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.

scholarly journals Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 367-377 ◽  
Author(s):  
Sandra Milena Bernal ◽  
Julia Heinzmann ◽  
Doris Herrmann ◽  
Bernd Timmermann ◽  
Ulrich Baulain ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Global Methylation ◽  
Oocyte Developmental Competence

SummaryCyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P<0.05). No statistical differences were found for blastocyst cell numbers. The mRNA expression for the EGR1 gene was down-regulated eight-fold in blastocysts that had been produced in vitro compared with their in vivo counterparts. Gene expression profiles for IGF2R, SLC2A8, COX2, DNMT3B and PCK2 did not differ among experimental groups. Bovine testis satellite I and Bos taurus alpha satellite methylation profiles from cAMP30aspiration protocol-derived blastocysts were similar to patterns that were observed in their in vivo equivalents (P > 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

scholarly journals Bovine blastocysts with developmental competence to term share similar expression of developmentally important genes although derived from different culture environments

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 551-564 ◽  
Author(s):  
N Ghanem ◽  
D Salilew-Wondim ◽  
A Gad ◽  
D Tesfaye ◽  
C Phatsara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Placental Development ◽  
Similar Expression ◽  
Maternal Interaction

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30–40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60–70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

scholarly journals Transcriptome analysis of gravitational effects on mouse skeletal muscles under microgravity and artificial 1 g onboard environment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Risa Okada ◽  
Shin-ichiro Fujita ◽  
Riku Suzuki ◽  
Takuto Hayashi ◽  
Hirona Tsubouchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Muscle Mass ◽  
Muscle Atrophy ◽  
Transcriptome Analysis ◽  
Fiber Type ◽  
Expression Profiles ◽  
Space Station ◽  
Gene Expression Profiles

AbstractSpaceflight causes a decrease in skeletal muscle mass and strength. We set two murine experimental groups in orbit for 35 days aboard the International Space Station, under artificial earth-gravity (artificial 1 g; AG) and microgravity (μg; MG), to investigate whether artificial 1 g exposure prevents muscle atrophy at the molecular level. Our main findings indicated that AG onboard environment prevented changes under microgravity in soleus muscle not only in muscle mass and fiber type composition but also in the alteration of gene expression profiles. In particular, transcriptome analysis suggested that AG condition could prevent the alterations of some atrophy-related genes. We further screened novel candidate genes to reveal the muscle atrophy mechanism from these gene expression profiles. We suggest the potential role of Cacng1 in the atrophy of myotubes using in vitro and in vivo gene transductions. This critical project may accelerate the elucidation of muscle atrophy mechanisms.

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.

Gene Expression Profiles in Rat Liver Slices Exposed to Hepatocarcinogenic Enzyme Inducers, Peroxisome Proliferators, and 17α-Ethinylestradiol

2006 ◽  
Vol 25 (5) ◽  
pp. 379-395 ◽  
Author(s):  
Gisela Werle-Schneider ◽  
Andreas Wölfelschneider ◽  
Marie Charlotte von Brevern ◽  
Julia Scheel ◽  
Thorsten Storck ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
Mode Of Action ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Peroxisome Proliferators ◽  
Liver Slices ◽  
Enzyme Inducers

Transcription profiling is used as an in vivo method for predicting the mode-of-action class of nongenotoxic carcinogens. To set up a reliable in vitro short-term test system DNA microarray technology was combined with rat liver slices. Seven compounds known to act as tumor promoters were selected, which included the enzyme inducers phenobarbital, α-hexachlorocyclohexane, and cyproterone acetate; the peroxisome proliferators WY-14,643, dehydroepiandrosterone, and ciprofibrate; and the hormone 17 α-ethinylestradiol. Rat liver slices were exposed to various concentrations of the compounds for 24 h. Toxicology-focused TOXaminer™ DNA microarrays containing approximately 1500 genes were used for generating gene expression profiles for each of the test compound. Hierarchical cluster analysis revealed that (i) gene expression profiles generated in rat liver slices in vitro were specific allowing classification of compounds with similar mode of action and (ii) expression profiles of rat liver slices exposed in vitro correlate with those induced after in vivo treatment (reported previously). Enzyme inducers and peroxisome proliferators formed two separate clusters, confirming that they act through different mechanisms. Expression profiles of the hormone 17 α-ethinylestradiol were not similar to any of the other compounds. In conclusion, gene expression profiles induced by compounds that act via similar mechanisms showed common effects on transcription upon treatment in vivo and in rat liver slices in vitro.

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

scholarly journals Rapid default transition of CD4 T cell effectors to functional memory cells

2007 ◽  
Vol 204 (9) ◽  
pp. 2199-2211 ◽  
Author(s):  
K. Kai McKinstry ◽  
Susanne Golech ◽  
Won-Ha Lee ◽  
Gail Huston ◽  
Nan-Ping Weng ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cd4 T Cell ◽  
Effector Memory ◽  
Memory Cells ◽  
Two Stages

The majority of highly activated CD4 T cell effectors die after antigen clearance, but a small number revert to a resting state, becoming memory cells with unique functional attributes. It is currently unclear when after antigen clearance effectors return to rest and acquire important memory properties. We follow well-defined cohorts of CD4 T cells through the effector-to-memory transition by analyzing phenotype, important functional properties, and gene expression profiles. We find that the transition from effector to memory is rapid in that effectors rested for only 3 d closely resemble canonical memory cells rested for 60 d or longer in the absence of antigen. This is true for both Th1 and Th2 lineages, and occurs whether CD4 T cell effectors rest in vivo or in vitro, suggesting a default pathway. We find that the effector–memory transition at the level of gene expression occurs in two stages: a rapid loss of expression of a myriad of effector-associated genes, and a more gradual gain of expression of a cohort of genes uniquely associated with memory cells rested for extended periods.

Differential gene-expression profiles from canine cumulus cells of ovulated versus in vitro-matured oocytes

2016 ◽  
Vol 28 (3) ◽  
pp. 278 ◽  
Author(s):  
Su-Jin Cho ◽  
Kyeong-Lim Lee ◽  
Yu-Gon Kim ◽  
Dong-Hoon Kim ◽  
Jae-Gyu Yoo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Differential Gene ◽  
Monitoring Gene Expression ◽  
Follicle Aspiration

We compared the nuclear maturation status and gene-expression profiles of canine cumulus cells (CCs) derived from cumulus–oocyte complexes (COCs) that were spontaneously ovulated versus those that were matured in vitro. Cumulus–oocyte complexes were retrieved from uteri by surgical flushing (after spontaneous ovulation) or by ovariectomy follicle aspiration and in vitro maturation. The objective of Experiment 1 was to investigate the nuclear maturation status of in vivo- versus in vitro-matured oocytes. The objective of Experiment 2 was to compare gene-expression profiles of CCs derived from in vivo- versus in vitro-matured COCs. Genes analysed are related to cell maturation, development and apoptosis, including GDF9, MAPK1, PTX3, CX43, Bcl2 and BAX; mRNA expression for all of these genes, except for GDF9, differed (P < 0.05) between in vivo- and in vitro-matured CCs. In conclusion, we found that gene-expression profiles are related to the quality of CCs and therefore posit that monitoring gene expression could be a useful strategy to guide attempts to improve in vitro culture systems.

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