RESEARCH ARTICLE Molecular Reproduction & Development (2009) Identification, Isolation, and RT-PCR Analysis of Single Stage-Specific Spermatogenetic Cells Obtained From Portions of Seminiferous Tubules Classified by Transillumination Microscopy CHIARA VASCO, 1 MAURIZIO ZUCCOTTI, 2 CARLO ALBERTO REDI, 3 AND SILVIA GARAGNA 1,4 * 1 Dipartimento di Biologia Animale, Universit a degli Studi di Pavia, Pavia, Italy 2 Dipartimento di Medicina Sperimentale, Sezione di Istologia ed Embriologia, Universit a degli Studi di Parma, Parma, Italy 3 Fondazione I.R.C.C.S. Policlinico San Matteo, Pavia, Italy 4 Centro di Ingegneria Tissutale, Universit a degli Studi di Pavia, Pavia, Italy SUMMARY The protocol here described allows the analysis of gene expression in single specific mouse spermatogenetic cells. Germ cells were singularly isolated by microdissection of portions of seminiferous tubules classified, based on their transillumination pattern, into four distinct zones along their length. Single portions of a seminiferous tubule, corresponding to specific zones, were mechanically disaggregated into single cells that were (1) identified as spermatogonia, spermatocytes, round or elongated sper- matids, (2) isolated using a micromanipulator, and (3) singularly transferred into a test tube for retro-transcription PCR analysis. On each single isolated cell, we have determined the quantitative profile of expression of Gapdh, an endogenous house- keeping gene known to be expressed throughout spermatogenesis. The protocol described allows an accurate analysis of the temporal and quantitative profile of gene expression throughout the whole male gamete differentiation process which so far has mainly been performed on enriched population of cells. Mol. Reprod. Dev. 2009. ß 2009 Wiley-Liss, Inc. Received 10 March 2009; Accepted 19 June 2009 * Corresponding author: Dipartimento di Biologia Animale Universit a degli Studi di Pavia Piazza Botta 9, Pavia PV 27100, Italy. E-mail: silvia.garagna@unipv.it Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.21086 INTRODUCTION Spermatogenesis is a dynamic process during which undifferentiated diploid stem cells mature to differentiat- ed haploid cells called spermatozoa (Parvinen, 1982; McCarrey et al., 1992; De Rooji and Grootegoed, 1998; Eddy, 2002; Wiederkehr et al., 2002, 2004). Mammalian spermatogenesis occurs within the seminiferous tubules and consists of three main phases: a first mitotic phase, in which spermatogonia A divide mitotically until giving rise to intermediate and these to type B spermatogonia; a second meiotic phase, in which spermatocytes divide to form haploid round spermatids and a third phase, spermio- genesis, in which spermatids encompass a series of drastic morphological changes including acrosome formation, Additional Supporting Information may be found in the online version of this article. ß 2009 WILEY-LISS, INC.