187 INCREASED NUMBER OF EXPANDED BOVINE BLASTOCYSTS IN SOF AND CR1 RELATIVE TO KSOM

2007 ◽  
Vol 19 (1) ◽  
pp. 210
Author(s):  
D. M. Kohl ◽  
R. L. Monson ◽  
L. E. Enwall ◽  
J. J. Rutledge
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Pregnancy Rates ◽  
Embryo Morphology ◽  
Bovine Embryos ◽  
Chi Square Analysis

Assessment of morphological stage grade is a subjective procedure. Stage grade is of vital importance to, among other things, recipient synchrony for the purpose of establishing successful pregnancies. Asynchronous embryo transfer has led to decreases in pregnancy rates (Farin et al. 1995 Biol. Reprod. 52, 676–682) and has been implicated in contributing to large offspring syndrome (Young et al. 1996 Theriogenology 45, 231). Differences in embryo kinetics based on culture conditions have been well documented (Mello et al. 2005 Reprod. Fert. Dev. 17, 221 abst). Whether such differences are the result of species, breed, metabolic stress, sire effects, or separation from an in vivo environment has yet to be determined. The correlation between oxygen respiration rates and embryo morphology as well as embryo diameter in bovine embryos produced in vitro has shown promise in the development of a more objective predictor of embryo quality and perhaps pregnancy initiation (Lopes et al. 2005 Reprod. Fert. Dev. 17, 151 abst). As well, recent examination of gene expression patterns of in vitro-derived bovine embryos seems to indicate that longer periods of in vitro culture are associated with lower rates of embryo survival (Lonergan et al. 2006 Theriogenology 65, 137–152). We hypothesize that differences do exist in the number, rate, and morphological appearance of blastocysts and that these parameters are in large part based on culture conditions in vitro. The objective of this experiment was to determine the timing and distribution of blastocyst formation of in vitro-produced bovine embryos cultured in SOF8, CR18AA, and KSOM8, under a standard incubation environment. Bovine ovaries from a local abattoir were aspirated and matured for 18-22. Oocytes were fertilized with frozen-thawed Percoll-separated semen from a Holstein bull. Presumptive zygotes were vortexed to remove cumulus cells and placed into 3 different culture media in a highly humidified atmosphere containing 20% oxygen, 5% carbon dioxide, and compressed air at 38.5�C. Embryos were evaluated specifically at 168 h post-insemination (Day 7) and assigned a morphological stage grade (IETS) to determine fixed time point differences. A total of 6 complete replicates were performed. Only embryos exhibiting the presence of a blastocoel at this time were documented (early blast, mid-blast, expanded blast). At 168 h post-insemination, there were no significant differences in the total number of embryos reaching early or mid-blast stage in any of the media. However, chi-square analysis revealed an increase in the number of expanded blastocysts in SOF (n = 813) and CR1 (n = 838) treatments compared to KSOM (n = 824; P < 0.0001). Expanded blastocysts in SOF were also greater in number than in CR1 (P < 0.05). Embryo selection based on development to the expanded blastocyst stage on Day 7 may prove useful in increasing pregnancy rates, and may validate qualitative correlations based on oxygen consumption and gene expression profiles for embryos produced in vitro.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

scholarly journals Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a Model for In Vivo Gene Expression

2006 ◽  
Vol 188 (2) ◽  
pp. 399-408 ◽  
Author(s):  
Jennifer A. Loughman ◽  
Michael Caparon
Keyword(s):  
Gene Expression ◽  
Streptococcus Pyogenes ◽  
Growth Phase ◽  
Soft Tissues ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Transcriptional Response ◽  
Culture Conditions

ABSTRACT For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo.

171 THE EFFECT OF TRICHOSTATIN A ON THE DEVELOPMENT AND THE GLOBAL GENE EXPRESSION PATTERNS IN MOUSE EMBRYOS DEVELOPING IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 165
Author(s):  
X. S. Cui ◽  
X. Y. Li ◽  
T. Kim ◽  
N.-H. Kim
Keyword(s):  
Gene Expression ◽  
Trichostatin A ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Mouse Embryos ◽  
Differentially Expressed ◽  
Gene Expression Patterns ◽  
Cell Stage

