ABSTRACT
PURPOSE: Foxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes. MATERIALS AND METHODS: Testes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed. RESULTS: Foxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter. CONCLUSION: FOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function.
Subject(s)
Animals , Humans , Male , Mice , Cellular Senescence/physiology , Cell Nucleus/metabolism , Cytoplasm/metabolism , Forkhead Transcription Factors/metabolism , HEK293 Cells , Leydig Cells/drug effects , Luteinizing Hormone/blood , Phosphatidylinositol 3-Kinases , Phosphoproteins/metabolism , Phosphorylation , Signal Transduction/drug effects , Testosterone/bloodABSTRACT
BACKGROUND: To elucidate the mechanism of interaction between depolarizing and nondepolarizing muscle relaxants, train-of-four (TOF) fade during onset of neuromuscular blockade of d-tubocurarine (dTC) with or without decamethonium (C10) was evaluated in a rat phrenic nerve hemidiaphragm preparation. METHODS: Phrenic nerve hemidiaphragm preparations from 250~300 g Sprague Dawley rats (n=20) were suspended in a Krebs solution bubbled with 5% CO2 in O2 at 32oC. Phrenic nerves were stimulated with supramaximal stimuli of 0.2 ms duration at 0.15 Hz single twitch and 2 Hz TOF by a Grass S88 stimulator and the contractions of the hemidiaphragm were detected by a Grass FT03 force transducer then recorded. Estimation of ED50 for the dose response data were performed by a linear regression. The statistical significance of the results was determined by Wilcoxon Rank Sum test. p<0.05 was considered significant. RESULTS: Mean ED50 values of dTC and C10 calculated from the dose response relations were 7.76 microgram/ml and 0.65 microgram/ml respectively. Compared to adminstration of 2xED50 of dTC alone, TOF ratios at 75% and 50% of twitch height were markedly decreased by combination of ED50 of C10 and ED50 of dTC with statistic significance (67 +/- 1.9% vs. 46 +/- 3.1% and 36 +/- 2.5% vs. 7 +/- 2.5%). Conclusion: If fade in response to TOF stimulation represents a prejunctional effect, the results from this study suggests that the presynaptic action of C10 has some role in the mechanism of the interaction between dTC and C10 in the rat.