267 TARGETED SUPPRESSION OF THE EXPRESSION OF MATERNAL AND EMBRYONIC GENES DURING IN VITRO DEVELOPMENT OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 241
Author(s):  
D. Tesfaye ◽  
K. Nganvongpanit ◽  
F. Rings ◽  
M. Gilles ◽  
D. Jennen ◽  
...  
Keyword(s):  
Free Water ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
First Polar Body ◽  
Vitro Development ◽  
Bovine Oocytes ◽  
E Cadherin

Despite enormous advances in the identification and sequencing of developmentally relevant bovine genes, the function of the majority of these transcripts is not yet known. Here we aimed to apply the RNA interference (RNAi) approach to suppress the expression of the maternal transcript c-mos (AY630920) and embryonic transcripts E-cadherin (AY508164) and Oct-4 (AY490804) during in vitro development of bovine embryos using microinjection of sequence-specific double-stranded RNA (dsRNA). For this 435-, 341- and 341-bp-long dsRNA specific to the coding sequences of c-mos, E-cadherin and Oct-4 transcripts, respectively, were synthesized using Promega RiboMax" T7 system (Promega, Madison, WI, USA), where sense and antisense strands were transcribed from the target DNA template. Slaughterhouse ovaries were used to aspirate bovine oocytes, which were matured in TCM-199 with 12% estrus cow serum (ECS), fertilized in Fert-TALP, and cultured in CR1 medium at 39�C under humidified atmosphere of 5% CO2 in air. In Experiment 1, immature oocytes were categorized into three groups, each containing 50-60 oocytes: those injected with c-mos dsRNA, those injected with RNase-free water, and uninjected controls. In Experiment 2, zygotes were categorized into four groups, each containing 50-60 zygotes: those injected with E-cadherin dsRNA, those injected with Oct-4 dsRNA, those injected with RNase-free water, and uninjected controls. Each experiment was repeated four times. The effect of dsRNA on in vitro development of oocytes or embryos was assessed after microinjection during culture. The level of mRNA and protein expression was investigated using real-time PCR and western blot analysis, respectively. Data were analyzed using SAS, version 8 (SAS Institute Inc., Cary, NC, USA). Microinjection of c-mos dsRNA resulted in a 70% reduction of c-mos transcript abundance after maturation compared to the water-injected and uninjected controls (P < 0.05). Similarly, microinjection of E-cadherin and Oct-4 dsRNA at the zygote stage resulted in 80% and 60% reduction in transcript abundance at the blastocyst stage, respectively, compared to the uninjected controls (P < 0.05). Decreases in the c-mos (39 kDa) and E-cadherin proteins (119 kDa) were observed in the c-mos and E-cadherin dsRNA-injected groups, respectively, compared to the control. A higher proportion of oocytes (75%) showed first polar body extrusion after maturation in c-mos dsRNA-injected groups, compared to 52% in water-injected and 57% in uninjected controls. Only 22% from E-cadherin dsRNA- and 24% from Oct-4 dsRNA-injected zygotes developed to the blastocyst stage compared to 39 and 37% blastocyst rates in water-injected and uninjected control groups, respectively. In conclusion, injection of sequence-specific dsRNA in bovine oocytes and embryos resulted in suppression of mRNA and their protein products, thereby affecting in vitro development of bovine embryos.

146 DETRIMENTAL EFFECTS OF PROSTAGLANDIN F2α ON IN VITRO EMBRYO DEVELOPMENT IN BOVINE ARE INHIBITED BY A RECEPTOR ANTAGONIST

2008 ◽  
Vol 20 (1) ◽  
pp. 153 ◽  
Author(s):  
F. N. Scenna ◽  
J. L. Edwards ◽  
F. N. Schrick
Keyword(s):  
Prostaglandin F2α ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Negative Effects ◽  
In Vitro Development ◽  
Ann Arbor ◽  
Reproductive Techniques ◽  
Vitro Development ◽  
Cayman Chemical

