The Impact of Two Embryo Culture Media, SOF and Commercial BO, on Pre- and Post-Implantation Development of Cloned Sannen Goat Embryos

2020 ◽  
Author(s):  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Sayed Morteza Aghamiri ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mohsen Rahimi ◽  
...  
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Specific Gene ◽  
Limited Information ◽  
Major Drawback ◽  
Animal Cloning ◽  
The Impact ◽  
Post Implantation

Abstract Background: The ingredients of embryo culture media developed by different companies are disclosed. Thus, it is impossible to determine which ingredients might be responsible for differences in pre-and post-implantation embryo development. To address this gap, we performed an experiment to compare two embryo culture media, namely, SOF and commercial BO, on pre- and post-implantation development of cloned Sannen goat embryos. Cumulus oocyte complexes derived from slaughterhouse ovaries were used for in vitro embryo production . In vitro development of IVF, parthenogenetic and SCNT embryos were assessed in both BO and SOF media. The expression of 16 genes, including AKT , OCT4 , SOX2 , BMPR1 , FGFR4 , CDC25 , CDX2 , GCN5 , PCAF , FOXD3 , SMAD5 , FZD , LIFR1 , CTNNB , ERK1 , and IFNT , belonging to 7 important pathways, i.e. pluripotency, FGF, TGFβ, cell cycle and proliferation, histone transferase, trophectoderm, and WNT, were examined in the goat SCNT and IVF blastocysts from both BO and SOF media. Results: The blastocyst rate in BO medium was significantly higher than that of the SOF medium in SCNT embryos ( P < 0.05). All of the genes examined showed increased expression levels in SCNT embryos compared to IVF embryos. In the IVF group, OCT4 , BMPR1 , and GCN5 showed significantly higher expression in the SOF medium compared to the BO medium. In this group, AKT , FGFR4 , SOX2 showed significantly lower expression in the SOF medium compared to the BO medium. In the SCNT group, FGFR4 , GCN5 , FZD , CTNNB , BMPR1 , and FGFR4 showed significantly higher expression in SOF medium compared to BO medium. In vivo development did not differ significantly between the two groups. Conclusions: Based on these results, we concluded that the limited information available on the allocations of ICM and TE cells in SCNT embryos and embryo-specific gene expression may be the major drawback IVC medium and an impediment to successful animal cloning.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group.Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

scholarly journals Gastrin and Nitric Oxide Production in Cultured Gastric Antral Mucosa Are Altered in Response to a Gastric Digest of a Dietary Supplement

2021 ◽  
Vol 8 ◽  
Author(s):  
Jennifer L. MacNicol ◽  
Wendy Pearson
Keyword(s):  
Nitric Oxide ◽  
Cell Viability ◽  
Dietary Supplement ◽  
Culture Media ◽  
Gastric Fluid ◽  
Simulated Gastric Fluid ◽  
Antral Mucosa ◽  
The Impact

In vitro organ culture can provide insight into isolated mucosal responses to particular environmental stimuli. The objective of the present study was to investigate the impact of a prolonged culturing time as well as the addition of acidic gastric fluid into the in vitro environment of cultured gastric antral tissue to evaluate how altering the commonly used neutral environment impacted tissue. Furthermore, we aimed to investigate the impact of G's Formula, a dietary supplement for horses, on the secretion of gastrin, interleukin1-beta (IL-1β), and nitric oxide (NO). These biomarkers are of interest due to their effects on gastric motility and mucosal activity. Gastric mucosal tissue explants from porcine stomachs were cultured in the presence of a simulated gastric fluid (BL, n = 14), simulated gastric fluid containing the dietary supplement G's Formula (DF, n = 12), or an equal volume of phosphate buffered saline (CO, n = 14). At 48 and 60 h, 10−5 M carbachol was used to stimulate gastrin secretion. Cell viability was assessed at 72 h using calcein and ethidium-homodimer 1 staining. Media was analyzed for gastrin, IL-1β, and NO at 48, 60, and 72 h. There were no effects of treatment or carbachol stimulation on explant cell viability. Carbachol resulted in a significant increase in gastrin concentration in CO and DF treatments, but not in BL. NO was higher in CO than in BL, and NO increased in the CO and DF treatments but not in BL. In conclusion, the addition of carbachol and gastric digests to culture media did not impact cell viability. The use of an acidic gastric digest (BL) reduced the effect of cholinergic stimulation with carbachol at a concentration of 10−5 M and reduced NO secretion. The addition of the dietary supplement to the gastric digest (DF) appeared to mediate these effects within this model. Further research is required to evaluate the specific effects of this dietary supplement on direct markers of mucosal activity and the functional relevance of these results in vivo.

