Components of the anti-Müllerian hormone signaling pathway in gonads.

Anti-Müllerian hormone (AMH) is a member of the Transforming Growth Factor-beta (TGF-beta) family implicated in the regression of Müllerian ducts in male fetuses and in the development and function of gonads of both sexes. Members of the TGF-beta family signal through two types of serine/threonine kinase receptors called type I and type II, and two types of Smad proteins, receptor-regulated Smad (R-Smad) and common Smad, Smad4. Components of the AMH signaling pathway have been identified in gonads and gonadal cell lines. The AMH type II receptor is highly specific. In contrast, the identity of the AMH type I receptor is not clear; three type I receptors of Bone Morphogenetic Proteins (BMPs), Alk2, Alk3 and Alk6 may transduce AMH signals, but none of them has all the characteristics of an AMH type I receptor. AMH activates BMP-specific R-Smads and reporter genes.

Autosomal recessive segregation of a truncating mutation of anti-Müllerian type II receptor in a family affected by the persistent Müllerian duct syndrome contrasts with its dominant negative activity in vitro.

Anti-Müllerian hormone belongs to the TGFbeta family whose members exert their effects by signaling through two related serine/threonine kinase receptors. Mutations of the anti-Müllerian hormone type II receptor occur naturally, causing the persistent Müllerian duct syndrome. In a family with two members with persistent Müllerian duct syndrome and one normal sibling, we detected two novel mutations of the anti-Müllerian hormone type II receptor gene. One, transmitted by the mother to her three sons, is a deletion of a single base leading to a stop codon, causing receptor truncation after the transmembrane domain. The other, a missense mutation in the substrate-binding site of the kinase domain, is transmitted by the father to the two sons affected by persistent Müllerian duct syndrome, indicating a recessive autosomal transmission as in other cases of persistent Müllerian duct syndrome. Truncating mutations in receptors of the TGFbeta family exert dominant negative activity, which was seen only when each of the mutant anti-Müllerian hormone receptors was overexpressed in an anti-Müllerian hormone-responsive cell line. We conclude that assessment of dominant activity in vitro, which usually involves overexpression of mutant genes, does not necessarily produce information applicable to clinical conditions, in which mutant and endogenous genes are expressed on a one to one basis.

Ovarian granulosa cell tumors express a functional membrane receptor for anti-Müllerian hormone in transgenic mice.

Anti-Müllerian hormone inhibits granulosa cell growth and function. Both anti-Müllerian hormone and its type II receptor are expressed in normal granulosa cells. We show by histologic and molecular analyses that ovarian tumors developing in transgenic mice, obtained by targeted oncogenesis using an anti-Müllerian hormone promoter-SV40 oncogene construct, are of granulosa-cell origin. Because tissue-specific, cell-surface molecules are of particular interest for the analysis and treatment of tumors, we examined the expression of anti-Müllerian hormone type II receptor in the ovaries of these transgenic mice. We demonstrate that the anti-Müllerian hormone type II receptor is expressed not only in normal ovarian follicles, but also in granulosa cell tumors. Using a cell line derived from one of these tumors, we show that the anti-Müllerian hormone type II receptor protein is present on the surface of tumor cells and binds anti-Müllerian hormone. Furthermore, we show that the anti-Müllerian hormone receptor is functional in the granulosa tumor cell line, with anti-Müllerian hormone treatment inducing selective activation of Smad1. In conclusion, in this study we present a new murine transgenic model of granulosa cell tumors of the ovary and, using this model, we demonstrate for the first time cell-surface expression of a highly tissue-specific molecule, anti-Müllerian hormone type II receptor, as well as the selective activation of Smad proteins by anti-Müllerian hormone, in granulosa tumor cells.

Anti-Müllerian hormone and its receptors.

Anti-Müllerian hormone (AMH), a member of the transforming growth factor-beta family, is an important factor of male sex differentiation. It is produced by Sertoli cells from the time of fetal sex differentiation to puberty. AMH is also produced by granulosa cells from the time of birth to the end of ovarian activity. As other members of the transforming growth factor-beta family, AMH signals through two related but distinct receptors, both serine/threonine kinases with a single transmembrane domain, called type II and type I. The type II receptor has been cloned in 1994 and is expressed solely in AMH target organs. Engagement of the type I receptor BMPR-IB and downstream effector Smad1 by AMH has recently been demonstrated, however, its role in AMH biological actions remains to be proven.

Molecular mechanisms of hormone-mediated Müllerian duct regression: involvement of beta-catenin.

