Intracellular communication between the nucleus and cytoplasm is critically important for coordinating cellular events during embryogenesis. The karyopherin α/β heterodimer is an intracellular nuclear trafficking system that mediates nuclear import of proteins that bear classical nuclear localization signals (NLSs). Seven karyopherin α subtypes have been identified in the domestic pig, and while each of these karyopherin α subtypes is able to bind to a nuclear localization signal, individual karyopherin α subtypes have been shown to transport specific NLS-bearing proteins. The objective of this study was to determine the developmental requirements of karyopherin α subtypes (KPNAs) during cleavage development in porcine embryos. The purpose of this dissertation was to test the hypothesis that the karyopherin α/β heterodimer-mediated nuclear trafficking pathway serves regulatory roles during cleavage development by selectively partitioning intracellular cargoes, thereby affecting epigenetic modifications, transcription, and embryo developmental potential. We tested our hypothesis via a combination of a series of in vivo and in vitro assays. Our microinjection assay revealed that POU domain, class 3, transcription factor 2 (BRN2, also referred to as POU3F2) adopts a nuclear localization in all nuclei through the 4-cell stage of development, while only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. Octamer-binding transcription factor 4 (OCT4) adopts a nuclear localization in all nuclei prior to the 2-cell stage of development, whereas OCT4 is undetectable in nuclei at the 4-cell stage. In vitro binding assays showed that both BRN2 and OCT4 are able to bind with multiple porcine karyopherin α subtypes. Moreover, we tested the impact of KPNA1-depletion in the intracellular localization of BRN2 and the embryo developmental competence via a series of co-microinjection assays. Our results showed that GFP-BRN2 accumulation was significantly reduced in the nuclei of KPNA1-depleted embryos, and KPNA1-depleted embryos possessed significantly fewer nuclei as well as a reduced proportion with the capacity to develop to the 8-cell stage and beyond as compared with the control embryos.^ In summary, the data discussed in this dissertation provide more evidence to support the conclusion that discrete classes of NLS-bearing nuclear proteins may be preferentially imported by individual karyopherin α subtypes. Our data also indicate that KPNA1 might be involved in the transport of transcription factor BRN2 in 4-cell stage embryos. Furthermore, the data suggest that KPNA1 may serve a critical role of partitioning intracellular cargoes that are directly involved in zygotic genome activation, or that direct development immediately after zygotic genome activation, hence affecting early cleavage embryo development.
","abstract_html":"<p>Intracellular communication between the nucleus and cytoplasm is critically important for coordinating cellular events during embryogenesis. The karyopherin α/β heterodimer is an intracellular nuclear trafficking system that mediates nuclear import of proteins that bear classical nuclear localization signals (NLSs). Seven karyopherin α subtypes have been identified in the domestic pig, and while each of these karyopherin α subtypes is able to bind to a nuclear localization signal, individual karyopherin α subtypes have been shown to transport specific NLS-bearing proteins. The objective of this study was to determine the developmental requirements of karyopherin α subtypes (KPNAs) during cleavage development in porcine embryos. The purpose of this dissertation was to test the hypothesis that the karyopherin α/β heterodimer-mediated nuclear trafficking pathway serves regulatory roles during cleavage development by selectively partitioning intracellular cargoes, thereby affecting epigenetic modifications, transcription, and embryo developmental potential. We tested our hypothesis via a combination of a series of <em>in vivo</em> and <em>in vitro</em> assays. Our microinjection assay revealed that POU domain, class 3, transcription factor 2 (BRN2, also referred to as POU3F2) adopts a nuclear localization in all nuclei through the 4-cell stage of development, while only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. Octamer-binding transcription factor 4 (OCT4) adopts a nuclear localization in all nuclei prior to the 2-cell stage of development, whereas OCT4 is undetectable in nuclei at the 4-cell stage. <em>In vitro</em> binding assays showed that both BRN2 and OCT4 are able to bind with multiple porcine karyopherin α subtypes. Moreover, we tested the impact of KPNA1-depletion in the intracellular localization of BRN2 and the embryo developmental competence via a series of co-microinjection assays. Our results showed that GFP-BRN2 accumulation was significantly reduced in the nuclei of KPNA1-depleted embryos, and KPNA1-depleted embryos possessed significantly fewer nuclei as well as a reduced proportion with the capacity to develop to the 8-cell stage and beyond as compared with the control embryos.^ In summary, the data discussed in this dissertation provide more evidence to support the conclusion that discrete classes of NLS-bearing nuclear proteins may be preferentially imported by individual karyopherin α subtypes. Our data also indicate that KPNA1 might be involved in the transport of transcription factor BRN2 in 4-cell stage embryos. Furthermore, the data suggest that KPNA1 may serve a critical role of partitioning intracellular cargoes that are directly involved in zygotic genome activation, or that direct development immediately after zygotic genome activation, hence affecting early cleavage embryo development.