- Email: [email protected]
683 MONO-(2-ETHYLHEXYL) PHTHALATE DECREASES CLAUDIN-11 AND OCCLUDIN IN RAT SERTOLI CELLS
resection (aRP) and external beam radiation therapy (EBRT) in men with clinical T3 prostate cancer (cT3 PCA). Materials & Methods: Between April 1994 and August 2006, a total of 231 Japanese patients with cT3 PCA underwent either aRP (n = 112) or EBRT (n = 119) at our hospital; their intermediate-term oncological outcomes were analyzed retrospectively. Eighty-six patients in the aRP group and 114 in the EBRT group received neoadjuvant androgen deprivation therapy (ADT) before each definitive therapy. Twenty-five patients in the EBRT group continued ADT after EBRT. The median total radiation dose was 70 Gy. Results: Median follow-up periods in the aRP and EBRT groups were 78 and 72 months, respectively. Although the median age in the aRP group (67 years) was significantly younger than that in the EBRT group (72 years) (p < 0.001), there were no significant differences between the groups in PSA level (24.3 ng/mL in aRP and 36.0 ng/mL in EBRT; p = 0.17), biopsy Gleason score (GS) (5-6/7/8-10 in 14/52/45 aRP patients and 14/46/55 EBRT patients; p = 0.225) or clinical T stage (cT3a/b in 92/22 aRP patients and 83/33 EBRT patients; p = 0.077). During follow-up, 8 patients in the aRP group and 26 in the EBRT group (a total of 34 patients) died. Of these 34 deaths, 5 in the aRP group and 7 in the EBRT group were cancer-related. The 10-year local progression-free and distant metastasis-free survival rates were 90.0% and 73.3% in the aRP group and 78.7% and 87.0% in the EBRT group (p = 0.23, p = 0.21), and the 10-year cancer-specific and overall survival rates were 92.1% and 87.4% in the aRP group and 87.7% and 58.1% in the EBRT group (p = 0.30, p = 0.0002). The aRP group exhibited a more favorable trend toward local cancer control and cancer-specific survival and the only significant difference between the groups was in overall survival rate. Oncological outcomes were also compared with respect to age and different GS categories. The 10-year cancerspecific and overall survival rates in aRP patients over the age of 70 were 100% and 84.6%, respectively. The 10-year cancer-specific survival rate of patients with GS≤ 6 was 100% in both groups, but those of patients with GS 8-10 were 96.0% in the aRP group and 76.0% in the EBRT group (p = 0.079). Conclusions: In cT3 PCA, surgery (aRP) gave results almost as good as those of EBRT (+ADT) in terms of oncological outcome, and yielded particularly good local cancer control. Because patients with cT3 PCA constitute a heterogeneous cohort, however, the decision to pursue optical treatment should be based on other factors, in particular, age and GS.
Poster Session 57 INFERTILITY Sunday, 20 March, 15.45-17.15, Hall F1
681
Co-localisation of two active epigenetic histone marks, H4K12ac and H3K9ac, in gene promoters of spermatozoa from fertile donors and subfertile patients
Paradowska A.1, Miller D.2, Bartkuhn M.3, Weidner W.1, Steger K.1 1 Justus Liebig University, Dept. of Urology, Pediatric Urology and Andrology, Giessen, Germany, 2University of Leeds, Reproduction and Early Development Unit, Institute of Genetics and Health Therapeutics, Leeds, United Kingdom, 3 Justus Liebig University, Institute for Genetics, Giessen, Germany Introduction & Objectives: During spermiogenesis, approximately 85% of somatic histones are replaced by protamines. The remaining histones have been proposed to carry essential marks for the establishment of epigenetic information in the offspring. This study aims at analysing the genome-wide binding pattern of two active epigenetic histone marks, namely histone H4 acetylated at lysine 12 (H4K12ac) and histone H3 acetylated at lysine 9 (H3H9ac) in order to identify aberrant binding patterns between fertile donors and subfertile patients. Materials & Methods: Using antibodies against H4K12ac and H3K9ac, we performed chromatin immunoprecipitation (ChIP) on DNA from ejaculated spermatozoa from both fertile donors (control) and subfertile patients exhibiting impaired sperm chromatin condensation as assessed by aniline blue staining. While H4K12ac immunoprecipitates were analysed by hybridisation on HG18 human promoter arrays (NimbleGen), H3K9ac immunoprecipitates were placed on ENCODE (encyclopaedia of coding elements, NimbleGen) arrays containing promoters and intergenic sequences of 375 genes. Peak finding algorithms and data overlapping were performed applying R software (http://www.r-project.org). Results: In fertile donors, co-localization of binding sites for H4K12ac and H3K9ac could be demonstrated in 38 gene promoters. Ten of these genes are involved in reproductive processes and embryonic development, e.g. AFF4 (AF4/FMR2 family, member 4), AXIN1, NCOA6 (nuclear receptor coactivator 6), OR52A1 (olfactory receptor, family 52, subfamily A, member 1). A global depletion of H4K12ac occupancy has been identified in the group of subfertile patients (496 from fertile donors vs. 146 from subfertile patients). While H3K9ac was associated with 84 gene promoters in fertile donors, only 15 gene promoters interacting with H3K9ac could be demonstrated in subfertile patients. In the latter group, no co-localization of H4K12ac and H3K9ac could be observed with any of the investigated gene promoters. Conclusions: Both our data and data from AFF4-deficient mice, the offsprings of
which have been demonstrated to die either in-utero or neonatally due to impaired embryonic development (Urano et al. 2005), suggest that aberrant acetylation of H4K12ac and/or H3K9ac within developmentally important gene promoters in subfertile men may reflect insufficient sperm chromatin compaction and, as a consequence, may lead to inappropriate transfer of epigenetic information to the oocyte.
