261. Disruption of mitochondrial function in the blastocyst alters expression of the chromatin remodeler ATRX

2008 ◽  
Vol 20 (9) ◽  
pp. 61
Author(s):  
S. L. Wakefield ◽  
A. N. Filby ◽  
M. Lane ◽  
M. Mitchell
Keyword(s):  
Mitochondrial Function ◽  
Metabolic Regulation ◽  
Culture Media ◽  
Maternal Diet ◽  
Cell Number ◽  
Early Embryo ◽  
Placental Development ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Embryo Metabolism

Exposure of an embryo to suboptimal environments, including poor embryo culture media or inadequate maternal diet, can disrupt fetal and placental development and whilst the exact mechanisms responsible remain unknown, perturbed embryo metabolism has been implicated. We propose that stress applied to an early embryo causes mitochondrial dysfunction, resulting in a permanent epigenetic change. Thus the aim of this study was to determine the affect of directly perturbing mitochondria in the embryo, on development, metabolism and expression of the ATP-dependant chromatin remodelling protein, ATRX. Zygotes collected from gonadotrophin stimulated C57BL/6xCBA mice were cultured to the two-cell stage and then exposed to one of three treatments; control medium (C), medium lacking pyruvate (-P; embryos dependant on the mitochondrial Malate Aspartate Shuttle, MAS) or medium lacking pyruvate plus 5µM amino-oxyacetate (AOA), a specific MAS inhibitor (-P+AOA). Blastocyst development and metabolism were assessed by determining cell number and allocation, glycolysis, and ATP:ADP ratio. Relative gene expression of ATRX, was examined using RT PCR. Embryos dependant on the MAS alone (-P) had significantly decreased blastocyst development (87.1% v. 98.2%, P < 0.05), with a compensatory increase in glycolysis (0.20 v. 0.07 pmol/cell/hr, P < 0.001) despite a decrease in ATP:ADP (0.10 v. 0.13, P < 0.06), relative to the control. Inhibition of the MAS (-P+AOA) further reduced blastocyst development,(77.3%, P < 0.001) and decreased ATP:ADP (0.08, P < 0.004), but there was no change in glycolysis relative to control embryos (0.09 pmol/cell/hr, P = 0.3). Expression of ATRX was significantly increased for –P+AOA embryos relative to the control (1.63 v. 1.0, P < 0.007) but did not differ for –P embryos (1.1). This study demonstrates that direct perturbations of mitochondrial function in the embryo compromises its metabolic regulation and blastocyst development, and the expression of the epigenetic modulator ATRX. Further studies are underway to elucidate the implications of disrupted metabolic control and this epigenetic modulator on pregnancy outcomes.

Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro

2021 ◽  
Author(s):  
Yulia N Cajas ◽  
Karina Cañón-Beltrán ◽  
Carolina Núñez-Puente ◽  
Alfonso Gutierrez-Adán ◽  
Encina M González ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Developmental Effect ◽  
Promote Cell Survival ◽  
Development In Vitro

Abstract During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2 to 8-cell stage, MNEGA) or major (8 to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 μM AKT-InhIII; 10 μM AKT-InhIV; 10 μM nobiletin; nobiletin+AKT-InhIII; nobiletin+AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors which infers that nobiletin probably uses another signalling cascade that PI3K/AKT during early embryo development in bovine.

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.)

