Developmental dynamics of nuclear trafficking in the porcine embryo

Many cellular proteins shuttle between nuclear and cytoplasmic compartments at different times during the cell cycle. Correct localization of cellular proteins enables cells to respond to the environment and proceed through development. Eukaryotic cells partition cellular proteins between the nucleus and cytoplasm through members of the importin beta family of transport receptors. The aim of the studies presented here was to characterize the developmental requirements of two nuclear trafficking systems, namely those nuclear export reactions mediated by CRM1 and nuclear import reactions mediated by the importin alpha/beta heterodimer, during cleavage development of the porcine embryo. Through a series of embryo culture experiments in which a selective chemical inhibitor of CRM1 function, leptomycin B (LMB), was added to developing porcine embryos, an LMB-insensitive period of development was observed. Porcine embryos appear to develop independently of CRM1 function from the pronuclear through the four-cell stage. Immunocytochemical analysis of CRM1 showed that CRM1 was present as a nuclear protein from the pronuclear to morula stages of development. During the LMB-insensitive period, CRM1 protein adopted a punctate staining pattern throughout the nucleus and perinuclear cytoplasm. A microinjection assay, in which labeled reporter proteins capable of export by CRM1 were introduced into the nuclei of pronuclear and 2-cell stage embryos, was used to determine if CRM1 was functional during the LMB-insensitive stages. Analysis of both control embryos and those treated with LMB revealed that CRM1-mediated export was functional during these stages. Immunocytochemistry was also employed to localize specific importin alpha homologues in cleavage stage porcine embryos. Results indicated that three importin alpha homologues (hSRP1alpha, hSRP1gamma and NPI-1) were present throughout development. Both hSRP1alpha and hSRP1gamma occupied the nuclear compartment from the pronuclear through morula stages. RNA interference experiments were carried out to determine the developmental requirements of hSRP1alpha and hSRP1gamma during early cleavage. RNA interference involves microinjection of double stranded RNA (dsRNA) molecules homologous to regions of target genes that ultimately mediate the degradation of the endogenous target mRNA. Embryos injected with hSRP1gamma dsRNA had significantly fewer blastomeres than embryos injected with hSRP1alpha dsRNA, bovine interferon tau dsRNA (a negative control dsRNA) and embryos injected with culture medium. In conclusion, these studies indicate that multiple nuclear trafficking pathways function during early cleavage in the porcine embryo and that embryos may have differing developmental requirements for individual nuclear trafficking pathways.