Inheritance of DNA methylation on imprinted genes depends on the Dnmt1 (cytosine-5-)
methyltransferase protein. Methylation patterns on imprinted genes are maintained by oocytespecific
Dnmt1o isoform at the 8-cell stage of preimplantation development. Methylation
patterns in postimplantation embryos are maintained by the Dnmt1s isoform. To determine if
Dnmt1s can functionally replace Dnmt1o, we expressed Dnmt1s in oocytes and discovered that
Dnmt1s can maintain genomic imprints in the absence of Dnmt1o. However, the ability of
Dnmt1s to maintain imprinting is dependant on the level of oocyte Dnmt1s. Though Dnmt1s and
Dnmt1o have equivalent maintenance methyltransferase functions in oocytes, the unstable nature
of oocyte Dnmt1s, in comparison to oocyte Dnmt1o, leads to levels lower than what are required
to maintain methylation at the 8-cell stage. We also determined that in cloned embryos, Dnmt1o
undergoes none of its expected trafficking to 8-cell stage nuclei. Instead, these embryos exhibit
a mosaic pattern of Dnmt1s expression. Defects in intracellular trafficking of Dnmt1o and
misexpression of Dnmt1s, along with the intrinsic instability of Dnmt1s, might contribute to
iv
aberrant DNA methylation in cloned embryos, thus raising concerns about the use of current
cloning technologies for therapeutic cloning.
Molecular mechanisms involved in the formation of ovarian teratomas were also analyzed.
Unfertilized oocytes arrest at the MII stage of meiotic maturation. After fertilization, oocytes
continue into cell division. Premature activation of MII oocytes without fertilization, can lead to
ovarian teratoma formation. To better understand mechanisms governing the prevention of
spontaneous oocyte activation, we investigated the molecular defects leading to formation of
ovarian teratomas in the Tgkd mouse model. Tgkd is a transgene insertional mutation that leads
to reduced levels of the Inpp4b protein in MII oocytes of hemizygous Tgkd females. Wildtype
GV oocytes have less Inpp4b protein than MII oocytes, and a significant decrease in Inpp4b is
also seen after fertilization. Also, the dependence of ovarian teratoma formation on the mouse
strain, emphasizes the role of a strain-specific modifier on chromosome 6, possibly IP31. Thus,
it is possible that oocyte Inpp4b normally suppresses spontaneous MII oocyte activation,
possibly by reducing levels of IP3, an intermediate in the oocyte activation mechanism, that
occurs following fertilization.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04222004-135832 |
| Date | 03 May 2004 |
| Creators | Ratnam, Sarayu |
| Contributors | Urvashi Surti, Martin Schmidt, Robert Ferrell, John Richard Chaillet |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04222004-135832/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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