Karyopherin alpha subtypes and porcine early stage embryo development

<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>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Animal Science

Year

2014

Author dc:creator

• Li, Yanfang

Contributors dc:contributor

• Ryan Cabot
• Zoltan Machaty
• Kolapo Ajuwon
• Shihuan Kuang
• Ann Kirchmaier

• Animal Culture and Nutrition
• Agriculture

Repository record dc:identifier

https://docs.lib.purdue.edu/open_access_dissertations/322

OAI identifier oai:identifier

oai:docs.lib.purdue.edu:open_access_dissertations-1415