Hormonal and paracrine influences on Leydig cell steroidogenesis

Abstract: Androgen production by Leydig cells is critical for development of the male reproductive organs and for maintaining normal body homeostasis and fertility later in life. Biosynthesis of testosterone is driven by secretion of the gonadotropin, luteinizing hormone (LH) from the pituitary. In addition to this endocrine control of steroidogenesis there are numerous locally produced factors that interfere with steroid production in a paracrine manner. This thesis evaluates the paracrine effects on the hormonal function of Leydig cells of several factors that are locally produced in the testis under normal physiological conditions. Further, signaling pathways and mechanisms that triggers activation of steroidogenesis induced by human chorionic gonadotropin (hCG) and interleukin-1¥á (IL-1¥á) are also investigated. M¡§©öllerian inhibiting substance (MIS) is produced by Sertoli cells and causes regression of the M¡§©öllerian ducts during male development. Moreover, it is produced at high levels until puberty and has been shown to inhibit Leydig cell steroidogenesis at the level of cytochrome P450c17¥á-hydroxylase/C17-20 lyase (Cyp17). Here, the results show that MIS reverses the cAMP-induced conversion of progesterone to androstendione by Cyp17, while increasing the production of 5¥á-reduced forms of progesterone without affecting 5a¥á-reductase in microsomes from MA-10 Leydig tumor cells. We speculate that MIS does this by inhibiting the lyase activity of Cyp17. At the same time, MIS up-regulates cAMP induced expression of steroidogenic acute regulatory protein (StAR), a keyregulator of steroidogenesis. The findings in this thesis show that this is a secondary effect due to the lack of local feedback inhibition of androgens on StAR expression. Furthermore, it is shown that androgens can inhibit the expression of StAR both in vitro and in vivo, suggesting an autocrine mechanism for regulation of steroidogenesis. In addition to the inhibitory effects of MIS on steroidogenesis, the signaling pathways evoked by two stimulators of steroidogenesis, hCG and the pro-inflammatory cytokine IL-1a were investigated. Both hCG and IL-1¥á activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in primary cultures of immature rat Leydig cells. After acute stimulation by hCG, inhibition of ERK1/2 with UO126 resulted in decreased de novo synthesis of StAR and protein phosphatase 2 activity together with a decrease in androgen production. These factors have previously proven to be important for the activity of StAR, suggesting that the ERK1/2 cascade is an important part of hCG-induced signaling that is involved in the acute stimulation of steroidogenesis in immature Leydig cells. In contrast, ERK1/2 seems to play another role in IL-I¥á induced steroidogenesis where UO126 increases both StAR expression and phosphorylation, but still attenuates androgen production. Further experiments showed that UO126 decreased the mitochondrial membrane potential in the cells indicating a site of action for attenuation of androgen synthesis. In conclusion, this thesis reveals several new ways for the Leydig cell to regulate androgen production. The importance of the ERK pathway in immature Leydig cell steroidogenesis and an autocrine mechanism for regulation of StAR expression and androgen production are demonstrated. In addition, these results indicate the importance of timing, duration and combination of stimuli.