Nathalie di Clemente

Anti-Müllerian Hormone: The Jost Factor

Publisher Summary This chapter describes anti-Mullerian hormone. It discusses the molecular mechanisms of gene expression and protein bioactivity and the clinical applications of AMH research. In Josts laboratory, the factor responsible for Mullerian regression was called hormone inhibitrice. The homozygosity of the mutated allele was established by the results of allele-specific hybridization, which showed that the patients DNA hybridized only with the mutant oligonucleotide probe and not with the wild-type one. Identical results were obtained with DNA from the two other affected siblings. Although involved in male sex differentiation, the AMH gene is not located on a sex chromosome, in keeping with the tenet that only the genetic trigger for gonadal sex determination needs to be sex specific. Recent data on the location of the AMH gene in other species are available at present. No association to genes located on human chromosome 19 has been detected, indicating complex rearrangements in chromosomal structure during evolution.

ANTI-MULLERIAN 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.

Anti-mullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow.

Abstract The major limitation to the development of embryo production in cattle is the strong between-animal variability in ovulatory response to FSH-induced superovulation, mainly due to differences in ovarian activity at the time of treatment. This study aimed to establish whether anti-Müllerian hormone (AMH) was an endocrine marker of follicular populations in the cow, as in human, and a possible predictor of the ovarian response to superovulation. Anti-Müllerian hormone concentrations in plasma varied 10-fold between cows before treatment and were found to be highly correlated with the numbers of 3- to 7-mm antral follicles detected by ovarian ultrasonography before treatment (r = 0.79, P < 0.001) and the numbers of ovulations after treatment (r = 0.64, P < 0.01). Between-animal differences in AMH concentrations were found to be unchanged after a 3-mo delay (r = 0.87, P < 0.01), indicating that AMH endocrine levels were characteristic of each animal on a long-term period. The population of healthy 3- to 7-mm follicles was the main target of superovulatory treatments, contained the highest AMH concentrations and AMH mRNA levels compared with larger follicles, and contributed importantly to AMH endocrine levels. In conclusion, AMH was found to be a reliable endocrine marker of the population of small antral gonadotropin-responsive follicles in the cow. Moreover, AMH concentrations in the plasma of individuals were indicative of their ability to respond to superovulatory treatments.

THE ROLE OF ANTI-MULLERIAN 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.

Anti-Mullerian Hormone, Its Receptor, FSH Receptor, and Androgen Receptor Genes Are Overexpressed by Granulosa Cells from Stimulated Follicles in Women with Polycystic Ovary Syndrome

CONTEXT In the polycystic ovary syndrome (PCOS), in addition to intrinsic thecal dysregulation leading to hyperandrogenism, a granulosa cell (GC) dysregulation may occur. Expression of anti-Müllerian hormone (AMH), FSH receptor (FSHR) and androgen receptor (AR) are suspected to be altered in PCOS GCs. DESIGN The aim of this prospective study was to analyze the expression of these genes at the last stages of follicular maturation in GCs from 17 patients with PCOS and 15 controls undergoing controlled ovarian hyperstimulation during a cycle with in vitro fertilization. MATERIALS AND METHODS On the day of oocyte retrieval, follicular fluids were collected from small follicles (SF; 8-13 mm) and large follicles (17-22 mm) in separate tubes. Total RNAs and proteins were extracted from GCs. Reverse transcription was performed and quantification of gene expression levels was achieved by real-time quantitative PCR. RESULTS AMH and FSHR mRNA levels were significantly higher in PCOS than in controls in GCs from both SF and large follicles. Likewise, AR and AMH receptor II mRNA levels in GCs from SF were significantly higher in PCOS compared with controls. In both PCOS patients and controls, AMH and AR mRNA levels correlated strongly, positively, and independently to FSHR mRNA levels. CONCLUSION Using quantitative RT-PCR, AMH, AMH receptor II, FSHR, and AR genes were shown to be overexpressed by GCs from stimulated follicles of women with PCOS undergoing controlled ovarian hyperstimulation. This could be the sign of a maturation defect or may reflect hyperandrogenism.

