scholarly journals Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

2021 ◽  
Vol 22 (3) ◽  
pp. 1222
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Survival Rates ◽  
Control Group ◽  
P Value ◽  
Transcriptome Profile ◽  
Embryo Viability ◽  
The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

scholarly journals Vitrification of in Vivo-derived Porcine Morulae and Blastocysts Alters Metabolic and Stress Response Pathway Alterations

2020 ◽  
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M Cambra ◽  
Inmaculada Parrilla ◽  
Heriberto Rodriguez-Martinez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Survival Rates ◽  
Enrichment Analysis ◽  
Control Group ◽  
Embryo Viability ◽  
Developmental Potential ◽  
Go Enrichment ◽  
Stress Response Pathway

Abstract BackgroundDespite reported promising farrowing rates after non-surgical and surgical transfers of vitrified in vivo-derived porcine morulae and blastocysts (range: 70-75%), their pregnancy loss is yet higher (10-20%) than it is for fresh embryos (< 2.5%). The present study was designed to investigate whether vitrification would affect the transcriptome of porcine morulae and blastocysts, using microarrays and qRT-PCR validation. Material and methods and resultsMorulae and blastocysts were collected from weaned sows (n=13) by laparotomy at Day 6 (Day 0=start of estrus). Of each embryo category, 50 morulae and 50 blastocysts were vitrified (Treatment group) while fresh morulae (n=40) and blastocysts (n=40) acted as control group. After one week of storage, vitrified embryos were cultured in vitrofor 24 h after warming. Non-vitrified morulae (n=40) and blastocysts (n=40), cultured in vitro for 24 h were used as controls. After the in vitro culture period, embryo viability was morphologically assessed. A total of 30 viable embryos per group and embryonic stage(three pools of 10), were subjected to gene expressionanalysis byusing a microarray approach. A fold change cut-off of ±1.5 and a p-unadjusted <0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed morulae and blastocysts were similar to those of the control (nearly 100%, n.s.).A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated) and 105 (112 upregulated and 93 downregulated) in vitrified blastocysts compared to the control group.Transcriptome DEG profiles were dependent on developmental stage, and GO enrichment analysis mainly related DEGs tobiological processes.The vitrification/warming impact on morulae was mostly repression of gene expression with the exception of metabolism-related pathways. In the case of blastocysts, we noted the activation of the cell cycle, cellular senescence andof signaling pathways for TFGβ, p53, FoxO and MAPK. Disruption ofmetabolic-related pathways in morulae and steroid biosynthesis and gap junctions in blastocysts could be related to impaired embryo quality and developmental potential, despite the rather high post-warming survival rates seen in vitro.ConclusionIn conclusion, vitrification modified the transcriptome of in vivo-derived porcine morulae and blastocysts, resulting in moderate gene expression changes, which may disturb subsequent embryo development and pregnancy after embryo transfer.

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

129 POST-HATCHING DEVELOPMENT AND GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS IN RECIPIENT UTERUS AFTER MULTIPLE TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 212
Author(s):  
G. Machado ◽  
A. Ferreira ◽  
I. Pivato ◽  
A. Fidelis ◽  
J. F. Srpicigo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
T Test ◽  
Molecular Profile ◽  
Control Group ◽  
Fluid Medium ◽  
Uterine Flushing

