Gene expression analysis and in vitro production procedures for bovine preimplantation embryos: Past highlights, present concepts and future prospects

2018 ◽  
Vol 53 ◽  
pp. 14-19 ◽  
Author(s):  
Christine Wrenzycki
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Preimplantation Embryos ◽  
Future Prospects ◽  
In Vitro Production ◽  
Vitro Production

175 Nobiletin enhances the quality of in vitro-matured bovine oocytes and blastocysts by altering the transcription of key developmental genes

2019 ◽  
Vol 31 (1) ◽  
pp. 212
Author(s):  
Y. N. Cajas ◽  
K. Cañón-Beltrán ◽  
M. E. González ◽  
P. Ramos-Ibeas ◽  
A. Gutierrez-Adán ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Statistical Significance ◽  
Housekeeping Genes ◽  
Cumulus Cells ◽  
Cell Junction ◽  
In Vitro Production

One of the problems associated with in vitro production of embryos in bovine is the increase in reactive oxygen species (ROS), which leads to cell alterations and death. Nobiletin is a polymethoxyflavone isolated from citrus fruits with various beneficial effects on cell cycle regulation and inhibition of ROS production. In a preliminary study, we demonstrated that supplementation of 25 or 50 µM nobiletin to the in vitro maturation (IVM) medium reduces oxidative stress and improves oocyte nuclear and cytoplasmic maturation and embryo development. Thus, in this study, we aimed to evaluate the antioxidant activity of nobiletin during IVM on bovine matured oocytes, their cumulus cells (CC), and blastocysts by quantitative changes of gene expression. Immature cumulus oocytes complexes (COC) were aspirated from ovaries of slaughtered heifers. Selected COC underwent IVM in TCM-199+10% FCS and 10ng mL−1 epidermal growth factor (EGF; Control) supplemented with 25 µM (Nob25) or 50 µM (Nob50) nobiletin (MedChemExpress, Monmouth Junction, NJ, USA) or 0.001% dimethyl sulfoxide (DMSO control), a vehicle for nobiletin dilution, in 5% CO2 in air at 38.5°C. After 24h, 50 matured oocytes/group and their CC were snap-frozen in LN2 for gene expression analysis. The remaining oocytes were fertilized (Day 0) and cultured in vitro. Blastocysts (Day 7; n=50/group) were snap-frozen in LN2 for gene expression analysis (5 replicates). The mRNA abundance of candidate genes related with oxidative stress (SOD2, CYP51); apoptosis (BAX); quality (BMP15, BMP7, CLIC1, MAPK1, ABCB1); and cell junction (GJA1) was measured by quantitative PCR; H2AFZ and 18S rRNA were used as housekeeping genes. Statistical significance was assessed by one-way ANOVA. Supplementation of IVM medium with Nob25 or Nob50 produced changes in the expression levels of genes related to oxidative stress and apoptosis during IVM compared with controls. SOD2 and CYP51 were down-regulated in oocytes and CC (P<0.05) but not in blastocysts, whereas BAX was down-regulated only in CC (P<0.05). Nobiletin supplementation in IVM increased the expression of MAPK1 in oocytes and blastocysts (P<0.05); however, no differences were observed in CC. BMP15 for oocytes and their CC and GJA1 for CC were up-regulated in Nob25 and Nob50 groups compared with controls (P<0.05). The relative abundance of CLIC1 decreased in blastocysts from both nobiletin groups compared with controls (P<0.05). No significant differences in the expression in ABCB1 and BMP7 were detected. In conclusion, our results suggest that supplementation of 25 or 50 µM nobiletin to the IVM medium reduces oxidative stress in oocytes and CC, decreases CC apoptosis, and provokes positive changes in the expression of genes related to oocyte and embryo quality. This research was supported by Spanish MINECO (AGL2015-70140-R and AGL2015-66145-R). Y. N. Cajas was supported by a grant from SENESCYT-Ecuador.

Embryonic gene expression profiling using microarray analysis

2009 ◽  
Vol 21 (1) ◽  
pp. 22 ◽  
Author(s):  
Sadie L. Marjani ◽  
Daniel Le Bourhis ◽  
Xavier Vignon ◽  
Yvan Heyman ◽  
Robin E. Everts ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiling ◽  
Nuclear Transfer ◽  
Lower Cost ◽  
Preimplantation Embryos ◽  
Microarray Technology ◽  
In Vitro Production ◽  
Vitro Production ◽  
Level Of Analysis

Microarray technology enables the interrogation of thousands of genes at one time and therefore a systems level of analysis. Recent advances in the amplification of RNA, genome sequencing and annotation, and the lower cost of developing microarrays or purchasing them commercially, have facilitated the analysis of single preimplantation embryos. The present review discusses the components of embryonic expression profiling and examines current research that has used microarrays to study the effects of in vitro production and nuclear transfer.