scholarly journals Do We Pay Enough Attention to Culture Conditions in Context of Perinatal Outcome after In Vitro Fertilization? Up-to-Date Literature Review

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Piotr Marianowski ◽  
Filip A. Dąbrowski ◽  
Aleksandra Zyguła ◽  
Mirosław Wielgoś ◽  
Iwona Szymusik
Keyword(s):  
Embryo Culture ◽  
Perinatal Outcome ◽  
Culture Media ◽  
Perinatal Outcomes ◽  
Culture Conditions ◽  
Embryo Survival ◽  
Vitro Fertilization ◽  
Specific Culture ◽  
The Impact

Adverse perinatal outcomes in singleton IVF pregnancies have been most often explained by parental underlying diseases and so far laboratory conditions during embryo culture are still not explored well. The following review discusses the current state of knowledge on the influence of IVF laboratory procedures on the possible perinatal outcome. The role of improved media for human embryo culture is unquestionable. Addition of certain components to culture media and their effect on embryo survival and implantation rates have been taken into consideration recently and studied on animal model. Impact of media on perinatal outcome in IVF offspring has also been studied. It has been discovered that epigenetic changes and neonatal birth weight are probably associated with the use of specific culture media, as is the relation between placental size and its influence on perinatal outcome. There are still questions in the discussion about duration of embryo culture (cleavage stage versus blastocyst transfer). Some of the IVF methods, such as in vitro maturation of oocytes and freezing/thawing procedures, also require well-powered randomized controlled trials in order to define their exact impact on perinatal outcome. Constant further research is needed to assess the impact of laboratory environment on fetal and postnatal development.

scholarly journals The Impact of the Extracellular Matrix Environment on Sost Expression by the MLO-Y4 Osteocyte Cell Line

2022 ◽  
Vol 9 (1) ◽  
pp. 35
Author(s):  
Robert T. Brady ◽  
Fergal J. O’Brien ◽  
David A. Hoey
Keyword(s):  
Extracellular Matrix ◽  
Cell Line ◽  
Three Dimensional ◽  
3D Culture ◽  
Specific Gene ◽  
Collagen Scaffolds ◽  
Sost Gene ◽  
The Impact

Bone is a dynamic organ that can adapt its structure to meet the demands of its biochemical and biophysical environment. Osteocytes form a sensory network throughout the tissue and orchestrate tissue adaptation via the release of soluble factors such as a sclerostin. Osteocyte physiology has traditionally been challenging to investigate due to the uniquely mineralized extracellular matrix (ECM) of bone leading to the development of osteocyte cell lines. Importantly, the most widely researched and utilized osteocyte cell line: the MLO-Y4, is limited by its inability to express sclerostin (Sost gene) in typical in-vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact on Sost expression. Therefore, this study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells, when cultured on a hydroxyapatite (HA)-containing two-dimensional (2D) film analogue, expressed Sost. Moreover, three-dimensional (3D) culture within HA-containing collagen scaffolds significantly enhanced Sost expression, demonstrating the impact of ECM composition and dimensionality on MLO-Y4 behaviour. Importantly, in this bone mimetic ECM environment, Sost expression was found to be comparable to physiological levels. Lastly, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, overcoming a major limitation of this model.

The impact of distinct culture media inLeishmania infantumbiology and infectivity

Parasitology ◽  
2013 ◽  
Vol 141 (2) ◽  
pp. 192-205 ◽  
Author(s):  
NUNO SANTARÉM ◽  
JOANA CUNHA ◽  
RICARDO SILVESTRE ◽  
CÁTIA SILVA ◽  
DIANA MOREIRA ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Culture Media ◽  
Rational Approach ◽  
Cycle Progression ◽  
The Media ◽  
Parasite Biology ◽  
Basic Characteristics ◽  
The Impact

SUMMARYAn ideal culture medium forLeishmaniapromastigotes should retain the basic characteristics of promastigotes found in sandflies (morphology and infectivity). Furthermore, the media should not create a bias in experimental settings, thus enabling the proper extrapolation of results. To assess this we studied several established media for promastigote growth. We analysed morphology, viability, cell cycle progression, metacyclic profile, capacity to differentiate into axenic amastigotes and infectivity. Furthermore, using a rational approach from the evaluated media we developed a simple serum-free medium (cRPMI). We report that parasites growing in different media present different biological characteristics and distinctin vitroandin vivoinfectivities. The developed medium, cRPMI, proved to be a less expensive substitute for traditional serum-supplemented media for thein vitromaintenance of promastigotes. In fact, cRPMI is ideal for the maintenance of parasites in the laboratory, diminishing the expected loss of virulence over time typical of the parasite cultivation. Ultimately this report is a clear warning that the normalization of culture media should be a real concern in the field as media-specific phenomena are sufficient to induce biological bias with consequences in infectivity and general parasite biology.