Numerous studies have demonstrated negative effects of prostaglandin F2α (PGF2α) on bovine reproduction. Discovery of a PGF2α receptor (FPr) in bovine embryos (Scenna et al. 2006 Reprod. Fertil. Dev. 18, 180) allows for development of new therapeutic strategies to improve success of embryo transfer. Therefore, two experiments were performed to investigate the occurrence of any toxic effect of AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA), an FPr antagonist, on in vitro development of bovine embryos. In Exp. 1, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in potassium simplex optimized medium with polyvinyl alcohol (KSOM-PVA); n = 94); (2) 50 AL (50 nm AL-8810 in KSOM-PVA; n = 94); (3) 25 AL (25 nm AL-8810 in KSOM-PVA; n = 94); and (4) CON (control: KSOM-PVA; n = 95). In Exp. 2, pre-compacted embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 282); (2) 500 AL (500 nm AL-8810 in KSOM-PVA; n = 274); (3) 250 AL (250 nm AL-8810 in KSOM-PVA; n = 274); and (4) CON (control: KSOM-PVA; n = 278). Embryos remained in treatments until blastocyst assessment. Next, two experiments were performed to determine the efficiency of AL-8810 on preventing detrimental effects of PGF2α on pre-compacted embryos. In Exp. 3, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in KSOM-PVA; n = 121); (2) 10 PGF (10 ng mL–1 of PGF2α (Cayman Chemical Inc.) in KSOM-PVA; n = 91); (3) AL100+PGF (100 nm AL-8810 and 10 ng mL–1 of PGF2� in KSOM-PVA; n = 116); (4) CON (control: KSOM-PVA; n = 96). In Exp. 4, embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 87); (2) 10 PGF (10 ng mL–1 of PGF2α in KSOM-PVA; n = 87); (3) AL1000+PGF (1000 nm AL-8810 and 10 ng mL–1 of PGF2α in KSOM-PVA; n = 84); (4) CON (control: KSOM-PVA; n = 84). In Exp. 3 and 4, embryos remained in treatments for 48 h when development to morula was assessed. Data for all experiments were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). For Exp. 1, results indicated that addition of 100, 50, and 25 nm did not compromise embryonic development to the blastocyst stage compared to controls (60.2%, 55.8%, 55.4%, and 49.9%, respectively). In addition, orthogonal contrasts indicated that 100 nm AL-8810 improved development to the blastocyst stage (100 AL = 61% v. CON = 50.6%, P = 0.01). Similarly for Exp. 2, 1000, 500, and 250 nm AL-8810 did not affect in vitro development to the blastocyst stage compared to controls (40%, 39%, 34.8%, and 37.7%, respectively). In Exp. 3 and 4, addition of 1000 nm AL-8810, but not 100 nm, to culture medium of pre-compacted embryos exposed to PGF2α increased the ability of embryos to undergo compaction 48 h later (1000 AL+PGF = 51% v. PGF = 40%; P = 0.05). In conclusion, AL-8810 at a concentration of 1000 nm inhibits detrimental effects of PGF2α on the development of pre-compacted bovine embryos and may prove beneficial for other assisted reproductive techniques in cattle. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

139 INTRACELLULAR GLUTATHIONE CONCENTRATION AND IN VITRO DEVELOPMENT OF BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

2008 ◽  
Vol 20 (1) ◽  
pp. 150
Author(s):  
H. S. Lee ◽  
D. B. Koo ◽  
K. K. Lee ◽  
X. J. Yin ◽  
I. K. Kong
Keyword(s):  
Blastocyst Stage ◽  
Negative Group ◽  
Brilliant Cresyl Blue ◽  
Positive Group ◽  
In Vitro Development ◽  
Cresyl Blue ◽  
Intracellular Glutathione ◽  
Vitro Development ◽  
Bovine Oocytes

During in vitro development of bovine embryos, a large proportion of embryos fail to develop to the blastocyst stage. Brilliant cresyl blue (BCB) stain determines the activity of glucose-6-phosphate dehydrogenase, an enzyme synthesized in growing oocytes. It was previously reported that BCB positive oocytes are better developed to the blastocyst stage than BCB-negative oocytes for IVF (Alm et al. 2005 Theriogenology 63, 2194–2205). The objective of this study was to compare the intracellular glutathione (GSH) level and the developmental competence of bovine oocytes selected by BCB. Bovine ovaries were obtained at a local slaughterhouse and cumulus–oocytes complexes (COCs) were collected from follicles. In this study, we only used COCs with a compact cumulus investment. The concentration of BCB was reported in a previous study (Rodriguez-Gonzalez et al. 2002 Theriogenology 57, 1397–1409). The COCs were exposed to 26 µm of BCB for 90 min at 38.5�C in humidified air. Treated oocytes were divided into BCB-positive (colored cytoplasm) and BCB-negative (colorless cytoplasm). The selected COCs (BCB-positive and negative COCs) were matured in TCM-199 supplemented with 10% FBS, and then inseminated using frozen semen (1 � 106 cells mL–1). Subsequently, presumptive zygotes were cultured in vitro in CR1-aa medium supplemented with 0.3% BSA. After 3 days of IVC, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days at 38.5�C, 5% CO2 in air. Before (geminal vesicle (GV) stage) and after IVM (metaphase II (MII) stage), the COCs were denuded mechanically to prepare the samples for GSH assay, and then stored at –70�C until used. In the GV- and MII-stage oocytes, intracellular concentration of GSH was measured according to Mertens et al. (2005 Reprod. Domest. Anim. 40, 126–130). This experiment was replicated at least three times. GSH concentrations were analyzed by Student's t-test and the blastocyst formation rates were analyzed by chi-square analysis using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Differences among treatment effects were considered significant at P < 0.05. The GSH concentration of BCB-negative oocytes (4.5 � 0.7 pmol/oocyte; n = 105) was significantly (P < 0.05) lower than that of BCB-positive oocytes (7.3 � 0.7 pmol/oocyte; n = 78) before IVM. After 24 h IVM, no differences in the GSH concentration were observed between the BCB-positive group (13.5 � 0.5 pmol/oocyte; n = 85) and the BCB-negative group (12.6 � 2.6 pmol/oocyte; n = 110). However, the percentage of embryos developed to the blastocyst stage in the BCB-positive group was significantly higher compared with that of the BCB-negative group (23.5 � 3.6 v. 9.8 � 2.1%, respectively; P < 0.05). Here, we concluded that intracellular GSH plays an important role in embryonic development to the blastocyst stage in vitro.

