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OXIDATIVE STRESS-INDUCED INHIBITION OF TESTOSTERONE PRODUCTION BY RAT LEYDIG CELLS.
Wang, Paulus 1, Lin, Chiu-Shuang1, Lin, Tzu-Miao1, 1
ABSTRACT- Oxidative stress is one of the major contributors to damage male reproduction. The present study was to investigate the direct effects of hydrogen peroxide (H2O2) on the production of testosterone (T) by Leydig cells. Leydig cells of male rats were purified by Percoll density gradient centrifugation method. Purified rat Leydig cells (100000 cells/ml) were cultured with or without human chorionic gonadotropin (hCG, 0.005 IU/ml), forskolin (an activator of adenylyl cyclase, 0.01 mM), 8-bromo-adenosine 3′:5′-cyclic monophosphate (8-Br-cAMP, a membrane permeable analog of cAMP, 0.1 mM), A23187 (a calcium ionophore, 0.1 mM), cyclopiazonic acid ( CPA, an activator of intracellular calcium release, 1 M), or steroidogenic presursors (1-10 M) including 25-hydroxy-cholesterol, pregnenolone, progesterone, and androstenedione in the presence or absence of H2O2 (0.2 mM) at 34 C for 1 h. At the end of incubation, the medium was collected and measured for T by RIA. Administration of H2O2 (0.04-0.4 mM) dose-dependently decreased the production of T by rat Leydig cells. Production of T was increased by hCG, forskolin, 8-Br-cAMP, A23187, CPA and steroidogenic precursors in vitro. However, H2O2 at 0.2 mM decreased T production in response to hCG by 68±5% (p<0.01), to forskolin by 43±9% (p<0.01), to 8-Br-cAMP by 88±3% (p<0.01), to A23187 by 91±3% (p<0.01), and to CPA by 78±10%. The enhanced levels of T in response to all precursors were reduced by co-incubation of H2O2. These results suggest that oxidative stress caused by H2O2 inhibits T production by acting directly on the Leydig cells via the mechanisms involving the decrease of (1) the generation and action of cAMP, (2) the intracellular calcium, and (3) the enzyme activities of steroidogenesis of T.
KEY WORDS: oxidative stress, testosterone, H2O2, rat Leydig cells
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