160 ATTEMPTS TO CULTURE BIOPSIED CELLS FROM IN VITRO BOVINE BLASTOCYSTS FOR GENOTYPING

2010 ◽  
Vol 22 (1) ◽  
pp. 238 ◽  
Author(s):  
G. Gamarra ◽  
D. Le Bourhis ◽  
L. Gall ◽  
L. Laffont ◽  
S. Ruffini ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Vero Cells ◽  
Culture Conditions ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Isolated Cells ◽  
Sodium Pyruvate ◽  
Air Atmosphere ◽  
Number Of Cells

Genomic tools have now become available for most livestock species and are being used routinely for marker-assisted selection in cattle. One major challenge in bovine selection is the possibility to detect multiple markers from biopsies of pre-implantation stage embryos which allows to transfer only selected embryos following genotyping. Preliminary studies have shown that 2 ng of DNA collected from 200 embryonic cells (hatched blastocyst) may be sufficient for genotyping based on few markers (<100). However, the present genotyping techniques are much more demanding in terms of DNA. The aim of this work was to test different in vitro culture conditions of biopsied cells issued from bovine blastocysts to produce a large number of cells for genotyping. Bovine embryos were produced in vitro according to a standard protocol (Menck M et al. 1997 Reprod. Nutr. Dev. 37, 141-150). Only grade 1 embryos were biopsied using a microblade under a stereomicroscope. Biopsies had from 5 to 10 cells. Biopsied embryos were in vitro cultured in B2 + 2.5% FCS seeded with VERO cells for 48 h to assess the survival rate. Individual biopsies were cultured in vitro in 4-well culture dishes (Nunc) coated with collagen type 1 at 39°C in a humidified air atmosphere and 5% CO2 under 3 medium conditions. Intact hatched Days 8 to 10 blastocysts were cultured under the same conditions as controls. In condition 1, 43 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 10% FCS and 0.25% ITS (insulin, rransferrin, selenium). In condition 2, 30 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 20% FCS supplemented with 1 mM sodium pyruvate, 1 μg mL-1 of heparin, and 1 μg mL-1 of FGF4. In condition 3, 30 biopsies and 43 control blastocysts were cultured in a complex medium composed of 30% of [DMEM/F12 + 20% FCS] and 70% [DMEM/F12 + 20% FCS conditioned medium using mitomycined VERO cells] supplemented with 1 mM sodium pyruvate, 1.5 μg mL-1 of heparin, and 1.5 μg mL-1 of FGF4 (adapted from Oda et al. 2006 Methods Enzymol. 419, 387-400). Medium was replaced every 3 days. Outgrowths were physically detached and isolated cells were cultured using condition 3. For further passages, monolayers were trypsinized (0.025%) and cells were analyzed by immunofluorescence using anti-cytokeratin 1-8 antibodies. After biopsy and 48 h of in vitro culture, 97.1% (100/103) of embryos survived. For all culture conditions, none of the biopsied cells attached to the coated dishes and no colony were observed after culture. Control intact blastocysts adhered and formed significantly lower rate of outgrowths for condition 1 v. 2 and 3: 77.1% v. 85.7% and 93%, respectively (P < 0.05). After several passages, 3 cell lines were produced and we observed a network of cytokeratin filaments by immunofluorescence suggesting an epithelial cell type for this network. These results show that production of a large number of cells from biopsies was not efficient enough for genotyping. However, the 3 tested culture conditions are favorable for the production and multiplication of cells from intact bovine blastocysts and condition 3 seems to be a suitable medium condition for embryonic cell culture.

scholarly journals In Vitro Microvibration Increases Implantation Rate after Embryonic Cell Transplantation

2017 ◽  
Vol 26 (5) ◽  
pp. 789-794 ◽  
Author(s):  
Vladimir Isachenko ◽  
Karl Sterzik ◽  
Robert Maettner ◽  
Evgenia Isachenko ◽  
Plamen Todorov ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Assisted Reproductive Technologies ◽  
Implantation Rate ◽  
Reproductive Technologies ◽  
Embryonic Cell ◽  
Embryonic Cells ◽  
Natural Conditions ◽  
High Quality ◽  
Oviductal Fluid