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.

23 EFFECT OF HISTONE ACETYLATION LEVELS ON THE IN VITRO DEVELOPMENT OF CLONED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 111
Author(s):  
L. Chacón ◽  
J. A. Jenkins ◽  
S. P. Leibo ◽  
G. Wirtu ◽  
B. L. Dresser ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Cultured Cells ◽  
Pearson Correlation ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Donor Cells ◽  
Cloned Bovine ◽  
Vitro Development

The epigenetic status of donor cells is an important factor for their successful reprogramming during somatic cell nuclear transfer (SCNT). Environmental factors partly influence DNA methylation and histone modifications (Fraga et al. 2005 PNAS USA 102, 10 604–10 609; Ke et al. 2006 Carcinogenesis 27, 1481–1488; Rodenhiser and Mann 2006 CMAJ 174, 341–348); low temperatures have altered epigenetic events in plants (Amasino 2004 Plant Cell; Hao et al. 2002 Cryo Letters 23, 37–46). Because cryopreservation alters histone acetylation levels in donor cells and subsequent viability of cloned embryos (Gómez et al. 2008 Cloning Stem Cells, in press), similar alterations may occur in bovine cloned embryos reconstructed with donor cells thawed immediately before SCNT. The objectives of the present study were (1) to measure the relative levels of nuclear histone acetylation in bovine fibroblasts immediately after thawing (frozen/thawed) or following a period of culturing (cultured) and (2) to determine the influence of the epigenetic status of donor cells on the in vitro development of reconstructed, cloned bovine embryos by gauging blastocyst development. Cell cultures lines were derived from the skin of 3 adult cows and analyzed at passage 1 (P1), 2 (P2), and 10 (P10). For each of 3 passages, cells were cultured until reaching 100% confluence, followed by an additional 3 days of culture during which time acetylation levels were measured in cultured and frozen/thawed cells. For cryopreservation, cells at P1, P2, and P10 were disaggregated and resuspended in CryoStor™ (CS10; BioLife Solutions, Bothell, WA, USA) and cooled at 1.0°C min–1 to –80°C prior to storage in liquid nitrogen. Cells were fixed with ethanol for 12 h and incubated for 30 min with antibody directed against acetylated lysine 9 on histone 3 (H3K9). The cells were then incubated with a fluorescein isothiocyanate conjugated secondary antibody and DNA stain and evaluated by flow cytometry. Cloned embryos were reconstructed with cultured or frozen/thawed cells at P1, P2, and P10 as described by Vajta et al. 2005 (Reprod. Fertil. Dev. 17, 791–797). Derived embryos were cultured until Day 8, and cleavage and development to the blastocyst stage were evaluated. Histone acetylation levels for all 3 cell lines, either fresh or frozen/thawed, were significantly higher at P1 than at P2 and P10 (Table 1), and cryopreservation reduced histone acetylation levels only in cell culture line 2 at P1. Higher development to the blastocyst stage (25%) was observed when embryos were reconstructed with cultured cells at P2 and with cells that had lower histone acetylation levels (Pearson correlation, r = –0.55; P = 0.01) Table 1.Relative levels of histone acetylation in bovine fibroblast culture and percentages of development to blastocyst stage after cloning

Transition of Cleavage Divisions During In Vitro Development of Bovine Embryos

2009 ◽  
Vol 26 (1) ◽  
pp. 42-47
Author(s):  
Hitoshi Ushijima ◽  
Kiyoshi Akiyama ◽  
Toshio Tajima
Keyword(s):  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development

In vitro development of bovine oocytes fertilized by sperm-head injection

1992 ◽  
Vol 37 (1) ◽  
pp. 216 ◽  
Author(s):  
K. Goto ◽  
T. Matsumoto ◽  
Y. Takuma ◽  
Y. Nakanishi
Keyword(s):  
Sperm Head ◽  
In Vitro Development ◽  
Vitro Development ◽  
Bovine Oocytes

Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells

2018 ◽  
Vol 54 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Carolina Gonzales da Silva ◽  
Carlos Frederico Martins ◽  
Heidi Christina Bessler ◽  
Álvaro Moraes da Fonseca Neto ◽  
Tereza Cristina Cardoso ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Trichostatin A ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development

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.)

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