Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin.

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB1.

Mancozeb affects mitochondrial activity, redox status and ATP production in mouse granulosa cells.

BACKGROUND: Mancozeb (MZ) is a fungicide that belongs to the subclass of metal (Mn/Zn) ethylene-bis-dithiocarbamate pesticides. In mouse and human granulosa cells (GCs) exposed to MZ (0.01 µg/ml), morphological modifications and significant alterations of p53 expression level in comparison with control GCs were recorded. OBJECTIVES: To investigate if MZ (0.01 µg/ml) induces oxidative stress and alters energy metabolism in exposed mouse GCs. RESULTS: Following fungicide exposure, GCs showed low p53 content, a depolarized mitochondrial membrane potential (ΔΨm), as well as low ATP and reduced glutathione (GSH) levels associated with increased reactive oxygen species (ROS) generation. No remarkable differences on other parameters such as ATP/ADP ratio, energy charge, as well as induction of apoptosis and DNA damage were found. The activation of AKT and PDK1 kinases in MZ-treated cells was observed. Inhibition of ROS generation by the antioxidant N-acetylcysteine (NAC) restored a normal expression level of p53. CONCLUSIONS: Our results demonstrate that the low dose of MZ here used induces a mild oxidative stress in GCs, and provides evidence for the possible involvement of AKT/PKB signaling pathway in triggering adaptive and survival response.

Endocannabinoid signaling in mammalian ovary.

The role of the endocannabinoid system (ECS) in mammalian reproduction is a rather active field of research, due to its potential exploitation to combat human infertility. Available data shows that the aberrant endocannabinoid signaling negatively affects embryo development, implantation and pregnancy. Although many efforts have been devoted to a better understanding of the ECS in these steps of female reproduction, very little is known about its role in regulating ovarian follicle development and production of mature oocytes. This is the subject of the present review where we discuss current knowledge about the impact and potential exploitation of the ECS and endocannabinoid signaling in mammalian ovary and folliculogenesis.

Repeated ovarian stimulation does not affect the expression level of proteins involved in cell cycle control in mouse ovaries and fallopian tubes.

PURPOSE: To understand if repeated cycles (2-4 rounds) of gonadotropin stimulation could affect intracellular localization/content of proteins controlling cell cycle progression in mouse fallopian tubes (FT) and ovaries. METHODS: FT and ovaries of estrous mice (control) and of stimulated mice were analyzed to detect Oct-3/4, Sox-2, p53, ß-catenin, pAKT and cyclin D1 localization/content. Spindles and chromosome alignment were analyzed in ovulated oocytes. RESULTS: After round 4, FT and ovaries of control and stimulated groups showed no differences in Oct-3/4, Sox-2 and ß-catenin localization nor in Oct-3/4, Sox-2, p53, ß-catenin and pAKT contents. Cyclin D1 level increased significantly in FT of treated mice. Oocytes number decreased meanwhile frequency of abnormal meiotic spindles increased with treatments. CONCLUSIONS: Repetitive stimulations affected oocyte spindle morphology but did not induce changes in a set of proteins involved in cell cycle progression, usually altered in ovarian cancer. The significant increase of cyclin D1 in the FT requires further investigation.

Ovarian toxicity: from environmental exposure to chemotherapy.

Unlike men, who have continuous spermatogenesis throughout most of their lifetime, women are born with a fixed supply of follicles, and this number progressively declines with age until the menopause. Beside age, the speed of follicle depletion can be regulated by genetic, hormonal and environmental influences. In the course of their lives, women are exposed to multiple chemicals and radiation sources that can increase the chance of developing permanent infertility and premature ovarian failure (POF). A wealth of experimental data indicate that iatrogenic (chemotherapy, radiotherapy) and xenobiotic agents (e.g., chemicals, pharmaceuticals) are potent ovotoxicants capable of accelerating ovarian reserve depletion. In the present review we reported the negative effects exerted on mammalian ovary by some widely diffused environmental chemicals, as polycyclic aromatic hydrocarbons (PAHs) and dithiocarbamate mancozeb, and by 1-3 butadiene and 4-vinylcycloexene, two occupational chemicals known to be capable of inducing ovarian cancer and infertility. Furthermore, attention has been devoted to the consequences of chemo- and radiotherapy on the ovary, both known to affect reproductive lifespan. Our increasing understanding of metabolic alterations induced by these agents is fundamental to individuate new therapeutic strategies aimed to prevent ovarian dysfunction in fertile women.