2005 MONO-(2-ETHYLHEXYL) PHTHALATE DECREASES CLAUDIN-11 AND OCCLUDIN IN RAT SERTOLI CELLS

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THE JOURNAL OF UROLOGY姞

laser. The contralateral testis was processed for routine hematoxylin and eosin staining. Two genitourinary pathologists reviewed the H&E stained slides to familiarize themselves with normal rat spermatogenesis. Subsequently, each pathologist, blinded to the results, examined the MPM images acquired from the same rats and attempted to identify the developmental age by placing the cases in order of advancing spermatogenesis. RESULTS: Both pathologists identifed the stage of development and degree of spermatogenesis in the MPM images with 100% accuracy, with no interobserver variability. The testis of the 4 day old rat was easily distinguishable due to the relatively larger gonocytes compared to other cells with virtually no luminal space(A&B). At 11 days, the tubules were slightly larger–yet still without lumen, and the spermatogonia were more organized adjacent to the basement membrane (C&D). At 25 days, the tubules were further enlarged due to an increase in primary spermatocytes, still with no obvious lumen formation (E & F). This stage would be comparable to maturation arrest at the level of the primary spermatocyte frequently seen in humans undergoing fertility treatment. During puberty (35 days), tubules acquired a definite lumen with immature germ cells arranged in 2–3 layers along the basement membrane (G & H). In adult rats (I&J), germ cells form concentric layers with the most immature germ cells(spermatogonia) closer to the basement membrane and the more developed germ cells (spermatids) occupying successive layers as they mature toward the lumen. The heads of spermatozoa were recognized easily as they produced a vibrant autofluorescence. CONCLUSIONS: MPM can be used to identify spermatogenesis in the testis in ex vivo tissue of healthy rats. MPM may be useful in the future as an intra-operative imaging modality during micro-TESE. A unique advantage of this technique is the real-time acquisition of high resolution images without necessitating any extrinsic labeling agent.

Vol. 185, No. 4S, Supplement, Wednesday, May 18, 2011

2005 MONO-(2-ETHYLHEXYL) PHTHALATE DECREASES CLAUDIN-11 AND OCCLUDIN IN RAT SERTOLI CELLS Koji Chiba*, Kobe, Japan; Yutaka Kondo, Akashi, Japan; Fuping Li, Makoto Ando, Kohei Yamaguchi, Kobe, Japan; Tomomoto Ishikawa, Himeji, Japan; Masato Fujisawa, Kobe, Japan INTRODUCTION AND 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. 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 (1␮M, 10␮M, or 100␮M). 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, 10␮M) 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. Source of Funding: None

2006 THE EXPRESSION AND LOCALIZATION OF TIGHT JUNCTION PROTEINS IN INFERTILE MEN Koji Chiba*, Kohei Yamaguchi, Kei Matsushita, Fuping Li, Makoto Ando, Masato Fujisawa, Kobe, Japan

Source of Funding: National Institutes of Health

INTRODUCTION AND OBJECTIVES: Sertoli cell tight junctions (TJs), which contribute to form blood-testis barriers (BTBs), are believed to be crucial for spermatogenesis. In humans, several TJ proteins including claudin-11, and ZO-1 have been identified. Claudin-11 is a transmembrane protein found in Sertoli cell TJs, but is not found in many other types of TJs. Mice invalidated for this gene are infertile, with the loss of claudin-11 expression causing a disruption of this barrier, leading to an arrest in spermatogenesis. ZO-1 is the first TJ-associated cytoplasmic protein subjected to extensive investigation. It is reported that ZO-1 is a linker protein coupling the transmembrane TJ proteins to the cytoskeleton. In this study, we investigated the expression of these TJ associated mRNAs and localization of these TJ proteins in male infertile patients. METHODS: This study included 62 male infertility patients with azoospermia who underwent testicular sperm extraction (TESE) in our institution. Among 62 azoospermic patients, 5 were with obstructive azoospermia (OA). Other 57 patients were due to non obstructive