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Dissertation - Santanu De.pdf (4.95 MB)

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Protein 14-3-3 (YWHA) isoforms and their roles in regulating mouse oocyte maturation

De, Santanu
http://rave.ohiolink.edu/etdc/view?acc_num=kent1401803571

Abstract Details

2014, PHD, Kent State University, College of Arts and Sciences / Department of Biological Sciences.
The 14-3-3 (YWHA) is a highly conserved, ubiquitously expressed protein family regulating important cellular processes including cell cycle. This work, for the first time, explored the differential expression and roles of 14-3-3 isoforms during mouse oocyte maturation. All seven mammalian 14-3-3 isoforms were identified in mouse eggs and ovarian follicular cells including oocytes, by Western blotting. Immunocytochemical and immunohistochemical staining confirmed the presence of all 14-3-3 isoforms in oocytes, eggs and ovarian follicles with characteristic similarities and differences in their distributions. Mammalian oocytes are arrested at meiosis prophase I by an inhibitory phosphorylation on Cyclin-Dependent Kinase I (CDK1), released by CDC25B phosphatase which is bound and inactivated in phosphorylated form by 14-3-3 in oocyte cytoplasm. Here, in situ Proximity Ligation Assays (PLA) revealed that all 14-3-3 isoforms interact with CDC25B in oocytes, with reduced interactions in eggs. Phosphorylation of CDC25B at Ser-149 was found to be reduced in eggs compared to oocytes. Microinjection of a translation-blocking morpholino oligonucleotide against 14-3-3eta mRNA caused germinal vesicle breakdown in significantly higher percentage of oocytes compared to oocytes injected with morpholinos targeting other 14-3-3 isoforms. Thus, interaction of 14-3-3eta with CDC25B is required for maintaining prophase I arrest in oocytes. Protein 14-3-3eta was observed to accumulate and co-localize with alpha-tubulin at both meiosis I and II spindles during mouse oocyte maturation in vivo as well as in vitro. It interacts directly with alpha-tubulin with an accumulation of the interactions at meiotic spindles, detected by in situ PLA. In a significant 76% of mouse oocytes microinjected with the morpholino against 14-3-3eta mRNA, meiotic spindles were deformed or absent with clumped chromosomes, no accumulation of 14-3-3eta and no polar body formation. All control eggs showed normal, bipolar spindles with accumulation of 14-3-3eta. Therefore, 14-3-3eta is essential for normal meiotic spindle formation during in vitro maturation of mouse oocytes, in part by interacting with alpha-tubulin, to regulate the assembly of microtubules. These studies reveal 14-3-3 isoform-specific interactions with key proteins involved in mouse oocyte maturation, such as CDC25B and alpha-tubulin. The results help to elucidate the roles of 14-3-3 proteins in mammalian oogenesis and reproductive development.
Douglas Kline (Advisor)
Derek Damron (Committee Member)
Colleen Novak (Committee Member)
Werner Geldenhuys (Committee Member)
Jennifer Marcinkiewicz (Other)
Nicola Brasch (Other)
175 p.
Animal Sciences; Biology; Biomedical Research; Cellular Biology; Developmental Biology; Molecular Biology; Organismal Biology; Physiology
14-3-3; YWHA; spindle; CDC25B; phosphorylation; interaction; mouse; ovary; oocyte; egg; oocyte maturation

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Citations

  • De, S. (2014). Protein 14-3-3 (YWHA) isoforms and their roles in regulating mouse oocyte maturation [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1401803571

    APA Style (7th edition)

  • De, Santanu. Protein 14-3-3 (YWHA) isoforms and their roles in regulating mouse oocyte maturation. 2014. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1401803571.

    MLA Style (8th edition)

  • De, Santanu. "Protein 14-3-3 (YWHA) isoforms and their roles in regulating mouse oocyte maturation." Doctoral dissertation, Kent State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=kent1401803571

    Chicago Manual of Style (17th edition)

kent1401803571
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© 2014, all rights reserved.
This open access ETD is published by Kent State University and OhioLINK.