Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3

Numerous cellular proteins are able to localize to the nucleus due to the fact that they possess a nuclear localization signal (NLS) in their amino acid sequence. Nuclear localization sequences recognized by the importin alpha/beta heterodimer are found in cellular proteins capable of performing many diverse functions, ranging from chromatin remodeling to cell cycle regulation. Evidence has been presented that suggests individual importin alpha homologues are present at varying levels in different adult tissues. Other data have shown that specific subsets of NLSs found in different cellular proteins are recognized by individual importin alpha homologues with varying affinities. This evidence led us to hypothesize that due to the specific cargoes they carry, the mammalian embryo has different developmental requirements for individual importin alpha homologues. The results of the studies presented here indicate that importin alpha/beta-mediated import occurs throughout early cleavage in the porcine embryo, as determined by a reporter protein microinjection assay, and that multiple importin alpha homologues are present throughout early cleavage, as determined by immunocytochemical analysis. An RNA interference approach was used in an attempt to determine the developmental requirements for specific importin alpha homologues during early cleavage in the porcine embryo. Results from this study showed that fertilized porcine embryos injected with double stranded RNA (dsRNA) corresponding to the importin alpha homologue karyopherin alpha3 had significantly fewer nuclei following four days of culture than did embryos injected with dsRNA for another importin alpha homologue, karyopherin alpha2, or two control groups. This is the first report indicating that mammalian embryos may have differential developmental requirements for specific nuclear trafficking pathways.