Regression of the Müllerian duct in the male embryo is one unequivocal effect of anti-Müllerian hormone, a glycoprotein secreted by the Sertoli cells of the testis. This hormone induces ductal epithelial regression through a paracrine mechanism originating in periductal mesenchyme. To probe the mechanisms of action of anti-Müllerian hormone, we have studied the sequence of cellular and molecular events involved in duct regression. Studies were performed in male rat embryos and in transgenic mice overexpressing or lacking anti-Müllerian hormone, both in vivo and in vitro. Anti-Müllerian hormone causes regression of the cranial part of the Müllerian duct whereas it continues to grow caudally. Our work shows that this pattern of regression is correlated with a cranial to caudal gradient of anti-Müllerian hormone receptor protein, followed by a wave of apoptosis spreading along the Müllerian duct as its progresses caudally. Apoptosis is also induced by AMH in female Müllerian duct in vitro. Furthermore, apoptotic indexes are increased in Müllerian epithelium of transgenic mice of both sexes overexpressing the human anti-Müllerian hormone gene, exhibiting a positive correlation with serum hormone concentration. Inversely, apoptosis is reduced in male anti-Müllerian hormone-deficient mice. We also show that apoptosis is a decisive but not sufficient process, and that epitheliomesenchymal transformation is an important event of Müllerian regression. The most striking result of this study is that anti-Müllerian hormone action in peri-Müllerian mesenchyme leads in vivo and in vitro to an accumulation of cytoplasmic beta-catenin. The co-localization of beta-catenin with lymphoid enhancer factor 1 in the nucleus of peri-Müllerian mesenchymal cells, demonstrated in primary culture, suggests that overexpressed beta-catenin in association with lymphoid enhancer factor 1 may alter transcription of target genes and may lead to changes in mesenchymal gene expression and cell fate during Müllerian duct regression. To our knowledge, this is the first report that beta-catenin, known for its role in Wnt signaling, may mediate anti-Müllerian hormone action.

Engagement of bone morphogenetic protein type IB receptor and Smad1 signaling by anti-Müllerian hormone and its type II receptor.

Anti-Müllerian hormone induces the regression of fetal Müllerian ducts and inhibits the transcription of gonadal steroidogenic enzymes. It belongs to the transforming growth factor-beta family whose members signal through a pair of serine/threonine kinase receptors and Smad effectors. Only the anti-Müllerian hormone type II receptor has been identified. Our goal was to determine whether anti-Müllerian hormone could share a type I receptor with another family member. Co-immunoprecipitation of known type I receptors with anti-Müllerian hormone type II receptor clearly showed that the bone morphogenetic protein type IB receptor was the only cloned type I receptor interacting in a ligand-dependent manner with this type II receptor. Anti-Müllerian hormone also activates the bone morphogenetic protein-specific Smad1 pathway and the XVent2 reporter gene, an anti-Müllerian hormone type II receptor-dependent effect abrogated by a dominant negative version of bone morphogenetic protein type IB receptor. Reverse amplification experiments showed that bone morphogenetic protein type IB receptor is co-expressed with anti-Müllerian hormone type II receptor in most anti-Müllerian hormone target tissues. Our data support a model in which a ligand, anti-Müllerian hormone, gains access to a shared type I receptor and Smad1 system through a highly restricted type II receptor.

Mutant isoforms of the anti-Müllerian hormone type II receptor are not expressed at the cell membrane.

Anti-Müllerian hormone, a member of the transforming growth factor beta superfamily, produces early regression of Müllerian ducts in the male fetus through binding to a serine/threonine kinase receptor, homologous to type II receptors of the transforming growth factor beta (TGF-beta) family. A splice mutation of this receptor, described in a patient with abnormal retention of Müllerian derivatives, generates two mutant isoforms, one lacking the second exon and the other bearing an insertion of 12 bases between exons 2 and 3. Using hemagglutinin-tagged recombinant receptors, we have visualized wild type and mutant receptors in COS cells by Western blotting and immunoprecipitation. The 82-kDa, endoglycosidase H-insensitive, mature form of the wild type receptor is reduced to 68 kDa by N-glycosidase F treatment. Mutant receptor isoforms, 73 and 63 kDa for the long and short form, respectively, are sensitive to endoglycosidase H, suggesting that they are retained in the endoplasmic reticulum. Indeed, only the wild type receptor was expressed on the cell surface and bound iodinated anti-Müllerian hormone. These results provide a biological explanation for the failure of the mutant receptor to induce Müllerian regression.