105 IN VIVO DEVELOPMENT OF CANINE PARTHENOTES AND THE EXPRESSION PATTERN OF Igf2/Igf2r GENES

Abstract

Parthenogenesis is the process by which an oocyte develops without fertilization, resulting in parthenogenetic embryos carrying only maternal chromosomes. Until now, little information has been available on the post-activation development of parthenogenetic embryos, and there are no reports about canine post-implantation development of parthenogenetically activated oocytes. Thus, the objective of this study was to investigate the development of parthenogenetic canine embryos when implanted in vivo, and the subsequent post-implantation development of such canine parthenogenetic fetuses. Also, we examined expression patterns of Igf2 and its receptor (Igf2r), which are reciprocally imprinted and expressed from the paternal and maternal genomes, respectively, in other mammalians, to gain insight into the role of genomic imprinting during uniparental development. In vivo matured dog oocytes were obtained by flushing oviducts of mixed breed bitches ∼72 h after ovulation. The denuded oocytes (n = 48; 5 replicates) were subjected to chemical activation by incubation in a culture medium containing 10 μM calcium ionophore (Sigma, St. Louis, MO, USA) for 4 min and then in a culture medium supplemented with 1.9 mM of 6- dimethylaminopurine (Sigma) for 4 h at 39°C. Parthenogenetic embryos were surgically transferred to synchronized recipient female dogs. The implantation rate of parthenogenetic embryos was compared with that of artificially inseminated controls. Normal and parthenogenetic fetuses, obtained from recipients on Day 28, 30, and 32 of pregnancy, were analysed for gross external morphology and Igf2/Igf2r gene expression examined. Data were analysed using SAS and means compared by Student’s t-test. The in vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rate of parthenotes was 56.3%, which was significantly lower than those of the control (79.5%; P < 0.05). The weight of parthenogenetic fetuses and placentae recovered from uteri at 28, 30, and 32 day of pregnancy were significantly lighter than those of the control (P < 0.05), whereas the appearance of recovered parthenogenetic fetuses were comparable to those of in vivo fertilized fetuses. We found that both Igf2 and Igf2r were expressed in canine parthenotes but the expression level of Igf2 in the parthenotes was significantly lower than the control (P < 0.05). The expression level of Igf2r in the parthenotes was comparable with the control. These results confirmed that the protocols used in our present study were suitable for activating the canine oocyte artificially and to support the viability and developmental potential of canine embryos up to the mid-gestation stage. It will provide an opportunity to determine the reason for developmental differences between parthenogenetic and fertilized embryos, and will be a useful model system for elucidating the roles of parental genomes in mammalian postimplantation development.