed in order to reveal crucial cycling elements.

198 THE EFFECT OF SOURCE AND IN VITRO MATURATION ON THE ABUNDANCE OF MATERNAL mRNA OF SELECTED GENES IN FOLLICULAR BOVINE OOCYTES AND THEIR INFLUENCE ON IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 199
Author(s):  
T. Somfai ◽  
K. Imai ◽  
M. Kaneda ◽  
S. Akagi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Before And After ◽  
Vitro Development ◽  
Bovine Oocytes

The aim of the present study was to investigate the effect of oocyte source and in vitro maturation (IVM) on the expression of selected genes in bovine oocytes and their contribution to in vitro embryo development. Follicular oocytes were collected either by ovum pick-up from live cows or by the aspiration of ovaries of slaughtered cows following storage in Dulbecco’s PBS at 15°C for overnight. In vitro maturation was performed according to the method of (Imai et al. 2006 J. Reprod. Dev. 52, 19–29 suppl.). Gene expression was assessed before and after IVM by real-time PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1, and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA). Gene expression was analysed by a Roche Light Cycler 480 device and software (Roche, Indianapolis, IN). Relative expression of each gene was normalized to CCNB1, which in preliminary experiments appeared the most stably expressed irrespective of oocyte source and meiotic stage. Three replications were performed. Data were analysed by paired t-test. In immature ovum pick-up oocytes, genes related to metabolism (GAPDH, G6PDH, GLUT8) and stress (MnSOD, HSP70), and also OCT4, ATP1A1, and DYNC1/1 showed significantly (P < 0.05) higher expression compared with immature oocytes collected from slaughtered-stored ovaries. The expression of GDF9, GLUT8, CTNNB1, and PMSB1 was significantly (P < 0.05) reduced during IVM irrespective of the oocyte source. In a second experiment, IVF IVM oocytes showing an early (at 22 to 25 h after IVF) or late (at 27 to 30 h after IVF) first cleavage were either cultured in vitro or analysed for gene expression at the 2-cell stage. A higher (P < 0.05) rate of early-cleaving oocytes developed to the blastocyst stage compared with the rate of late-cleaving ones (46.2% v. 15.6%, respectively). Nevertheless, only ATP1A1 showed significantly reduced (P < 0.05) expression in late-cleaving embryos compared with early-cleaving ones. Our results suggest that although removal and storage of ovaries and IVM caused a reduction in the relative abundance of several genes in oocytes, in most cases, this did not affect embryo development. Among the genes studied, only ATP1A1 was correlated with in vitro development.

Glutamine and hypotaurine improves intracellular oxidative status and in vitro development of porcine preimplantation embryos

Zygote ◽  
2007 ◽  
Vol 15 (4) ◽  
pp. 317-324 ◽  
Author(s):  
C. Suzuki ◽  
K. Yoshioka ◽  
M. Sakatani ◽  
M. Takahashi
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
Production System ◽  
Oxidative Status ◽  
Preimplantation Embryos ◽  
In Vitro Development ◽  
Vitro Development ◽  
Number Of Cells

SummaryWe previously developed an in vitro-production system for porcine embryos and reported that the addition of glutamine (Gln) and hypotaurine (HT) during in vitro culture improved embryo development. This study examined the effects of Gln and HT on in vitro development, intracellular oxidative status and DNA damage of porcine preimplantation embryos. Porcine zygotes produced by in vitro maturation (IVM) and in vitro fertilization (IVF) were cultured until day 2 (day 0 = day of IVF) in porcine zygote medium (PZM) including 2 mM Gln and 5 mM HT, namely PZM-5. On day 2, the cleaved embryos were selected and cultured for 24 h in PZM-5 to which one of the following substances was added: (1) none (control); (2) Gln; (3) HT; or (4) Gln + HT. After 24 h of culture in each medium, the embryos were then returned to PZM-5 and cultured until day 5. Day-5 blastocyst yield was significantly higher in the Gln and Gln + HT groups (p < 0.05) than in the control and HT groups. In addition, Gln + HT significantly increased the total number of cells in blastocysts (p < 0.05) compared with the control. Although the number of cells and the intracellular GSH levels in day-3 cleaved embryos did not differ among treatments, addition of Gln, HT or Gln + HT significantly (p < 0.05) reduced the intracellular H2O2 content and the extent of DNA damage compared with the control. These results indicate that the presence of Gln and HT in PZM-5 from day 2 to day 3 promotes the development of porcine embryos by improvement of intracellular oxidative status.

