Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Nganvongpanit, Korakot; Müller, Heike; Rings, F.; Hoelker, M.; Jennen, Danyel; Tholen, Ernst; Havlíček, V.; Besenfelder, U.; Schellander, K.; Tesfaye, Dawit

doi:10.1530/rep.1.01040

Cited by 44 publications

(37 citation statements)

References 68 publications

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“…Low expression of CX43 in blastocysts has been associated with low quality and reduced survival after cryopreservation [47,48]. E-CADHERIN intervenes in cell to cell adhesion as it is associated with compaction [49,50] and blastocyst formation [51]. In our study, the levels of E-CAD-HERIN did not differ between the IVF blastocysts and parthenotes, suggesting that the E-CADHERIN contribution to the compaction process and blastocyst formation is not altered in parthenotes.…”

Section: Gene Expression In Parthenotesmentioning

confidence: 46%

Gene Expression in Early Expanded Parthenogenetic and In Vitro Fertilized Bovine Blastocysts

Gómez

Caamaño

Bermejo-Álvarez³

et al. 2009

Journal of Reproduction and Development

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Abstract. Mammalian oocytes can undergo artificial parthenogenesis in vitro and develop to the blastocyst stage. In this study, using real-time PCR, we analyzed the expression of genes representative of essential events in development. In vitro matured oocytes were either fertilized or activated with ionomycin + 6-DMAP and cultured in simple medium. The pluripotency-related gene Oct3/4 was downregulated in parthenotes, while the de novo methylation DNMT3A gene was unchanged. Among the pregnancy recognition genes, IFN-t was upregulated, PGRMC1 was downregulated and PLAC8 was unchanged in parthenotes. Among the metabolism genes, SLC2A1 was downregulated, while AKR1B1, COX2, H6PD and TXN were upregulated in parthenotes; there was no difference in SLC2A5. Among the genes involved in compaction/blastulation, GJA1 expression increased in parthenotes, but no differences were detected within ATP1A1 and CDH1. Expression of p66 shc and the Bax/Bcl2 ratio were higher in parthenotes, and there was no difference in p53. Parthenotes and embryos may differ in the way they stimulate apoptosis, with a preponderant role for p66 shc within parthenotes. Differentially affected functions may also include pluripotency, de novo methylation and early embryonic signalling. Key words: Bovine, Embryo, Gene expression, Parthenote (J. Reprod. Dev. 55: [607][608][609][610][611][612][613][614] 2009) arthenogenesis, a form of reproduction not spontaneous in mammals, is a process by which the oocyte develops without the male gamete. Mammalian parthenotes are unable to develop to term in utero (developing for a maximum of 48 days in the cow; [1]), and this is thought to be due to alterations in their genomic imprinting, a mechanism that provides complementary, but not equivalent, specific expression between maternal and paternal genomes [2]. During fertilization, the spermatozoon triggers multiple and rhythmic oscillations of intracellular free calcium that release the oocyte from this blocked stage [3]. These intracellular calcium patterns can be mimicked by a number of chemicals that activate the oocyte without sperm, yielding parthenogenetic embryos [4]. In cattle, production of reconstructed embryos by somatic cell nuclear transfer (SCNT) [5] and intracytoplasmic sperm injection (ICSI) [6] depends on successful oocyte activation. Therefore, procedures involving SCNT and ICSI could benefit from a better knowledge of mechanisms that differ between embryos and parthenotes.Contrary to the research in mice and humans, most studies have failed to detect putatively imprinted genes in bovine early development [7,8], suggesting that monoallelic expression may not develop until after Day 21 of development, as occurs in ovine species [9]. In fact, a recent study found imprinted expression of bovine Mest-1 in Day-21 but not Day-14 embryos, while 7 other putatively imprinted genes were shown to be not imprinted at the two stages analyzed [10]. However, gene expression does differ between blastocysts from embryos and parthenotes in domestic species [11...