In natural conditions the oocyte and embryo are subjected to ever-changing dynamic processes. However, the routine assisted reproductive technologies today involve the use of static in vitro culture systems. The objective was to determine whether there is any difference in the viability of embryos after in vitro culture under static and mechanical microvibration conditions. The viability of embryonic cells (9,624 embryos) generated from 4,436 couples after in vitro culture was evaluated. For groups ≤29, 30-34, 35-39, and ≥40 years, the following rates of high-quality embryos without fragmentation (two to four blastomeres on day 2; six to eight blastomeres and compacting morula on day 3; blastocyst, expanded and hatching blastocyst on day 5) were detected (static vs. vibration, respectively): 65% versus 71%, 44% versus 69%, 67% versus 76% (for statistically significant differences between respective rates in these three groups, p <0.05), and 67% versus 66% (p > 0.1). The following baby-take-home rates were determined for groups ≤29, 30-34, 35-39, and ≥40 years (static vs. vibration, respectively): 30% versus 31% (p > 0.1, increasing only on the level of tendency), 28% versus 37%, 23% versus 29%, and 9% versus 15% (differences between respective rates in these three groups with p < 0.05). It was concluded that in vitro culture of embryos under microvibration (with a mimic of conditions in nature whereby oviductal fluid is mechanically agitated by the epithelial cilia) significantly increases the baby-take-home rate for patients 30 years and older.

Circus movements and blebbing locomotion in dissociated embryonic cells of an amphibian, Xenopus laevis

1976 ◽  
Vol 22 (3) ◽  
pp. 575-583
Author(s):  
K.E. Johnson
Keyword(s):  
Xenopus Laevis ◽  
Cell Contact ◽  
Embryonic Cells ◽  
Gastrula Stage ◽  
Isolated Cells ◽  
Daughter Cells ◽  
Cytoplasmic Protrusion ◽  
Endodermal Cells ◽  
In Cells

Circus movements, which involve the circumferential rotation of a hyaline cytoplasmic protrusion, occur in cells obtained by EDTA dissociation of gastrula-stage Xenopus laevis embryos. Only a few dissociated blastula-stage cells show circus movements, more early gastrula-stage cells show them, and nearly all late gastrula-stage cells show them. Circus movements cease in cells prior to mitosis and begin again in daughter cells after mitosis is completed. In early gastrulae, only 17% of prospective endodermal cells show circus movements while 79% of prospective mesodern, archenteric roof, and posterior neural ectoderm do so. Isolated cells as well as groups of cells in vitro are often propelled by circus movements. There is an obvious antagonism between cell contact and circus movements. The morphogenetic significance of circus movements and blebbing locomotion is discussed.

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

2014 ◽  
Vol 26 (1) ◽  
pp. 123
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
P. Hassel ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

53 IMPACTS OF USING PROCAINE AS A DNA-DEMETHYLATING AGENT IN IN VITRO CULTURE OF BOVINE CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 132
Author(s):  
V. A. Michalczechen-Lacerda ◽  
F. C. Rodrigues ◽  
R. V. de Sousa ◽  
R. Rumpf ◽  
M. M. Franco
Keyword(s):  
Dna Methylation ◽  
Cell Viability ◽  
In Vitro Culture ◽  
Cytogenetic Analysis ◽  
Imprinted Genes ◽  
Demethylating Agent ◽  
Chromosome Integrity ◽  
Methylation Patterns ◽  
Number Of Cells

Euchromatin and heterochromatin organisation define the specificity of each cell type. This structure is controlled by epigenetic modifications and the DNA methylation is one of the best known for inducing transcriptional repression. Recently, procaine was uncovered as a DNA-demethylating agent, but there are few reports about its dynamic epigenetic action on somatic cells. Mono-allelic expression of imprinted genes is controlled by DNA methylation and inherited to somatic tissues of a sex-specific manner. The aim was to investigate the effects of using procaine, a DNA-demethylating agent, in in vitro culture of bovine (Bos taurus indicus) fibroblast for 72 h (passage 4). We have evaluated cell viability, chromosome integrity, and DNA methylation patterns. To evaluate cell viability, we have used trypan blue 0.4%. To evaluate chromosome integrity, we have used conventional cytogenetic analysis. To investigate DNA methylation patterns, we have analysed 2 differentially methylated regions (DMR) located into the exon 10 of IGF2 and exon 1 of XIST imprinted genes, using the bisulfite sequencing method (EZ DNA methylation kit, Zymo Research, Orange, CA, USA). After bisulfite treatment and nested-PCR, the amplicons were separated in agarose gel electrophoresis, purified with GenClean III kit (MP Biomedicals, Irvine, CA, USA), cloned in a pGEM-T easy vector system (Promega, Madison, WI), and sequenced. The DNA sequences were analysed using the BiQ Analyzer v. 2.0 (2008) software. The cell viability data were analysed using ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney tests, and the methylation status were analysed using Student’s t-test or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies). Cell culture using 0.1 mM or 0.5 mM of procaine were viable and the number of cells with intact membrane was higher than the control and 2.0 mM of procaine groups (P ≤ 0.05). The total number of cells was lower in the group with 2.0 mM of procaine (P ≤ 0.01). Cytogenetic analysis showed no differences among the groups, with no chromosome abnormalities detected. The methylation pattern was not different for both DMR evaluated among the groups. We have observed that there was a beneficial effect to the cells that have received supplementation with 0.1 mM or 0.5 mM of procaine, because there was an increase in the number of viable cells without chromosomal abnormalities. We cannot ignore that a global DNA demethylation may have occurred, which was not detected in the specific analysed regions. The results obtained here may contribute to improving the efficiency of animal cloning, transgenic animal production, and the knowledge about stem cells. Supported by Embrapa Genetic Resources and Biotechnology and CAPES.

