Functional study of a germ cell-specific Y-box protein MSY2 in mouse oocytes and early embryos / Junying Yu.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Yu, Junying.

Contributor:

Schultz, Richard M., advisor.

Hecht, Norman B., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Cell and Molecular Biology.

Academic Dissertations as Topic.

Medical Subjects:

Cell and Molecular Biology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Physical Description:

xiii, 148 pages : color illustrations ; 29 cm

Production:

2002.

Summary:

mRNA regulation plays a key role in gene expression during mammalian oogenesis and early embryogenesis. In this dissertation, I have examined the role of a germ cell-specific Y box protein MSY2 in the regulation of translation and stability of maternal mRNAs in mouse oocytes and early embryos. RT-PCR and Western blot analyses showed that both MSY2 mRNA and protein accumulated during oocyte growth, and was degraded by the mid-two cell stage. Quantification of MSY2 indicated that it was a very abundant protein, representing about 2% of total oocyte protein in the fully-grown oocyte, suggesting a rather global role for MSY2 in the regulation of maternal mRNAs. This was supported by results from RNA gel shift and filtration assays using MSY2 recombinant protein, which showed nonsequence-specific binding of MSY2 to full-length mRNAs even though the binding of MSY2 to short RNAs can be sequence-dependent. In in vitro translation assays with rabbit reticulocyte lysate, MSY2 moderately repressed the translation of mRNAs when present at the protein/mRNA molar ratio found in mouse oocytes (∼73:1). Microinjection of MSY2-EGFP chimeric mRNAs followed by confocal microscopy, immunofluorescence and Western blot analysis showed that the majority of MSY2 (∼75%) was associated with an oocyte-specific, Triton-insoluble cytoskeletal structure. This association requires its intact cold-shock domain (CSD) and one additional basic/aromatic (B/A) island, as indicated by deletion analysis. Binding of MSY2 to intact maternal mRNA appears to be required for such association, since degradation of mRNAs by RNaseA released almost all the MSY2 protein. ZP3 promoter-mediated expression of MSY2 hairpin dsRNA in transgenic females successfully reduced both MSY2 mRNA and protein (60%) levels in mouse oocytes. Decreased MSY2 expression resulted in abnormal meiotic spindle formation and high incidence of egg activation failure, which in turn led to a markedly decreased litter size. Results from two-dimensional gel analysis indicated a decrease in global protein synthesis in both fully-grown oocytes and metaphase II eggs from MSY2 transgenic females. Quantification of total poly(A) mRNAs in transgenic fully-grown oocytes showed a decrease of 22.5%. In summary, these data support a global role for MSY2 in translational repression and stabilization of maternal mRNAs in growing oocytes.

Notes:

Advisers: Richard M. Schultz; Norman B. Hecht.

Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 2002.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 3073080.

OCLC:

244972900