Abstract
Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), a ubiquitin-ribosomal fusion gene, is a major source of ubiquitin protein for covalent modification of proteinaceous substrates recycled by ubiquitin-proteasome system (UPS). Its role in early embryo development has not been studied. Using the CRISPR/Cas9 gene editing tool, the objective of this study was to determine if UBA52 protein is required for mammalian embryogenesis. Matured metaphase II porcine oocytes were injected with CRISPR Cas9+guide RNAs (Uba52 gRNA) or Cas9 without gRNAs as control, followed by in vitro fertilization (IVF) and embryo culture to day 7. Injection of Cas9+gRNAs affected embryo development. On day 4 of embryo culture, the proportion of 2-, 4- and 8-cell stage embryos was significantly different between the Uba52 gRNA and control group (P<0.05), with more 8-cell stage embryos in the control and more 4- and 2-cell stage embryos in the Uba52g RNA group. This delay in the development of Uba52 gRNA embryos occurred at the transition from the 4- to 8-cell stages, around the time of major zygotic genomic activation. The percentage of blastocyst formation on day 7 and the cell number per blastocyst were significantly lower in the Uba52 gRNA group than in the control (P<0.05). Genotyping by PCR and DNA gel electrophoresis analysis showed that 91.8% of embryos that failed to develop to blastocyst had either a monoallelic or a biallelic modification of the Uba52 gene. In comparison, only 24.4% of embryos that reached blastocyst had a monoallelic modification and biallelic editing was not found in any of the blastocysts. Based on immuno-labeling intensity, both UBA52 and proteasome protein levels on days 4 and 7 of culture were significantly lower in the Uba52 gRNA group than in the control (P<0.05), in agreement with UBA52 western blotting-densitometry of day 4 embryos. Morphological examination of blastomere nuclei revealed abnormal nuclear structure in the Uba52 gRNA group, such as reduced size, irregular shapes, nucleus fragmentation and uneven DNA distribution at all stages of embryo development. Nuclear morphology studies of embryos injected with Cas9+gRNAs and co-injected with plasmid DNA encoding nuclear localized GFP further supported these observations. In conclusion, our data indicate that the Uba52 gene is essential in early embryogenesis.