The effects of brain-derived neurotrophic factor and metformin on in vitro developmental competence of bovine oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 187-193 ◽  
Author(s):  
So Gun Hong ◽  
Goo Jang ◽  
Hyun Ju Oh ◽  
Ok Jae Koo ◽  
Jung Eun Park ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Embryo Development ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Early Embryo ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryBrain-derived neurotrophic factor (BDNF) signalling via tyrosine kinase B receptors may play an important role in ovarian development and function. It has been reported that metformin elevates the activity of Tyrosine kinase receptors and may amplify BDNF signalling. The objective of this study was to investigate the effect of BDNF during in vitro maturation (IVM) and/or in vitro culture (IVC) (Experiment 1), and to evaluate the collaborative effect of BDNF and metformin treatment on the developmental competence of bovine in vitro fertilized (IVF) embryos (Experiment 2). In Experiment 1, BDNF, which was added to our previously established IVM systems, significantly increased the proportions of MII oocytes at both 10 ng/ml (86.7%) and 100 ng/ml (85.4%) compared with the control (64.0%). However, there was no statistically significant difference in blastocyst development between the control or BDNF-supplemented groups. In Experiment 2, in order to investigate the effect of BDNF (10 ng/ml) and/or metformin (10−5 M) per se, TCM-199 without serum and hormones was used as the control IVM medium. The BDNF (48.3%) and BDNF plus metformin (56.5%) significantly enhanced the proportions of MII oocytes compared with the control (34.4%). Although, BDNF or metformin alone had no effect in embryo development, BDNF plus metformin significantly improved early embryo development to the 8–16-cell stage compared with the control (16.5 vs. 5.5%). In conclusion, the combination of BDNF and metformin may have a collaborative effect during the IVM period. These results could further contribute to the establishment of a more efficient bovine in vitro embryo production system.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

126. IMPLICATIONS FOR FETAL AND PLACENTAL DEVELOPMENT FOLLOWING MITOCHONDRIAL PERTURBATION IN THE EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 45
Author(s):  
S. Wakefield ◽  
M. Lane ◽  
M. Mitchell
Keyword(s):  
Mitochondrial Function ◽  
Weight Ratio ◽  
Threshold Effect ◽  
Blastocyst Stage ◽  
Placental Weight ◽  
Placental Development ◽  
Cell Stage ◽  
Crown Rump Length ◽  
Fetal Survival ◽  
Placental Efficiency

The environment an embryo is exposed to can profoundly influence peri- and post-natal development despite having some capacity to adapt. Whilst the mechanisms responsible remain largely unknown, mitochondria are a likely target. In this study we deliberately perturbed mitochondrial function in the mouse embryo, using a model we have established that shows step-wise changes in embryo metabolism and development. The aim of this study was to provide direct evidence implicating mitochondrial dysfunction in the embryo with perturbed fetal and placental development. Zygote stage embryos were recovered from superovulated female mice and cultured in control conditions to the 2-cell stage. Embryos were then allocated to one of three treatments; control media (0μM-AOA), 5μM or 50μM of the known mitochondrial inhibitor, Amino-Oxyacetate, in the absence of pyruvate (5μM-AOA, 50μM-AOA). Embryos were cultured to the blastocyst stage, then transferred to pseudopregnant recipients, with fetal and placental parameters measured on day 18 of pregnancy. Implantation rates and fetal survival for both 5μM-AOA and 50μM-AOA was comparable to control embryos. For 5μM-AOA there was a significant reduction in placental weight (P=0.02) but normal fetal weight, and a significant increase in fetal: placental weight ratio (P=0.002) relative to the control, suggesting increased placental efficiency. When mitochondria were further perturbed (50μM-AOA), the fetuses and placentas were both considerably compromised: that is, decreased fetal and placental weights (P=0.002), reduced placental diameter (P=0.03) and decreased fetal crown rump length (P=0.07). This study demonstrates that mitochondrial function in the embryo impacts on peri-natal development, providing compelling evidence for mitochondrial function involvement in the mechanisms underpinning “embryo programming”. This data suggests a threshold effect, whereby embryos can only adapt up until a point after which development is compromised. Further elucidating these mechanisms is important for understanding how maternal environments and embryo culture systems determine development of future offspring.

