Early expression of the androgen receptor in the Sertoli cells of a marsupial coincides with downregulation of anti-Müllerian hormone at the time of urogenital virilization.

Anti-Müllerian hormone (AMH), responsible for the regression of Müllerian ducts, is strongly expressed by eutherian fetal and postnatal Sertoli cells. Both AMH and testosterone levels are high during the period of fetal reproductive tract virilization which occurs largely in utero in eutherian mammals. Taking advantage of the fact that differentiation of the urogenital tract occurs after birth in marsupials, we studied the ontogeny and regulation of AMH in the tammar wallaby testis and related it to the expression of the androgen receptor in Sertoli cells. Testicular AMH expression was high between days 10-30 post partum, then fell to basal levels by day 60 and remained low until day 90, the oldest age examined. AMH expression was repressed by treatment of male pouch young with the potent androgen androstanediol. Thus, in the tammar, AMH expression decreases in response to androgen at the time of initial urogenital masculinization, in contrast to the situation in humans in which AMH is repressed by testosterone only at the time of puberty. The difference might be explained by the timing of androgen receptor expression which appears in tammar Sertoli cells at around day 40 of pouch life but only at a later developmental stage in eutherians.

[Two immunoassays for antimullerian hormone measurement: analytical and clinical performances]. / Deux dosages de l'hormone antimüllérienne : performances analytiques et cliniques.

Today, serum antimullerian hormone (AMH) is considered as an interesting marker of fertility potential in women to determine follicular status and in men to evaluate testicular function. We study analytical and clinical characteristics of two AMH commercialized immunoassays: Immunotech and DSL methods. The detection limits (close to 0.13 ng/mL), functional sensitivities (close to 0.30 ng/mL) are equivalent, and imprecision results are acceptable for entirely manual assays. The Immunotech method is linear within the calibration range (up to 21 ng/mL) and the DSL method presents a lack of linearity making it accurate only up to 11 ng/mL (and not up to 14 ng/mL as it is indicated by the manufacturer). The two methods allow to measure human AMH, don't cross react with TGF-beta superfamily proteins and the DSL immunoassay recognize mouse (25%), rat (68%) and calf (100%) AMH. The comparison between the two methods (from 0.3 to 200 ng/mL) shows a good correlation (r = 0.979) with not statistically different results (p = 0.31). From a clinical point of view, the two methods allow the evaluation of follicular status in normo-ovulatory women and in women with polycystic ovary syndrome. Results are in agreement with studies showing that AMH serum concentration is strongly correlated with the number of antral follicles. In conclusion, the Immunotech method seems to be more efficient than the DSL method even if the two methods are suitable for clinical applications needing AMH measurements.

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.

TGF-beta Family Members and Gonadal Development.

Several members of the transforming growth factor beta (TGF-beta) family are involved in gonadal development; namely, TGF-beta itself, inhibins, activins, anti-Müllerian hormone (AMH) and GDF-9. These proteins do not affect initial gonadal organogenesis but play either a stimulatory or inhibitory role in the division and differentiation of gonadal cells and in meiotic maturation in the female. Furthermore, as shown by transgenic mouse technology, both AMH and inhibin act as tumor suppressors.

Receptors for anti-müllerian hormone on Leydig cells are responsible for its effects on steroidogenesis and cell differentiation.

Strong overexpression of anti-Müllerian hormone (AMH) in transgenic mice leads to incomplete fetal virilization and decreased serum testosterone in the adult. Conversely, AMH-deficient mice exhibit Leydig cell hyperplasia. To probe the mechanism of action of AMH on Leydig cell steroidogenesis, we have studied the expression of mRNA for steroidogenic proteins in vivo and in vitro and performed a morphometric analysis of testicular tissue in mice overexpressing the hormone. We show that overexpression of AMH in male transgenic mice blocks the differentiation of Leydig cell precursors. Expression of steroidogenic protein mRNAs, mainly cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P450c17), is decreased in transgenic mice overexpressing AMH and in AMH-treated purified Leydig cells. In contrast, transgenic mice in whom the AMH locus has been disrupted show increase expression of P450c17. In vitro, but not in vivo, AMH also decreases the expression of the luteinizing hormone receptor. The effect of AMH is explained by the presence of its receptor on Leydig cells. Our results provide insight into the action of AMH as a negative modulator of Leydig cell differentiation and function.

The role of anti-Müllerian hormone in gonadal development.

Anti-Müllerian (AMH), a member of the transforming growth factor beta produced by immature Sertoli cells and, to a lesser degree, by granulosa cells from birth to the end of reproductive life, does not affect gonadal determination but has a negative effect upon gonadal development in both sexes. It blocks meiosis in fetal ovaries, leading to loss of germ cells and subsequent fibrous degeneration, and inhibits the transcription of aromatase and LH receptor. AMH also affects the development and function of the adult testis by blocking the differentiation of mesenchymal into Leydig cells and by independently decreasing the expression of steroidogenic enzymes.

Serine/threonine kinase receptors and ligands.

Serine/threonine receptors transduce signals for the TGF-beta family, several members of which, such as decapentaplegic and bone morphogenetic proteins, are involved in early patterning of the embryo. The gene encoding the anti-Müllerian hormone (AMH) receptor has recently been cloned; gene targeting produces the same effects as targeting of the AMH gene itself. Another divergent member of the TGF-beta family, GDNF, signals through Ret, a tyrosine kinase receptor; binding to Ret requires the cooperation of GDNFR-alpha. The signal transduction pathway of serine/threonine receptors is now being intensively studied; the immunophilin FKBP-12 and MAD proteins are known to be involved.