65 Co-culture of porcine epithelial oviductal cells and invitro-produced embryos: Effect on embryo development and quality

2021 ◽  
Vol 33 (2) ◽  
pp. 139
Author(s):  
M. S. Lorenzo ◽  
G. M. Teplitz ◽  
P. R. Cruzans ◽  
C. G. Luchetti ◽  
J. Ghersa ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Embryo Development ◽  
Bovine Serum ◽  
Embryo Quality ◽  
Culture Conditions ◽  
Sodium Pyruvate ◽  
Apoptosis Index ◽  
Chi Squared ◽  
Blastocyst Rate ◽  
Number Of Cells

The oviduct is involved in many reproductive functions, including early embryo development. The epithelial cells that cover the oviduct produce oviducal fluid and could be used to recreate the invivo environment into which embryo development takes place. This study aimed to evaluate the co-culture of porcine embryos with a monolayer of porcine oviducal epithelial cells (POEC) and its effect on embryo development and quality. The POEC were obtained by pressing the isthmus (from diestrus sow oviducts) using slides and performing 3 cycles of vortexing and decanting in DMEM-F12 medium. Passage 1 cells were used for these experiments (POEC-1). Oocytes were obtained from follicular aspiration of slaughterhouse ovaries. Oocytes were invitro matured for 44h in TCM-199 supplemented with human menopausal gonadotrophin and cyclic AMP during the first 22h. Invitro fertilization was performed with 17°C-refrigerated boar semen for 4h in 100-µL drops of TCM-199 with caffeine, bovine serum albumin, sodium lactate, and sodium pyruvate (20 denuded oocytes per drop, 1×106 spermatozoa mL−1). Presumptive zygotes were washed and randomly assigned to one of the following groups for invitro culture: control (50-µL drop of NCSU-23 with sodium pyruvate and lactate), POEC-1 (same as the control+POEC-1 50 000 cells mL−1), POEC-1+FBS (same as the control+POEC-1 50 000 cells mL−1 and 2.5% of fetal bovine serum). Culture conditions were 7% O2, 5% CO2, 39°C, and humidity. On Day 2, the cleavage rate was recorded, and embryos were transferred to NCSU-23 drops with glucose and without cells. The blastocyst rate was recorded on Day 7. Embryo quality was assessed by counting the number of cells per blastocyst (Hoechst) and the apoptosis index (TUNEL-positive cells/total cells). Co-culture with POEC-1 significantly increased the blastocyst rate (control: 14%; POEC-1+FBS: 10%; POEC-1: 28%; P&lt;0.05 Chi-squared test) and allowed embryo hatching (control: 0; POEC-1+FBS: 22.2%; POEC-1: 7; P&lt;0.05 Chi-squared test). However, there was no significant difference in the number of cells per blastocyst (control: 58.6±6; POEC-1+FBS: 50.3±3.7; POEC-1: 50.6±4.8; nonparametric ANOVA) or in the apoptosis index (control: 8.1; POEC+FBS 8.3; POEC: 7.4; nonparametric ANOVA). The use of POEC-1 during the first 2 days of embryo culture enhanced embryo development and improved culture conditions, allowing embryo hatching. The effect on embryo development could be due to an effect of POEC itself or the effect of feeder cells. Other parameters of embryo quality should be evaluated in the future.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

Influence of in Vitro Culture Conditions on Tumor Cell Differentiation

1986 ◽  
Vol 72 (1) ◽  
pp. 15-20 ◽  
Author(s):  
Giuliana Porro ◽  
Elda Tagliabue ◽  
Sylvie Ménard ◽  
M.I. Colnaghi
Keyword(s):  
Cell Differentiation ◽  
Tumor Cell ◽  
In Vitro Culture ◽  
Culture Conditions ◽  
Tumor Cell Differentiation
Sign in / Sign up

Export Citation Format

Share Document