</p>","abstract_has_math":false,"creators":["Li, Yanfang"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Animal Science","degree_department":null,"school":null,"contributors":["Ryan Cabot","Zoltan Machaty","Kolapo Ajuwon","Shihuan Kuang","Ann Kirchmaier"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-10-01T07:00:00Z","date_published":"2014-10-01T07:00:00Z","updated_at":"2026-07-24T03:53:34Z","subjects":["Animal Culture and Nutrition","Agriculture"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://docs.lib.purdue.edu/open_access_dissertations/322","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Ryan Cabot","Zoltan Machaty","Kolapo Ajuwon","Shihuan Kuang","Ann Kirchmaier"]},{"key":"dc:creator","label":"Author","values":["Li, Yanfang"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"thesis:degree_discipline","label":"Discipline","values":["Animal Science"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Animal Culture and Nutrition","Agriculture"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://docs.lib.purdue.edu/open_access_dissertations/322"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Intracellular communication between the nucleus and cytoplasm is critically important for coordinating cellular events during embryogenesis. The karyopherin α/β heterodimer is an intracellular nuclear trafficking system that mediates nuclear import of proteins that bear classical nuclear localization signals (NLSs). Seven karyopherin α subtypes have been identified in the domestic pig, and while each of these karyopherin α subtypes is able to bind to a nuclear localization signal, individual karyopherin α subtypes have been shown to transport specific NLS-bearing proteins. The objective of this study was to determine the developmental requirements of karyopherin α subtypes (KPNAs) during cleavage development in porcine embryos. The purpose of this dissertation was to test the hypothesis that the karyopherin α/β heterodimer-mediated nuclear trafficking pathway serves regulatory roles during cleavage development by selectively partitioning intracellular cargoes, thereby affecting epigenetic modifications, transcription, and embryo developmental potential. We tested our hypothesis via a combination of a series of in vivo and in vitro assays. Our microinjection assay revealed that POU domain, class 3, transcription factor 2 (BRN2, also referred to as POU3F2) adopts a nuclear localization in all nuclei through the 4-cell stage of development, while only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. Octamer-binding transcription factor 4 (OCT4) adopts a nuclear localization in all nuclei prior to the 2-cell stage of development, whereas OCT4 is undetectable in nuclei at the 4-cell stage. In vitro binding assays showed that both BRN2 and OCT4 are able to bind with multiple porcine karyopherin α subtypes. Moreover, we tested the impact of KPNA1-depletion in the intracellular localization of BRN2 and the embryo developmental competence via a series of co-microinjection assays. Our results showed that GFP-BRN2 accumulation was significantly reduced in the nuclei of KPNA1-depleted embryos, and KPNA1-depleted embryos possessed significantly fewer nuclei as well as a reduced proportion with the capacity to develop to the 8-cell stage and beyond as compared with the control embryos.^ In summary, the data discussed in this dissertation provide more evidence to support the conclusion that discrete classes of NLS-bearing nuclear proteins may be preferentially imported by individual karyopherin α subtypes. Our data also indicate that KPNA1 might be involved in the transport of transcription factor BRN2 in 4-cell stage embryos. Furthermore, the data suggest that KPNA1 may serve a critical role of partitioning intracellular cargoes that are directly involved in zygotic genome activation, or that direct development immediately after zygotic genome activation, hence affecting early cleavage embryo development.
"]},{"key":"dc:title","label":"Title","values":["Karyopherin alpha subtypes and porcine early stage embryo development"]}]}],"canonical_facts":{"dc:contributor":["Ryan Cabot","Zoltan Machaty","Kolapo Ajuwon","Shihuan Kuang","Ann Kirchmaier"],"dc:creator":["Li, Yanfang"],"dc:description.abstract":["Intracellular communication between the nucleus and cytoplasm is critically important for coordinating cellular events during embryogenesis. The karyopherin α/β heterodimer is an intracellular nuclear trafficking system that mediates nuclear import of proteins that bear classical nuclear localization signals (NLSs). Seven karyopherin α subtypes have been identified in the domestic pig, and while each of these karyopherin α subtypes is able to bind to a nuclear localization signal, individual karyopherin α subtypes have been shown to transport specific NLS-bearing proteins. The objective of this study was to determine the developmental requirements of karyopherin α subtypes (KPNAs) during cleavage development in porcine embryos. The purpose of this dissertation was to test the hypothesis that the karyopherin α/β heterodimer-mediated nuclear trafficking pathway serves regulatory roles during cleavage development by selectively partitioning intracellular cargoes, thereby affecting epigenetic modifications, transcription, and embryo developmental potential. We tested our hypothesis via a combination of a series of in vivo and in vitro assays. Our microinjection assay revealed that POU domain, class 3, transcription factor 2 (BRN2, also referred to as POU3F2) adopts a nuclear localization in all nuclei through the 4-cell stage of development, while only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. Octamer-binding transcription factor 4 (OCT4) adopts a nuclear localization in all nuclei prior to the 2-cell stage of development, whereas OCT4 is undetectable in nuclei at the 4-cell stage. In vitro binding assays showed that both BRN2 and OCT4 are able to bind with multiple porcine karyopherin α subtypes. Moreover, we tested the impact of KPNA1-depletion in the intracellular localization of BRN2 and the embryo developmental competence via a series of co-microinjection assays. Our results showed that GFP-BRN2 accumulation was significantly reduced in the nuclei of KPNA1-depleted embryos, and KPNA1-depleted embryos possessed significantly fewer nuclei as well as a reduced proportion with the capacity to develop to the 8-cell stage and beyond as compared with the control embryos.^ In summary, the data discussed in this dissertation provide more evidence to support the conclusion that discrete classes of NLS-bearing nuclear proteins may be preferentially imported by individual karyopherin α subtypes. Our data also indicate that KPNA1 might be involved in the transport of transcription factor BRN2 in 4-cell stage embryos. Furthermore, the data suggest that KPNA1 may serve a critical role of partitioning intracellular cargoes that are directly involved in zygotic genome activation, or that direct development immediately after zygotic genome activation, hence affecting early cleavage embryo development.
"],"dc:identifier":["https://docs.lib.purdue.edu/open_access_dissertations/322"],"dc:subject":["Animal Culture and Nutrition","Agriculture"],"dc:title":["Karyopherin alpha subtypes and porcine early stage embryo development"],"thesis:degree_discipline":["Animal Science"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T03:53:34Z"}