Abstract:
Partitioning intracellular proteins between nuclear and cytoplasmic compartments is critically important for coordinating major cellular events involved in transcription and differentiation. Import of cytoplasmic proteins bearing classical nuclear localization signals (NLSs) into the nucleus is mediated by the importin α/β heterodimer. Importin α, also called karyopherin α (KPNA), serves to recognize the NLS-bearing cytoplasmic cargo. Six KPNA molecules have been characterized in human (KPNA1-6). Select KPNA molecules are known to be differently expressed in specific tissues; individual KPNA molecules also have specificity for unique NLS-bearing cargos. We hypothesized that transcripts for individual porcine KPNA molecules would be present at differing levels at specific stages of oocyte maturation and cleavage development, thereby reflecting the changing requirements for particular import pathways during this window of development. To test this hypothesis, we first identified the porcine orthologs of KPNA1-6. We also identified the open reading frame of a potentially novel KPNA, KPNA7. KPNA7 was highly represented in the porcine EST database from expressed sequence tags derived from oocytes and ovarian tissue. Transcript abundance of KPNA1-7 was determined in germinal vesicle (GV) and MII-stage (MII) porcine oocytes and 4-cell (4C) and blastocyst-stage (BL) porcine embryos using quantitative real-time PCR. mRNA was isolated from pools (50 200) of GV and MII oocytes and 4C and BL embryos produced by IVF. Transcripts for KPNA1-7 and YWHAG (internal control for transcript normalization) were amplified in duplicate across 3 to 5 replicates. Relative transcript abundance of these genes was measured using the comparative CT method; GV was taken as the calibrator stage. Data were analyzed using GLM procedures in SAS (SAS Institute Inc., Cary, NC, USA) with the significance level at 0.05, and differences were compared by Tukey's post test. Our results showed that KPNA1 had a significant decrease in MII oocytes (4-fold, GV v. MII). Transcript abundance of KPNA2 was significantly higher in GV oocytes and 4C embryos than in MII oocytes (2-fold GV v. MII; 3-fold 4C v. MII); KPNA2 transcripts were not detectable in BL embryos. KPNA3 transcripts were reduced in BL embryos compared to GV oocytes (8-fold, BL v. GV). KPNA4 transcripts were increased at the 4-cell stage (18-fold, GV v. 4C). The transcripts of KPNA5 were detectable only in GV and MII oocytes. No significant changes in the amount of KPNA6 transcripts were detectable at the stages analyzed. Transcript levels of KPNA7 were reduced in BL embryos as compared to the GV oocytes (1165-fold, BL v. GV). Throughout all these stages examined, KPNA5 had the lowest transcript abundance and was not detectable in 4C and BL stages. Transcripts levels of KPNA7 were in higher abundance than KPNA1-6 in GV and MII oocytes. Results suggest that KPNA7 is a new member of the KPNA family. Our results also suggest that porcine oocytes and embryos, at discrete stages of development, have differing requirements for individual KPNA molecules.