Abstract
Mature oocytes can be parthenogenetically activated by a variety of methods and the resulting embryos are valuable for studies of the respective roles of paternal and maternal genomes in early mammalian development. In the present study, we report the first successful development of parthenogenetic canine embryos to the post-implantation stage. Nine out of ten embryo transfer recipients became pregnant and successful in utero development of canine parthenotes was confirmed. For further evaluation of these parthenotes, their fetal development was compared with artificially inseminated controls and differentially expressed genes (DEGs) were compared using ACP RT-PCR, histological analysis and immunohistochemistry. We found formation of the limb-bud and no obvious differences in histological appearance of the canine parthenote recovered before degeneration occurred; however canine parthenotes were developmentally delayed with different cell cycle regulating-, mitochondria-related and apoptosis-related gene expression patterns compared with controls. In conclusion, our protocols were suitable for activating canine oocytes artificially and supported early fetal development. We demonstrated that the developmental abnormalities in canine parthenotes may result from defective regulation of apoptosis and aberrant gene expression patterns, and provided evidence that canine parthenotes can be a useful tool for screening and for comparative studies of imprinted genes.
Highlights
Parthenogenesis is the process by which oocytes can develop without fertilization, and the resulting parthenogenetic embryos, called parthenotes, carry only maternal chromosomes [1]
The pregnancy rate of the Artificial insemination (AI) group was 100% based on the number of inseminated dogs and 79.5% based on the number of ovulated oocytes which was calculated from the number of Corpora lutea (CL) (Table 2)
Various stages of in vivo matured oocytes including early mature, mature and moderately aged were used for parthenogenetic activation, and unlike results reported for SCNT embryos [31], pregnancy was achieved after embryo transfer regardless of the oocyte stage used for activation
Summary
Parthenogenesis is the process by which oocytes can develop without fertilization, and the resulting parthenogenetic embryos, called parthenotes, carry only maternal chromosomes [1]. In order to further understand the molecular basis for embryo development in utero, we performed detailed profiling of differentially expressed genes (DEGs) in canine parthenotes and in normally fertilized fetuses. Several methodologies such as cDNA microarrays, serial analysis of gene expression (SAGE), suppression subtractive hybridization (SSH) and annealing control primer (ACP) based RT-PCR were utilized for screening of DEGs [22,23,24,25,26] The latter method uses ACPs that target sequence hybridization to the template via a polydeozyinosie [poly(dI)] linker and allows only genuine products to be amplified [24,27]. We investigated the ability of parthenogenetic canine embryos to implant in vivo, and the molecular basis for development of parthenotes in utero by detailed profiling of DEGs, histological examinations and gene expression analyses on canine parthenotes and age-matched control fetuses derived from normal fertilization in vivo to gain insights into the underlying mechanisms of impaired fetal development
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