212 INTERSPECIFIC CLONING AND EMBRYO AGGREGATION INFLUENCE THE EXPRESSION OF oct4, nanog, sox2, AND cdx2 IN CHEETAH AND DOMESTIC CAT BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 196
Author(s):  
L. N. Moro ◽  
D. Veraguas ◽  
L. Rodriguez-Alvarez ◽  
M. I. Hiriart ◽  
C. Buemo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Internal Control ◽  
Gene Expression Analysis ◽  
Early Embryo ◽  
Domestic Cat ◽  
Nuclear Reprogramming ◽  
Acinonyx Jubatus ◽  
Standard Curve

The cheetah (Ch, Acinonyx jubatus) is a species considered globally endangered and cloning is one of the assisted reproductive techniques that can help to preserve it and to study early embryo development. However, the production of cloned felid embryos remains inefficient, probably because of the difficulty to control the process of nuclear reprogramming and obtain adequate gene expression. Embryo aggregation has been demonstrated to improve the cloning efficiency in several species and to normalise cdx2 in the mouse by lowering its expression (Balbach et al. 2010), but it has not been evaluated in felids before. To better understand the effect of interspecific somatic-cell nuclear transfer (iSCNT) and embryo aggregation in nuclear reprogramming, we analysed the expression of oct4, sox2, nanog, and cdx2 in cheetah blastocysts generated by iSCNT, domestic cat blastocysts (Dc) generated by SCNT, and IVF blastocysts as control. To achieve this, domestic cat oocytes were in vitro matured and zona-free SCNT or iSCNT was performed, as previously described (Moro et al. 2014, Reprod. Fertil. Dev.). Zona-free reconstructed embryos were then cultured individually (1X) or two embryo were cultured together (2X) in microwells, in synthetic oviductal fluid (SOF) medium. The experimental groups were Dc1X, Dc2X, Ch1X, Ch2X, and IVF. After 8 days of in vitro culture the blastocysts obtained were stored in RNA-later at –20°C. For gene expression analysis, blastocysts were pooled as follows: Dc1X, 4 replicates of 3 blastocysts each; Dc2X, 4 replicates of 3 blastocysts each; Ch1X, 2 replicates of 2 blastocysts and 1 replicate of 1 blastocyst; Ch2X, 4 replicates of 3 blastocysts each; IVF 3 replicates of 3 blastocysts each. Embryos were treated with a Cells-to-cDNA TM II kit (Life Technologies, Carlsbad, CA, USA) lyses buffer and treated with DNase I (0.04 U μL–1) for genomic DNA digestion. Gene expression analysis was performed by real-time qPCR using the standard curve method. In all qPCRs, GAPDH was used as an internal control. The statistical analysis was performed using a non-parametric Kruskal–Wallis test (P < 0.05). We observed that Dc1X blastocysts overexpressed the 4 genes evaluated respect to the IVF control. However, the gene expression of the aggregated group (Dc2X) was lower for all the genes, achieving the same levels of nanog and sox2 as the IVF blastocysts. The expression of oct4 and cdx2 were also closer to the expression levels of the control in the Dc2X group than in the Dc1X group. With respect to interspecific embryos, the amount of oct4 and cdx2 was also significantly reduced in the Ch2X blastocysts respect to Ch1X blastocysts. Both cheetah groups showed significantly lower expression of oct4, cdx2, and nanog than the IVF control. In conclusion, transcription of pluripotent and early differentiation factors in cheetah embryos was not as efficient as in the domestic cat embryos, probably caused by interspecific transfer. Our study demonstrated for the first time that defects in gene expression of domestic cat embryos can be corrected by embryo aggregation, providing a simple strategy to improve felid cloning.

