| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 87 === Amphetamine has been used to treat narocolepsy and obesity. It could act on dopaminergic neurons indirectly to induce dopamine release in brain. However, the mechanism of amphetamine in reproduction is not clear. In 1996, some papers indicated that amphetamine could induce cyclic AMP (cAMP) production and suppress testosterone production in rat testes. It is possible that amphetamine might directly act on Leydig cells to affect steroidogenesis. It is well established that steroidogenesis in Leydig cells is regulated by luteinizing hormone/chorionic gonadotropin (LH/CG) via the second messenger cAMP signal transduction pathway. In this process, steroidogenic acute regulatory protein (StAR protein) plays a rate-limiting step. To study the mechanism of amphetamine on Leydig cell function, MA-10 mouse Leydig tumor cell line and purified normal mouse Leydig cells were used. The results showed that amphetamine (10-6M) significantly enhanced hCG-induced progesterone production at 3 hours in MA-10 cells. However, in dose response curve, we found that amphetamine at 10-10M could increase hCG-induced progesterone production more than at 10-6M in MA-10 cells. Interestingly, the StAR protein and P450 side chain cleavage enzyme (P450scc) expressions were not significantly difference between hCG treatment alone and hCG in combination with amphetamine treatment in MA-10 cells. The result indicated that amphetamine with hCG treatment could not affect enzyme and protein expression in MA-10 cells. It is possible that amphetamine can enhance enzyme activity to induce progesterone production in MA-10 cells. To clarify the effect of amphetamine on the function of enzymes in steroidogenesis process, pregnenolone and 22(R)-hydroxycholesterol were used to determine the function of P450scc and 3β-hydroxysteroid dehydrogenase (3β-HSD). The results showed that progesterone production was increased by amphetamine only with 22(R)-hydroxycholesterol treatment in MA-10 cells, which indicated that amphetamine could enhance P450scc activity. However, amphetamine could not affect the activity of 3β-HSD in MA-10 cells. In contrast to hCG treatment, amphetamine could decrease dbcAMP-induced progesterone production at 10-10 M in MA-10 cells. Inversely, the StAR protein and P450scc expressions were increased by amphetamine with dbcAMP treatment in MA-10 cells. It is possible that progesterone can be converted to become another steroid in MA-10 cells. The concentration of 17α-hydroxy-progesterone、androstenedione、testosterone and estradiol were detected by radioimmunoassay. The results show that the concentrations of 17α-hydroxy-progesterone、androstenedione、testosterone and estradiol were not enhanced by the treatment of amphetamine with dbc-AMP in MA-10 cells. The time course response curve show that testosterone productions were not significantly different between hCG alone treatment and hCG treatment with amphetamine (10-6M) in normal Leydig cells as the purity was 82%. However, the treatment of low dose amphetamine (10-10M) with hCG could enhance testosterone production more than hCG treatment alone at 120 minutes in normal Leydig cells. Amphetamine could also enhance hCG-induced testosterone production in normal Leydig cells at 60 minutes as the purity was 90%. These results indicated that amphetamine could enhance testosterone production in mouse Leydig cells. Taken together, the data suggested that amphetamine could directly affect the activity of steroidogenic enzyme to influence the steroidogenesis in Leydig cells.
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