Karyopherin alpha seven and porcine embryo development
Abstract
Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localization signals into the nucleus. Seven karyopherin α subtypes have been identified in mammals. The aim of these studies were to determine the roles karypopherin alpha 7 (KPNA7) served during early porcine embryo development. Quantitative RT-PCR results revealed that the seven KPNA subtypes have various transcript abundance at discrete stages of porcine embryo development. KPNA7 has the highest level of transcripts in early porcine cleavage stage embryos and the expression reduce significantly by the blastocyst stage. Immunocytochemical analysis showed that KPNA7 adopted nuclear localization in GV-stage oocytes, 2-cell and 4-cell stage embryos, but not in blastocyst stage embryos.The targeted reduction of KPNA7 led to reduced developmental competence in porcine embryos. An in vitro binding assay revealed that KPNA7 could bind to karyopherin β and suppressor of variation 39 homolog 2 (SUV39h2). A GST pull-down assay was performed in order to screen for KPNA7-specific cargo proteins. Here a liquid chromatography tandem mass spectroscopy (LC-MS/MS) approach was used to determine the proteins that interact with recombinant KPNA7, twenty-two proteins were presumed to be the cargoes for KPNA7, and DAVID analysis predicted that these proteins were related to critical events such as transcription, histone modification, chromatin organization and DNA binding. Together, our data suggest that KPNA7 serves a critical role in early porcine embryo development.
Degree
Ph.D.
Advisors
Cabot, Purdue University.
Subject Area
Molecular biology|Animal sciences|Developmental biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.