137 DIFFERENTIAL GENE EXPRESSION OF IN VITRO-MATURED BOVINE OOCYTES WITH OR WITHOUT A POLAR BODY
| Autor: | S. Wohlres-Viana, Luiz Sérgio de Almeida Camargo, J. H. M. Viana, N. C. Rabelo, M. F. M. Guimarães, C. C. R. Quintão, L. T. Iguma, A. J. R. Camargo, Bruno Campos de Carvalho, M. M. Pereira, J. N. S. Sales |
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| Rok vydání: | 2012 |
| Předmět: |
medicine.medical_specialty
Theriogenology Reproductive technology Biology Oocyte Oogenesis Molecular biology In vitro maturation Polar body Endocrinology medicine.anatomical_structure Reproductive Medicine Complementary DNA Gene expression Immunology Genetics medicine Animal Science and Zoology Molecular Biology Developmental Biology Biotechnology |
| Zdroj: | Reproduction, Fertility and Development. 24:181 |
| ISSN: | 1031-3613 |
| Popis: | Oocyte competence is associated with the amount of transcripts stored in the ooplasm and oocyte ability to extrude polar bodies (PB). To our knowledge, however, no data comparing mRNA levels between bovine oocytes maturated in vitro with or without PB are available. The aim of the present study was to compare the relative abundance of transcripts of glucose transporter 1 (GLUT1), insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor (IGF2R), growth differentiation factor-9 (GDF9) and aquaporin 3 (AQP3) genes between oocytes with and without PB (PB and NPB groups, respectively) following in vitro maturation. Immature bovine oocytes were obtained by follicular aspiration and matured in TCM-199 (Gibco Life Technologies, New York, NY, USA) containing 10% of oestrus cow serum and 20 μg mL–1 of FSH (Pluset, Serono, Italy) for 24 h under 5% CO2 in air at 38.5°C. Subsequently, oocytes were visually classified according to the presence or absence of PB and then denuded and rapidly frozen in liquid nitrogen. Three pools of 10 oocytes for each group were subjected to total RNA extraction using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions and treated with DNase. Reverse transcription and cDNA amplification were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (WTA2, Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Relative abundance of the target transcripts was performed by quantitative RT-PCR (Applied Biosystems Prism 7300 Sequence Detection Systems, Foster City, CA, USA) using a mixture of SYBR® Green PCR Master Mix (Applied Biosystems), 200 ng of cDNA, nuclease-free water and specific primers for each reaction. Expression of the β-actin gene was used as an endogenous reference. Relative gene expression analysis was performed using the software REST© 2005 using the Pair Wise Fixed Reallocation Randomization Test©. The relative expression values are presented as mean ± standard error. The relative abundance of GLUT1 (0.81 ± 0.07), IGF2R (0.72 ± 0.07) and GDF9 (0.82 ± 0.10) genes was lower (P 0.05) in relative abundance between PB and NPB groups for the other genes. The results suggest that the amount of some transcripts stored in the matured ooplasm is associated with the presence of PB. The authors acknowledge FAPEMIG, CNPq and Innovation Network Project on Animal Reproduction (01.07.01.002). |
| Databáze: | OpenAIRE |
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