Trichostatin A (TSA) is an inhibitor of histone deacetylase and is able to alter gene expression patterns by interfering with the removal of acetyl groups from histones. The aim of this study was to determine the effect of TSA treatment on the development and gene expression patterns of mouse zygotes developing in vitro. The addition of 100 nm TSA to the culture medium did not affect the cleavage of mouse embryos (TSA treatment, 148/150 (99%) v. control, 107/107 (100%)); however, embryos that were treated with TSA arrested at the 2-cell stage (145/148, 98%). We estimated the number of nuclei in control and TSA-treated embryos by propidium iodide staining, taking into account the presence of any cells with two or more nuclei. At 62–63 h post-hCG stimulation, control zygotes had developed to the 4-cell stage and exhibited one nucleus in each blastomere, indicative of normal development. In contrast, we observed tetraploid nuclei in at least one blastomere in 20.8% (11/53) of the embryos that had been treated with TSA. At 28–29 h post-hCG stimulation (metaphase of the 1-cell stage), there was no difference in the mitotic index (as determined by analyzing the microtubule configuration) in the TSA group compared to the control group. At the 2-cell stage, however, we did not observe mitotic spindles and metaphase chromatin in embryos in the TSA treatment group compared to the controls. Interestingly, when embryos were cultured in TSA-free medium from 35 h post-hCG stimulation (S- or early G2-phase of the 2-cell stage) onward, almost all of them (47/50) developed to the blastocyst stage. In contrast, when embryos were cultured in TSA-free medium from 42 h post-hCG stimulation (middle G2-phase of the 2-cell stage) onward, they did not develop to the 4-cell stage. We used Illumina microarray technology to analyze the gene expression profiles in control and TSA-treated late 2-cell-stage embryos. Applied Biosystems Expression System software was used to extract assay signals and assay signal-to-noise ratio values from the microarray images. Our data showed that 897 genes were significantly (P < 0.05; 2-sample t-test) up- or down-regulated by TSA treatment compared to controls. Analysis using the PANTHER classification system (https://panther.appliedbiosystems.com) revealed that the 575 genes that were differentially expressed in the TSA group compared to the control were classified as being associated with putative biological processes or molecular function. Overall, in terms of putative biological processes, more nucleoside, nucleotide, and nucleic acid metabolism, protein metabolism and modification, signal transduction, developmental process, and cell cycle genes were differentially expressed between the TSA and control groups. In terms of putative molecular function, more nucleic acid-binding transcription factor and transferase genes were differentially expressed between the groups. The results collectively suggest that inhibition of histone acetylation in mouse embryos affects gene expression profiles at the time of zygotic genome activation, and this subsequently affects further development.

scholarly journals Effect ofEx VivoCulture Conditions on Immunosuppression by Human Mesenchymal Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Myoung Woo Lee ◽  
Dae Seong Kim ◽  
Somi Ryu ◽  
In Keun Jang ◽  
Hye Jin Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Human Mesenchymal Stem Cells ◽  
Gene Expression Profiles ◽  
Culture Conditions ◽  
Seeding Density ◽  
Initial Seeding ◽  
Density Treatment

A microarray analysis was performed to investigate whetherex vivoculture conditions affect the characteristics of MSCs. Gene expression profiles were mainly influenced by the level of cell confluence rather than initial seeding density. The analysis showed that 276 genes were upregulated and 230 genes downregulated in MSCs harvested at~90% versus~50% confluence (P<0.05,FC>2). The genes that were highly expressed in MSCs largely corresponded to chemotaxis, inflammation, and immune responses, indicating direct or indirect involvement in immunomodulatory functions. Specifically, PTGES and ULBP1 were up-regulated in MSCs harvested at high density. Treatment of MSCs withPTGESorULBP1siRNA reversed their inhibition of T-cell proliferationin vitro. The culture conditions such as cell confluence at harvest seem to be important for gene expression profile of MSCs; therefore, the results of this study may provide useful guidelines for the harvest of MSCs that can appropriately suppress the immune response.

scholarly journals Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Andrea Jurisicova ◽  
Beth M Acton
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Cell Fate ◽  
Embryo Development ◽  
Preimplantation Embryo ◽  
Culture Media ◽  
Culture Conditions ◽  
Early Embryo ◽  
Preimplantation Embryo Development