682
Reduced expression of chromatin remodelling factors: A reason for round spermatid maturation arrest and idiopathic male infertility?
Schagdarsurengin U.1, Steilmann C.1, Bergmann M.2, Kliesch S.3, Weidner W.1, Steger K.1 1 Justus-Liebig-University Giessen, Dept. of Urology, Paediatric Urology and Andrology, Giessen, Germany, 2Justus-Liebig-University Giessen, Dept. of Veterinary Anatomy, Histology and Embryology, Giessen, Germany, 3University of Münster, Center of Reproductive Medicine and Andrology, Münster, Germany Introduction & Objectives: Human spermiogenesis is associated with histone to protamine exchange and a high grade chromatin condensation. These processes are supported by nuclear transition proteins and chromatin remodelling factors. Ten chromatin remodelling factor families are known. This study aims to analyse whether a different chromatin remodelling factor expression pattern exists between normal spermatogenesis and round spermatid maturation arrest as potential reason for impaired spermatogenesis and idiopathic male infertility. Materials & Methods: Laser capture microdissection was used to excise seminiferous tubules from testicular biopsies with normal spermatogenesis (n=7) and round spermatid maturation arrest (n=7). RNA was isolated, first strand cDNA synthesis and pre-amplification were performed using Epigenetic Chromatin Remodelling Factors PCR arrays with 84 genes. Results: Applying hierarchical cluster analysis, three gene expression clusters with six subgroups were identified. The expression pattern ranged from a few genes upregulated in round spermatid maturation arrest to a multitude of genes (74) with a higher expression in normal round spermatids. 22 genes exhibited a significant difference: 21 genes showed a crucial reduced expression in round spermatid maturation arrest (e.g. BMI1, PCGF1/F5/F6, EZH2, MBD4 and CTCF) compared to normal round spermatids; one gene, BRWD3, revealed a significant higher expression in round spermatid maturation arrest. Conclusions: Reduced expression of chromatin remodelling factors may lead to aberrant chromatin condensation during spermatogenesis and to round spermatid maturation arrest, respectively.
683
Mono-(2-ethylhexyl) phthalate decreases claudin-11 and occludin in rat Sertoli cells
Chiba K.1, Kondo Y.2, Li F.1, Ando M.1, Yamaguchi K.1, Ishikawa T.3, Fujisawa M.1 1 Kobe University Graduate School of Medicine, Dept. of Urology, Kobe, Japan, 2 Hyogo Cancer Center, Dept. of Urology, Akashi, Japan, 3Ishikawa Hospital, Dept. of Urology, Himeji, Japan Introduction & Objectives: Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Sertoli cell tight junctions (TJs) are believed to be crucial for spermatogenesis, and several TJ proteins including claudin-11, occludin, and ZO-1 have been identified. To date, it has been reported that several endocrine disruptors may affect Sertoli cell TJ integrity. In this study, we examined the association of Mono-(2-etylhexyl) Phthalate (MEHP) and TJ proteins in cultured rat Sertoli cells. We also investigated whether Sertoli cells respond to MEHP treatment by activating mitogen-activated protein kinases (MAPK) signaling pathways. Materials & Methods: Sertoli cells were isolated and purified from the testes of 18-d-old Sprague Dawley rats and incubated at 34 °C. On day 3 ex vivo, Sertoli cells were treated with MEHP (1µM, 10µM, or 100µM). At 0.5, 1, 3, 6, and 24 h after either vehicle (control) or MEHP, whole cell lysates for protein and total RNA were isolated from each replicate. Claudin-11, occludin, and ZO-1 mRNAs were evaluated by quantitative real-time PCR (QRT-PCR) analysis. Western blot analysis was used to determine the activation of extracellular signal-related kinases 1 and 2 (ERK1/2), stress-activated protein kinase/Jun-N-terminal kinase (SAPK/ JNK), p38 mitogen activated protein kinase (p38 MAPK). We also examined nitric oxide synthases (NOSs), the members of the tumor necrosis factor (TNF) ligands (Fas L and TRAIL) and its receptor (Fas) expressions by Western blot analysis. Results: In each concentration, MEHP treatment led to significant decreases in claudin-11 and occludin mRNA, but not in ZO-1. Fas, Fas L, TRAIL, and NOSs proteins were not affected by MEHP. The MEHP exposure induced the phosphorylation of ERK1/2, whereas the phosphorylation of p38 MAPK and SAPK/ JNK were not induced. When specific ERK1/2 activity inhibitor (PD98059, 10µM) was added, decreases of claudin-11 and occludin by MEHP were prevented, indicating that these effects depend on the activation of the ERK pathway. Conclusions: These data show that MEHP exposure alters claudin-11 and occludin mRNAs in Sertoli cells through the activation of the ERK pathway. Impairment of the integrity of the blood-testis barrier may contribute to alterations in spermatogenic function.
