3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 82
Author(s):  
M. Paczkowski ◽  
C. Bidwell ◽  
D. Spurlock ◽  
J. Waddell ◽  
R. L. Krisher
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Dna Methyltransferase ◽  
In Vitro Maturation ◽  
Fold Increase ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Dna Methyltransferase 1

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P < 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (>7-fold increase in vivo), caspase 8 (>4-fold increase in vivo), and eukaryotic translation initiation factor 1B (>4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (>3-fold and >5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

6 EXPRESSION PROFILING OF SINGLE BOVINE EMBRYOS REVEALS SIGNIFICANT EFFECTS OF IN VITRO MATURATION, FERTILIZATION AND CULTURE

2006 ◽  
Vol 18 (2) ◽  
pp. 111
Author(s):  
S. L. Smith ◽  
L.-Y. Sung ◽  
R. Page ◽  
B. Henderson ◽  
F. Du ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Oocyte Maturation ◽  
Expression Profiling ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Differentially Expressed ◽  
In Vitro Oocyte Maturation

Cattle and sheep embryos transferred after in vitro production are often afflicted by large offspring syndrome (LOS), which has been correlated with the presence of serum and/or cell co-culture. Previous research indicates that post-fertilization culture affects blastocyst quality and gene expression, and in vitro oocyte maturation and fertilization impact developmental competence. To dissect the effects of in vitro maturation, fertilization, and culture, we compared the expression profiles of single bovine blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF, n = 15); (2) in vivo maturation, in vivo fertilization, and in vitro culture (IVD, n = 14); and (3) in vivo maturation, fertilization, and development (AI, n = 14). For in vitro culture, the embryos were cultured for 2 days in CR1aa medium with bovine serum albumin (BSA) and then transferred to CR1aa with 10% fetal bovine serum (FBS) with cumulus cells until Day 7, at which time the embryos were vitrified. IVD zygotes were surgically collected from two superovulated Holstein donor cows 24 h post-insemination and cultured in the same system. To conduct expression profiling, total RNA was isolated from individual thawed embryos. The RNA was subjected to three rounds of amplification utilizing a previously adapted and validated T7 linear amplification protocol. Amplified RNA from each embryo and from a standard reference was indirectly labeled with Cy3 or Cy5 by dye swap and hybridized to a custom bovine cDNA microarray containing ~6300 unique genes. After Loess normalization, an ANOVA model (GeneSpring 6.1 and SAS 9.0) was used to identify differentially expressed genes. The P-values were adjusted for multiple comparisons using the false discovery rate approach, and a e2-fold differential criterion was applied. A subset of the differentially expressed genes was verified by real-time RT-PCR. The blastocyst rates for IVF and IVD embryos were 37% and 75%, respectively. There were 305, 365, and 200 genes differentially expressed between the AI and IVD, the IVF and IVD, and the AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and the IVD embryos, making these potential candidates for LOS. There were 61 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology categories 'RNA processing' and 'RNA binding' were over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression. This work was supported by USDA grants to X.Y., H.A.L., and X.C.T.

163 EFFECTS OF DIFFERENT IN VITRO MATURATION SYSTEMS ON EMBRYO DEVELOPMENT IN BOVINE PREPUBERTAL AND ADULT DONORS

2014 ◽  
Vol 26 (1) ◽  
pp. 195
Author(s):  
S. M. Bernal ◽  
J. Heinzmann ◽  
D. Herrmann ◽  
U. Baulain ◽  
A. Lucas-Hahn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Lactating Cows