Intrafollicular Steroids and Anti-Müllerian Hormone During Normal and Cystic Ovarian Follicular Development in the Cow

Abstract Development of follicular cysts is a frequent ovarian dysfunction in cattle. Functional changes that precede cyst formation are unknown, but a role for anti-Müllerian hormone (AMH) in the development of follicular cysts has been suggested in humans. This study aimed to characterize intrafollicular steroids and AMH during follicular growth in a strain of beef cows exhibiting a high incidence of occurrence of follicular cysts. Normal follicular growth and cyst development were assessed by ovarian ultrasonography scanning during the 8 days before slaughtering. Experimental regression of cysts was followed by rapid growth of follicles that reached the size of cysts within 3–5 days. These young cysts exhibited higher intrafollicular concentrations of testosterone, estradiol-17beta, and progesterone than large early dominant follicles did in normal ovaries, but they exhibited similar concentrations of AMH. Later-stage cysts were characterized by hypertrophy of theca interna cells, high intrafollicular progesterone concentration, and high steroidogenic acute regulatory protein mRNA expression in granulosa cells. Progesterone and AMH concentrations in the largest follicles (≥10 mm) and cysts were negatively correlated (r == −0.45, P << 0.01). Smaller follicles (<<10 mm) exhibited higher intrafollicular testosterone and estradiol-17beta concentrations in ovaries with cysts compared to normal ovaries. During follicular growth, AMH concentration dropped in follicles larger than 5 mm in diameter and in a similar way in ovaries with and without cysts. In conclusion, enhanced growth and steroidogenesis in antral follicles <<10 mm preceded cyst formation in cow ovaries. Intrafollicular AMH was not a marker of cystic development in the cow, but low AMH concentrations in cysts were associated with luteinization.

Processing of Anti-Müllerian Hormone Regulates Receptor Activation by a Mechanism Distinct from TGF-β

TGF-β family ligands are translated as prepropeptide precursors and are processed into mature C-terminal dimers that signal by assembling a serine/threonine kinase receptor complex containing type I and II components. Many TGF-β ligands are secreted in a latent form that cannot bind their receptor, due to the pro-region remaining associated with the mature ligand in a noncovalent complex after proteolytic cleavage. Here we show that anti-Müllerian hormone (AMH), a TGF-β family ligand involved in reproductive development, must be cleaved to bind its type II receptor (AMHRII), but dissociation of the pro-region from the mature C-terminal dimer is not required for this initial interaction. We provide direct evidence for this interaction by showing that the noncovalent complex binds to a soluble form of AMHRII in an ELISA format and to AMHRII immobilized on Sepharose. Binding of the noncovalent complex to Sepharose-coupled AMHRII induces dissociation of the pro-region from the mature C-terminal dimer, whereas no dissociation occurs after binding to immobilized AMH antibodies. The pro-region cannot be detected after binding of the AMH noncovalent complex to AMHRII expressed on COS cells, indicating that pro-region dissociation may occur as a natural consequence of receptor engagement on cells. Moreover, the mature C-terminal dimer is more active than the noncovalent complex in stimulating Sma- and Mad-related protein activation, suggesting that pro-region dissociation contributes to the assembly of the active receptor complex. AMH thus exemplifies a new mechanism for receptor engagement in which interaction with the type II receptor promotes pro-region dissociation to generate mature ligand.

