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The Licensing Protein ORC4 is Required for Polar Body Extrusion During Murine Meiosis.

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Title:The Licensing Protein ORC4 is Required for Polar Body Extrusion During Murine Meiosis.
Authors:Nguyen, Hieu T.
Contributors:Developmental & Reprod Biology (department)
Keywords:DNA replication
chromosomes
embryo development
ORC4
Polar body extrusion
Date Issued:Dec 2017
Publisher:University of Hawaiʻi at Mānoa
Abstract:Six proteins, ORC1-6, make up the origin recognition complex (ORC) that initiates licensing of the DNA replication origin. We have previously shown that subunit ORC1, ORC2, ORC3, and ORC5 are localized between the separating maternal chromosomes at anaphase II just after fertilization. During investigation, we identified ORC4 as having an unexpected localization in the polar body wherein ORC4 surrounds one set of chromosomes during both female meiotic divisions. The ORC4 structure, or ORC4 cage as we have termed it, eventually is discarded in the polar bodies while the chromosome set that does not interact with ORC4 segregate into the oocyte. Interestingly, none of the other five ORC proteins were found to be involved in this structure. In Zygotic G1, ORC4 surrounds the nuclei of the polar bodies, but was not detectable in the pronuclei. When the zygote entered mitosis, ORC4 was only detected in the polar body. At this point, the ORC4 that was in the polar body also migrated into the nuclei, suggesting that ORC4 or an associated protein is modified during the first embryonic cell cycle to allow it to bind DNA. We experimentally forced oocytes to extrude sperm chromatin as a pseudo-polar body and found that under these conditions, the sperm chromatin did become enclosed in an ORC4 cage. Next, we attempted to prevent the formation of the ORC4 cage by injecting peptides that contained sequences of different ORC4 protein domains into metaphase II oocytes just before typical cage formation. Our rationale was that the ORC4 peptides would block protein-protein interactions required for cage formation. Two out of six tested peptides prevented the ORC4 cage formation and simultaneously inhibited polar body extrusion, resulting in the formation of two pronuclei that were retained in the oocyte. Our previous results demonstrated that recombinantly expressed ORC4, which contained histidine tag at the C terminus, could be utilized by oocytes to form the ORC4 cage at one set of chromosomes. Using immunocytochemistry (ICC), we were able to show that ORC4-His tags incorporated with endogenous ORC4. However, we could not use these tags to study on live cells because they required cell fixation to identify by ICC. To test the localization of ORC4 in live cells, we generate a fusion ORC4-eGFP and ORC4 labeling with FlAsH. These constructs were then used to synthesize mRNA and microinjected into MII oocyte. Through confocal fluorescence fluctuation spectroscopy (FFS) measurements, we are able to record the movement of ORC4 signals moving forward to form a cage or PB. This work provides the first evidence that the ORC4 plays a necessary role for polar body extrusion.
Description:Ph.D. Thesis. University of Hawaiʻi at Mānoa 2017.
URI:http://hdl.handle.net/10125/62302
Rights:All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections: Ph.D. - Developmental and Reproductive Biology


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