scholarly journals 77CLONED EMBRYO DEVELOPMENT IN VITRO: COMPARISON OF CONVENTIONAL AND INDUCED ENUCLEATION PROCEDURES FOR BOVINE CYTOPLAST PREPARATION

2004 ◽  
Vol 16 (2) ◽  
pp. 160
Author(s):  
M.-K. Wang ◽  
E.W. Overstrom
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Blastocyst Stage ◽  
In Vitro Development ◽  
Uv Illumination ◽  
Development In Vitro ◽  
Second Polar Body ◽  
Vitro Development

Induced enucleation (IE) of oocytes with demecolcine produces competent ooplasts for SCNT as demonstrated previously in mouse, goat, cow and pig. Whether bovine IE cytoplasts are more or less competent than conventionally enucleated MII oocytes to support nuclear reprogramming of somatic chromatin and embryo development in vitro is not known. This study compared in vitro development of cloned bovine embryos produced by conventional and IE enucleation methods. Three experimental groups were: (1) Parthenogenetic controls. In vitro-matured, MII-arrested bovine oocytes were activated by a single (1×Act, 10μM ionomycin in Tyrodes-HEPES, 5min) or double activation (2×Act; 1×Act, wash 5min, 10μgmL−1 cycloheximide [CHX] 20min, repeat 1×Act) followed by incubation in CHX and 5μgmL−1 cytochalasin B (CB) for 6h, and then culture (BARC medium) for 7 days. (2) Conventional SCNT. MII oocytes were enucleated by micromanipulation in HEPES-buffered enucleation medium (BARC containing 7.5μgmL−1 CB, 5μgmL−1 Hoechst 33342, 10% FBS) under UV illumination (3–5s). Donor cells (fibroblasts, passage 7–9) were inserted into the perivitelline space, and the reconstructed couplets activated (1×Act). Reconstructed couplets were then electrofused, placed in BARC medium containing 10μgmL−1 CHX and 5μgmL−1 CB (6h), and then cultured for 7 days. (3) IE SCNT. MII oocytes were activated (1×Act), placed into BARC-5% FBS containing 0.4μgmL−1 demecolcine (DEME), 10μgmL−1 CHX, 2μgmL−1 cytochalasin D for 20min, then 20min without DEME, then returned to DEME. At 1–1.5h post-activation, the extruding second polar body (PB2) containing nuclear chromatin was removed by micromanipulation, couplets were reconstructed and fused as above, and additionally activated (two pulses, 20–30V/mm, 20μs). Embryos were cultured in 10μgmL−1 CHX and 5μgmL−1 CB medium for 4–5 hour, then BARC for 7 days. The results (Table 1) reveal that 2×Act increases embryo development at Day 2, but not Day 7. Further, there are no significant differences in embryo development rates between conventional and IE SCNT protocols. Respectively, 46%, 32% and 21% of cleaved control (1×Act), conventional and IE embryos developed to 16 cells on Day 7. In vitro development of cleavage embryos to the blastocyst stage was greater in controls (25–32%) than in conventional (22%) and IE (17%) SCNT groups on Day 7. Further comparisons of in vivo development between conventional and IE SCNT methods following embryo transfer are warranted. Supported by ACT, Cyagra and USDA NRI \#2001-35205-09966. Table 1 Embryo development: Conventional v. induced enucleation

scholarly journals 160EFFECTS OF EXTRACELLULAR ION CONCENTRATIONS ON IN VITRO DEVELOPMENT OF DOMESTIC CAT IVF EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 202 ◽  
Author(s):  
W.F. Swanson ◽  
A.L. Manharth ◽  
J.B. Bond ◽  
H.L. Bateman ◽  
R.L. Krisher ◽  
...  
Keyword(s):  
Culture Media ◽  
Ionic Composition ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Vitro Development

