Reconstruction of the heteroparental diploid condition in rabbit zygotes by nuclear transfer

Studies on genomic imprinting showed that parental genomes have complementary roles during embryogenesis, are both essential and need to be synchronized in their embryonic stage for successful development to term. To our knowledge, these studies have not been performed in species other than mice. We studied the in vitro and in vivo development of reconstructed zygotes by combining female haploid nuclear donors and androgenetic hemizygous recipients. Haploid donor embryos at the 8- or 32-cell stage were obtained from electroactivated young rabbit ova (eight pulses maximum, consisting of 0 6 kVcm(-1) for 60 microsec each, 38 min apart) which were further cultured for 24 h or 32 h. Couplets formed by both the haploid male hemizygous recipients and haploid female donor cells were electrofused (2.2 kVcm(-1) for 60 microsec duration each, 30 min apart) and their nuclear configuration determined 122 of those fused (43%: 122/286) were diploid. Reconstructed diploid zygotes developed in vitro up to the compacted morula, blastocyst and hatched stages (1/8-nuclei x 50%, 18% and 9% vs. 1/32-nuclei: 47%, 25% and 19%; P > 0.05), respectively. In embryo transfer assays, both 1/32-reconstructed zygotes and control, non-manipulated zygotes were transferred to synchronized does Four live reconstructed fetuses (4/49: 8 1% survival rate) and five in regression stage (9/49: 18% implantation rate) were observed on Day 21 post-ovulation, whereas from control zygotes, 11 fetuses were alive (11/53 21% fetal survival rate) and 2 degenerated (13/53 x 24 5% implantation rate). Similar results were obtained from a final experiment, in which development was allowed to progress to term. Six live rabbit pups derived front experimentally reconstructed zygotes (11%; 6/54) and three fetuses in regression stage were obtained; values slightly lower than those derived from non-manipulated and transferred control zygotes (18% 9/50, live born rate).