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Section: Gene Expression In Parthenotesmentioning

confidence: 46%

Gene Expression in Early Expanded Parthenogenetic and In Vitro Fertilized Bovine Blastocysts

Gómez

Caamaño

Bermejo-Álvarez³

et al. 2009

Journal of Reproduction and Development

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“…5) and only rare embryo-survivors further developed. Similarly, POU5F1 (Oct-4)-depleted embryos are able to develop until the morula stage without any significant differences in developmental competence, although the maternal mRNA can be detected in embryos before EGA and the first embryonic transcript in bovine can be detected at the morula stage (Nganvongpanit et al 2006a(Nganvongpanit et al , 2006b). In our previous study (Kanka et al 2009), we have shown that CENPF transcription from embryonic genome is activated during major genome activation, i.e.…”

Section: Discussionmentioning

confidence: 99%

Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage

Toralova¹,

Šušor²,

Němcová³

et al. 2009

Reproduction

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Identification of genes that are important for normal preimplantation development is essential for understanding the basics of early mammalian embryogenesis. In our previous study, we have shown that CENPF (mitosin) is differentially expressed during preimplantation development of bovine embryos. CENPF is a centromere-kinetochore complex protein that plays a crucial role in the cell division of somatic cells. To our best knowledge, no study has yet been done on either bovine model, or oocytes and preimplantation embryos. In this study, we focused on the fate of bovine embryos after injection of CENPF double-stranded RNA (dsRNA) into the zygotes. An average decrease of CENPF mRNA abundance by 94.9% or more and an extensive decline in immunofluorescence staining intensity was detected relative to controls. There was no disparity between individual groups in the developmental competence before the 8-cell stage. However, the developmental competence rapidly decreased then and only 28.1% of CENPF dsRNA injected 8-cell embryos were able to develop further (uninjected control: 71.8%; green fluorescent protein dsRNA injected control: 72.0%). In conclusion, these results show that depletion of CENPF mRNA in preimplantation bovine embryos leads to dramatic decrease of developmental competence after embryonic genome activation.

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“…In the pig, parthenotes showed increased blastocyst yields and cell numbers in coincidence with decreased TUNELpositive nuclei and BAX expression, while increasing BCL2L1 and POU5F1 expression (Choi et al 2008). Knockdown of POU5F1 mRNA causes a reduction in the ICM cells in bovine IVF embryos (Nganvongpanit et al 2006a), which could be a sign of diminished pluripotency. However, the ICM of our parthenotes showed no reduced cell counts and produces outgrowths (data not shown).…”

Section: Development and Gene Expression In Parthenotesmentioning

confidence: 99%

Biological differences between in vitro produced bovine embryos and parthenotes

Gómez¹,

Gutiérrez‐Adán²,

Díez³

et al. 2009

Reproduction

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Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes and in vitro produced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage. In vitro matured oocytes were either fertilized or activated with ionomycin C6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-related POU5F1 and the methylation DNMT3A genes were downregulated in parthenotes. Among pregnancy recognition genes, TP-1 was upregulated in parthenotes, while PGRMC1 and PLAC8 did not change. Expression of p66 shc and BAX/BCL2 ratio were higher, and p53 lower, in parthenotes. Among metabolism genes, SLC2A1 was downregulated, while AKR1B1, PTGS2, H6PD, and TXN were upregulated in parthenotes, and SLC2A5 did not differ. Among genes involved in compaction/blastulation, GJA1 was downregulated in parthenotes, but no differences were detected within ATP1A1 and CDH1. Within parthenotes, the expression levels of SLC2A1, TP-1, and H6PD, and possibly AKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, through p66 shc and p53respectively, and in their mechanisms to control pluripotency and de novo methylation.Reproduction (2009) 137 285-295

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Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Cited by 44 publications

References 68 publications

Gene Expression in Early Expanded Parthenogenetic and In Vitro Fertilized Bovine Blastocysts

Gene Expression in Early Expanded Parthenogenetic and In Vitro Fertilized Bovine Blastocysts

Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage

Biological differences between in vitro produced bovine embryos and parthenotes

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