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GDNF Signaling Regulates YBX1/mRNA Interactions in Mouse Spermatogonia

Phillips, Bart (2012) GDNF Signaling Regulates YBX1/mRNA Interactions in Mouse Spermatogonia. Doctoral Dissertation, University of Pittsburgh.

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Abstract

Spermatogenesis is a highly coordinated and productive process that maintains continuous sperm production in post-pubertal males. Spermatogonial stem cells (SSCs) are the basis of spermatogenesis and balance self-renewal with differentiation to maintain the stem cell pool while producing differentiating progenitors that give rise, through several transit-amplifying mitotic divisions, two meiotic divisions and morphological differentiation to sperm. The molecular mechanisms driving these fate decisions are poorly understood, though numerous RNA regulatory pathways are thought to be critical. We used microarray analysis of distinct testis models to identify genes whose expression correlates with SSC activity. Fourteen RNA binding proteins were identified, and their testis expression was validated using RT-PCR and immunohistochemistry. The multi-functional RNA regulator YBX1 was expressed only by cells located on the basement membrane of seminiferous tubules, consistent with the location of SSCs. YBX1 was of special interest because of its heretofore unexplored role in the testis, as well as its possible phosphorylation by AKT. The AKT signaling pathway is activated in mouse SSCs by GDNF, a factor essential for SSC survival and self-renewal. We hypothesized that YBX1 was phosphorylated by AKT signaling downstream of GDNF, and that this phosphorylation would affect YBX1 interaction with target messenger RNAs (mRNAs). We show that YBX1 is phosphorylated in spermatogonia, and that GDNF signaling modulates YBX1 phosphorylation. Inhibiting either PI3K or AKT in SSC cultures also causes a loss of YBX1 phosphorylation, suggesting that GDNF works through the PI3K/AKT signaling pathway to phosphorylate YBX1. To identify mRNA targets of YBX1, we immunoprecipitated YBX1 from SSC cultures grown in the presence or absence of GDNF, and isolated co-immunoprecipitated mRNAs. In normal growth conditions (containing GDNF), YBX1 associated with mRNA for genes expressed by all germ cells (Dazl and Ddx4), but not those expressed specifically by more undifferentiated germ cells (GFRα1, Ngn3 and Plzf). Furthermore, association of YBX1 with a specific mRNA correlates with an increase in protein levels for that gene. These data collectively lead us to conclude that YBX1 mediates GDNF-dependent translational regulation of specific transcripts that may be important for coordinating SSC maintenance, renewal and/or differentiation.


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Details

Item Type: University of Pittsburgh ETD
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Phillips, Bartbtp3@pitt.eduBTP3
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairOrwig, Kyleorwigke@upmc.edu
Committee MemberRich, Chailletchaillet@pitt.edu CHAILLET
Committee MemberEric , Lagasselagasse@pitt.edu LAGASSE
Committee MemberChristine, Milcarekmilcarek@pitt.edu MILCAREK
Committee MemberWill, Walkerwalkerw@pitt.edu WALKERW
Date: 13 December 2012
Date Type: Publication
Defense Date: 9 August 2012
Approval Date: 13 December 2012
Submission Date: 11 December 2012
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 99
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Biochemistry and Molecular Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: RNA Regulation, RNA Binding Proteins, Spermatogonial Stem Cell, Testis, YBX1, GDNF
Date Deposited: 13 Dec 2012 13:04
Last Modified: 15 Nov 2016 14:08
URI: http://d-scholarship.pitt.edu/id/eprint/16870

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