Epigenetic reprogramming of somatic cells by zygotic factors
Sowry, B. G. (2009). Epigenetic reprogramming of somatic cells by zygotic factors (Thesis). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/3260
Permanent Research Commons link: https://hdl.handle.net/10289/3260
Cloning cattle using somatic cell nuclear transfer (SCNT) is an inefficient process, with approximately only 5% of transferred embryos developing to live offspring. SCNT produced cattle have a high mortality rate due to a number of developmental abnormalities caused mainly by the incorrect epigenetic reprogramming of the donor cell to a pluripotent state. Conventional bovine SCNT involves fusion of a somatic donor cell into an enucleated metaphase II (MII) arrested oocyte. The resulting embryo is cultured to the blastocyst stage before being transferred to a surrogate cow to produce live offspring. Zygotes were initially thought to be unsuitable as SCNT recipients in bovine, until it was revealed that using telophase II (TII) zygotes as opposed to MII oocytes results in improved in vivo development. Metaphase zygotes have also been used successfully as SCNT recipients in mouse to produce cloned blastocysts and it has been proposed that reprogramming factors sequester in the pronuclei of interphase zygotes. Little is known about the nature the nuclear reprogramming, however a few candidate reprogramming factors have emerged recently. TCTP is known to activate key pluripotency genes (POU5F1 and NANOG) in somatic cell nuclei. It has also been identified as present in bovine oocytes with a high potential to reprogram somatic cells. Reprogramming of cell nuclei by Xenopus egg extract has found to require BRG1. Immunodepletion of BRG1 was shown to decrease the reprogramming ability of the egg extract, whist its over-expression increased reprogramming potential. HDAC1 has been found to initiate a transcriptionally repressive state in preimplantation mouse embryos possibly inhibiting transcription of reprogramming factors. Knockdown of HDAC1 using TSA is known to increase development of mouse NT embryos. The aim of this study was to produce blastocysts using metaphase zygotes as recipients for SCNT in bovine. In addition, localisation and abundance of candidate reprogramming factors TCTP (TPT1 gene), BRG1 and HDAC1 were examined in MII oocytes as well as TII, interphase and metaphase zygotes. This study found metaphase zygotes are unsuitable as recipients for bovine SCNT using current methodologies, possibly due to the premature cleavage of the embryos. Control embryos produced using MII oocytes as recipients developed to blastocyst with an efficiency of ~ 11%. mRNA analysis of zygotes and oocytes did not reveal any significant differences in the relative concentrations of TPT1 or BRG1 between the samples. The TCTP and HDAC1 proteins showed a similar pattern of localisation in the MII oocytes and all stages of zygotes. Both proteins clearly localise to the maternal chromatin in the second polar body of TII zygotes. This finding has never previously been described and may in part explain why there is increased cloning efficiency observed when using TII zygotes as SCNT recipients. This increased understanding of these reprogramming factors may increase our knowledge of the processes which occur during NT and lead to greater efficiency of bovine cloning for commercial and therapeutic purposes.
The University of Waikato
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