A successful model to assess embryo development after transplantation of prophase nuclei.
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.
Takeuchi
Takumi
T
The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, 505 East 70th Street, HT-336, New York, NY 10021, USA. gdpalerm@med.cornell.edu
Rosenwaks
Zev
Z
Palermo
Gianpiero D
GD
D000818:Animals; D002970:Cleavage Stage, Ovum; D020732:Cytogenetic Analysis; D010064:Embryo Implantation; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000187:drug effects / Q000601:surgery*; D005314:Embryonic and Fetal Development; D005260:Female; D005306:Fertilization; D005307:Fertilization in Vitro; D006064:Gonadotropins, Equine / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology; D011247:Pregnancy; D011418:Prophase; D020554:Sperm Injections, Intracytoplasmic
D006064:Gonadotropins, Equine