This study aimed to compare post-hatching development of Day 7 in vitro and in vivo embryos cultured in recipient uterus until Day 14. For producing in vitro embryos (IVP), oocytes were matured, fertilized (Day 0) and cultured in vitro for 6 days (Day 7) in synthetic oviduct fluid medium supplemented with 5% of fetal bovine serum and incubated at 39°C in 5% CO2 in air. At Day 7, part of IVP blastocysts was transferred to recipient uterus and part was stored for gene expression analysis. As a control group, in vivo embryos were produced after ovarian stimulation, insemination and uterine flushing on Day 7 post insemination. Similarly to the IVP embryos, part of embryos was transferred to recipient uterus and part was stored for gene expression analysis. Day 7 in vivo (n = 53) and IVP (n = 64) expanded blastocysts were transferred to synchronized recipients (10/horn) and were collected by uterine flushing 7 days after transfer (Day 14). Recovered embryos were measured using Motic Image Plus software and evaluated for presence and size of embryonic disc (ED). A trophoblast biopsy was removed and stored for gene expression analysis. For the molecular profile evaluation of Day 7 and Day 14 in vivo and in vitro embryos, 8 genes related with placentation, implantation, oxidative stress, and glucose metabolism (PLAC8, CD9, GLUT-1, GLUT-3, KRT8, MnSOD, HSP70, and INFT, respectively) were quantified by RT-qPCR using ΔΔCT method and CYC-A gene as endogenous control. The recovery rate of Day 14 embryos, analyzed by chi-square test, was higher (P < 0.05) for in vitro than for in vivo embryos, being 50.0% (64/128) and 38.6% (53/137), respectively. No differences (P > 0.05; t-test) were observed in embryo length when comparing Day 14 in vitro (19.1 ± 2.4 mm) and in vivo embryos (24.2 ± 3.7 mm). ED was detected in 25% (16/64) of in vitro and in 26% (14/53) of in vivo embryos. No differences were found (P > 0.05; t-test) in diameter between the two types of embryos (0.3 ± 0.0 mm/in vitro and 0.3 ± 0.0 mm/in vivo). Regarding gene expression, Day 7 IVP embryos showed higher (P < 0.05, Mann–Whitney test) expression of HSP70 and SCL2A1 than in vivo embryos. However, at Day 14 no differences between embryos were observed in transcript levels for any of the studied genes. Therefore, the present study showed that although differences in Day 7 in vitro embryos were observed at the molecular level compared to in vivo counterpart, after transfer to the uterine environment, they showed similar morphology and gene expression profile. These results highlight the importance of evaluating embryos produced by assisted reproductive techniques in later stages of development to have a more precise evaluation of their quality. Financial support: Embrapa, CNPq, CAPES.

Gene Expression Analysis of Troglitazone Reveals Its Impact on Multiple Pathways in Cell Culture: A Case for In Vitro Platforms Combined with Gene Expression Analysis for Early (Idiosyncratic) Toxicity Screening

2006 ◽  
Vol 25 (2) ◽  
pp. 85-94 ◽  
Author(s):  
Gordon Vansant ◽  
Patrick Pezzoli ◽  
Robert Saiz ◽  
Aaron Birch ◽  
Chris Duffy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Type 2 Diabetes ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Peroxisome Proliferator ◽  
Toxicity Screening ◽  
The Impact

Peroxisome proliferator-activated receptor gamma (PPAR γ) agonists of the thiazolidinedione family are used for the treatment of type 2 diabetes mellitus due to their ability to reduce glucose and lipid levels in patients with this disease. Three thiazolidinediones that were approved for treatment are Rezulin (troglitazone), Avandia (rosiglitazone), and Actos (pioglitazone). Troglitazone was withdrawn from the market due to idiosyncratic drug toxicity. Rosiglitazone and pioglitazone are still on the market for the treatment of type 2 diabetes. The authors present data from a gene expression screen that compares the impact these three compounds have in rats, in rat hepatocytes, and in the clone 9 rat liver cell line. The authors monitored the changes in expression in multiple genes, including those related to xenobiotic metabolism, proliferation, DNA damage, oxidative stress, apoptosis, and inflammation. Compared to the other two compounds, troglitazone had a significant impact on many of the pathways monitored in vitro although no major perturbation was detected in vivo. The changes detected predict not only general toxicity but potential mechanisms of toxicity. Based on gene expression analysis, the authors propose there is not just one but multiple ways troglitazone could be toxic, depending on a patient’s environment and genetic makeup, including immune response-related toxicity.

scholarly journals Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

2021 ◽  
Vol 8 ◽  
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Alejandro González-Plaza ◽  
Inmaculada Parrilla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Pathways ◽  
Survival Rates ◽  
Gene Expression Pattern ◽  
Control Group ◽  
High Survival ◽  
Embryo Survival ◽  
Developmental Potential

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted &lt;0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

scholarly journals Anti-Obesity and Anti-Adipogenic Effects of Chitosan Oligosaccharide (GO2KA1) in SD Rats and in 3T3-L1 Preadipocytes Models

