Mono-(2-ethylhexyl) Phthalate rapidly decreases claudin-11 in Sertoli cells

Abstract

Phthalates are ubiquitous environmental contaminants that target the fetal and pubertal testis and lead to alterations in endocrine and spermatogenic function. Claudin-11 is involoved in membrane interactions at Sertoli cells tight-junction and with the extracellular matrix. The function of tight-junction is under the control of paracrine and endocrine as well as physiochemical factors in various tissues. So, we examined the association of Mono-(2-etylhexyl) Phthalate (MEHP) and claudin-11 in cultured rat Sertoli cells. We also investigated whether Sertoli cells respond to MEHP by activating mitogen-activated protein kinases (MAPK) signaling pathways. Controlled animal experiment in vitro. 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 μM, 10 μM, or 100 μM). 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 mRNAs were evaluated by quantitative real-time RT-PCR analysis and phosphorylated proteins (p38, p44/42, and SAPK/JNK) were assayed by Western blot analysis. 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 RT-PCR and Western blot analysis. MEHP treatment led to a significant time- and dose-dependent decrease in Claudin-11 mRNA. Fas, Fas L, TRAIL, and NOSs protein and mRNA were not changed. The MEHP exposure induced the phosphorylation of p44/42, not p38 and SAPK/JNK. When p44/42 MAPK activity inhibitors are used, decrease of claudin-11 by MEHP is prevented, indicating that these effects depend on activation of enzymes. These data show that MEHP exposure rapidly alters claudin-11 in Sertoli cells, which might contribute to defects in intact junctional complex formation of sertoli cells leading to impairment of the integrity of the blood-testis barrier and alterations in endocrine and spermatogenic function.