The Niche-Derived Glial Cell Line-Derived Neurotrophic Factor (GDNF) Induces Migration of Mouse Spermatogonial Stem/Progenitor Cells Lisa Dovere 1 , Stefania Fera 1 , Margherita Grasso 2 , Dante Lamberti 1 , Cesare Gargioli 3 , Barbara Muciaccia 1 , Anna Maria Lustri 1 , Mario Stefanini 1 , Elena Vicini 1 * 1 Fondazione Pasteur Cenci Bolognetti, Department of Anatomical, Histological, Forensic and Orthopaedic Sciences - Section of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy, 2 Centre for Integrative Biology, University of Trento, Trento, Italy, 3 Department of Biology, University of Rome Tor Vergata, Rome, Italy Abstract 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. Citation: Dovere L, Fera S, Grasso M, Lamberti D, Gargioli C, et al. (2013) The Niche-Derived Glial Cell Line-Derived Neurotrophic Factor (GDNF) Induces Migration of Mouse Spermatogonial Stem/Progenitor Cells. PLoS ONE 8(4): e59431. doi:10.1371/journal.pone.0059431 Editor: Eduardo Moreno, University of Bern, Switzerland Received November 14, 2012; Accepted February 14, 2013; Published April 22, 2013 Copyright: ß 2013 Dovere et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Italian Ministry of University (FIRB, grant number: RBIN06E9Z8_008) and Sapienza University (Ateno 2011) to E.V. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: elena.vicini@uniroma1.it Introduction A paradigm of the adult unipotent stem cell is the spermato- gonial stem cell (SSC), which sustains the daily production of millions of mature sperm throughout the male adult life through spermatogenesis. SSCs belong to a class of spermatogonia defined as ‘‘undifferentiated’’ type A spermatogonia, a hallmark of which is their typical nuclear morphology and the expression of markers such as PLZF, neurogenin3, E-cadherin, Lin-28, and GFRA1 [1;2]. Spermatogenesis is a cyclic process that in the mouse is divided into 12 stages (I-XII), each stage representing a unique association of germ cells at different steps of differentiation. The relationship between the spermatogenic stages and the kinetics of proliferation and differentiation of the spermatogonia have been analyzed in different mammalian species [2]. In all the stages, undifferentiated spermatogonia can be found as single cells (type Asingle, As) or as interconnected chains of cells composed by two (defined as Apaired: Apr) up to 32 undifferentiated spermatogonia (defined as Aaligned: Aal). Subsequently, during stages VII and VIII of the cycle, almost all of the larger chains (Aal4–Aal32) differentiate into A1 spermatogonia. In mammals, spermatogonia are located in the basal region of the seminiferous tubules, in contact with the Sertoli cells and basement membrane that separate them from the peritubular myoid cells. Interestingly, spermatogonia are not immotile, they change their relative position. Migration of undifferentiated spermatogonia was first suggested by detailed morphological analysis of the topography of spermatogonia in the mouse testis [3]. More recently, this conclusion was supported by a time-lapse analysis of GFP-labeled undifferentiated spermatogonia that were tracked in vivo for several days and were found to migrate over the basal lamina [4;5]. Migration of undifferentiated spermatogonia could ensure even distribution of germ cell progeny over the basal compartment of the seminiferous tubules [3] or may be essential to keeping stem or progenitor cells in the right environment to ensure the self-renewal of the SSCs [6]. Cell migration may be random or directed toward a chemoat- tractant gradient. Direct migration, or chemotaxis, is activated by extracellular ligands that bind to cell surface receptors, and this process can lead to reorganization of the actin and myosin cytoskeletons and, finally, to cell movement. It has been recently shown that Sertoli cells chemoattract only stem/progenitor PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e59431