RESUMEN
OBJECTIVES: To highlight important clinical aspects of Persistent Müllerian duct syndrome (PMDS). PMDS belongs to the group of differences of sex development. It is attributed to mutations in genes encoding for the anti-Müllerian hormone or its type II receptor (AMHR2) and inherited via an autosomal recessive transmission. CASE PRESENTATION: An 18-day-old male infant with known bilateral cryptorchidism, presented with left-sided obstructed inguinal hernia. The diagnosis of PMDS was considered during inguinal exploration as both testes together with uterus and fallopian tubes were recognized in the hernial sac. Histology confirmed the presence of Müllerian-derived tissues. Genetic testing revealed two different mutations of the AMHR2 gene, both with autosomal recessive transmission: a frequently encountered deletion of 27 pairs bases on exon 10 of this 11 exon gene and a novel deletion of 2 pairs bases on exon 6. CONCLUSIONS: This case is notable being the rarest type of PMDS, that of transverse testicular ectopia and associated with a novel AMHR2 gene mutation.
Asunto(s)
Criptorquidismo , Hernia Inguinal , Femenino , Humanos , Masculino , Criptorquidismo/complicaciones , Criptorquidismo/genética , Exones , Hernia Inguinal/complicaciones , Hernia Inguinal/genética , Pelvis , Recién NacidoRESUMEN
STUDY QUESTION: Can mutations of genes other than AMH or AMHR2, namely PPP1R12A coding myosin phosphatase, lead to persistent Müllerian duct syndrome (PMDS)? SUMMARY ANSWER: The detection of PPP1R12A truncation mutations in five cases of PMDS suggests that myosin phosphatase is involved in Müllerian regression, independently of the anti-Müllerian hormone (AMH) signaling cascade. WHAT IS KNOWN ALREADY: Mutations of AMH and AMHR2 are detectable in an overwhelming majority of PMDS patients but in 10% of cases, both genes are apparently normal, suggesting that other genes may be involved. STUDY DESIGN, SIZE, DURATION: DNA samples from 39 PMDS patients collected from 1990 to present, in which Sanger sequencing had failed to detect biallelic AMH or AMHR2 mutations, were screened by massive parallel sequencing. PARTICIPANTS/MATERIALS, SETTING, METHODS: To rule out the possibility that AMH or AMHR2 mutations could have been missed, all DNA samples of good quality were analyzed by targeted next-generation sequencing. Twenty-four samples in which the absence of AMH or AMHR2 biallelic mutations was confirmed were subjected to whole-exome sequencing with the aim of detecting variants of other genes potentially involved in PMDS. MAIN RESULTS AND THE ROLE OF CHANCE: Five patients out of 24 (21%) harbored deleterious truncation mutations of PP1R12A, the gene coding for the regulatory subunit of myosin phosphatase, were detected. In addition to PMDS, three of these patients presented with ileal and one with esophageal atresia. The congenital abnormalities associated with PMDS in our patients are consistent with those described in the literature for PPP1R12A variants and have never been described in cases of AMH or AMHR2 mutations. The role of chance is therefore extremely unlikely. LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study is the lack of experimental validation of the role of PPP1R12A in Müllerian regression. Only circumstantial evidence is available, myosin phosphatase is required for cell mobility, which plays a major role in Müllerian regression. Alternatively, PPP1R12A mutations could affect the AMH transduction pathway. WIDER IMPLICATIONS OF THE FINDINGS: The study supports the conclusion that failure of Müllerian regression in males is not necessarily associated with a defect in AMH signaling. Extending the scope of molecular analysis should shed light upon the mechanism of the initial steps of male sex differentiation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by la Fondation Maladies Rares, GenOmics 2021_0404 and la Fondation pour la Recherche Médicale, grant EQU201903007868. The authors report no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.