Eur Urol Suppl 2011;10(2):219
Poster Session 57 INFERTILITY Sunday, 20 March, 15.45-17.15, Hall F1
681
Co-localisation of two active epigenetic histone marks, H4K12ac and H3K9ac, in gene promoters of spermatozoa from fertile donors and subfertile patients
Paradowska A.1, Miller D.2, Bartkuhn M.3, Weidner W.1, Steger K.1 1 Justus Liebig University, Dept. of Urology, Pediatric Urology and Andrology, Giessen, Germany, 2University of Leeds, Reproduction and Early Development Unit, Institute of Genetics and Health Therapeutics, Leeds, United Kingdom, 3 Justus Liebig University, Institute for Genetics, Giessen, Germany Introduction & Objectives: During spermiogenesis, approximately 85% of somatic histones are replaced by protamines. The remaining histones have been proposed to carry essential marks for the establishment of epigenetic information in the offspring. This study aims at analysing the genome-wide binding pattern of two active epigenetic histone marks, namely histone H4 acetylated at lysine 12 (H4K12ac) and histone H3 acetylated at lysine 9 (H3H9ac) in order to identify aberrant binding patterns between fertile donors and subfertile patients. Materials & Methods: Using antibodies against H4K12ac and H3K9ac, we performed chromatin immunoprecipitation (ChIP) on DNA from ejaculated spermatozoa from both fertile donors (control) and subfertile patients exhibiting impaired sperm chromatin condensation as assessed by aniline blue staining. While H4K12ac immunoprecipitates were analysed by hybridisation on HG18 human promoter arrays (NimbleGen), H3K9ac immunoprecipitates were placed on ENCODE (encyclopaedia of coding elements, NimbleGen) arrays containing promoters and intergenic sequences of 375 genes. Peak finding algorithms and data overlapping were performed applying R software (http://www.r-project.org). Results: In fertile donors, co-localization of binding sites for H4K12ac and H3K9ac could be demonstrated in 38 gene promoters. Ten of these genes are involved in reproductive processes and embryonic development, e.g. AFF4 (AF4/FMR2 family, member 4), AXIN1, NCOA6 (nuclear receptor coactivator 6), OR52A1 (olfactory receptor, family 52, subfamily A, member 1). A global depletion of H4K12ac occupancy has been identified in the group of subfertile patients (496 from fertile donors vs. 146 from subfertile patients). While H3K9ac was associated with 84 gene promoters in fertile donors, only 15 gene promoters interacting with H3K9ac could be demonstrated in subfertile patients. In the latter group, no co-localization of H4K12ac and H3K9ac could be observed with any of the investigated gene promoters. Conclusions: Both our data and data from AFF4-deficient mice, the offsprings of
which have been demonstrated to die either in-utero or neonatally due to impaired embryonic development (Urano et al. 2005), suggest that aberrant acetylation of H4K12ac and/or H3K9ac within developmentally important gene promoters in subfertile men may reflect insufficient sperm chromatin compaction and, as a consequence, may lead to inappropriate transfer of epigenetic information to the oocyte.
682
Reduced expression of chromatin remodelling factors: A reason for round spermatid maturation arrest and idiopathic male infertility?
