THE ROLE OF GDNF SIGNALING IN MAINTENANCE OF SPERMATOGONIAL STEM CELLS AND PROGENITOR SPERMATOGONIA IN THE MATURE MOUSE TESTIS
Parker, Nicole A
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Infertility affects about 10% of couples around the world and roughly half of the cases are due to infertility of men. Amongst those cases are men who are infertile due to the lack and/or dysfunction of spermatogonial stem cells (SSCs). As a consequence, the testes of these men display the nonobstructive azoospermia (NOA)/ Sertoli cell only (SCO) histological phenotype. Recent analyses indicate that substantial numbers of SSCs remain in some human SCO testes, raising the possibility they are therapeutic targets. But development of new therapies requires that we first obtain a precise and detailed understanding of the regulation of SSCs in the normal adult testes. A goal of this dissertation is to contribute to this effort by using a novel, chemical-genetic approach that reversibly inhibits signaling to mouse SSCs and their immediate progeny, the progenitor spermatogonia through glial cell line-derived neurotrophic factor (GDNF). In mice SSCs are classically defined as a subset of morphologically defined A single (As) spermatogonia, which can produce more SSCs or they can give rise to As cells that act as progenitors and generate A paired (Apr) and then A aligned (Aal) spermatogonia. It is well established that GDNF is essential for sustaining SSCs in culture and for their formation in newborn mouse testis. However, when we initiated our experiments, the specific function of GDNF in the normal adult testis was not well explored. Our experiments use three different experimental approaches to demonstrate that GDNF is critical for maintaining SSCs and progenitor spermatogonia in vivo in the mature mouse testis. We reveal that GDNF stimulates the replication and suppresses the differentiation of these cells. We also demonstrate that the response to altered GDNF signaling changes as As cells form Apr and then Aal spermatogonia. When GDNF signaling is inhibited, numbers of As spermatogonia decrease more slowly than Apr and Aal spermatogonia. This is consistent with data from other experiments indicating that when GDNF signaling is inhibited, numbers of SSCs decrease more slowly than numbers of progenitor spermatogonia. We then shifted our focus to explore the response of SSCs and progenitor spermatogonia to restoration of GDNF signaling after its inhibition for 9 days. Our data demonstrates that while all of these cells are gradually restored, numbers of Aal spermatogonia increase before numbers of As and Apr spermatogonia. This restoration of As, Apr, and Aal cells is achieved by a rapid increase in cell replication and a slower suppression of differentiation. Taken together, our results demonstrate that GDNF is an essential regulator for SSC and progenitor spermatogonia number, replication, and differentiation in the mature mouse testis. In addition the data reveals there are significant changes in the responses to GDNF as SSCs give rise to progenitor spermatogonia. We then asked the question, what drives the rebuilding of the SSCs and progenitor spermatogonia once GDNF signaling is restored? We demonstrated that this rebuilding process is not associated with increased transcriptional expression of GDNF or any other known extrinsic regulators of SSCs and its progenitors. However, transcriptional analysis of control and treated testes lead us to hypothesize that regulators intrinsic to these cells play an important role in their recovery once GDNF signaling is restored. Fully elucidating the function of GDNF and molecular mechanisms involved in maintaining and restoring SSC and progenitor spermatogonia number may provide a better understanding of the biological basis of infertility of men with deficient or dysfunctional SSCs.