Prepubertal bovine females have been suggested as a source of oocytes in order to accelerate genetic gain and decrease the generation interval. However, prepubertal oocytes have a lower developmental competence than their adult counterparts. In vitro maturation (IVM) systems using cyclic AMP (cAMP) regulators and 30-h culture have been suggested to improve blastocyst in vitro production rates from bovine oocytes (Albuz et al., 2010). The present study evaluated the effects of an addition of the cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX), and cilostamide during extended IVM on blastocyst yields and gene expression in prepubertal and adult bovine females. Holstein-Friesian donors were submitted to ovum pick-up twice per week. Oocytes from groups of 12 animals, including lactating cows (>2 lactations) and prepubertal donors (6–10 months old) were used in the following treatment groups: TCM24 (24-h IVM, routine protocol/control), cAMP30 (2-h pre-IVM culture using forskolin-IBMX and 30-h IVM adding cilostamide), DMSO30 [2-h pre-IVM culture and 30-h IVM with dimethyl sulfoxide (DMSO)/vehicle control]. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. In vivo blastocysts were produced from superovulated cows and used for gene expression analysis. Cleavage rates, blastocyst formation, and mRNA abundance of selected genes were evaluated. The Glimmix procedure from SAS/STAT (SAS Institute Inc., Cary, NC, USA) was performed to compare blastocyst and cleavage rates. One-way ANOVA was implemented to evaluate gene expression. A total of 793 oocytes from the different sources were submitted to the IVM treatments. Cleavage rates (prepubertal donors: 64.6 ± 4%, 59.1 ± 6.4%, 53 ± 4.4%, cows: 55.1 ± 4.3%, 59 ± 6.5%, 50.8 ± 4.4%, for TCM24, cAMP30, and DMSO30, respectively; P > 0.05) and blastocyst/zygotes rates (prepubertal donors: 27 ± 6%; 21.8 ± 3.5%; 17.6 ± 2.4%; cows: 28 ± 3.3%; 27.7 ± 2.9%; 22.7 ± 3.2% for TCM24, cAMP30, and DMSO30, respectively; P > 0.05) did not differ among in vitro treatments. The mRNA relative abundance of the EGR1 gene was down-regulated 6-fold in all in vitro-produced blastocysts compared with their in vivo counterparts (P < 0.05). Gene expression profiles for SLC2A8, DNMT3B, BCL-XL, and PRDX1 were similar in in vitro and in vivo blastocysts. These results show similar embryo production patterns in prepubertal and adult donors. Furthermore, DMSO did not show effects on embryo developmental rates when used during IVM. The gene expression levels of EGR1 confirm our recent findings in blastocysts obtained from oocytes from slaughterhouse ovaries (data not presented), showing its usefulness as an embryo quality marker. These preliminary results indicate that oocyte developmental capacity in prepubertal donors can be similar to that of the adult donors without addition of cAMP modulators.

280 INDISTINGUISHABLE TRANSCRIPTIONAL PROFILES BETWEEN IN VIVO- AND IN VITRO-PRODUCED BOVINE FETUSES

2010 ◽  
Vol 22 (1) ◽  
pp. 297
Author(s):  
L. Jiang ◽  
S. L. Marjani ◽  
M. Bertolini ◽  
H. A. Lewin ◽  
G. B. Anderson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Gene Expression ◽  
Expression Profiles ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Differential Gene

