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J. Biol. Chem., Vol. 282, Issue 35, 25842-25851, August 31, 2007
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Glial Cell Line-derived Neurotrophic Factor Regulation of Genes Essential for Self-renewal of Mouse Spermatogonial Stem Cells Is Dependent on Src Family Kinase Signaling*
From the Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Self-renewal and differentiation by spermatogonial stem cells (SSCs) is the foundation for continual spermatogenesis. SSC self-renewal is dependent on glial cell line-derived neurotrophic factor (GDNF); however, intracellular mechanisms stimulated by GDNF in SSCs are unknown. To investigate these mechanisms we utilized a culture system that maintains a mouse undifferentiated germ cell population enriched for self-renewing SSCs. In these cultures mRNA for the transcription factors Bcl6b, Erm, and Lhx1 are up-regulated by GDNF and decreased in its absence. The expression of all three molecules was further identified in undifferentiated spermatogonia in vivo. Using small interfering RNA to reduce expression and transplantation to quantify stem cell numbers, Bcl6b, Erm, and Lhx1 were shown to be important for SSC maintenance in vitro. Next, GDNF was shown to activate both Akt and Src family kinase (SFK) signaling in SSCs, and culture of SSCs with inhibitors to Akt or SFKs followed by transplantation analysis showed significant impairment of SSC maintenance in vitro. Apoptosis analysis revealed a significant increase in the percentage of apoptotic cells when Akt, but not SFK, signaling was impaired, indicating that multiple signaling pathways are responsible for SSC self-renewal and survival. Biochemical and gene expression experiments revealed that GDNF up-regulated expression of Bcl6b, Erm, and Lhx1 transcripts is dependent on SFK signaling. Overall, these data demonstrate that GDNF up-regulation of Bcl6b, Erm, and Lhx1 expression through SFK signaling is a key component of the intracellular mechanism for SSC self-renewal.
Received for publication, April 25, 2007 , and in revised form, June 26, 2007.
* The research was supported by the NICHD, National Institutes of Health Grants HD044445 and HD052728 and by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-5, Table 1, and experimental procedures.
1 Current address: Dept. of Dairy and Animal Sciences, Pennsylvania State University, University Park, PA 16802.
2 To whom correspondence should be addressed. Tel.: 215-898-8805; Fax: 215-898-0667; E-mail: cpope{at}vet.upenn.edu.
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