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 203-213 ◽  
Author(s):  
S. Eswari ◽  
G. Sai Kumar ◽  
G. Taru Sharma
Keyword(s):  
Culture Media ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Preimplantation Embryos ◽  
Leukaemia Inhibitory Factor ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryThe objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus–oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8–16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

scholarly journals 159EFFECT OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) AND SERUM, ALONE OR IN COMBINATION, ON INTERFERON-TAU (IFNT) PRODUCTION BY OVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 201
Author(s):  
J.A. Rooke ◽  
M. Ewen ◽  
M.E. Staines ◽  
T.G. McEvoy ◽  
C.J. Ashworth
Keyword(s):  
Group Treatment ◽  
Culture Media ◽  
Cell Number ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Factorial Anova ◽  
Gm Csf ◽  
Individual Culture ◽  
Chi Square Analysis ◽  
Embryo Grade

The objectives were to establish whether GM-CSF, which may stimulate IFNT production by blastocysts at implantation, influences IFNT production by ovine embryos in culture and whether GM-CSF can explain stimulation of IFNT production by ovine serum (Rooke et al., 2003 Reprod. Abstr. Series 30:56). Oocytes, aspirated from sheep ovaries obtained from a local abattoir on 4 separate days, were matured and fertilized by standard procedures. On Day 1, cleaved zygotes (approximately 10 per 0.05-mL−1 drop; total, 175 per treatment) were cultured in synthetic oviductal fluid containing either 0.3% bovine serum albumin and amino acids (SOFA) or 10% sheep serum (SOFS) in the presence (+) or absence (−) of 5ngmL−1 ovine GM-CSF. Medium was changed every 2 days. On Days 6 and 7, blastocysts were removed from their group drops and assigned in a balanced manner individually to 0.05-mL−1 drops of one of the 4 media for a further 24h. Embryo grade, diameter and cell number were recorded. IFNT concentrations in conditioned media samples were determined by ELISA. The presence of serum accelerated blastocyst development (cross-tabulation; chi-square analysis; P=0.039; Table 1). Both serum and GM-CSF in group culture media increased (2×2 factorial ANOVA, P&lt;0.001) IFNT concentrations (ngmL−1; SOFA−, 0.5; SOFA+, 1.7; SOFS−, 2.7; SOFS+, 4.2; SED, 0.47) with no interaction between serum and GM-CSF. After 24h individual culture of Day 6 blastocysts, IFNT concentrations (2×2 factorial ANOVA; Table 1) were influenced mainly by the origin of the blastocyst (group treatment covariate, serum, P&lt;0.001; GM-CSF, P&lt;0.001) rather than by individual blastocyst culture media (GM-CSF, NS; serum, P&lt;0.001). The pattern of results was similar for Day 7 blastocysts. Incubation of zygotes in the presence of GM-CSF had no effect on embryo grade or diameter but blastocysts (both day 6 and 7) incubated individually for 24h in the presence of GM-CSF had more cells (128 v. 119; SED, 3.52; P=0.001) than blastocysts incubated concurrently without GM-CSF. However, the pattern of IFNT production after correction for cell number was unchanged. In conclusion, culture of zygotes in medium containing GM-CSF or serum, alone or in combination, from Days 1 to 7 increased IFNT production, and IFNT production by individually cultured blastocysts depended on their origin. SAC receives financial support from the Scottish Executive Environment and Rural Affairs Department. Table 1 Blastocyst yields (% of zygotes) on Days 6 and 7 and IFNT concentrations (ngML−1) in media after individual culture of Day 6 blastocysts

scholarly journals 233.Ammonium affects mitochondrial distribution and function in mouse 2-cell embryos

2004 ◽  
Vol 16 (9) ◽  
pp. 233 ◽  
Author(s):  
D. L. Zander ◽  
D. A. Froiland ◽  
M. Lane
Keyword(s):  
Amino Acids ◽  
Membrane Potential ◽  
Intensity Ratio ◽  
Culture Media ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Pixel Intensity ◽  
Essential Components ◽  
Mitochondrial Distribution

