Müllerian inhibiting substance blocks the protein kinase A-induced expression of cytochrome p450 17alpha-hydroxylase/C(17-20) lyase mRNA in a mouse Leydig cell line independent of cAMP responsive element binding protein phosphorylation.

Müllerian inhibiting substance (MIS) is produced by fetal Sertoli cells and causes regression of the Müllerian duct in male fetuses shortly after commitment of the bipotential embryonic gonad to testes differentiation. MIS is also produced by the Sertoli cells and granulosa cells of the adult gonads where it plays an important role in regulating steroidogenesis. We have previously shown that MIS can dramatically reduce testosterone synthesis in Leydig cells by inhibiting the expression of cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (Cyp17) mRNA in vitro and in vivo. To characterize the signal transduction pathway used by MIS to control expression of endogenous Cyp17 in a mouse Leydig cell line, we demonstrate that MIS inhibits both LH- and cAMP-induced expression of Cyp17 at concentrations as low as 3.5 nM and for as long as 18 h. The induction of steroidogenic acute regulatory protein (StAR) mRNA by cAMP, however, was slightly increased by addition of MIS. Protein kinase A (PKA) inhibition with H-89 blocked Cyp17 mRNA induction, suggesting that MIS interferes with the PKA signal transduction pathway. Inhibition of Cyp17 induction was not seen with added U0126, and wortmannin inhibited the induction incompletely. In addition, phosphorylation of the cAMP responsive element binding protein (CREB) was not detected following 50 micro M cAMP exposure, a concentration sufficient for Cyp17 mRNA induction. Moreover, CREB phosphorylation, which was observed with addition of 500 micro M cAMP, was not inhibited by coincubation with MIS. Taken together, these results suggest that cAMP induces expression of Cyp17 by a PKA-mediated mechanism and that this induction, which is inhibited by MIS signal transduction, does not require CREB activity, and is distinct from that used to induce steroidogenic acute regulatory protein expression.

Müllerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents.

Müllerian Inhibiting Substance (MIS) expression is inversely proportional to the serum concentration of testosterone in males after birth and in vitro studies have shown that MIS can lower testosterone production by Leydig cells. Also, mice overexpressing MIS exhibited Leydig cell hypoplasia and lower levels of serum testosterone, but it is not clear whether this is a result of MIS affecting the development of Leydig cells or their capacity to produce testosterone. To examine the hypothesis that MIS treatment will result in decreased testosterone production by mature Leydig cells in vivo, we treated luteinizing hormone (LH)-stimulated adult male rats and mice with MIS and demonstrated that it can lead to a several-fold reduction in testosterone in serum and in testicular extracts. There was also a slight decrease in 17-OH-progesterone compared to the more significant decrease in testosterone, suggesting that MIS might be regulating the lyase activity of cytochrome P450c17 hydroxylase/lyase (Cyp17), but not its hydroxylase activity. Northern analysis showed that, in both MIS-treated rats and mice, the mRNA for Cyp17, which catalyzes the committed step in androgen synthesis, was down-regulated. In rats, the mRNA for cytochrome P450 side-chain cleavage (P450scc) was also down-regulated by MIS. This was not observed in mice, indicating that there might be species-specific regulation by MIS of the enzymes involved in the testosterone biosynthetic pathway. Our results show that MIS can be used in vivo to lower testosterone production by mature rodent Leydig cells and suggest that MIS-mediated down-regulation of the expression of Cyp17, and perhaps P450scc, contributes to that effect.

Anti-Müllerian hormone produces endocrine sex reversal of fetal ovaries.

We have previously reported that anti-Müllerian hormone (AMH), also known as Müllerian-inhibiting substance, the testicular glycoprotein involved in regression of the Müllerian ducts of the male fetus, induces the formation of seminiferous cord-like structures in fetal ovaries exposed to it in organ culture. We have now investigated the effect of bovine AMH, purified to homogeneity, on ovarian endocrine differentiation. Ovine fetal ovaries exposed to AMH release testosterone instead of estradiol, an endocrine sex reversal due to suppression of aromatase activity. AMH dramatically decreases the conversion rate of testosterone to estradiol and also decreases total aromatase activity, as measured by the tritiated water technique. AMH acts by decreasing aromatase biosynthesis rather than by blocking enzyme activity, as suggested by the relatively long period of AMH exposure required to produce an effect. In the rabbit fetal ovary, aromatase activity is AMH-responsive during the whole gestational period. The basal steroidogenic activity of rat fetal ovaries is extremely low but can be markedly increased by cAMP. AMH completely blocks the effect of cAMP. Taken together, our results suggest that AMH plays a pivotal role in both morphological and endocrine gonadal sex differentiation.