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.

A 27 base-pair deletion of the anti-müllerian type II receptor gene is the most common cause of the persistent müllerian duct syndrome.

The persistent müllerian duct syndrome, characterized by the lack of regression of müllerian derivatives, uterus and tubes in otherwise normally masculinized males, is a genetically transmitted disorder implicating either anti-müllerian hormone (AMH), a member of the transforming growth factor-beta superfamily, or its type II receptor, a serine/threonine kinase homologous to the receptors of other members of the transforming growth factor-beta superfamily. We have now performed molecular studies in a total of 38 families. The basis of the condition, namely 16 AMH and 16 AMH receptor mutations, was identified in 32 families. The type of genetic defect could be predicted from the level of serum AMH which is very low or undetectable in patients with AMH mutations and at the upper limit of normal in receptor mutations. Whereas AMH mutations are extremely diverse, patients from 10 out of 16 families with receptor mutations had a 27 bp deletion in exon 10 on at least one allele. This deletion is thus implicated in approximately 25% of patients with persistent müllerian duct syndrome. All AMH and AMH receptor mutations were consistent with an autosomal recessive mode of transmission.

Cloning and expression of the chick anti-Müllerian hormone gene.

Müllerian duct regression in male embryos is due to early production by fetal Sertoli cells of anti-Müllerian hormone, a homodimeric protein of the transforming growth factor- beta superfamily. In mammals, both female Müllerian ducts develop into the uterus and Fallopian tubes, whereas in birds, the right oviduct does not develop. To gain insight into sex differentiation in birds, we have cloned the cDNA for chick anti-Müllerian hormone using antibodies raised against the partially purified protein. Expression cloning was required because of the lack of cross-hybridization between mammalian and chick anti-Müllerian hormone DNA. The chick DNA and protein are significantly longer, due to insertions that abolish nucleotide homology, except in the cDNA coding for the C-terminal, bioactive part of the protein. Nevertheless, the general structure of the gene, sequenced from the transcription initiation to the polyadenylation site, and the main features of the protein are conserved between the chick and mammals. The chick anti-Müllerian hormone gene is expressed at high levels in Sertoli cells of the embryonic testes and in lower amounts in both ovaries, higher levels being reached on the left side after 10 days of incubation.

Insensitivity to anti-müllerian hormone due to a mutation in the human anti-müllerian hormone receptor.

Anti-Müllerian hormone (AMH) and its receptor are involved in the regression of Müllerian ducts in male fetuses. We have now cloned and mapped the human AMH receptor gene and provide genetic proof that it is required for AMH signalling, by identifying a mutation in the AMH receptor in a patient with persistent Müllerian duct syndrome. The mutation destroys the invariant dinucleotide at the 5' end of the second intron, generating two abnormal mRNAs, one missing the second exon, required for ligand binding, and the other incorporating the first 12 bases of the second intron. The similar phenotypes observed in AMH-deficient and AMH receptor-deficient individuals indicate that the AMH signalling machinery is remarkably simple, consisting of one ligand and one type II receptor.

Cloning, expression, and alternative splicing of the receptor for anti-Müllerian hormone.

Anti-Müllerian hormone, also called Müllerian-inhibiting substance or factor, is a glycoprotein dimer belonging to the transforming growth factor-beta superfamily and synthesized by immature Sertoli cells and postnatal granulosa cells. Anti-Müllerian hormone plays a key role in sex differentiation by inducing the regression of Müllerian ducts in the male fetus. It is also responsible for the stunting and masculinization of fetal ovaries in bovine freemartin fetuses and may be involved in the control of follicular maturation in the postnatal ovary. Using a degenerate probe for a consensus region of the transforming growth factor-beta receptor superfamily to screen a complementary DNA library from rabbit fetal ovaries, we cloned a complementary DNA coding for a transmembrane serine/threonine kinase, which is expressed around the fetal Müllerian duct, in fetal and adult granulosa cells, and in immature Sertoli cells. Two transcripts, generated by alternative splicing of an exon coding for an N-terminal 61-amino acid domain, are strongly expressed in anti-Müllerian hormone target organs and Sertoli cells. The longer, 569-amino acid, isoform binds anti-Müllerian hormone when transiently expressed in COS cells and is believed to encode its functional receptor.

Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth factor-beta superfamily.

Mullerian inhibiting substance (MIS)/anti-Mullerian hormone is a differentiation factor that causes regression of the Mullerian duct in the developing male fetus and an apparent sex reversal of the fetal ovary when inappropriately exposed to it. The purified product is a 140-kilodalton glycoprotein composed of two identical subunits. We show that a C-terminal fragment of MIS, which shares homology with transforming growth factor-beta, causes regression of the Mullerian duct and inhibits the biosynthesis of aromatase in the fetal ovary. However, both activities are enhanced dramatically by addition of the N-terminal portion of MIS. Under conditions where potentiation occurs, the N- and C-terminal domains of MIS reassociate. These results indicate that the N-terminus of MIS, unlike that of the other members of the transforming growth factor-beta family, plays a role in maintaining the biological activity of the C-terminus.