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.

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

scholarly journals ID: 1087 Effects of oocytes collection and electro-activation protocols on maturation and preimplantation development of parthenogenetic diploid porcine embryos

2017 ◽  
Vol 4 (S) ◽  
pp. 175
Author(s):  
Le Thi Thu Thuy ◽  
Nguyen Ba Tu ◽  
Bui Hong Thuy ◽  
Nguyen Van Thuan
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Activation Condition ◽  
Parthenogenetic Embryo ◽  
Morula Stage ◽  
Collection Methods ◽  
Dissection Method

Advances in porcine embryos production have been impaired by the in vitro maturation (IVM) system and in vitro developments (IVD) are still not optimized. The present study was undertaken to determine the effects of different oocytes collection methods (aspiration and dissection) on oocytes maturation and combine with the different electro-activation protocols on in vitro pre-implantation development of parthenogenetic diploid porcine embryos. The results showed that dissection method was significantly higher both in quantity (87.23%, p <0.05) of matured oocytes and in the rate of oocytes with very good quality (53.00%) compared with aspiration method (62.00%, 30.22%; respectively). We further accessed the quality of matured oocytes competence via parthenogenetic embryo development. Using four electro-activation protocols, we found that the electro-activation condition had no effect on the development of embryos to the 2-cell, 4-cell and 8-cell stages. However, the oocytes activated by the 60V/cm amplitude of pulse group had less (41.33%; p <0.05) development at the morula stage than 90V/cm, 100V/cm and 150V/cm amplitude of pulse groups (73.99%, 67.62%, 72.55%; respectively). The embryos develop to blastocyst stage was highest in the group induced by 90V/cm electro pulse was 71.76% compared to 100V/cm, 150V/cm, and 60V/cm (62.86%, 62.05%, and 37.63%, p<0.001, respectively). We conclude that the combining of dissection method for porcine oocytes collection and electro-activation with the amplitude of pulses 90V/cm can enhance both the activation success and development competition in in vitro pre- implantation parthenogenetic diploid porcine embryos.

scholarly journals Effects of Progesterone on in Vitro Developmental Competence of Bovine Embryos

2020 ◽  
Vol 8 (1) ◽  
pp. 42
Author(s):  
Orhan Örnek ◽  
Yusuf Ziya Güzey
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Early Embryo ◽  
Developmental Competence ◽  
Direct Role ◽  
Vitro Fertilization ◽  
Morula Stage ◽  
Vitro Development

Progesterone plays a key role in the establishment and maintenance of pregnancy in mammalian. Increasing levels of circulating progesterone in the post-conception period are associated with conceptus elongation and high pregnancy rates in cattle. Contradictory results are available on the direct role of progesterone in early embryo development. The objective of this study was to evaluate direct effects of progesterone on in vitro development of cattle embryos. Immature oocytes collected from slaughtered animals and cultured in the presence of different concentrations of progesterone (25, 50, 100 ng/mL) following in vitro fertilization. Cleavage rates in 25 and 50 ng/mL concentrations of progesterone were significantly higher than those in controls and 100 ng/mL. Rate of embryos that reached to the morula stage was similar in all groups. Supplementation of 25 and 50 ng/mL progesterone to the culture media significantly increased blastocyst yield while 100 ng/mL progesterone resulted in a decrease. As a conclusion, we can suggest that progesterone supplementation in in vitro culture may support embryo development at low levels.

scholarly journals 111. THE ROLE OF L-PROLINE IN PREIMPLANTATION MOUSE EMBRYO DEVELOPMENT IN VITRO

2010 ◽  
Vol 22 (9) ◽  
pp. 29 ◽  
Author(s):  
S. Ozsoy ◽  
M. B. Morris ◽  
M. L. Day
Keyword(s):  
Amino Acids ◽  
Embryo Development ◽  
Preimplantation Embryo ◽  
Blastocyst Stage ◽  
Low Density ◽  
Mouse Embryo Development ◽  
Preimplantation Mouse Embryo ◽  
Preimplantation Mouse ◽  
Development In Vitro

Amino acids are known to play important roles in preimplantation embryo development, including regulation of cell volume and metabolism. Inclusion of l-glutamine, glycine and betaine in embryo culture medium has been shown to improve development in vitro by acting as organic osmolytes, thereby regulating cell volume. The purpose of the present study was to examine the effect of l-proline on preimplantation mouse embryo development in vitro. One-cell stage embryos were cultured in modified HTF, at low density (1 embryo/100 μL) and high density (1 embryo/μL) in the presence and absence of amino acids. Development of the embryos was scored every 24 h until the blastocyst stage. At low density, l-proline significantly increased the proportion of embryos developing to the blastocyst stage. This effect was abolished by culture at high density, suggesting that l-proline was activating a pathway similar to that involved in autocrine signalling by trophic factors in the preimplantation embryo. The improvement in development observed in the presence of l-proline was not due to its action as an organic osmolyte since the osmolality of the medium was 270 mOsm. Furthermore, glycine and betaine, which are known to act as osmolytes in embryos, had no effect on blastocyst development. In embryonic stem cells L-proline is taken up by an amino acid transporter and is involved in the regulation of growth and differentiation (1). The present data suggest that l-proline may have a similar, important role in preimplantation development. (1) JM Washington, J Rathjen, F Felquer, A Lonic, MD Bettess, N Hamra, L Semendric, BSN Tan, J-A Lake, RA Keough, MB Morris and PD Rathjen (2010) Am J Physiol Cell Physiol 298: C982–C992.

