title: Production of fertile offspring from oocytes grown in vitro by nuclear transfer in cattle.
abstract: 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.
authors.affiliation: Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hirao
authors.forename: Kenji
authors.initials: K
authors.lastname: Naruse
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kaneda
authors.forename: Tamas
authors.initials: T
authors.lastname: Somfai
authors.forename: Kosuke
authors.initials: K
authors.lastname: Iga
authors.forename: Manabu
authors.initials: M
authors.lastname: Shimizu
authors.forename: Satoshi
authors.initials: S
authors.lastname: Akagi
authors.forename: Feng
authors.initials: F
authors.lastname: Cao
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kono
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagai
authors.forename: Naoki
authors.initials: N
authors.lastname: Takenouchi
mesh_terms: D000818:Animals; D002417:Cattle; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods* / Q000662:veterinary; D046149:Embryo Culture Techniques / Q000662:veterinary; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D005298:Fertility; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary*; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D011247:Pregnancy
keywords: cattle; cell culture; oocyte development; oocyte growth; reprogramming; somatic cell nuclear transfer