Maternal exposure to dioxin imprints sexual immaturity of the pups through fixing the status of the reduced expression of hypothalamic gonadotropin-releasing hormone.

Our previous studies have shown that treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 µg/kg) at gestational day (GD) 15 reduces the pituitary synthesis of luteinizing hormone (LH) during the late fetal and early postnatal period, leading to the imprinting of defects in sexual behaviors at adulthood. However, it remains unclear how the attenuation of pituitary LH is linked to sexual immaturity. To address this issue, we performed a DNA microarray analysis to identify the gene(s) responsible for dioxin-induced sexual immaturity on the pituitary and hypothalamus of male pups, born of TCDD-treated dams, at the age of postnatal day (PND) 70. Among the reduced genes, we focused on gonadotropin-releasing hormone (GnRH) in the hypothalamus because of published evidence that it has a role in sexual behaviors. An attenuation by TCDD of GnRH expression emerged at PND4, and no subsequent return to the control level was seen. A change in neither DNA methylation nor histone acetylation accounted for the reduced expression of GnRH. Intracerebroventricular infusion of GnRH to the TCDD-exposed pups after reaching maturity restored the impairment of sexual behaviors. Supplying equine chorionic gonadotropin, an LH-mimicking hormone, to the TCDD-exposed fetuses at GD15 resulted in a recovery from the reduced expression of GnRH, as well as from the defects in sexual behavior. These results strongly suggest that maternal exposure to TCDD fixes the status of the lowered expression of GnRH in the offspring by reducing the LH-assisted steroidogenesis at the perinatal stage, and this mechanism imprints defects in sexual behaviors at adulthood.

Inulavosin, a melanogenesis inhibitor, leads to mistargeting of tyrosinase to lysosomes and accelerates its degradation.

The melanosome is a highly specialized organelle where melanin is synthesized. Tyrosinase and tyrosinase-related protein-1 (Tyrp1) are major melanosomal membrane proteins and key enzymes for melanin synthesis in melanocytes. Inulavosin, a melanogenesis inhibitor isolated from Inula nervosa (Compositae), reduced the melanin content without affecting either the enzymatic activities or the transcription of tyrosinase or Tyrp1 in B16 melanoma cells. To our knowledge, this inhibitor is previously unreported. Electron-microscopic analyses revealed that inulavosin impaired late-stage development of melanosomes (stages III and IV), in which melanin is heavily deposited. However, it did not alter the early stages of melanosomes (stages I and II), when filamentous structure is observed. Immunofluorescence analyses showed that tyrosinase, but not Tyrp1, was specifically eliminated from melanosomes in cells treated with inulavosin. Unexpectedly, inulavosin specifically accelerated the degradation of tyrosinase but not other melanosomal/lysosomal membrane proteins (Tyrp1, Pmel17, and LGP85). The degradation of tyrosinase induced by inulavosin associated with lysosomes but not the proteasome. Interestingly, lysosomal protease inhibitors restored the melanogenesis but not the targeting of tyrosinase to melanosomes in the cells treated with inulavosin. Instead, colocalization of tyrosinase with lysosome-associated membrane protein-1 at late endosomes/multivesicular bodies and lysosomes was accentuated. Taken together, inulavosin inhibits melanogenesis as a result of mistargeting of tyrosinase to lysosomes.