During the past several decades, in vitro fertilization (IVF) has been increasingly used in animal production and human infertility treatment. In vitro production (IVP) has been shown to cause reduced developmental competence, aberrant gene expression, and developmental abnormalities. Our objective was to determine how in vitro procedures influence global gene expression during fetal development. To this end, we analyzed the gene expression profiles of liver and placentome tissue samples (n = 18) from IVP and in vivo-derived fetuses at Days 90 and 180 of gestation (n = 5 IVP and n = 4 in vivo-derived pregnancies for each day of gestation). Standard in vitro maturation and fertilization protocols were employed. Putative zygotes were co-cultured with bovine oviductal epithelial cells to the blastocyst stage. In vivo embryos were collected 7 days after AI by nonsurgical uterine flushing. Blastocyst-stage IVP and in vivo embryos were transferred to synchronized recipients and monitored until collection at Day 90 or 180. The pregnancy rate at Day 90 was 12% and 27% for IVP and in vivo pregnancies, respectively (Bertolini et al. 2004 Reproduction 128, 341-354). To conduct expression profiling, total RNA from each tissue sample and a standard reference was indirectly labeled with Cy3 and Cy5, respectively, and hybridized in duplicate to custom, bovine 13 K oligonucleotide microarrays. After Loess normalization, a two-way (origin and day) ANOVA model (GeneSpring 7.3.1) was used to identify differentially expressed genes in each tissue. The P-values were adjusted for multiple comparisons using a 5% false discovery rate (FDR). The expression of 11 candidate genes was confirmed independently by quantitative RT-PCR. Surprisingly, in both the liver and placentome tissues, no differential gene expression was detected between the IVP and in vivo fetuses at Day 90 and 180. This was observed even when the FDR was relaxed to 10% and 20%. A total of 879 genes (523 genes ≥ 1.5-fold) were differentially expressed during liver development from 90 to 180 days of gestation. Conversely, no differential gene expression was detected in the placentomes during this developmental period. Our findings show that during early and mid gestation, surviving IVP fetuses had normal patterns of gene expression. It is possible that embryos with less severe perturbations may survive with their gene expression normalized as development proceeds. Additionally, initial changes in gene expression caused by IVP may affect subsequent development, but do not necessarily persist throughout gestation. Present addresses: L. Jiang, Columbia University, New York, NY, USA; S. L. Marjani, Yale University, New Haven, CT, USA; M. Bertolini, University of Fortaleza, CE, Brazil. This work was supported by USDA grants to X.Y, H.A.L., and X.C T.

scholarly journals CircFAM13B promotes the proliferation of hepatocellular carcinoma by sponging miR-212, upregulating E2F5 expression and activating the P53 pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ying Xie ◽  
Xiaofeng Hang ◽  
Wensheng Xu ◽  
Jing Gu ◽  
Yuanjing Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Gene Expression Omnibus ◽  
Differentially Expressed ◽  
In Vivo Studies ◽  
Circular Rnas ◽  
Novel Target ◽  
Underlying Mechanisms

Abstract Background Most of the biological functions of circular RNAs (circRNAs) and the potential underlying mechanisms in hepatocellular carcinoma (HCC) have not yet been discovered. Methods In this study, using circRNA expression data from HCC tumor tissues and adjacent tissues from the Gene Expression Omnibus database, we identified out differentially expressed circRNAs and verified them by qRT-PCT. Functional experiments were performed to evaluate the effects of circFAM13B in HCC in vitro and in vivo. Results We found that circFAM13B was the most significantly differentially expressed circRNA in HCC tissue. Subsequently, in vitro and in vivo studies also demonstrated that circFAM13B promoted the proliferation of HCC. Further studies revealed that circFAM13B, a sponge of miR-212, is involved in the regulation of E2F5 gene expression by competitively binding to miR-212, inhibits the activation of the P53 signalling pathway, and promotes the proliferation of HCC cells. Conclusions Our findings revealed the mechanism underlying the regulatory role played by circFAM13B, miR-212 and E2F5 in HCC. This study provides a new theoretical basis and novel target for the clinical prevention and treatment of HCC.

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.

Abstract 41: Explant-Derived Cells from Chronic Heart Failure Activated Endothelial-Mesenchymal Transition and Attenuated Pluripotency Genes Expression

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Liudmila Zakharova ◽  
Hikmet Nural ◽  
Mohamed A Gaballa
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Smooth Muscle Actin ◽  
Fold Increase ◽  
Gene Expressions ◽  
Mesenchymal Transition ◽  
Cell Outgrowth ◽  
Pluripotency Genes

