1886 MSH6 MISMATCH REPAIR DEFICIENCIES IN MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA (NOA)

Vol. 183, No. 4, Supplement, Wednesday, June 2, 2010

lencing of several developmentally important genes. Identification of H4K12ac-binding genes in sperm was followed by a comparison of our data with sperm promoter methylation datasets. METHODS: Chromatin immunoprecipitation (ChIP) assay with an anti-H4K12ac antibody was performed on ejaculated sperm from healthy donors. For microarray analysis (chip), co-hybridization of enriched probe with Cy5 and input with Cy3 was performed and log2-ratio was measured for each feature on the array (HG18, NimbleGene). Dataset for sperm methylation was obtained from Kim et al., 2007 and Weber et al., 2007. RESULTS: Alignments of our ChIP-on-chip data with custom tracks along chromosomes 6, 11, 16, 17 and 19 clearly showed H4K12ac association with regions of high gene density and regions of promoter DNA hypermethylation. While PAX genes exhibited consistent binding to H4K12ac, their promoters have been demonstrated to be hypermethylated in spermatozoa. HOX genes, however, was found to be strongly unmethylated. CONCLUSIONS: Comparison of our data with a sperm DNAmethylation database revealed that histone-packaged DNA, in general, is methylated. However, there are important exceptions to this rule including genes that are essential for embryonic development. These results strongly suggest that, I addition, a global epigenetic marking of spermatozoal chromatin is established that may direct chromatin repackaging events during spermiogenesis. Source of Funding: German Research Foundation

1886 MSH6 MISMATCH REPAIR DEFICIENCIES IN MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA (NOA) Kathleen Hwang*, Sarmistha Mukherjee, John W. Weedin, Josephine B. Addai, Larry I. Lipshultz, Dolores J. Lamb, Houston, TX INTRODUCTION AND OBJECTIVES: Mutations in mismatch repair (MMR) genes are associated with hereditary nonpolyposis colorectal cancer (HNPCC), which is an autosomal dominant cancer susceptibility syndrome. Deletion of mismatch repair genes such as mlh1 and msh2 (and other DNA repair genes) in rodents results in susceptibility to cancer development and male infertility with testicular pathologies similar to that in men with NOA. Earlier work from our laboratory identified defects in MSH2 gene expression and subcellular localization in some men with NOA. MSH2 and MSH6 form a heterodimeric MutS␣ complex, are phosphorylated, and are responsible for removal of mismatch base pairs. Mutations in MMR (common in HNPCC) lead to microsatellite instability and are associated with increased incidence of other malignancies. We tested the hypothesis that defects in MSH6 are associated with NOA. METHODS: A group of 63 men with idiopathic NOA, without evidence of Y microdeletion, provided testis biopsies for fibroblast culture. All these patients underwent testicular sperm extraction and intracytoplasmic sperm injection (ISCI) with in vitro fertilization to attempt to conceive a pregnancy. The control group consisted of fibroblast culture, derived from vasectomy specimens of 56 normal fertile men. Genomic DNA and RNA are extracted from confluent plates for characterization of mutations and gene expression studies of MSH6 proteins. Cell lysates were further analyzed for immunoblots and immunoprecipitated with MSH6 antibodies. RESULTS: Genomic DNA analysis of the 15 exons of the MSH6 gene spanning 24072 bp by DHPLC revealed the presence of polymorphism and genetic aberrations in exons 4 and 6 in 4/63 patients total and none of the fertile controls. Immunoprecipitation with MSH6 antibodies revealed a single band at 163 kDa. Gene expression studies by real time RT-PCR of the MSH6 gene will reveal changes in the overall levels of MSH6 mRNA. CONCLUSIONS: When mismatch repair proteins are damaged, the cells have a “mutator” phenotype with greatly increased rates of spontaneous mutation and may exhibit microsatellite instability. By elucidating the pathway for these combined genetic aberrations, one can better identify the associated health risk for children conceived by

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ICSI and whether their male factor fathers are at increased risk for cancer development. Our long-term goal is to define the molecular cause of NOA. We also hope to better define the interactions between the heterodimeric formation of MSH2 and MSH6. Source of Funding: Supported in part by NIH grants P01 HD36289 and K12 DK0083014, the Multidisciplinary K12 Urologic Research (KURe) Career Development Program to KH and DJL

1887 THE INTERACTION OF MODIFIED HISTONES WITH THE BROMODOMAIN TESTIS-SPECIFIC (BRDT) GENE AND ITS MRNA LEVEL IN SPERM OF FERTILE AND INFERTILE MEN Cornelia Steilmann, Marcia Cavalcanti, Marek Bartkuhn, Wolfgang Weidner, Klaus Steger, Agnieszka Paradowska*, Giessen, Germany INTRODUCTION AND OBJECTIVES: Binding of acetylated histones to BRDT gene in sperm may cause gene expression while methylation contributes to the silencing of developmentally important genes after fertilization. There is no evidence for histones binding sites in BRDT gene locus in sperm. Here, we analyzed the interaction between BRDT and differentially modified histones. Futhermore, the mRNA level of BRDT has been studied in order to find correlation between epigenetic changes and infertility. METHODS: A ChIP (Chromatin Immunoprecipitation)assay was performed with ejaculate of fertile (n⫽10) and infertile men (n⫽10) using antibodies against acetylated (H3K9ac, H4K12ac) and methylated (H3K9dm, H3K9tm, H3K27tm) histones. Immunoprecipitated DNA was analysed by real-time quantitative PCR (RTquPCR) with primer pairs for BRDT in promoter and in exon region.The BRDT mRNA level was screened by real-time reverse transcriptase PCR in spermatozoa from 6 healthy donors and 49 infertile patients. Finally,Spearman correlation coefficients were calculated for evaluation of proportional values between BRDT mRNA level and diagnostic parameters. RESULTS: In spermatozoa from fertile men, H3K9ac exhibited a relative enrichment to the input in all regions of the BRDT gene. The highest enrichment was found in promoter region 2 (9%) and the lowest in promoter region 1 (2%). Histones H3K9dm, H3K9tm and H3K27tm showed an enrichment to the BRDT binding site (2-21%) with the lowest values in the exon. In spermatozoa of infertile H3K9 were lower in the promoter regions (1-4%), but 4 times higher in the exon than in fertile men. Methylated histones exhibited a lower enrichment than in fertile men (0-5%), except for H3K27tm (3-24%). Within the exon, H3K27tm exhibit an enrichment 12 times higher than in fertile men. BRDT mRNA was identified in sperm from both fertile and infertile men, however BRDT fold change was 2 times higher in a group of infertile patients(Tuckey’s test p⬍ 0.0466). Correlation trend was observed between BRDT mRNA level and sperm morphology, (Spearman Correlation Coefficients r⫽0.07)as wll as aniline blue staining (2⫹3)(r⫽0.08). CONCLUSIONS: Reduced histone methylation in promoter of BRDT may be associated with increased level of its transcript in infertile patients. As normal sperm morphology and efficient chromatin condensation are standard parameters used by intracytoplasmatic morphologically selected sperm injection, to improve fertilization, the correspondence between BRDT level with sperm morphology and chromatin condensation is not unexpected. Source of Funding: Financial support for the study was provided by German Society of Andrology and Bayer Vital GmbH