Mono-(2-ethylhexyl) phthalate directly alters the expression of leydig cell genes and CYP17 lyase activity in cultured rat fetal testis

Exposure to phthalates in utero alters fetal rat testis gene expression and testosterone production, but much remains to bedone to understand the mechanisms underlying the direct action of phthalate within the fetal testis. We aimed toinvestigate the direct mechanisms of action of mono-(2-ethylhexyl) phthalate (MEHP) on the rat fetal testis, focusing onLeydig cell steroidogenesis in particular. We used an in vitro system based on the culture for three days, with or withoutMEHP, of rat fetal testes obtained at 14.5 days post-coitum. Exposure to MEHP led to a dose-dependent decrease intestosterone production. Moreover, the production of 5 alpha-dihydrotestosterone (5a-DHT) (268%) and androstenedione(254%) was also inhibited by 10 mM MEHP, whereas 17 alpha-hydroxyprogesterone (17a-OHP) production was found toincrease (+41%). Testosterone synthesis was rescued by the addition of androstenedione but not by any of the otherprecursors used. Thus, the hormone data suggested that steroidogenesis was blocked at the level of the 17,20 lyase activityof the P450c17 enzyme (CYP17), converting 17a-OHP to androstenedione. The subsequent gene expression and proteinlevels supported this hypothesis. In addition to Cyp17a1, microarray analysis showed that several other genes important fortestes development were affected by MEHP. These genes included those encoding insulin-like factor 3 (INSL3), which isinvolved in controlling testicular descent, and Inha, which encodes the alpha subunit of inhibin B. These findings indicatethat under in vitro conditions known to support normal differentiation of the fetal rat testis, the exposure to MEHP directlyinhibits several important Leydig cell factors involved in testis function and that the Cyp17a1 gene is a specific target toMEHP explaining the MEHP-induced suppression of steroidogenesis observed.