MP76-10 MSH5 DEFICIENCIES IN A SUBSET OF NOA MEN

Abstract

You have accessJournal of UrologyInfertility: Basic Research, Physiology, Pathophysiology1 Apr 2015MP76-10 MSH5 DEFICIENCIES IN A SUBSET OF NOA MEN Koji Chiba, Alex Ridgeway, Larry Lipshultz, Masato Fujisawa, and Dolores Lamb Koji ChibaKoji Chiba More articles by this author , Alex RidgewayAlex Ridgeway More articles by this author , Larry LipshultzLarry Lipshultz More articles by this author , Masato FujisawaMasato Fujisawa More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2795AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Spermatogenesis requires prolific cellular division and high fidelity DNA replication, as well as homologous recombination during meiosis. DNA repair systems are needed to correct cellular insults or to commit the cell to apoptosis if repair is not possible. MSH5, which is mainly expressed in testis, is a key component of the DNA repair pathways required for meiosis. MSH5 plays an important role in the repair of DNA double-strand breaks (DSB). When DSB occur, MSH5 interacts with c-abl. The activation of C-ABL then induces p73 accumulation. Because MSH5 and p73 are required in mouse models for spermatogenesis, we sought to define their expression in infertile men. METHODS Fibroblasts were cultured using testicular biopsy samples obtained from non-obstructive azoospermia (NOA) men (n=31). For fertile controls (n=14), men who underwent vasectomy reversal provided the fibroblasts. A global DNA methylation profile using the Infinium HumanMethylation450 BeadChip was obtained on these patients. MSH5 gene expression was quantified by qPCR and Western blot. Fibroblasts were then treated with Neocarzinostatin (NCS), a potent drug that induces DSB. Cell growth after NCS treatment was evaluated using WST assay, and comet assay was performed to evaluate DSB formation. MSH5, p73 gene expression was quantified by qPCR before and after treatment with NCS. RESULTS DNA methylation was significantly increased at a CpG island that spans the promoter region to exon 2 of MSH5. Specifically, five terminal CpGs in exon 2 of MSH5 were hypermethylated in a subset of NOA men tested (6 out of 31). This cohort also displayed decreased MSH5 gene expression and reduced MSH5 protein. The cell growth assay showed that NCS severely inhibited fibroblast growth after treatment in NOA- hypermethylated MSH5 group, whereas only mild cell growth inhibition was evident in fertile group. A comet assay showed a significant comet halo after NCS treatment in both groups, showing that DSB was induced by NCS treatment. Fertile control cells rapidly repaired the DSB whereas cells with hypermethylated MSH5 did not. NCS administration to the fertile group increased MSH5 and p73 gene expression. In contrast, expression of these genes remained low after NCS treatment of the NOA group. CONCLUSIONS NOA patients frequently show increased DNA methylation of MSH5 with down-regulation of its gene expression. In these men, MSH5 and p73 gene expression failed to respond to DSB formation caused by NCS. Down-regulation of the MSH5 and p73 pathways may be involved in spermatogenic failure in NOA patients and underlie other additional health concerns for the patient. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e987-e988 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Koji Chiba More articles by this author Alex Ridgeway More articles by this author Larry Lipshultz More articles by this author Masato Fujisawa More articles by this author Dolores Lamb More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...