Recovery of Spermatogonial Stem Cells and Progenitors Post Inhibition of GDNF Signaling

The testicular histology of some men with nonobstructive azospermia (NOA) suggests they have lost significant numbers of spermatogonial stem cells (SSCs) and their immediate progeny, progenitor spermatogonia (PS). In order to understand the dysfunction in those cases of NOA, we used a chemical-genetic approach to study how mouse SSCs and PS are restored after their numbers are depleted due to inhibition of glial cell line-derived neurotrophic factor (GDNF) signaling. GDNF signaling is required for the formation and maintenance of mouse SSCs and PS, and we hypothesized that this restoration of SSCs and PSs is correlated with increased expression of GDNF. To test this hypothesis, we temporarily inhibited GDNF signaling for 9 days and testes were collected on days 10 through 28 of the experiment. One day prior to collection, mice were injected with the thymidine analogue EDU to label replicating cells. We then determined the numbers of spermatogonia expressing GFRα1, a marker of SSCs and PS, the fraction replicating, and the message levels of GDNF. Measurement of GFRα1+ cell number following treatment showed significant GFRα1+ cell loss at day 10 where cell number was reduced 11-fold in respect to controls. However, by day 28, cell number was reduced 2-fold in respect to controls displaying recovery of cell number over time. This recovery was associated with an increase in GFRα1+ spermatogonia replication, especially in the subset that contains SSCs. Surprisingly this recovery was not associated with an increase in testis content of GDNF transcripts or protein. Neither was there an increase in expression of transcripts encoding FGF2 and CXCL12, two other paracrine factors required by mouse SSCs. As a result, we conclude that while temporary loss of GDNF signaling depletes SSCs and PS, the restoration of these cells is not dependent on increased expression of any known paracrine factors that regulate SSCs. Thus, this restoration may be driven by intrinsic factors to these cells or by other unknown paracrine factors. Discovery of the mechanisms driving this restoration may provide valuable insight to the etiology of the human NOA phenotype. Supported by (R01HD074542−01).