Schagdarsurengin U.1, Steilmann C.1, Bergmann M.2, Kliesch S.3, Weidner W.1, Steger K.1 1 Justus-Liebig-University Giessen, Dept. of Urology, Paediatric Urology and Andrology, Giessen, Germany, 2Justus-Liebig-University Giessen, Dept. of Veterinary Anatomy, Histology and Embryology, Giessen, Germany, 3University of Münster, Center of Reproductive Medicine and Andrology, Münster, Germany Introduction & Objectives: Human spermiogenesis is associated with histone to protamine exchange and a high grade chromatin condensation. These processes are supported by nuclear transition proteins and chromatin remodelling factors. Ten chromatin remodelling factor families are known. This study aims to analyse whether a different chromatin remodelling factor expression pattern exists between normal spermatogenesis and round spermatid maturation arrest as potential reason for impaired spermatogenesis and idiopathic male infertility. Materials & Methods: Laser capture microdissection was used to excise seminiferous tubules from testicular biopsies with normal spermatogenesis (n=7) and round spermatid maturation arrest (n=7). RNA was isolated, first strand cDNA synthesis and pre-amplification were performed using Epigenetic Chromatin Remodelling Factors PCR arrays with 84 genes. Results: Applying hierarchical cluster analysis, three gene expression clusters with six subgroups were identified. The expression pattern ranged from a few genes upregulated in round spermatid maturation arrest to a multitude of genes (74) with a higher expression in normal round spermatids. 22 genes exhibited a significant difference: 21 genes showed a crucial reduced expression in round spermatid maturation arrest (e.g. BMI1, PCGF1/F5/F6, EZH2, MBD4 and CTCF) compared to normal round spermatids; one gene, BRWD3, revealed a significant higher expression in round spermatid maturation arrest. Conclusions: Reduced expression of chromatin remodelling factors may lead to aberrant chromatin condensation during spermatogenesis and to round spermatid maturation arrest, respectively.
683
Mono-(2-ethylhexyl) phthalate decreases claudin-11 and occludin in rat Sertoli cells
Chiba K.1, Kondo Y.2, Li F.1, Ando M.1, Yamaguchi K.1, Ishikawa T.3, Fujisawa M.1 1 Kobe University Graduate School of Medicine, Dept. of Urology, Kobe, Japan, 2 Hyogo Cancer Center, Dept. of Urology, Akashi, Japan, 3Ishikawa Hospital, Dept. of Urology, Himeji, Japan Introduction & Objectives: Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Sertoli cell tight junctions (TJs) are believed to be crucial for spermatogenesis, and several TJ proteins including claudin-11, occludin, and ZO-1 have been identified. To date, it has been reported that several endocrine disruptors may affect Sertoli cell TJ integrity. In this study, we examined the association of Mono-(2-etylhexyl) Phthalate (MEHP) and TJ proteins in cultured rat Sertoli cells. We also investigated whether Sertoli cells respond to MEHP treatment by activating mitogen-activated protein kinases (MAPK) signaling pathways. Materials & Methods: Sertoli cells were isolated and purified from the testes of 18-d-old Sprague Dawley rats and incubated at 34 °C. On day 3 ex vivo, Sertoli cells were treated with MEHP (1µM, 10µM, or 100µM). At 0.5, 1, 3, 6, and 24 h after either vehicle (control) or MEHP, whole cell lysates for protein and total RNA were isolated from each replicate. Claudin-11, occludin, and ZO-1 mRNAs were evaluated by quantitative real-time PCR (QRT-PCR) analysis. Western blot analysis was used to determine the activation of extracellular signal-related kinases 1 and 2 (ERK1/2), stress-activated protein kinase/Jun-N-terminal kinase (SAPK/ JNK), p38 mitogen activated protein kinase (p38 MAPK). We also examined nitric oxide synthases (NOSs), the members of the tumor necrosis factor (TNF) ligands (Fas L and TRAIL) and its receptor (Fas) expressions by Western blot analysis. Results: In each concentration, MEHP treatment led to significant decreases in claudin-11 and occludin mRNA, but not in ZO-1. Fas, Fas L, TRAIL, and NOSs proteins were not affected by MEHP. The MEHP exposure induced the phosphorylation of ERK1/2, whereas the phosphorylation of p38 MAPK and SAPK/ JNK were not induced. When specific ERK1/2 activity inhibitor (PD98059, 10µM) was added, decreases of claudin-11 and occludin by MEHP were prevented, indicating that these effects depend on the activation of the ERK pathway. Conclusions: These data show that MEHP exposure alters claudin-11 and occludin mRNAs in Sertoli cells through the activation of the ERK pathway. Impairment of the integrity of the blood-testis barrier may contribute to alterations in spermatogenic function.
Eur Urol Suppl 2011;10(2):219