Gonadotropin-releasing hormone （GnRH） is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an im...Gonadotropin-releasing hormone （GnRH） is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase （MAPK） pathways are involved in GnRH agonist （GnRHa）-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3β-hydroxysteroid dehydrogenase （3β-HSD） and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay （RIA）. The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3β-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L-1 after 24 h, activation of ERKI/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERKI/2, the upregulation of 3β-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3β-HSDgene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.展开更多
This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig ceils were treated with annexin A5. Testosterone produc...This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig ceils were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory （STAR）, P450scc, 3β-hydroxysteroid dehydrogenase （3β-HSD）, 17β-hydroxysteroid dehydrogenase （17β-HSD）, and 17β-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of STAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 （50 μmol l^-1） for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated （P 〈 0.05）. Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.展开更多
文摘Gonadotropin-releasing hormone （GnRH） is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase （MAPK） pathways are involved in GnRH agonist （GnRHa）-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3β-hydroxysteroid dehydrogenase （3β-HSD） and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay （RIA）. The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3β-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L-1 after 24 h, activation of ERKI/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERKI/2, the upregulation of 3β-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3β-HSDgene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.
文摘This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig ceils were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory （STAR）, P450scc, 3β-hydroxysteroid dehydrogenase （3β-HSD）, 17β-hydroxysteroid dehydrogenase （17β-HSD）, and 17β-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of STAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 （50 μmol l^-1） for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated （P 〈 0.05）. Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.
