Lei Wang

Date of Graduation

2015

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Animal and Nutritional Sciences

Committee Chair

Jianbo Yao

Committee Co-Chair

Vagner A Benedito

Committee Member

Kenneth P Blemings

Committee Member

Jorge Flores

Committee Member

E Keith Inskeep

Abstract

The finely orchestrated development and maturation of the oocytes has been the focus of many studies in which oocyte-specific genes essential for folliculogenesis and early embryonic development have been identified. Studies using knockout mice models of Nobox, Sohlh1, Sohlh2, Figla and Lhx8 have revealed that these oocyte specific transcriptional factors form networks in which they regulate the expression of each other and downstream targets. However, little is known about the mechanisms that control the nuclear transport of these essential factors in oocytes and early embryos. The nuclear envelope separates the nuclear and cytoplasmic compartment in the cell and uncouples transcription and translation; therefore, nucleo-cytoplasmic transport is necessary and highly regulated in eukaryotic cells. The soluble proteins that play key roles in mediating the bidirectional trafficking of macromolecules across the nuclear envelope are collectively called Karyopherins (KPNs), with those involved in nuclear import referred to as KPNAs. The first oocyte-specific KPNA gene, KPNA7, was discovered in bovine oocytes; later the homologous genes were found in mice, human and pig. Bovine KPNA7 is highly expressed in germinal vesicle (GV) and metaphase II oocytes, as well as in early stage embryos collected before embryonic genome activation, but is barely detectable in morula and blastocyst stage embryos after the maternal to embryonic transmission. Female mice lacking KPNA7 gene are affiliated with reproductive reduction and sex imbalance by inducing preferential fetal lethality. Induced abnormalities of gene expression (Dppa2, Dppa4 and Piwil2) and epigenetic modifications (down-regulation of histone H3K27me3) are also part of the Kpna7 knock out effects. Although it is known that KPNA7 is a maternal transcript critical for development in mammals, little is known about mechanism of regulation its expression and the nuclear translocation function of KPNA7 protein in bovine embryos or the expression and functions of KPNA7 in rainbow trout.;A series of experiments to study KPNA7 was performed in both cattle and rainbow trout. The first study characterized rainbow trout Kpna7, analyzed its expression, and identified its interacting partners. The cDNA for rainbow trout kpna7 encodes a protein of 519 amino acids, which contains a conserved IBB (importin beta binding domain) domain and seven ARM (armadillo/beta-catenin-like repeat) motifs. RT-PCR and Western blot analyses revealed that Kpna7 is specifically expressed in mature oocytes. Real time PCR analysis demonstrated that expression of kpna7 mRNA was high in unfertilized oocytes and decreases gradually in early stage embryos until 3 day post fertilization followed by a sharp decreased reaching a level barely detectable in day 4 embryos and thereafter. Using a yeast two-hybrid screening system, two Kpna7-interacting proteins were identified from a rainbow trout oocyte cDNA library: Stl3 (rhamnose-binding lectin 3) and an uncharacterized protein. Both genes appeared to be expressed specifically in ovary/testis as revealed by RT-PCR analysis. Co-immunoprecipitation assays confirmed the interaction between Kpna7 and Stl3, and co-transfection experiments using EGFP-tagged Stl3 showed that Kpna7 facilitated the nuclear transportation of Stl3 through interaction with the predicted NLS cluster at the C-terminus of Stl3. Results indicated that Kpna7 may function as a unique nuclear transport receptor for oocyte-specific proteins important for early embryonic development. (Abstract shortened by UMI.).

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