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 Benedito

Committee Member

Melanie Clemmer

Committee Member

Robert Dailey

Committee Member

Jorge Flores

Committee Member

Michael Vernon

Abstract

Much of the loss of potential offspring in cattle is concentrated in the early embryonic period. Maternal mRNAs that accumulate in the oocyte during oogenesis have important functional roles during the initial stages of embryonic development, before embryonic genome activation. It is well regarded that the oocyte plays an active role in regulation of key aspects of the reproductive process required for fertility. What is more, oocyte-specific transcription factors seem to be the controlling feature influencing germ cell success throughout oogenesis, fertilization, and early embryonic development. Of the remarkably diverse array of transcription factors encoded by mammalian genomes, about two-thirds encode C2H2 zinc-finger proteins.;Zinc finger proteins exclusively expressed in mammalian oocytes have not been reported. Deep sequencing of a bovine oocyte library revealed a highly abundant transcript that matches an uncharacterized gene in the NCBI database. cDNA cloning of the novel ZNFO gene revealed a transcript containing a 2,145 bp open reading frame that codes for a protein of 714 amino acids with a conserved KRAB domain at the N-terminus and nine zinc finger motifs at the C-terminus. The individual ZNF motifs fit the conserved two cysteine-two histidine sequence model. ZNFO mRNA was detectable in fetal ovaries and was undetectable in all somatic tissues analyzed, including granulosa and theca cells. Real-time PCR analysis revealed ZNFO mRNA is highly abundant in GV and MII stage oocytes as well as in pronuclear to 8-cell stage embryos but undetectable in blastocyst stage embryos. Immunohistochemical analysis detected ZNFO protein in oocytes throughout folliculogenesis. Identification and characterization revealed the novel ZNFO is a KRAB-containing maternal-effect gene found exclusively in bovine oocytes.;To elucidate the functional role of ZNFO, zygotes were generated by in vitro maturation and fertilization of oocytes and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was reduced significantly relative to the uninjected and negative control siRNA-injected embryos. Furthermore, interaction of ZNFO with the highly conserved transcriptional repressor co-factor (KAP1) was demonstrated by GST pull-down, and evidence supporting transcriptional repression by ZNFO using a GAL4-luciferase assay. In addition, transfection studies verified that a ZNFO-GFP fusion protein localizes specifically to the nucleus, further supporting the proposed function in transcriptional regulation. These studies demonstrate that ZNFO is a maternally-derived oocyte-specific factor required for early embryonic development in cattle, which has a functional role as a transcriptional regulator required during early embryogenesis by repressing transcription, possibly controlling activation of the embryonic genome.

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