Maturation of the reconstructed oocytes by germinal vesicle transfer in rabbits and mice

Abstract

The present study was designed to evaluate the feasibility of germinal vesicle (GV) transfer in rabbits and mice. The GV oocytes were collected from ovaries and cultured in 20 μg/mL 3-isobutyl-1-methylxanthin (IBMX) in TCM199 medium, which caused oocytes to shrink, enlarging the perivitelline space to facilitate the GV removal and transfer. Pairs of GV — cytoplast complexes were fused with electric pulses, and the fused, reconstructed oocytes were cultured in TCM199 for 24 h. Results are as follows: 1) The exposure time of rabbit GV oocytes to IBMX medium affected the success of GV removal. For oocytes cultured for 2 and 3 h in IBMX medium, removed rates were 56% and 44, respectively, significantly higher (P < 0.05) than removal rates of GV oocytes cultured for 1 and 4 h (27% and 27%, respectively); 2) There was no significant difference (P > 0.1) in fusion and maturation rates of rabbit reconstructed oocytes collected at 72 and 84 h after initiation of FSH injection to donors; 3) eCG in the maturation media improved development of rabbit-to-rabbit GV transferred oocytes but had no positive effect on mouse-to-rabbit GV transferred oocytes; 4) When mouse GV-karyoplasts were injected into enucleated rabbit oocytes, fusion rates of GV-karyoplasts measuring 40- to 50-μm and 80- to 90-μm in diameters obtained were 84% and 93%, respectively. The rates were significantly higher (P < 0.05) than fusion rates after transferring GV-karyoplasts measuring 30- to 35-μm in diameter (63%). The maturation rate (89%) of reconstructed oocytes composed of 80- to 90-μm mouse GV-karyoplasts and rabbit GV-enucleated cytoplasts was higher than that seen for oocytes composed of 40- to 50-μm (77%, P<0.05) or 30- to 35-μm (59%, P<0.01) mouse karyoplasts. Thirty-five of the 63 (56%) mature mouse-to-rabbit reconstructed oocytes had the normal complement of 20 chromosomes.