In vivo vs. in vitro models for studying the effects of elevated temperature on the GV-stage oocyte, subsequent developmental competence and gene expression

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.

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

Reproduction Fertility and Development ◽

10.1071/rd15553 ◽

2017 ◽

Vol 29 (9) ◽

pp. 1667 ◽

Cited By ~ 10

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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3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab3 ◽

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.

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6 GENE EXPRESSION ANALYSIS OF IN VIVO- AND IN VITRO-MATURED PORCINE METAPHASE II OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab6 ◽

2014 ◽

Vol 26 (1) ◽

pp. 117 ◽

Cited By ~ 2

Author(s):

L. Cox ◽

G. Saunders ◽

J. Stevens ◽

S. C. Isom

Keyword(s):

Gene Expression ◽

Rna Degradation ◽

Functional Gene ◽

Gene Set Enrichment Analysis ◽

Developmental Competence ◽

Mrna Levels ◽

Multiple Testing Correction ◽

Metaphase Ii Oocytes

In vitro-matured (IVM) oocytes lack the same developmental competence as oocytes that are matured in vivo (IVV), yet no compelling explanation for this discrepancy has been provided at the molecular level. The aim of this study was to quantify and compare mRNA levels in IVM and IVV oocytes for genes from a wide variety of functional gene categories, including RNA degradation, pluripotency, epigenome modification, oocyte-specific, and apoptosis. Quantitative real-time PCR (qPCR) was used to evaluate the relative gene expression levels of 70 genes in each of 33 individual IVM oocytes from 4 different collection days and 29 individual IVV oocytes from 4 different donor animals. The qPCR data were analysed using ANOVA and significance was assigned at P < 0.05. After a multiple testing correction was applied, relative transcript abundances for 32 of the 70 genes tested were found to be significantly different (q < 0.05) between the IVM and IVV oocytes. Of these significantly different genes, 23 were higher in the IVM oocytes and only 9 were higher in the IVV oocytes. The 32 significantly differentially expressed genes were then evaluated in relation to their corresponding functional gene categories. Of particular interest, transcripts for 7/14 RNA degradation-related genes (CNOT3, DCP1A, DDX6, LSM1, PABPN1, PABPN1L, PARN) and 3/9 oocyte specific genes (BMP15, YBX2, H1FOO) were significantly more abundant in the IVM oocytes. In contrast, transcripts for 4/8 epigenetic related transcripts (ASH2l, DNMT1, EHMT2, EZH2), 2/2 apoptosis related genes (BCL2, XIAP), and 1/4 pluripotency factors (LIN28) were significantly more abundant in the IVV oocytes. Gene set enrichment analysis confirmed that, within the context of this experimental design, RNA degradation and chromatin remodelling pathways are significantly perturbed in IVM oocytes. We conclude that in vitro maturation has profound effects on transcript populations of metaphase-II oocytes, with most transcripts being higher in IVM oocytes. We expect that this data will lead to a better understanding of how we can improve the quality of oocytes that are matured in vitro as well as provide information to help to identify markers that could be indicative of oocyte quality.

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Abstract P141: Cardiac TRH Partly Mediates Angiotensin II-induced Fibrotic and Hypertrophy Effects in "in vivo" and "in vitro" Models

Hypertension ◽

10.1161/hyp.66.suppl_1.p141 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Silvia I García ◽

Ludmila S Peres Diaz ◽

Maia Aisicovich ◽

Mariano L Schuman ◽

María S Landa

Keyword(s):

Gene Expression ◽

Body Weight ◽

Cardiac Hypertrophy ◽

Structural Changes ◽

In Vitro Models ◽

Heart Weight ◽

Saline Control ◽

Control Group

Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p<0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p<0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p<0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p<0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.

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Cyclosporine A treated in vitro models induce cholestasis response through comparison of phenotype-directed gene expression analysis of in vivo Cyclosporine A-induced cholestasis

Toxicology Letters ◽

10.1016/j.toxlet.2013.06.236 ◽

2013 ◽

Vol 221 (3) ◽

pp. 225-236 ◽

Cited By ~ 11

Author(s):

Anne S. Kienhuis ◽

Alexa P. Vitins ◽

Jeroen L.A. Pennings ◽

Tessa E. Pronk ◽

Ewoud N. Speksnijder ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Cyclosporine A ◽

Gene Expression Analysis ◽

In Vitro Models

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

Zygote ◽

10.1017/s0967199413000658 ◽

2014 ◽

Vol 23 (3) ◽

pp. 367-377 ◽

Cited By ~ 14

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.

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Gene expression patterns of the liver in response to alcohol: In vivo and in vitro models compared

Experimental and Molecular Pathology ◽

10.1016/j.yexmp.2005.12.006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 241-251 ◽

Cited By ~ 28

Author(s):

Fawzia Bardag-Gorce ◽

Barbara A. French ◽

Jennifer Dedes ◽

Jun Li ◽

Samuel W. French

Keyword(s):

Gene Expression ◽

Expression Patterns ◽

In Vitro Models ◽

Gene Expression Patterns

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Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation

Reproduction ◽

10.1530/rep-16-0426 ◽

2017 ◽

Vol 153 (3) ◽

pp. R109-R120 ◽

Cited By ~ 16

Author(s):

Hannah M Brown ◽

Kylie R Dunning ◽

Melanie Sutton-McDowall ◽

Robert B Gilchrist ◽

Jeremy G Thompson ◽

...

Keyword(s):

Gene Expression ◽

Cell Metabolism ◽

Cumulus Cell ◽

Cumulus Cells ◽

Developmental Competence ◽

Oocyte Competence ◽

Oocyte Developmental Competence ◽

Growth Factor Signalling

In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

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

Reproduction ◽

10.1530/rep-10-0476 ◽

2011 ◽

Vol 142 (4) ◽

pp. 551-564 ◽

Cited By ~ 48

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.

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