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Koltunow A.M.G.,CSIRO | Johnson S.D.,CSIRO | Rodrigues J.C.M.,CSIRO | Rodrigues J.C.M.,Embrapa Genetic Research and Biotechnology | And 10 more authors.
Plant Journal | Year: 2011

Asexual seed formation, or apomixis, in the Hieracium subgenus Pilosella is controlled by two dominant independent genetic loci, LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP). We examined apomixis mutants that had lost function in one or both loci to establish their developmental roles during seed formation. In apomicts, sexual reproduction is initiated first. Somatic aposporous initial (AI) cells differentiate near meiotic cells, and the sexual pathway is terminated as AI cells undergo mitotic embryo sac formation. Seed initiation is fertilization-independent. Using a partially penetrant cytotoxic reporter to inhibit meioisis, we showed that developmental events leading to the completion of meiotic tetrad formation are required for AI cell formation. Sexual initiation may therefore stimulate activity of the LOA locus, which was found to be required for AI cell formation and subsequent suppression of the sexual pathway. AI cells undergo nuclear division to form embryo sacs, in which LOP functions gametophytically to stimulate fertilization-independent embryo and endosperm formation. Loss of function in either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in total reversion to sexual reproduction. Therefore, in these apomicts, sexual reproduction is the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode genes essential for sexual reproduction, but may function to recruit the sexual machinery at specific time points to enable apomixis. © 2011 Blackwell Publishing Ltd.

Franco M.M.,Embrapa Genetic Research and Biotechnology | Fagundes N.S.,Federal University of Uberlandia | Michalczechen-Lacerda V.A.,University of Brasilia | Caixeta E.S.,University of Jose do Rosario Vellano | And 4 more authors.
Journal of Assisted Reproduction and Genetics | Year: 2014

Purpose: The aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1-3 mm and ≥8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern. Methods: Genomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised. Results: There were no differences in the methylation pattern among groups (P>0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17±14.11 % and 82.93±5.86 %, respectively, and those from large follicles showed methylation levels of 81.81±10.40 % and 79.64±13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17±12.01 % and 81.19±10.15 %. Conclusions: In conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis, and to be a non-invasive method for determining oocyte quality, the identification of new epigenetic markers in cumulus cells has great potential to be used to support reproductive biotechniques in humans and other mammals. © 2013 Springer Science+Business Media.

Filho E.S.,Embrapa Genetic Research and Biotechnology | Filho E.S.,University of Brasilia | Caixeta E.S.,University of Brasilia | Pribenszky C.,Szent Istvan University | And 5 more authors.
Reproduction, Fertility and Development | Year: 2011

Sublethal stress treatment has been reported to enhance gametes' performance in subsequent procedures, such as cryopreservation. The aim of the present study was to evaluate the effect of different equilibration times between the termination of a sublethal hydrostatic pressure (HP) stress treatment and the initiation of vitrification on the post-thaw survival, continued in vitro development, hatching rate and gene expression of selected candidate genes of in vitro-produced (IVP) expanded bovine blastocysts. Day 7 IVP blastocysts were subjected to 600 bar pressure for 60min at 32°C. Immediately after pressure treatment (HP0h) or after 1 or 2h incubation (HP1h and HP2h groups, respectively), embryos were either vitrified and warmed using the open pulled straw method, followed by 72h in vitro culture or were stored at -80°C until gene expression analysis. Re-expansion and hatching rates after vitrificationwarming were significantly (P<0.05) higher in the HP0h (88 and 76%, respectively) and HP1h (90 and 75%, respectively) groups than in the untreated (82 and 63%, respectively) and HP2h groups (79 and 70%, respectively). Moreover, the HP1h group showed further improvement in the speed of re-expansion and resumption of normal in vitro development. Cumulative analysis of all genes (SC4MOL, HSP1A1A, SOD2 and GPX4) revealed a similar pattern of expression, with a tendency for peak transcript abundance 1h after HP treatment. Application of HP stress treatment was found to be efficient in increasing the in vitro developmental competence of vitrified bovine embryos. © 2011 CSIRO.

Fagundes N.S.,Federal University of Uberlandia | Fagundes N.S.,Embrapa Genetic Research and Biotechnology | Michalczechen-Lacerda V.A.,Embrapa Genetic Research and Biotechnology | Michalczechen-Lacerda V.A.,University of Brasilia | And 12 more authors.
Molecular Human Reproduction | Year: 2011

Oocyte quality is one of the most important aspects of in vitro embryo development. Extensive epigenetic programming must occur during oocyte growth and maturation. A specific DNA methylation pattern of the imprinted genes must be established on differentially methylated regions (DMR). The insulin-like growth factor 2 (IGF2) gene is an important growth factor, and it is imprinted in several mammalian species. The aim of this study was to evaluate the methylation pattern on the DMR of the last exon of IGF2 in immature and mature bovine oocytes with different developmental competencies. Mature oocytes from large follicles were less methylated (28.93%) than immature oocytes from large follicles (77.38% P = 0.002), and there was also a tendency towards lower methylation in mature oocytes from large follicles (28.93%) compared with mature oocytes from small follicles (52.58% P = 0.07). Immature oocytes from small and large follicles showed 53.85% (7/13) and 91.66% (11/12) hypermethylated sequences, respectively, whereas mature oocytes from small and large follicles showed 61.11% (11/18) and 40% (4/10), respectively. The hypomethylation pattern in mature oocytes from large follicles may be related to the higher competence of these oocytes. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a molecular marker for oocyte competence in cattle and as a model for studies in other species. © The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

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