A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with <i>in vitro</i> developmental outcome

Biase, Fernando H.; Martelli, Lúcia Regina; Merighe, Giovana Krempel Fonseca; Biase, W. Karyna Freitas Santos; Miranda, M. S.; Smith, Lawrence C.; Meirelles, Flávio Vieira

doi:10.1017/s0967199409005395

Cited by 6 publications

(8 citation statements)

References 25 publications

(24 reference statements)

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“…RT-qPCR is a frequently used technology for specific gene expression evaluation and comparative analysis of stable RGs determined for each specific cell type increases reliability (Vandesompele et al, 2002;Radonic et al, 2004;Pfaffl et al, 2004;Andersen et al, 2004). ACTB, GAPDH, H2AFZ, H2A, HPRT1, PPIA, and RNA18S are the most common genes used in such studies involving the bovine model (Patel et al, 2007;Biase et al, 2009;Macabelli et al, 2014). In other mammals, especially human, sheep, and mouse, different reference genes are more common, such as TBP, GUSB, and B2M (Ragni et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

Caetano¹,

Furtado²,

Batista³

et al. 2019

Anim Reprod

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Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.

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Section: Discussionmentioning

confidence: 99%

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

Caetano¹,

Furtado²,

Batista³

et al. 2019

Anim Reprod

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“…At the end of G1, cells are committed to division and lose the ability to respond to external factors. The nonacquisition of cytoplasmic competence during oocyte maturation may result in a longer G2 phase, which will result in a greater sensitivity to external factors and may explain the intense embryo blockage that occurs in this embryonic stage (Biase et al, 2009;Lonergan et al, 2003). Unlike the IVF 8-16C embryos, all the transcripts were downregulated in the PA group, suggesting a failure in the mechanism of EGA, at least in relation to the events related to cell cycle.…”

Section: Discussionmentioning

confidence: 98%

The Mechanism of Oocyte Activation Influences the Cell Cycle–Related Genes Expression During Bovine Preimplantation Development

Milazzotto

Feitosa

Paula-Lopes

et al. 2012

Cellular Reprogramming

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The first cleavage divisions and preimplantation embryonic development are supported by mRNA and proteins synthesized and stored during oogenesis. Thus, mRNA molecules of maternal origin decrease and embryonic development becomes gradually dependent on expression of genetic information derived from the embryonic genome. However, it is still unclear what the role of the sperm cell is during this phase and whether the absence of the sperm cell during the artificial oocyte activation affects subsequent embryonic development. The objective of this study was to determine, in bovine embryos, changes in cell cycle-associated transcript levels (cyclin A, cyclin B, cyclin E, CDC2, CDK2, and CDK4) after oocyte activation in the presence or absence of the sperm cell. To evaluate that, in vitro-produced (IVP) and parthenogenetically activated (PA) embryos (2-4 cells (2-4C), 8-16 cells (8-16C) and blastocysts) were evaluated by real-time PCR. There was no difference in cleavage and blastocyst rates between IVP and PA groups. Transcript level was higher in oocytes than in IVP and PA embryos. Cleaved PA embryos showed higher expression of cyclin A, cyclin B, cyclin E, and CDK2 and lower expression of CDC2 when compared with that from the IVP group. At the time of activation, all transcripts were expressed less in PA than in IVP embryos, whereas at the blastocyst stage, almost all genes were expressed at a higher level in the PA group. These results suggest that in both groups there is an initial consumption of these transcripts in the early stages of embryonic development. Furthermore, 8-16C embryos seem to synthesize more cell cycle-related genes than 2-4C embryos. However, in PA embryos, activation of the cell cycle genes seems to occur after the 8-to 16-cell stage, suggesting a failure in the activation process.

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“…The chemicals used for in vitro culture were purchased from Sigma-Aldrich Co. (Oakville, Ontario, Canada) unless otherwise stated. Oocyte collection and biopsy were conducted as described elsewhere (Biase et al ., 2009). Briefly, bovine cumulus–oocyte complexes (COCs) were aspirated from antral follicles of 3–8 mm in diameter.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we proposed a new experimental model that collected a fraction of the ooplasm for experimental analysis, allowing the remaining of the oocyte to be chemically activated and cultured in vitro. The developmental competence of the oocytes is then classified based on actual embryo development (Biase et al ., 2009). This experimental model has been used to demonstrate that the total amount of the polyadenylated RNA present in MII oocytes does not correlate with developmental competence (Biase et al ., 2009).…”

Section: Introductionmentioning

confidence: 99%

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Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage

Biase

Everts

Oliveira

et al. 2012

Zygote

Self Cite

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The mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8-16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: 'RNA processing', 'translation' and 'mRNA metabolic process'. Genes that are important to the molecular functions of 'RNA binding' and 'translation factor activity, RNA binding' were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

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A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

Cited by 6 publications

References 25 publications

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

The Mechanism of Oocyte Activation Influences the Cell Cycle–Related Genes Expression During Bovine Preimplantation Development

Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage

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