375 Gene expression analysis of galactosamine-induced hepatotoxicity in-vivo and in-vitro

2003 ◽  
Vol 144 ◽  
pp. s102
Author(s):  
H. Hildebrand ◽  
G. Kempka ◽  
H. Ellinger ◽  
B. Stuart ◽  
B. Wahle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis

Global Gene Expression Analysis Demonstrates that Transforming Growth Factor β1 (TGF-β1) Signals Exclusively through Receptor Complexes Involving TGF-β Receptor I and Identifies Numerous Targets of TGF-β Signaling.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2178-2178
Author(s):  
Goran Karlsson ◽  
Yingchun Liu ◽  
Marie-José Goumans ◽  
Jonas Larsson ◽  
Ju-Seog Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Target Genes ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Global Gene Expression Analysis ◽  
Β Receptor ◽  
Tgf Β1

Abstract In the hematopoietic system, TGF-β1 is one of the most potent extrinsic regulators, affecting both early progenitors and committed cells. At the top of the hematopoietic hierarchy, TGF-β1 maintains hematopoietic stem cells (HSCs) in quiescence in vitro through transcriptional regulation of genes encoding proteins important in the cell cycle. We have shown that TGF-β receptor I (TβRI) −/− HSCs exhibit increased proliferative capacity in vitro and that TβRII−/− mice develop a multifocal autoimmune disease, mainly mediated by T-cells (Larsson et al, 2003, Levéen et al 2002). The mechanisms of TGF-β signaling in hematopoietic cells are poorly understood and many target genes of TGF-β signaling remain elusive. In this study we have used global gene expression analysis to investigate whether all TGF-β signaling is mediated by TβRI and II. Furthermore, we asked what target genes are affected upon TGF-β stimulation in normal and TGF-β signaling deficient murine embryonic fibroblasts (MEFs). MEFs were grown with and without TGF-β1 stimulation and proliferation, transcriptional responses and expression analysis were performed. We demonstrate through Western Blot analysis, luciferase reporter assays and cell expansion experiments how these cells lack functional TβRI. Additionally, transcriptional assays show that no other Smad activity is triggered by TGF-β1 stimulation. Furthermore, we demonstrate through quantitative RT-PCR that the inhibitor of differentiation family of genes, known targets of TGF-β signaling, are not affected by TGF-β1 in TβRI−/− MEFs, while wt cells downregulate these genes 4–8.5 fold in response to stimulation. In order to completely exclude alternative receptors outside the TGF-β superfamily and signaling pathways activated through TβRII alone, we performed global gene expression profiling on TGF-β1 stimulated TβRI−/− MEFs with unstimulated TβRI deficient cells as reference. Very few (0.05 %) of the more than 37,000 spots on the microarray had a >2 fold differential expression in the two experiments conducted. Similar experiments performed on wt cells resulted in differential expression of between 2.6–3.9 % of the genes printed. From this data we conclude that no signaling affecting gene expression occur in the absence of TβRI in these cells. Additionally we present transcriptional profiles of MEF cell lines that either are normal or are TβRI deficient. By means of cDNA microarray technology, we have identified genes that were differentially expressed when TβRI deficient fibroblasts were compared to wt cells stimulated with TGF-β1. Our results create a data base of 461 significantly differentially expressed (p<0.01) target genes of TGF-β signaling. These include genes potentially responsible for the growth arrest induced by TGF-β1, like Gadd45g, Gas5, Id1, Id2 and Id3. However, the most significantly enriched number of differentially expressed genes are involved in protein folding and chaperone activities (Hspa9a, Hsp105, Hspe1, Hsp60, Cct2, Cct3, Cct8, Tcp1 and Dnaja1. Studies to identify TGF-β signaling responsive genes in HSCs are in progress.

In vitro gene expression analysis of nephrotoxic drugs in rat primary renal cortical tubular cells

2008 ◽  
Vol 28 (2) ◽  
pp. 237-248 ◽  
Author(s):  
Hiromi Suzuki ◽  
Tomoaki Inoue ◽  
Tomochika Matsushita ◽  
Kazuko Kobayashi ◽  
Ikuo Horii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Tubular Cells ◽  
Nephrotoxic Drugs

TRAC gene expression analysis in in vitro drug metabolism screening

2010 ◽  
Vol 196 ◽  
pp. S145-S146
Author(s):  
J. Rautio ◽  
J. Kotimaa ◽  
M. Sirkiä
Keyword(s):  
Gene Expression ◽  
Drug Metabolism ◽  
Expression Analysis ◽  
Gene Expression Analysis
Sign in / Sign up

Export Citation Format

Share Document