Previous studies show that prenatal di‐(2‐ethylhexyl) phthalate (DEHP) exposure induces premature testicular aging. However, the evidence is weak, and the underlying mechanisms remain unclear. p38/extracellular signal‐regulated kinase (ERK)/c‐Jun NH(2)‐terminal kinase (JNK) MAPK pathways participate in aging. Leydig cell (LC) senescence results in testicular aging. Whether prenatal DEHP exposure induces premature testicular aging by promoting LC senescence warrants further study. Here, male mice undergo prenatal exposure to 500 mg per kg per day DEHP, and TM3 LCs are treated with 200 µm mono (2‐ethylhexyl) phthalate (MEHP). MAPK pathways, testicular toxicity, and senescent phenotypes (β‐gal activity, p21, p16, and cell cycle) of male mice and LCs are explored. Prenatal DEHP exposure induces premature testicular aging in middle‐aged mice (poor genital development, reduced testosterone synthesis, poor semen quality, increased β‐gal activity, and upregulated expression of p21 and p16). MEHP induces LCs senescence (cell cycle arrest, increased β‐gal activity, and upregulated expression of p21). p38 and JNK pathways are activated, and the ERK pathway is inactivated. In conclusion, prenatal DEHP exposure induces premature testicular aging by promoting LC senescence through MAPK signaling pathways.

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Epidemiologic studiessuggested the association between prenatal Di-(2-ethylhexyl) phthalate (DEHP) exposure and disorders of sex development (DSD), adult male disorders, and reproductive aging. However, the evidence is still weak, and the underlying mechanism hasn’t been revealed. Mono-(2-ethylhexyl) phthalate (MEHP) is the main bioactive metabolite of DEHP. Inhibiting testosterone synthesis by interfering with steroidogenic gene expression induces testicular toxicity. So prenatal DEHP exposure may induce lifelong testicular toxicity by continuously interfering with steroidogenic gene expression. In this study, male mice underwent different doses (0, 100, 500, 1000mg/kg) of prenatal DEHP exposure, the testicular toxicity (genital development, testosterone, semen quality, and morphology of testis tissue) in the neonatal, post-puberal and middle-aged stages was observed, and the steroidogenic gene (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd3b2) expression was analyzed by qPCR and WB. We also explored the interference of steroidogenic gene expression in TM3 Leydig cells after MEHP exposure. As a result, prenatal DEHP exposure induced lifelong testicular toxicity including instant testicular injury, DSD, and reproductive aging. The male mice with prenatal exposure manifested as poor genital development and reduced testosterone synthesis, poor semen quality, and phylogeneticseminiferous tubules, especially in the high dose (1000mg/kg). Prenatal DEHP exposure continuously interfered with steroidogenic gene expression. MEHP reduced testosterone synthesis of TM3 Leydig cells by interfering with steroidogenic gene expression. In conclusion, prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression, thus indicating the association between prenatal exposure and DSD, adult male disorders, and reproductive aging. Environmental Implication DEHP, a widely applied plasticizer, is easily contacted by pregnant women and causes prenatal exposure of male offspring. Epidemiologic and animal studies indicate that prenatal DEHP exposure is associated with male genital malformation, as well as adult male disorders (infertility, low testosterone). Our study explores the lifelong testicular toxicity in male mice and the key role of interfering with steroidogenic gene expression on testicular toxicity following prenatal DEHP exposure. As a result, prenatal DEHP exposure induced lifelong testicular toxicity including instant testicular injury, DSD, and reproductive aging by continuously interfering with steroidogenic gene expression.