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Hum. Reprod. Advance Access published online on March 11, 2004
Human Reproduction, doi:10.1093/humrep/deh149
© 2004 by European Society of Human Reproduction and Embryology
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Received September 1, 2003
Accepted November 24, 2003

Article

A successful model to assess embryo development after transplantation of prophase nuclei

Takumi Takeuchi 1, Zev Rosenwaks 1, and Gianpiero D. Palermo 1*

1 The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA

* To whom correspondence should be addressed. E-mail: gdpalerm{at}med.cornell.edu.


  Abstract

BACKGROUND: Germinal vesicle transplantation (GVT) provides a means of investigating interactions between karyoplasts and cytoplasts isolated from different cells. Technically, GVT can be accomplished with a high degree of efficiency without compromising the maturation of either the human or mouse oocyte nucleus. Although maturation, fertilization and preimplantation development have been established using GVT, full-term development has been reported only after supplementation with fresh mature ooplasm. In this study, we assess the ability of immature oocytes collected from gonadotrophin-primed ovaries to mature in vitro after GVT and develop to full-term. METHODS: GV oocytes were retrieved from either non-stimulated or pregnant mare’s serum gonadotrophin (PMSG)-primed female mice. Microsurgically isolated GV karyoplasts were transplanted into previously enucleated oocytes. Oocytes successfully reconstituted by electrofusion were cultured for 14 h to allow nuclear maturation. Metaphase II oocytes were subjected to Piezo-ICSI, and those fertilized normally were cultured to the blastocyst stage. Some such embryos were transferred to pseudopregnant female mice to examine their potential for normal development. Cumulus-denuded non-manipulated oocytes that were matured in vitro served as controls. RESULTS: The reconstitution and maturation rates were comparable in oocytes isolated from PMSG-primed and from unstimulated ovaries. The rate of normal fertilization in oocytes from primed ovaries was significantly higher than that of their non-primed counterparts (63.5 versus 39.6%; P < 0.01). This difference was also confirmed in terms of blastocyst development (31.8 versus 7.9%; P < 0.01). Of a total of 70 embryos transferred to the oviduct of five recipient mice, 21.4% developed to normal live offspring. All developed as normal adults and proved to be fertile. The live birth rate was comparable to that obtained using non-manipulated control oocytes (22.3%). CONCLUSIONS: Higher rates of fertilization and blastocyst formation were obtained after GVT of mouse oocytes isolated from PMSG-primed ovaries compared with their non-primed counterparts. These represent the first mouse offspring derived from in vitro matured, cumulus-denuded oocytes treated by allo-GVT and fertilized by ICSI. Thus, GVT appears not to impair oocyte maturation, fertilization and pre- and post-implantation development and, after gonadotrophin priming, allows generation of healthy mouse offspring without mature ooplasm supplementation.

Key words: Key words: embryo development/germinal vesicle transplantation/ICSI/in vitro maturation/nuclear transfer


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