VASP is regulated by GDNF both at transcriptional and post-transcriptional levels.

<p>Intact seminiferous tubules (a–b) or GS cells (c–d) were cultured in the presence of an increasing concentration of GDNF. VASP expression level was analyzed at the mRNA level by real time PCR (a, c) and at the protein level by western blot analysis (b, d). (b, d) Representative blots are shown, and histograms at the bottom of the blots show the average data obtained by densitometric analysis of the Ser153 p-VASP (left) and the VASP (right) immunoreactive bands from three different experiments. The results (mean ± SEM) are expressed as density in arbitrary units. *P<0.05 vs. control; **P<0.001 vs. control; ns: not significant vs. control.</p

The Niche-Derived Glial Cell Line-Derived Neurotrophic Factor (GDNF) Induces Migration of Mouse Spermatogonial Stem/Progenitor Cells

<div><p>In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.</p></div

VASP correlates with GFRA1 expression in undifferentiated spermatogonia.

<p>(a–c) Intact seminiferous tubules were immunostained with anti-VASP (red), anti-GFRA1 (cyan) and anti-PLZF antibodies (green), and single Z-sections were acquired at the confocal microscope. A merging of the pictures is shown in the right column. (a) The intensity of the VASP staining in GFRA1-expressing cells (arrowheads) is higher than GFRA1 dull/negative (arrows). (b–c) Seldom we found a pair of cells (identified by PLZF expression) that were VASP and GFRA1 co-segregated in one of the two cells. Arrowhead: PLZF-positive, GFRA1-high, VASP-high cells; arrows: PLZF-positive, GFRA1-dull/negative, VASP- dull/negative. Dashed lines: outline of the seminiferous tubules. Scale bars: 20 µm.</p

GDNF-induced migration in the GC-1 spermatogonial cell line is rescued by GFRA1 expression.

<p>(a) Western blot analysis of protein extracts from immature and adult testis and GC-1 cell lines. GC-1 cells express Ret but not GFRA1. (b) Immunofluorescence detection of GFRA1 (red) in GC-1 cells and GFRA1-transduced GC-1 cells. Nuclei were stained with TOTO-3. (c, d) GDNF-induced migration in GC-1 cells is rescued by expression of GFRA1. (c) Parental GC-1 cells, n = 3; (one-way ANOVA). (d) GFRA1-transduced GC-1 cells, n = 3; *P<0.05 vs. control (one-way Anova, Student-Newman-Keuls post-hoc).</p

GDNF induces the migration of undifferentiated spermatogonia.

<p>Cell migration was evaluated using the Boyden chamber assay, as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059431#s2" target="_blank">Materials and Methods</a> section. GS cells subcultured on laminin (a) or Thy-1-selected cells (b) were treated at 100 ng/ml GDNF, 15 µg/ml neutralizing antibody, or, to abrogate the GDNF gradient in the Boyden chamber, 100 ng/ml GDNF was added in both the upper and lower chambers. The results are shown as the migration index, which was calculated as the fold increase over the control with no GDNF. Data are expressed as the mean ± SEM from n = 3 experiments, measured in triplicate. *P<0.05 vs. control with no GDNF (one-way ANOVA, Student-Newman-Keuls post hoc).</p

DataSheet_1_cAMP-specific phosphodiesterase 8A and 8B isoforms are differentially expressed in human testis and Leydig cell tumor.pdf

Cyclic adenosine monophosphate/Protein kinase A (cAMP/PKA) signaling pathway is the master regulator of endocrine tissue function. The level, compartmentalization and amplitude of cAMP response are finely regulated by phosphodiesterases (PDEs). PDE8 is responsible of cAMP hydrolysis and its expression has been characterized in all steroidogenic cell types in rodents including adrenal and Leydig cells in rodents however scarce data are currently available in humans. Here we demonstrate that human Leydig cells express both PDE8A and PDE8B isoforms. Interestingly, we found that the expression of PDE8B but not of PDE8A is increased in transformed Leydig cells (Leydig cell tumors-LCTs) compared to non-tumoral cells. Immunofluorescence analyses further reveals that PDE8A is also highly expressed in specific spermatogenic stages. While the protein is not detected in spermatogonia it accumulates nearby the forming acrosome, in the trans-Golgi apparatus of spermatocytes and spermatids and it follows the fate of this organelle in the later stages translocating to the caudal part of the cell. Taken together our findings suggest that 1) a specific pool(s) of cAMP is/are regulated by PDE8A during spermiogenesis pointing out a possible new role of this PDE8 isoform in key events governing the differentiation and maturation of human sperm and 2) PDE8B can be involved in Leydig cell transformation.</p