scholarly journals Combinations of Trolox and ascorbic acid have a beneficial effect on in vitro maturation of pig oocytes

2021 ◽  
Author(s):  
Ileana Miclea ◽  
Marius Zahan
Keyword(s):  
Ascorbic Acid ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Membrane Damage ◽  
High Concentration ◽  
First Polar Body ◽  
Morula Stage ◽  
Vitro Development

Abstract: The poor in vitro development of pig oocytes and embryos has been blamed on oxidative stress. We sought to find out if combinations of Trolox (T), a synthetic and cell-permeable derivative of vitamin E, and ascorbic acid (AA) could improve the maturation rates of in vitro cultured pig oocytes. Pig oocytes underwent maturation for 44–45 h in medium M 199 supplemented with 0 μM T + 0 μM AA, 100 μM T + 250 μM AA, 300 μM T + 250 μM AA, 100 μM T + 750 μM AA or 300 μM T + 750 μM AA. These combinations were chosen based on previous research conducted in our laboratory and on the available literature. After maturation, several parameters were assessed: cumulus oophorus expansion, oocyte viability (based on the presence of metabolic activity versus membrane damage), extrusion of the first polar body, mitochondrial membrane potential (MMP), pronucleus formation, and embryo development after fertilization. All antioxidant combinations significantly improved cumulus expansion and formation of the first polar body. The best was 300 μM T + 250 μM AA for the first characteristic and 300 μM T + 750 μM AA for the second. Antioxidant presence in the maturation media increased the percentages of viable oocytes but not significantly. MMP was not significantly modified by the addition of antioxidant combinations. We also found that a low concentration of T (100 µM) mixed with a high concentration of AA (750 µM) in the oocyte maturation media led to significantly higher rates of both female and male pronuclei formation and also enhanced embryo development to the morula stage. Therefore, we recommend this combination to improve the in vitro maturation media of pig oocytes.  

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.

Early mouse preimplantation development is unaffected by microinjection of metallothionein antibodies

Zygote ◽  
1995 ◽  
Vol 3 (1) ◽  
pp. 81-84 ◽  
Author(s):  
Elena Ibánez ◽  
Francesca Vidal ◽  
Juan Hidalgo
Keyword(s):  
Embryo Development ◽  
Mouse Embryo ◽  
Normal Mouse ◽  
Polyclonal Antibodies ◽  
Experimental Treatment ◽  
Mouse Embryos ◽  
Mouse Embryo Development ◽  
Morula Stage ◽  
Early Mouse

SummaryPolyclonal antibodies that cross-react with rodent metallothionein I (MT I) and metallothionein II (MT II) were microinjected in 1-cell and 2-cell mouse embryos, into either the cytoplasm or the nucleus. Regardless of the experimental treatment, mouse embryo development in vitro was not affected and most of the embryos cleaved normally until the morula stage. The results suggest that metallothionein is not essential for normal mouse early preimplantational development, in agreement with recent studies in mice with inactivated MT I and MT II genes.

Effect of zinc and copper on preimplantation mouse embryo development in vitro and metallothionein levels

Zygote ◽  
1993 ◽  
Vol 1 (3) ◽  
pp. 225-229 ◽  
Author(s):  
Francesca Vidal ◽  
Juan Hidalgo
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Mrna Levels ◽  
Mouse Embryo Development ◽  
Preimplantation Mouse Embryo ◽  
Preimplantation Mouse ◽  
Development In Vitro

The effect of zinc and copper on the in vitro development of mouse preimplantation embryos and on metallothionein (MT) levels was studied by exposing the embryos to 100 μM concentrations of the metals for 24 h at the 1-cell,2-cell, 6-8-cell, morula and blastocyst stages. Zinc affected embryo development in the early but not in the late stages, whereas copper affected it more generally. The combined presence of both metals caused a stronger embryotoxicity. MT levels were measured by radioimmunoassay and were found to be similar at all developmental stages, though possibly higher at the blastocyst stage. The exposure of embryos to zinc and copper increased MT levels significantly only at the blastocyst stage, supporting previously published results on MT mRNA levels.

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