Methyl tert-butyl ether inhibits pubertal development of Leydig cells in male rats by inducing mitophagy and apoptosis

Methyl tert-butyl ether (MTBE) is a widely used gasoline additive. It is considered an endocrine-disrupting chemical. Whether MTBE affects the development of Leydig cells in late puberty of males and its underlying mechanism remains unclear. Twenty-four male Sprague-Dawley rats (35 days old) were randomly allocated into four groups and were orally given MTBE (0, 300, 600, and 1200 mg/kg/day) from postnatal day (PND) 35–56. MTBE markedly reduced serum testosterone levels at 300 mg/kg and higher doses without altering the serum levels of luteinizing hormone and follicle-stimulating hormone. It mainly inhibited cell proliferation, induced mitochondrial autophagy and apoptosis, and indirectly stimulated Sertoli cells to secrete anti-Müllerian hormones, thereby significantly reducing the number of Leydig cells at 1200 mg/kg. MTBE also markedly down-regulated the expression of mature Leydig cell biomarker Cyp11a1 and Hsd3b1 and their proteins, while up-regulating the expression of immature Leydig cell biomarker Akr1c14 and its protein at 600 mg/kg and higher. MTBE significantly down-regulated the expression of cell cycle gene Ccnd1, antioxidant gene Gpx1, and anti-apoptotic gene Bcl2, while increasing pro-apoptotic gene Bax level at 1200 mg/kg. In vitro study further confirmed that MTBE can inhibit testosterone synthesis by inducing reactive oxygen species (ROS) generation, mitophagy, and apoptosis at 200 and 300 mM. In conclusion, exposure to MTBE compromises the development of Leydig cells in late puberty in male rats. Illustration of action of MTBE exposure on Leydig cell (LC) development in the late puberty. MTBE induces oxidative stress by lowering the transcript of GPX1 to cause the following actions: 1) activating AMPK and inactivating mTOR to induce mitophagy (a kind of autophagy); 2) inhibiting BCL2 expression and increasing the expression of Bax, thus increasing the mitochondrial membrane permeability to low the MMP and then the activation of CASP3, thereby inducing apoptosis. Finally, exposure to MTBE compromises the development of Leydig cells. [Display omitted] •MTBE reduces serum testosterone level in male rats after late pubertal exposure.•MTBE inhibits Leydig cell proliferation and number.•MTBE down-regulates the expression of maturity biomarker of Leydig cells.•MTBE induces ROS, apoptosis, and mitophagy.•MTBE reduces phosphorylation of mTOR and induces phosphorylation of AMPK in the testis.