Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer.
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.
Hall
V J
VJ
Centre for Stem Cell Biology and Developmental Genetics, Institute of Human Genetics, University of Newcastle upon Tyne, UK.
Compton
D
D
Stojkovic
P
P
Nesbitt
M
M
Herbert
M
M
Murdoch
A
A
Stojkovic
M
M
D000375:Aging / Q000502:physiology*; D034961:Antigens, Nuclear / Q000096:biosynthesis; D018797:Cell Cycle Proteins; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D016547:Kinesins / Q000096:biosynthesis; D034521:Nuclear Matrix-Associated Proteins / Q000096:biosynthesis; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology*; D010312:Parthenogenesis / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020554:Sperm Injections, Intracytoplasmic; D014404:Tubulin / Q000096:biosynthesis
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C470243:KIF11 protein, human; C486566:KIFC1 protein, human; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D014404:Tubulin; D016547:Kinesins