Domestic cat embryos typically are cultured in media formulated for somatic cells or embryos from rodents or livestock species. Under these conditions, blastocyst development has been inconsistent and delayed relative to embryos grown in vivo, and embryo viability following transfer has been low. Our goal is to systematically define the culture requirements of the feline embryo to improve embryo development and viability. The objective of this study was to determine the ionic (NaCl, KCl, KH2PO4, and CaCl2:MgSO4) preferences of domestic cat IVF embryos. Anestral female cats were injected (i.m.) with 150IU eCG followed 84h later by 100IUhCG. Oocytes were recovered via laparoscopic follicular aspiration approximately 24h post-hCG injection (Day 0). Semen was collected from one of two males by means of an artificial vagina and washed once in HEPES-buffered IVF medium. Mature cumulus-oocyte complexes were co-incubated with 2.5–5×105 motile sperm mL−1 in IVF medium (100mM NaCl, 4.0mM KCl, 1.0mM KH2 PO4, 2.0mM CaCl2, 1.0mM MgSO4-7H2O, 25.0mM NaHCO3, 3.0mM glucose, 0.1mM pyruvate, 6.0mM L-lactate, 1.0mM glutamine, 0.1mM taurine, 1×MEM nonessential amino acids, 50μgmL−1 gentamicin, and 4.0mgmL−1 BSA) for 19 to 22h in 6% CO2 in air (38.7°C). Cumulus cells were removed and embryos cultured (8–11 embryos/50μL drop; 6% CO2, 5% O2, 89% N2, 38.7°C) in media containing 100.0 or 120.0mM NaCl, 4.0 or 8.0mM KCl, 0.25 or 1.0mM KH2PO4, and 1.0mM:2.0mM or 2.0mM:1.0mM CaCl2:MgSO4 (2×2×2×2 factorial design). The remaining components of the culture medium were identical to the IVF medium (but w/o gentamicin). Development to the blastocyst stage by Day 6, metabolism (glycolysis and pyruvate) of each blastocyst, and final cell number (Hoechst 33342 staining) of all embryos were evaluated. Final cell number of cleaved embryos and development to the blastocyst stage were analyzed using analysis of variance in the GLIMMIX macro of SAS. A total of 236 oocytes were inseminated, yielding 128 cleaved embryos (54%), including 6 blastocysts (4.7% of cleaved embryos). Cell number was not (P&gt;0.05) affected by NaCl, KCl, or KH2PO4 concentrations, but tended (P=0.057) to be higher after culture in 2.0mM:1.0mM CaCl2:MgSO4. Treatments did not significantly affect (P&gt;0.05) development to the blastocyst stage, but numerically more blastocysts were produced in 100.0mM NaCl (4/6), 8.0mM KCl (5/6), or 1.0mM KH2PO4 (5/6). Both CaCl2:MgSO4 ratios resulted in 3 blastocysts. Blastocysts contained 61.08±5.1 (mean±SEM, n=6) cells and actively metabolized glucose (glycolysis, 3.7±0.8pmol/embryo/3h or 0.06±0.01pmol/cell/3h) and pyruvate (0.75±0.27pmol/embryo/3h or 0.013±0.005pmol/cell/3h). These results suggest that the ionic composition of culture media influences the in vitro development of cat IVF embryos. (Supported by NIH grant RR15388.)

scholarly journals In-vitro development of preimplantation embryos recovered from IUD-bearing mice

Reproduction ◽  
1978 ◽  
Vol 54 (2) ◽  
pp. 413-417 ◽  
Author(s):  
P. R. Hurst ◽  
K. Jefferies ◽  
K. J. Dawson ◽  
P. Eckstein
Keyword(s):  
Preimplantation Embryos ◽  
In Vitro Development ◽  
Vitro Development
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