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Hum. Reprod. Advance Access published online on September 6, 2006
Human Reproduction, doi:10.1093/humrep/del345
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received March 3, 2006
Revised July 24, 2006
Accepted August 1, 2006

Article

Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer

V.J. Hall 1 *, D. Compton 2, P. Stojkovic 3, M. Nesbitt 4, M. Herbert 4, A. Murdoch 4, and M. Stojkovic 3

1 Centre for Stem Cell Biology and Developmental Genetics, Institute of Human Genetics, University of Newcastle upon Tyne, UK
2 Department of Biochemistry, Dartmouth Medical School, Hanover, NH, USA
3 Centre for Stem Cell Biology and Developmental Genetics, Institute of Human Genetics, University of Newcastle upon Tyne, UK; Principe Felipe, Centro de Investigación, C/E.P. Avda. Autopista del Saler, Valencia, Spain
4 Newcastle Fertility Centre at Life, International Centre for Life, Newcastle upon Tyne, UK

* To whom correspondence should be addressed.
V.J. Hall, E-mail: Vanessa.Hall{at}med.lu.se


  Abstract

BACKGROUND: Improving human nuclear transfer (NT) efficiencies is paramount for the development of patient-specific stem cell lines, although the opportunities remain limited owing to difficulties in obtaining fresh mature oocytes. METHODS: Therefore, the developmental competence of aged, failed-to-fertilize human oocytes as an alternate cytoplasmic source for NT was assessed and compared with use of fresh, ovulation-induced oocytes. To further characterize the developmental potential of aged oocytes, parthenogenetic activation, immunocytochemical analysis of essential microtubule proteins involved in meiotic and mitotic division, and RT-PCR in single oocytes (n = 6) was performed to determine expression of oocyte-specific genes [oocyte-specific histone 1 (H1FOO), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zygote arrest 1 (ZAR1)] and microtubule markers [nuclear mitotic arrest (NuMA), minus-end directed motor protein HSET and the microtubule kinesin motor protein EG5]. RESULTS: For NT, enucleation and fusion rates of aged oocytes were significantly lower compared with fresh oocytes (P < 0.05). Cleavage rates and subsequent development were poor. In addition, parthenote cleavage was low. Immunocytochemical analysis revealed that many oocytes displayed aberrant expression of NuMA and EG5, had disrupted meiotic spindles and tetrapolar spindles. One of the six oocytes misexpressed GDF9, BMP15 and ZAR1. Two oocytes expressed EG5 messenger RNA (mRNA), and HSET and NuMA were not detectable. RT-PCR of mRNA for oocyte specific genes and microtubule markers in single aged oocytes. CONCLUSIONS: Thus, aneuploidy and spindle defects may contribute to poor parthenogenetic development and developmental outcomes following NT.

Keywords: gene expression/human/microtubule/oocyte/somatic cell nuclear transfer.
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