Clinical aspects and molecular genetics of the persistent Müllerian duct syndrome

Sexual differentiation is implemented by the actions of the testicular hormones, testosterone and anti-Mu ̈llerian hormone (AMH), on the undifferentiated bipotential fetal reproductive tract (Jost, 1947). In the absence of AMH, Mu ̈llerian ducts develop into the uterus, Fallopian tubes and upper vagina, while lack of testosterone synthesis or action allows the external genitalia and urogenital sinus to assume the constitutive female appearance. Intersex conditions in XY subjects may be due to the impaired secretion of both testicular hormones; persistence of Mu ̈llerian derivatives is then associated with incomplete or absent virilization of the external genitalia, as in gonadal dysgenesis, for example. Alternatively, insufficient production of, or lack of sensitivity to, a single testicular hormone may lead to dissociation between AMH and testosterone-dependent steps of sexual differentiation. Mutations in genes involved in testosterone metabolism result in an external female appearance, while allowing AMH-mediated Müllerian regression to occur. The lack of production or action of AMH has the opposite effect: external virilization is achieved normally, but uterus and Fallopian tubes fail to regress, defining the so-called persistent Mu ̈llerian duct syndrome (PMDS). PMDS is allegedly a rare condition, with approximately 150 cases described in the literature, but we believe that its incidence has been underestimated because, in the past, surgical correction of undescended testes was not always undertaken, and because the presence of Mu ̈llerian structures is often missed or not correctly identified (Zeller et al., 1994).

Differential regulation of ovarian anti-müllerian hormone (AMH) by estradiol through α- and β-estrogen receptors.

BACKGROUND Anti-müllerian hormone (AMH) is a member of the TGF-β family, which limits follicle maturation. Recently serum AMH has been recognized as a useful diagnostic and prognostic tool in human reproductive endocrinology. OBJECTIVE The aim of this study was to investigate the regulation of human ovarian AMH by estradiol and FSH. METHODS AMH mRNA were quantified by real time RT-PCR in human granulosa cells (GC). AMH transcription was studied in KK1 GC cotransfected with estrogen receptors (ER)-β or ERα, and normal human AMH promoter-luciferase construct (hAMH-luc) or mutated AMH promoter reporter constructs. Binding sites for estradiol (estrogen response element half-site) and steroidogenic factor 1 were disrupted by targeted mutagenesis. The level of ER in GC was determined by quantitative RT-PCR and Western blotting. RESULTS In KK1 cells, estradiol up-regulated and inhibited hAMH-luc in the presence of ERα and ERβ respectively. Disruption of estrogen response element half-site and/or steroidogenic factor 1 binding sites did not modify ERβ-mediated effect of estradiol on hAMH-luc, whereas it affected that conveyed by ERα. The FSH enhancement of hAMH-luc was abolished by estradiol in cells overexpressing ERβ. When both ER were transfected, estradiol inhibited hAMH-luc or had no effect. Estradiol repressed AMH mRNAs in human GC, which express a little more ERα than ERβ mRNA. CONCLUSIONS Our results show that AMH expression can be differentially regulated by estradiol depending on the ER and suggest that its decrease in GC of growing follicles, which mainly express ERβ, and during controlled ovarian hyperstimulation is due to the effect of estradiol.

Hyperproliferation of mitotically active germ cells due to defective anti-Müllerian hormone signaling mediates sex reversal in medaka

The function of AMH (Anti-Müllerian hormone), a phylogenetically ancient member of the TGFβ family of proteins, in lower vertebrates is largely unknown. Previously, we have shown that the gene encoding the type II anti-Müllerian hormone receptor, amhrII, is responsible for excessive germ cell proliferation and male-to-female sex reversal in the medaka hotei mutant. In this study, functional analyses in cultured cells and of other amhrII mutant alleles indicate that lack of AMH signaling causes the hotei phenotype. BrdU incorporation experiments identified the existence of both quiescent and mitotically active germ cells among the self-renewing, type I population of germ cells in the developing gonad. AMH signaling acts in supporting cells to promote the proliferation of mitotically active germ cells but does not trigger quiescent germ cells to proliferate in the developing gonad. Furthermore, we show that the male-to-female sex reversal phenotype in hotei mutants is not a direct consequence of AMH signaling in supporting cells, but is instead mediated by germ cells. Our data demonstrate that interfollicular AMH signaling regulates proliferation at a specific stage of germ cell development, and that this regulation is crucial for the proper manifestation of gonadal sex directed by sex determination genes.