Human preimplantation embryo development is prone to high rates of early embryo wastage, particularly under currentin vitroculture conditions. There are many possible underlying causes for embryo demise, including DNA damage, poor embryo metabolism and the effect of suboptimal culture media, all of which could result in an imbalance in gene expression and the failed execution of basic embryonic decisions. In view of the complex interactions involved in embryo development, a thorough understanding of these parameters is essential to improving embryo quality. An increasing body of evidence indicates that cell fate (i.e. survival/differentiation or death) is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins, many of which are expressed during oocyte and preimplantation embryo development. The recent availability of mutant mice lacking expression of various genes involved in the regulation of cell survival has enabled rapid progress towards identifying those molecules that are functionally important for normal oocyte and preimplantation embryo development. In this review we will discuss the current understanding of the regulation of cell death gene expression during preimplantation embryo development, with a focus on human embryology and a discussion of animal models where appropriate.

scholarly journals 232 SPECIES-RELATED DIFFERENCES IN BLASTOCYST QUALITY ARE ASSOCIATED WITH DIFFERENCES IN RELATIVE mRNA EXPRESSION

2005 ◽  
Vol 17 (2) ◽  
pp. 266
Author(s):  
D. Rizos ◽  
A. Gutierrez-Adan ◽  
P. Moreira ◽  
C. O'Meara ◽  
T. Fair ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Culture Media ◽  
Survival Rates ◽  
Qualitative Difference ◽  
Transcript Level ◽  
Transcript Abundance ◽  
Major Influence ◽  
Embryo Morphology ◽  
Bovine Embryos

We have previously reported a species-related qualitative difference (in terms of ultrastructural morphology and cryotolerance) between bovine and ovine blastocysts produced under identical conditions of in vitro culture in synthetic oviduct fluid (SOF1, Rizos et al. 2002 Mol. Reprod. Dev. 62, 320–327). The overall objective of this study was to see if these differences were reflected at the transcript level. From each of five IVF replicates, groups of 10 bovine and 10 ovine blastocysts were used. The objective of Experiment 1 was to compare the relative transcript abundance of eight candidate genes between ovine and bovine blastocysts cultured in SOF1. Following real-time quantitative RT-PCR, transcript levels for MnSOD, survivin, and Glut-5 were significantly higher in ovine than in bovine blastocysts (ANOVA, P < 0.05), while transcripts for Cx31, IFN-tau and SOX were significantly more abundant in bovine blastocysts (P < 0.01). For the two remaining transcripts, E-cad and Na/K, there was no difference. The objective of Experiment 2 was to examine the possibility of modifying the pattern of expression in both types of blastocysts by changing the culture medium. Culture took place in SOF1 or SOF2 (Holm et al. 1999 Theriogenology 52, 683–700). Culture of bovine embryos in SOF2 resulted in a significant increase in the level of expression of MnSOD and Glut-5 (P < 0.05) compared to culture in SOF1. For all the other transcripts except survivin, there was a significant decrease in the relative abundance. Culture of ovine embryos in either SOF1 or SOF2 did not have a major influence on transcript abundance; of the eight transcripts examined, the relative abundance of only one, SOX, was significantly altered. Based on the above, the objective of Experiment 3 (3 replicates) was to determine whether the changed pattern of expression in bovine blastocysts produced in SOF2 was associated with an improvement in cryotolerance. Bovine blastocysts produced in both culture media were vitrified and warmed, and survival was assessed by re-expansion and hatching. Blastocysts produced in SOF2 had significantly higher survival rates at 24, 48, and 72 h and significantly higher hatching rates following vitrification and warming than those produced in SOF1 (P < 0.001). In conclusion, we have demonstrated that the apparent differences between ovine and bovine embryos in their adaptability to culture conditions, manifested in differences in embryo morphology and cryotolerance, are related to differences in mRNA relative abundance. This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78 and by Ministerio de Ciencia y Tecnologia from Spain under Grant No. AGL2003-05783.

scholarly journals Mesenchymal Stromal Cell Differentiation for Generating Cartilage and Bone-Like Tissues In Vitro

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2165
Author(s):  
Graziana Monaco ◽  
Yann D. Ladner ◽  
Alicia J. El Haj ◽  
Nicholas R. Forsyth ◽  
Mauro Alini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Osteogenic Differentiation ◽  
Type I Collagen ◽  
Specific Growth ◽  
Culture Media ◽  
Culture Conditions ◽  
Osteogenic Potential ◽  
Type I

In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFβ1 or BMP2, as well as low (2% O2) or high (20% O2) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O2 significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O2 tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O2; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O2 tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population.

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