The objective of the present study is to determine whether di-(2-ethylhexyl) phthalate (DEHP) exposure at adulthood affects regeneration of rat Leydig cells. 90-day-old Long-Evans rats received intraperitoneal injection of 75 mg/kg ethane dimethanesulfonate (EDS) to eliminate mature Leydig cells, and then were randomly divided into 3 groups, in which rats were gavaged with the corn oil (control) or 10 or 750 mg/kg DEHP daily for 35 days. Serum testosterone and luteinizing hormone levels were assessed by RIA, Leydig cell numbers and proliferation rate were evaluated, and the mRNA levels of Leydig cell specific genes were measured by qPCR. Both 10 and 750 mg/kg DEHP treatments increased Leydig cell numbers on day 14, 21 and 35 post-EDS, due to significant increase of the number of Leydig cell precursors from day 14 to 21 post-EDS. However, serum testosterone levels were halved in 10 and 750 mg/kg DEHP groups compared to control on day 35 post-EDS despite the increased Leydig cell numbers. Quantitative PCR showed that Leydig cell specific genes including Lhcgr, Cyp11a1, Hsd3b1, and Insl3 were significantly down-regulated in 750 mg/kg DEHP-treated testes on post-EDS day 21 and beyond. The present study suggests that DEHP increases Leydig cell proliferation but inhibits differentiation during the regeneration of Leydig cells.
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