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24 July 2013 Production of Fertile Offspring from Oocytes Grown In Vitro by Nuclear Transfer in Cattle
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Because of recent advancements in reproductive technology, oocytes have attained an increasingly enriched value as a unique cell population in the production of offspring. The growing oocytes in the ovary are an immediate potential source that serve this need; however, complete oocyte growth before use is crucial. Our research objective was to create in vitro-grown (IVG) oocytes that would have the ability to perform specialized activities, including nuclear reprogramming, as an alternative to in vivo-grown oocytes. Bovine oocyte–granulosa cell complexes with a mean oocyte diameter of approximately 100 μm were cultured on Millicell membrane inserts, with culture medium supplemented with 4% polyvinylpyrrolidone (molecular weight, 360 000), 20 ng/ml androstenedione, 2 mM hypoxanthine, and 5 ng/ml bone morphogenetic protein 7. Oocyte viability after the 14-day culture period was 95%, and there was a 71% increase in oocyte volume. Upon induction of oocyte maturation, 61% of the IVG oocytes extruded a polar body. Eighty-four percent of the reconstructed IVG oocytes that used cumulus cells as donor cells underwent cleavage, and half of them became blastocysts. DNA methylation analyses of the satellite I and II regions of the blastocysts revealed a similar highly methylated status in the cloned embryos derived from in vivo-grown and IVG oocytes. Finally, one of the nine embryos reconstructed from the IVG oocytes developed into a living calf following embryo transfer. Fertility of the offspring was confirmed. In conclusion, the potential of a proportion of the IVG oocytes was comparable to that of in vivo-grown oocytes.

Yuji Hirao, Kenji Naruse, Masahiro Kaneda, Tamas Somfai, Kosuke Iga, Manabu Shimizu, Satoshi Akagi, Feng Cao, Tomohiro Kono, Takashi Nagai, and Naoki Takenouchi "Production of Fertile Offspring from Oocytes Grown In Vitro by Nuclear Transfer in Cattle," Biology of Reproduction 89(3), (24 July 2013).
Received: 18 March 2013; Accepted: 1 July 2013; Published: 24 July 2013

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