Cardiac progenitor cells are generated from atria explants; however the cellular origin and the mechanisms of cell outgrowth are unclear. Using transgenic tamoxifen-induced Willms tumor 1 (Wt1)-Cre/ERT and Cre-activated GFP reporter mice, we found approximately 40% of explant-derived cells and 74% of explant-derived c-Kit+ cells originated from the epicardium. In atria from sham hearts, Wt1+ cells were located in a thin epicardial layer, while c-Kit+ cells were primarily found within both the sub-epicardium and the myocardium, albeit at low frequency. No overlap between c-Kit+ and Wt1+ cells was observed, suggesting that epicardial Wt1+ cells do not express c-Kit marker in vivo, but more likely the c-Kit marker was acquired in culture. Compared with 4 days in culture, at day 21 we observed 7 folds increase in Snail gene expression; 32% increase in α-smooth muscle actin (SMA) marker, and 30% decrease in E-cadherin marker, suggesting that the explant-derived cells underwent epithelial to mesenchymal transition (EMT) in vitro. Cell outgrowths released TGF-β (1036.4 ± 1.18 pm/ml) and exhibited active TGF-β signaling, which might triggered the EMT. Compared to shams, CHF cell outgrowths exhibited elevated levels of EMT markers, SMA (49% vs. 34%) and Snail (2 folds), and reduced level of Wt1 (11% vs. 22%). In addition, CHF cell outgrowths had two folds increase in Pai1 gene expression, a direct target of TGF-β signaling. In c-Kit+ cells derived from CHF explants, Nanog gene expression was 4 folds lower and Sox 2 was 2 folds lower compared with cells from shams. Suppression of EMT in cell outgrowth increased the percentage of c-Kit+ and Wt1+ cells by 17%, and 15%, respectively. Also suppression of EMT in c-Kit+ cells resulted in 4 folds increase in Nanog and 3 fold increase in Sox2 gene expressions. Our results showed that CHF may further exuberates EMT while diminishes the re-activation of pluripotency genes. Thus, EMT modulation in CHF is a possible strategy to regulate both the yield and the pluripotency of cardiac-explant-derived progenitor cells.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

scholarly journals In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development

2017 ◽  
Vol 29 (9) ◽  
pp. 1667 ◽  
Author(s):  
M. Arias-Álvarez ◽  
R. M. García-García ◽  
J. López-Tello ◽  
P. G. Rebollar ◽  
A. Gutiérrez-Adán ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rabbit Model ◽  
Developmental Competence ◽  
Gene Markers ◽  
Developmental Potential ◽  
Junction Protein ◽  
Mitochondrial Distribution ◽  
Potential Biomarkers

In vivo-matured cumulus–oocyte complexes are valuable models in which to assess potential biomarkers of rabbit oocyte quality that contribute to enhanced IVM systems. In the present study we compared some gene markers of oocytes and cumulus cells (CCs) from immature, in vivo-matured and IVM oocytes. Moreover, apoptosis in CCs, nuclear maturation, mitochondrial reallocation and the developmental potential of oocytes after IVF were assessed. In relation to cumulus expansion, gene expression of gap junction protein, alpha 1, 43 kDa (Gja1) and prostaglandin-endoperoxide synthase 2 (Ptgs2) was significantly lower in CCs after in vivo maturation than IVM. In addition, there were differences in gene expression after in vivo maturation versus IVM in both oocytes and CCs for genes related to cell cycle regulation and apoptosis (V-Akt murine thymoma viral oncogene homologue 1 (Akt1), tumour protein 53 (Tp53), caspase 3, apoptosis-related cysteine protease (Casp3)), oxidative response (superoxide dismutase 2, mitochondrial (Sod2)) and metabolism (glucose-6-phosphate dehydrogenase (G6pd), glyceraldehyde-3-phosphate dehydrogenase (Gapdh)). In vivo-matured CCs had a lower apoptosis rate than IVM and immature CCs. Meiotic progression, mitochondrial migration to the periphery and developmental competence were higher for in vivo-matured than IVM oocytes. In conclusion, differences in oocyte developmental capacity after IVM or in vivo maturation are accompanied by significant changes in transcript abundance in oocytes and their surrounding CCs, meiotic rate, mitochondrial distribution and apoptotic index. Some of the genes investigated, such as Gja1, could be potential biomarkers for oocyte developmental competence in the rabbit model, helping improve in vitro culture systems in these species.

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