REPRODUCTION RESEARCH Establishment of long-term monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen Gillian Cowan, Andrew J Childs, Richard A Anderson 1 and Philippa T K Saunders MRC Human Reproductive Sciences Unit, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK and 1 Division of Development and Reproductive Sciences, Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK Correspondence should be addressed to P T K Saunders; Email: p.saunders@ed.ac.uk Abstract The somatic (Sertoli cell (SC), Leydig cell (LC), and peritubular myoid (PTM) cell) cells play key roles in development of the fetal testis. We established monolayer cultures from second trimester human testes and investigated the pattern of expression of cell-lineage characteristic mRNAs. Expression of some SC-associated genes (SRY , SOX9, WT1, GATA4, and SF1) was detectable up to and including passage 3 (P3), while others (anti-Mu ¨llerian hormone; desert hedgehog) present prior to dissociation were not expressed in the cultured cells. Transcripts encoding the androgen receptor were expressed but addition of dihydrotestosterone (DHT) had no impact on expression of mRNAs expressed in SC or LC. Total concentrations of mRNAs encoding smooth muscle actin (ACTA2) and desmin increased from P1 to P3; an increasing proportion of the cells in the cultures were immunopositive for ACTA2 consistent with proliferation/differentiation of PTM cells. In conclusion, somatic cell monolayer cultures were established from human fetal testes; these cultures could form the basis for future studies based on isolation of purified populations of somatic cells and manipulation of gene expression that is difficult to achieve with organ culture systems. Our results suggest that fetal SC do not maintain a fully differentiated phenotype in vitro, yet PTM (ACTA2 positive) cells readily adapt to monolayer culture conditions in the presence of DHT. This culture system provides an opportunity to study the impact of regulatory factors on gene expression in PTM cells, a population thought to play a key role in mediating androgen action within the developing testis. Reproduction (2010) 139 749–757 Introduction Normal maturation of the germ cell population and establishment of the male phenotype is regulated by factors produced by the somatic cell populations in the fetal testis including Sertoli cell (SC), Leydig cell (LC), and peritubular myoid (PTM) cell types. In the human fetal gonad, testis cords consisting of SC and fetal germ cells (gonocytes) form between the 7th and 9th weeks of gestation. In the second trimester, the cords are surrounded by a layer of PTM cells and a substantial interstitial compartment containing steroidogenically active LC and blood vessels (Gaskell et al. 2004). Specification and differentiation of the SC lineage are central to male gonadal development and begins when cells within the genital ridge differentiate into SC precursors in response to the expression of a protein encoded by the sex-determining region on the Y chromosome (SRY) gene (Hacker et al. 1995, Bullejos & Koopman 2001) expression of which is associated with upregulation of male-specific genes including the SRY-like HMG-box protein SOX9 (Bishop et al. 2000, Vidal et al. 2001). The fetal SC also synthesise anti- Mu ¨llerian hormone (AMH): a secreted glycoprotein that plays a critical role in remodeling of the reproductive tract in males (Hacker et al. 1995, De Santa Barbara et al. 1998, Rajpert-De Meyts et al. 1999). The expression of AMH is in turn regulated by transcription factors including the orphan nuclear receptor, steroidogenic factor 1 (SF1; Sekido & Lovell-Badge 2008), a member of the GATA family of zinc finger proteins, GATA4 (Manuylov et al. 2007), and the product of the Wilms’ tumor suppressor gene (WT1; Gao et al. 2006) all of which are expressed in SC in vivo. Functional LC characterised by the expression of enzymes required for biosynthesis of testosterone including side-chain cleavage p450 (CYP11A1/SCCp450) and 3-b-hydroxysteroid dehydrogenase (HSD3B/3bHSD; Murray et al. 2000) are evident from 9 weeks gestation in the human (Gaskell et al. 2004, Ostrer et al. 2007). The interstitial compartment also contains many cells thought to be undifferentiated fibroblasts as well as cells of the vasculature including endothelial cells q 2010 Society for Reproduction and Fertility DOI: 10.1530/REP-09-0532 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org