Amino acids are key regulators of embryo function and are essential components in embryo culture media. Amino acids spontaneously breakdown and are metabolised by embryos resulting in ammonium build-up in the medium. While ammonium does not affect blastocyst development, the ability of these blastocysts to implant was reduced along with subsequent fetal growth rates. However, the mechanism for the inhibitory effect of ammonium is currently not known. It has been demonstrated in other tissues that mitochondrial bioenergetics can be disrupted by the presence of ammonium in the media which subsequently affects cellular viability. Therefore, the aim of this study was to examine the effects of ammonium on the mitochondria of mouse embryos cultured in the presence of ammonium. Mouse zygotes from superovulated females were cultured in medium G1.2 with or without 300 μM ammonium for 22 h at 37oC in 6%CO2�:�5%O2�:�89%N2. In vivo-developed 2-cell embryos were flushed from the reproductive tract and assessed immediately. At the 2-cell stage mitochondrial distribution (Mitotracker) and membrane potential (JC-1) were assessed using confocal microscopy and images were quantitated using IP Lab software package. Differences between treatments were determined using ANOVA and Bonferroni's multiple comparison procedure. Culture of zygotes to the 2-cell stage in medium G1.2 did not affect mitochondrial distribution compared to in vivo controls. However, 2-cell embryos cultured with ammonium had a decrease in their mitochondrial nuclear�:�cortical ratio (97���1 compared to 106���1; P�<�0.05) indicating that mitochondria were dispersing away from the nuclei. Culture with ammonium also significantly decreased the mitochondrial membrane potential (0.50���0.01 mean pixel intensity ratio) compared to those cultured without ammonium (0.72���0.3 mean pixel intensity ratio, P�<�0.001). The data presented demonstrates that culture for only 24�h with ammonium disrupts both mitochondrial distribution and membrane potential and supports our hypothesis that mitochondria are an early target for the inhibitory action of ammonium.

299. Sensitivity of embryos to an environmental stressor, ammonium, is dependent on stage of temporal exposure

2005 ◽  
Vol 17 (9) ◽  
pp. 127
Author(s):  
D. L. Zander ◽  
J. G. Thompson ◽  
M. Lane
Keyword(s):  
Gene Expression ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Early Stage ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Stages Of Development ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Stage Embryo

Extended embryo culture in vitro may cause increased cellular perturbations resulting in poorer developmental outcomes. Exposure of embryos to ammonium throughout the entire pre-implantation period decreased cell number and ICM development, increased apoptosis and perturbs glucose metabolism. The aim of this study was to examine the relative susceptibility of the pre- and post-compaction stage embryo to these perturbations resulting from temporal exposure to ammonium. Mouse embryos (n = 350 per treatment) were collected from F1 female mice. Embryos were exposed to either control medium or medium with 300 μM ammonium for the entire culture period. Temporal treatments involved culture with or without ammonium, from the zygote to 2-cell stage, 2-cell to 8-cell stage, or the 8-cell to the blastocyst stage. At the blastocyst stage, ICM development, apoptosis, gene expression and glucose metabolism were assessed. Differences between treatments were determined using generalised linear modelling and LSD post-hoc tests. Exposure to ammonium at any stage did not affect blastocyst development. Exposure to ammonium pre-compaction significantly decreased both blastocyst and ICM cell number while these were unaffected when exposure occurred post-compaction. Levels of apoptosis were significantly increased when exposure to ammonium was continual to the blastocyst stage (6.5% compared to control 2.4%, P < 0.05) or from the zygote to the 2-cell stage (5.8%, P < 0.05). However, apoptosis was not altered during post-compaction exposure (2.8%). Glucose uptake was decreased by culture with ammonium at all stages of development (P < 0.001). Gene expression of GLUT1 in the blastocyst was not altered by ammonium while GLUT3 expression was significantly reduced by exposure at all stages of development (P < 0.01). The data presented suggests that the pre-compaction stage embryo is most susceptible to ammonium stress and the effects of this early stage exposure appear irreversible. Intriguingly, glucose uptake and GLUT3 expression at the blastocyst stage appear to be markers of ammonium exposure.

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 &lt; 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.

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