Role of importin alpha8, a new member of the importin alpha family of nuclear transport proteins, in early embryonic development in cattle

Abstract

Nuclear proteins such as transcription and chromatin remodeling factors are required for initiation of transcription in early embryos before embryonic genome activation. The nuclear transport of these proteins is mediated by transport factors such as importins. Through analysis of expressed sequence tags from a bovine oocyte cDNA library, we identified a new member of the importin alpha family (named importin alpha8). The cloned cDNA for bovine importin alpha8 (KPNA7) is 1817 base pair in length, encoding a protein of 522 amino acids that contains a conserved importin beta-binding domain and seven armadillo motifs. The RT-PCR analysis revealed that KPNA7 mRNA is specifically expressed in ovaries and mature oocytes. Real-time PCR demonstrated that KPNA7 expression in germinal vesicle (GV) oocytes is 33 to 2396 times higher than that of other importin alpha genes and that KPNA7 mRNA is abundant in GV and metaphase II oocytes, as well as in earlystage embryos collected before embryonic genome activation, but is barely detectable in morula- and blastocyst-stage embryos. Similarly, expression of KPNA7 protein is very high in oocytes and early embryos but is low in blastocysts. A glutathione S-transferase pull-down assay revealed that KPNA7 has a strong binding affinity for the nuclear protein nucleoplasmin 2 relative to that of other importin alphas. RNA interference experiments demonstrated that knockdown of KPNA7 in early embryos results in a decreased proportion of embryos developing to the blastocyst stage. These results suggest that KPNA7 may have an important role in the transport of essential nuclear proteins required for early embryogenesis. © 2009 by the Society for the Study of Reproduction, Inc.