(1)] is a glycoprotein hormone member of the transforming growth factor ␤ (TGF␤) family of growth and differentiation factors and is well known for its role in the regression of Müllerian ducts. Normally, if an embryo is 46XY, the bipotential gonads commit to testes development and MIS begins to be expressed by differentiating Sertoli cells, leading to regression of the Müllerian ducts at 7-10 weeks in humans and 13-17 days in rodents during male embryonal development. In the absence of MIS, the Müllerian ducts differentiate into the female internal reproductive organs: the uterus, fallopian tubes, and upper vagina (reviewed in ref.2).MIS continues to be expressed in males well after Müllerian duct regression and persists at high levels even after birth until puberty, with levels inversely correlated with the increase in serum testosterone. Females begin expressing low levels of MIS in ovarian granulosa cells after birth and reach the same serum levels as those of postpubescent boys. Recent studies suggest that there may be several aspects of physiological significance to the continued MIS expression, including effects on gonadal steroidogenesis (3), follicle recruitment (4, 5), and inhibition of prostate and breast cell proliferation (6, 7).Male mice overexpressing MIS have reduced levels of testosterone and Leydig cell hypoplasia, and are often undervirilized (8). Conversely, mice with null mutations in either MIS or the MIS type II receptor (MISRII) have Leydig cell hyperplasia (9, 10). Indeed, Leydig cells express MISRII, and MIS inhibits Leydig cell testosterone production directly, at least in part by inhibiting the expression of one or more of the steroidogenic enzymes (11-14). MIS and MISRII are expressed in the granulosa cells of developing follicles (15-18), and a role for MIS in gonadal function in the female has also been demonstrated. In addition to the lack of an internal reproductive tract, the ovaries of female transgenic mice that overexpress MIS become largely depleted of germ cells by 2 weeks postnatal and are not detectable in many of the adult mice (8,11). Subsequent studies have shown that primordial follicles of mice deficient for MIS are recruited to develop at a faster rate than in wild-type mice (5) and that addition of MIS to cultured ovaries inhibits primordial follicle growth (4). These observations suggest that MIS plays a significant role in ovarian function, although the exact mechanisms have yet to be deduced.A functional hypothalamic-pituitary-gonadal axis is also critical to mammalian reproductive development and function. The biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by the pituitary is tightly controlled in the hypothalamic-pituitary-gonadal axis by an interconnected system of stimulatory and inhibitory mechanisms. In addition to major roles for gonadotropin-releasing hormone (GnRH) and gonadal steroids, several members of the TGF␤ family modulate gonadotropin production. Among these, the effects of activins and inhibins ha...

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.

Mü llerian 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 hydroxylase͞lyase (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 downregulated 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.