MP70-03 RHOXF2 AS A GUARDIAN OF THE HUMAN MALE GERMLINE STEM CELLS

Abstract

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2016MP70-03 RHOXF2 AS A GUARDIAN OF THE HUMAN MALE GERMLINE STEM CELLS Hye-Won Song, David Skarbrevik, Eric Babajanian, Tung-Chin Hsieh, and Miles Wilkinson Hye-Won SongHye-Won Song More articles by this author , David SkarbrevikDavid Skarbrevik More articles by this author , Eric BabajanianEric Babajanian More articles by this author , Tung-Chin HsiehTung-Chin Hsieh More articles by this author , and Miles WilkinsonMiles Wilkinson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1428AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The human germline is essential for the maintenance of our species and thus must be protected from insults. A candidate to have a role in germline protection is the X-linked reproductive homeobox (RHOX) gene cluster, which encodes a large set of transcription factors expressed in the reproductive tract, including male and female germ cells. While the RHOX transcription factors have been well studied in rodents, little is known about their function in humans. Of particular interest is human RHOXF2, which we previously showed is expressed in human spermatogonia and regulates genes that have the potential to provide genome protective roles, including GDAP1 and UNC5C. Here we further test the hypothesis that RHOXF2 serves as a genome protector in male germ cells. We examine its effect on LINE1 elements, which are deleterious transposable elements constituting 17% of the human genome that must be suppressed to maintain genome integrity METHODS Human germ cells were obtained from testicular biopsies and cultured under different conditions. RHOXF2 expression was depleted using a RHOXF2 small hairpin (sh)RNA we cloned into the lentiviral vector. Immunostaining and quantitative (q)RT-PCR were used to test the expression of RHOXF2, as well as spermatogonial and proliferation markers. LINE1 transposition was assayed in vitro in HEK-293T cells using LINE1 luciferase reporters RESULTS Modest forced RHOXF2 expression (to a level similar to that in germ cells) repressed LINE1 transposition, as assayed using both mouse and human in vitro transposition assays, in HEK-293 cells. A mutant version of RHOXF2 present in human infertility patients failed to have this activity. Loss of the mouse RHOXF2 putative ortholog, Rhox10, led to increased expression of LINE1 and substantial reduction in the number of germline stem cells in vivo. To elucidate the underlying mechanism of RHOXF2 action, we optimized conditions for culturing spermatogonia from human testicular biopsies. We successfully obtained germ cells expressing spermatogonial markers that undergo proliferation in vitro. Using RNA interference (RNAi), we depleted RHOXF2 in these spermatogonia cultures CONCLUSIONS We provide evidence that human RHOXF2 and its putative mouse ortholog, Rhox10, serve to repress the transposition of deleterious transposable elements in male germ cells. Coupled with previous finding that RHOXF2 regulates genes critical for genome defense mechanisms, it suggests that RHOXF2 serves as a guardian of genome integrity in male germ cells © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e906-e907 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hye-Won Song More articles by this author David Skarbrevik More articles by this author Eric Babajanian More articles by this author Tung-Chin Hsieh More articles by this author Miles Wilkinson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...