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 331
Author(s):  
Jung-Yun Lee ◽  
Tae Yang Kim ◽  
Hanna Kang ◽  
Jungbae Oh ◽  
Joo Woong Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Body Weight ◽  
Density Lipoprotein ◽  
Treated Group ◽  
Control Group ◽  
Chitosan Oligosaccharide ◽  
Sd Rats ◽  
Adipogenic Gene

Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

153 SUPPLEMENTATION WITH LINOLENIC ACID AND L-CARNITINE DURING IVM REDUCED THE EXPRESSION OF GENES RELATED TO LIPOGENESIS BUT DID NOT ALTER THE LIPID CONTENT AND CRYOTOLERANCE OF IN VITRO-PRODUCED EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 185 ◽  
Author(s):  
B. C. S. Leao ◽  
N. A. S. Rocha Frigoni ◽  
P. C. Dall'Acqua ◽  
M. Ambrogi ◽  
G. B. Nunes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Linolenic Acid ◽  
Lipid Content ◽  
Control Group ◽  
Intracellular Lipid ◽  
Chi Square ◽  
Sudan Black B ◽  
Expression Of Genes ◽  
The Impact

This study was conducted to evaluate the impact of supplementation during in vitro maturation (IVM) with linolenic acid (ALA), l-carnitine (L-car), or the combination of both supplements on the embryo intracellular lipid content and cryotolerance, as well as in the embryo expression of genes involved in lipid metabolism (lipogenesis regulation: SCD1, FASN, and SREBP1; and β-oxidation pathway: CPT1B and CPT2). Cumulus-oocyte complexes (n = 1076) were IVM for 22 h at 38.5°C and 5% CO2 in air, in TCM-199 medium with bicarbonate, hormones, and 10% FCS (control group), supplemented with 100 μM ALA (ALA group), 5 mM L-car (L-car group), or a combination of 100 μM ALA + 5 mM L-car (ALA + L-car group). After IVF, presumptive zygotes were in vitro cultured in SOFaa medium supplemented with 5 mg mL−1 BSA and 2.5% FCS, at 38.5°C and 5% CO2 in air during 7 days. Cleavage and blastocyst rates were evaluated on Day 3 and 7, respectively (IVF = Day 0). At Day 7, the blastocysts were stained with the lipophilic dye Sudan Black B (n = 60), vitrified/warmed (n = 260; Ingámed® protocol, Maringa-PR, Brazil), or collected for analysis of gene expression (n = 180). Embryonic development were analysed by ANOVA and the multiple comparisons of means were determined by Tukey’s test. The embryonic re-expansion data were subjected to chi-square test and the differences in gene expression among groups were evaluated by Duncan’s multiple range test (P < 0.05). Data are presented as means ± standard error means. There was no effect (P > 0.05) of the supplements used during IVM on cleavage (79.54 ± 2.76% to 82.16 ± 1.13%) and blastocyst rates (29.03 ± 3.07% to 30.46 ± 2.01%). Similarly, the intracellular lipid content in Day-7 blastocysts (1.03 ± 0.04 to 1.15 ± 0.07 pixels) and the embryonic cryotolerance, assessed by the re-expansion rates after 24 h (67.3 to 78.3%) hatching rates after 48 h (11.5 to 25.5%) of post-warming culture, were unaffected (P > 0.05) by the supplements of IVM medium. Although the treatments did not alter (P > 0.05) the expression of CPT1B and CPT2 genes, the expression of FASN gene was decreased (P < 0.05) in the ALA group and the expression of SREBP1 gene was decreased (P < 0.05) in the ALA and L-car groups. The expression of the gene SCD1 was reduced (P < 0.05) in all treatments compared with the control group. Thus, despite the lack of effects of the treatments performed during IVM on the intracellular lipid content and cryotolerance of the embryos derived from the treated oocytes, a reduction in the expression of genes related to lipogenesis was observed in Day-7 blastocysts. These results suggest that treatments performed in the oocytes during IVM may have prolonged effects, affecting the subsequent expression of genes in embryos. Further studies are needed to determine the mechanisms related to the differentiation of the oocyte machinery during maturation. Financial support was provided by FAPESP (#2012/10084–4 and #2013/07382–6).

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