MECHANISMS OF SPERMATOGONIAL STEM CELL DIFFERENTIATION

Open Access
- Author:
- Kaucher, Amy V
- Graduate Program:
- Animal Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- July 12, 2010
- Committee Members:
- Dr Jon Oatley, Thesis Advisor/Co-Advisor
Jon M Oatley, Thesis Advisor/Co-Advisor - Keywords:
- SPERMATOGONIAL STEM CELL
SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3
NEUROGENIN 3
DIFFERENTIATION - Abstract:
- In mammalian testes, the actions of spermatogonial stem cells (SSC) provide a foundation for spermatogenesis during adult life. Like other tissue-specific stem cell populations, SSC initiate differentiation while also maintaining a cohort of undifferentiated stem cells via self-renewal. Decisions to self-renew or differentiate are controlled both extrinsically and intrinsically. These decisions must be tightly controlled, as dysregulation can lead to loss of tissue homeostasis, infertility or cancer. Currently, understanding of mechanisms regulating SSC fate decisions, particularly differentiation, is limited. Therefore, the goal of the studies herein was to identify and examine molecular factors required for fate decisions of SSC. Two proteins, signal transducer and activator of transcription 3 (Stat3) and Neurogenin 3 (Ngn3), were investigated. Stat3, a transcription factor, is part of the Jak/Stat signal transduction pathway. The Drosophila Stat3 homolog, Stat92E is necessary for self-renewal germline stem cells. Also, embryonic stem cells require Stat3 signaling to maintain pluripotency. Ngn3 is a transcription factor needed for the differentiation of neurons and ƒÒ-cell progenitors. In the mammalian germline, expression of Ngn3 coincides with early germ cell differentiation. Furthermore, in rodents, the cytokine Glial cell line-derived neurotrophic factor (Gdnf) regulates SSC self-renewal and Ngn3 is known to be a Gdnf down-regulated gene. Despite this, to our knowledge, the functional roles of Stat3 or Ngn3 have yet to be examined in SSC fate decisions. In Chapter II: Regulation of SSC differentiation by Stat3, the objective was to determine the effect of Stat3 mRNA reduction on SSC fate decisions. To address the objective, Stat3-specific shRNA was used to permanently reduce Stat3 expression in cultured Thy1+ germ cells. After functional germ cell transplantation, it was apparent that short hairpin RNA-mediated stable reduction of STAT3 expression in cultured SSCs abolished SSC ability to differentiate beyond the undifferentiated spermatogonial stage following transplantation into recipient testes. Therefore, these results demonstrate that STAT3 promotes the differentiation of SSC. The objective of Chapter III: Neurogenin 3 is a regulator of mouse spermatogonial stem cell differentiation was to determine if NGN3 plays a functional role in SSC differentiation. To address this, we studied the SSC-enriched Thy1+ germ cell population, which is also composed of non-SSC spermatogonia produced by differentiation. Withdrawal and replacement of Gdnf from cultured THY1+ germ cells resulted in an increase followed by down-regulation of Ngn3 gene expression, indicating that Ngn3 transcription is repressed by GDNF signaling. Within the THY1+ germ cell population, NGN3 expression was found in a sub-fraction of cells both in vivo and after long-term culture. Transient reduction of Ngn3 expression in cultured THY1+ germ cells by siRNA treatment increased SSC content after multiple self-renewal cycles without impacting spermatogonial proliferation overall, suggesting alteration of the balance between SSC fate decisions in favor of self-renewal. Lastly, it was found that the SSC differentiation factor STAT3 binds the Ngn3 promoter to regulate Ngn3 transcription in THY1+ germ cells. Collectively, these results indicate NGN3 plays a key role in regulating differentiation of SSCs via a STAT3-mediated mechanism. Collectively, the data of these two studies indicate that Stat3 and Ngn3 work in conjunction to regulate SSC differentiation.