scholarly journals Characterizing Transcriptional and Epigenetic Signatures Induced By FLT3-ITD Activation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2186-2186
Author(s):  
Barbara Spitzer ◽  
Olga A Guryanova ◽  
Omar Abdel-Wahab ◽  
Nicole Kucine ◽  
Mazhar Adli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Drug Exposure ◽  
Chromatin State ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Flt3 Inhibitor ◽  
The Impact

Abstract Molecular studies have shown that specific somatic mutations impact therapeutic response and overall outcome in acute myeloid leukemia (AML) and have informed the development of molecularly targeted therapies. Previous studies have shown that the FLT3-ITD mutant disease allele predicts a poor prognosis in AML. Despite this important insight and the established role of FLT3-ITD mutations in AML pathogenesis, the impact of this mutation on gene regulation has not been extensively investigated. We hypothesized that transcriptional and epigenetic studies using genetically accurate murine models, cell lines, and primary AML samples would allow us to identify how FLT3 activation induces changes in gene expression and chromatin state. To assess the impact of FLT3-ITD associated FLT3 activation on gene expression, we performed RNA-sequencing studies on two FLT3-ITD cell lines (MOLM-13 and MV4-11) in the presence/absence of AC-220, a potent, specific FLT3 inhibitor. We also treated mice expressing a constitutive FLT3-ITD knock-in allele with AC-220 or vehicle, and performed RNA-sequencing on purified granulocyte-macrophage progenitors (GMPs). We assessed the impact of transient (4-12 hours drug treatment) and chronic (10-14 days) FLT3 inhibition on gene expression; we hypothesized that chronic drug exposure would result in more robust FLT3-mutant gene expression changes. In each case, the effects of FLT3-ITD activation/inhibition on gene expression were compared to RNA-seq data from FLT3-ITD mutant patients from TCGA. We first investigated the impact of short-term and chronic drug exposure on FLT3-ITD dependent gene expression in vitro. Comparison of short-term drug and vehicle treated cells revealed 159 differentially expressed (DE) genes (Benjamini-Hochberg false discovery rate (BH FDR) p < 0.05 and an absolute log2 fold change (FC) > 0.8). By contrast, we found that chronic FLT3 inhibition identified 743 DE genes. Comparison between the acutely and chronically treated cell lines revealed overlap of only 19 genes, suggesting important differences between the acute and steady-state effects of FLT3-inhibition. We found more significant overlap between chronic FLT3-inhibitor gene expression and FLT3-ITD specific gene expression in TCGA, demonstrating that long-term drug exposure more robustly delineates mutant-specific effects on gene expression. We next investigated the impact of short and long term FLT3-inhibition on gene expression in vivo. Analysis of DE genes identified 93 genes in the acutely treated mice vs. vehicle, and 274 genes in chronically treated mice (BH FDR p < 0.05 and absolute log2 FC of > 0.5). Only 12 DE genes were shared between these two perturbations compared with vehicle control. We then compared these gene expression signatures to FLT3-ITD specific gene expression from TCGA; we noted a significant inverse correlation between the signatures of chronic FLT3 inhibition in vivo with FLT3-ITD specific gene expression in TCGA (R2=0.47), but no correlation between the gene expression changes of acute FLT3 inhibition and FLT3-ITD TCGA patients (R2=0.09). We next integrated the FLT3 signatures from our in vivo work and TCGA with ChIP-sequencing for H3K4me3 and H3K27me3 in primary samples with FLT3-ITD compared to normal controls. We found that 3.6% of DE genes in our in vivo system, and 7.2% of DE genes in TCGA, had significant changes in H3K4me3 or H3K27me3. The most common alteration in chromatin state observed with FLT3 activation was an increase in H3K4me3 and transcriptional activation, with a smaller set of genes showing increased H3K27me3 and reduced expression, consistent with FLT3-mediated epigenetic repression. Motif analysis showed that DE loci with significant changes in chromatin state were enriched for ELF5, NF-E2, Pu.1, and Bach1 binding sequences, implicating these transcription factors in mediating FLT3-dependent gene expression effects. By studying the global transcriptional changes that occur with chronic, steady-state FLT3 inhibition in in vitro and in vivo systems, we identified a set of gene expression changes characteristic of FLT3-activation. In addition, integrating changes in gene expression and chromatin state allowed us to identify loci with alterations in epigenetic state in the setting of FLT3-ITD associated FLT3 activation, and to identify candidate transcription factors that mediate FLT3-dependent effects on gene expression. Disclosures No relevant conflicts of interest to declare.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
E. París-Oller ◽  
S. Navarro-Serna ◽  
C. Soriano-Úbeda ◽  
J. S. Lopes ◽  
C. Matás ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits. Results The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group. Conclusions The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

scholarly journals Amylase-Producing Myeloma Cells Reduced Sensitivity to Dexamethasone and Bortezomib.

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5690-5690
Author(s):  
Shohei Mizuno ◽  
Ichiro Hanamura ◽  
Akinobu Ota ◽  
Karnan Sivasundaram ◽  
Tomoko Narita ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Conflicts Of Interest ◽  
Constitutive Expression ◽  
Proliferation Rate ◽  
Specific Gene ◽  
Significant Difference ◽  
Mock Control ◽  
The Impact

Abstract Despite recent progress in treatment for multiple myeloma (MM), a complete cure remains elusive. To further improve the therapeutic outcome of patients with MM, elucidation of the pathology of refractory cases is important. Hyperamylasemia, which is associated with ectopic amylase (AMY) production by MM cells, is a rare condition, and it has been reported to present with poor prognosis showing rapid tumor growth, extramedullary tumor mass formation, and refractoriness of the condition. However, to date, there have been no biological analyses of MM cells ectopically producing AMY. In this study we generated transfectants that stably expressed AMY with human MM cells, and investigated the impact that ectopic AMY production has on tumor proliferation and changes in drug susceptibility in vitro and in vivo. Two human MM cell lines (RPMI8226 and KMS11) and the cDNA encoding AMY1 were used to establish transfectants with ViraPower™ Lentiviral Gateway Expression Kit (Invitrogen), because the increased AMY isotype was salivary type, which is coded in AMY1, in all MM patients previously reported. The constitutive expression and production of AMY1 were confirmed in the AMY-transfectants (8226/AMY and KMS11/AMY), while they were not in the mock controls. These transfectants were assayed for proliferation and apoptosis after exposure to dexamethasone (Dex), bortezomib (Bz) and lenalidomide (Len) in vitro. The anti-myeloma activity of Bz was also tested in vivo in a xenograft model generated by injecting 8226/AMY or the mock cells into NOD-SCID mice. 8226/AMY had no growth advantage in vitro but grew rapidly when subcutaneously transplanted in mice compared with the mock control (2,177±878 vs 970±131 mm3, p = 0.044). 8226/AMY showed a higher cell proliferation rate than the mock control in vitro when treated with Dex (40uM), Bz (2nM), and Len (1mM). The number of apoptotic 8226/AMY cells decreased after exposure to Bz and Len, but the number after exposure to Dex was equivalent compared with the mock control by the Annexin / Propidium Iodide assay. Therefore, 8226/AMY became less sensitive to Bz and Len partly through the inhibition of apoptosis induced by these drugs. 8226/AMY grew rapidly subcutaneously in mice compared with the mock control when treated with Bz (0.3mg/kg, twice weekly) (p = 0.017). As for KMS11/AMY, the AMY-transfectant showed a higher proliferation rate than the mock control in vitro. KMS11/AMY showed reduced susceptibility to Dex, no change in the susceptibility to Bz, and an enhanced susceptibility to Len unexpectedly in comparison with the mock control. The reason for a difference in the effect of ectopic AMY expression on the susceptibility to anti-MM drugs between 8226/AMY and KMS11/AMY is unclear; however, it might be due to the nature of their parental cells. No significant difference was observed in the gene expression profiling between both AMY-transfectants and each of the respective mock controls, except for AMY1, suggesting that ectopic AMY expression did not affect the expression level of the specific gene in MM. In conclusion, we found that 8226/AMY had reduced susceptibility to Dex, Bz, and Len in vitro and also rapid tumor growth with a weakened anti-tumor effect of Bz in vivo. All of these were consistent with the clinical course of previously reported patients with ectopic AMY-producing MM. On the other hand, KMS11/AMY showed an enhanced susceptibility to Len compared with the mock control, indicating that Len might be effective for some patients with AMY-producing MM. Our data provided beneficial clues for elucidating the molecular pathology and developing a treatment strategy for this clinical setting. Disclosures No relevant conflicts of interest to declare.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of an improved IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimal invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and improved (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group. Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but can mitigate the impact of artificial procedures in the offspring.

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