Asunto(s)
Trastorno del Desarrollo Sexual 46,XY , Humanos , Masculino , Fosfatasa de Miosina de Cadena Ligera/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Hormona Antimülleriana/genética , Hormona Antimülleriana/metabolismo , ADNRESUMEN
Anti-Müllerian hormone (AMH) is a member of the TGF-ß family produced essentially by the supporting somatic cells of the testis. Initially known for its inhibiting role upon the development of female internal organs, AMH has been shown to exert many other effects namely upon germ cells. Circulating AMH reflects the ovarian reserve of young developing follicles and is used to evaluate the fertility potential in assisted reproduction. The signaling pathway of AMH is both similar and different from that of other members of the TGF-ß family. Like these, it signals through two distinct serine/threonine receptors, type 1 and type 2, that phosphorylate cytoplasmic effectors, the Smads. It also shares type 1 receptors and Smads with other members of the family. However, AMH is the only family member with its own, dedicated, ligand-specific type 2 receptor, AMHR2. The monogamic relationship between AMH and AMHR2 is supported by molecular studies of the Persistent Müllerian Duct Syndrome, characterized by the presence of Müllerian derivatives in otherwise normally virilized males: mutations of AMH or AMHR2 are clinically indistinguishable.
Asunto(s)
Trastorno del Desarrollo Sexual 46,XY , Hormonas Peptídicas , Hormona Antimülleriana/genética , Hormona Antimülleriana/metabolismo , Trastorno del Desarrollo Sexual 46,XY/genética , Trastorno del Desarrollo Sexual 46,XY/metabolismo , Femenino , Humanos , Masculino , Transducción de Señal/genética , Testículo/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The persistent Müllerian duct syndrome (PMDS) is a 46,XY disorder of sexual development characterized by the persistence of Müllerian duct derivatives, uterus and tubes, in otherwise normally masculinized males. The condition, transmitted as a recessive autosomal trait, is usually due to mutations in either the anti-Müllerian hormone (AMH) gene or its main receptor. Many variants of these genes have been described, all targeting the coding sequences. We report the first case of PMDS due to a regulatory mutation. The AMH promoter contains two binding sites for steroidogenic factor 1 (SF1), one at -102 and the other at -228. Our patient carries a single base deletion at -225, significantly decreasing its capacity for binding SF1, as measured by the electrophoresis mobility shift assay. Furthermore, by linking the AMH promoter to the luciferase gene, we show that the transactivation capacity of the promoter is significantly decreased by the mutation, in contrast to the disruption of the -102 binding site. To explain the difference in impact we hypothesize that SF1 could partially overcome the lack of binding to the -102 binding site by interacting with a GATA4 molecule linked to a nearby response element. We show that disruption of both the -102 SF1 and the -84 GATA response elements significantly decreases the transactivation capacity of the promoter. In conclusion, we suggest that the distance between mutated SF1 sites and potentially rescuing GATA binding motifs might play a role in the development of PMDS.
Asunto(s)
Hormona Antimülleriana/química , Hormona Antimülleriana/metabolismo , Trastorno del Desarrollo Sexual 46,XY/genética , Mutación , Factores de Empalme de ARN/metabolismo , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Hormona Antimülleriana/genética , Sitios de Unión/genética , Línea Celular , Niño , Preescolar , Femenino , Humanos , Recién Nacido , Masculino , Linaje , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
BACKGROUND/OBJECTIVES: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain, and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances follicle-stimulating hormone synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored. METHOD/RESULTS: Here, we demonstrated in perifused murine LßT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LßT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (-53/-37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knockdown of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and ß-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells. CONCLUSIONS: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína Forkhead Box O1/metabolismo , Gonadotrofos/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína Forkhead Box O1/genética , Regulación de la Expresión Génica , Gonadotrofos/metabolismo , Ratones , Regiones Promotoras Genéticas , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/genéticaRESUMEN
Male sex differentiation is driven by two hormones, testosterone and anti-Müllerian hormone (AMH), responsible for regression of Müllerian ducts in male fetuses. Mutations inactivating AMH or AMH receptor type 2 (AMHR2) are responsible for persistent Müllerian duct syndrome (PMDS) in otherwise normally virilised 46,XY males. This review is based on published cases, including 157 personal ones. PMDS can present in one of three ways: bilateral cryptorchidism, unilateral cryptorchidism with contralateral hernia and transverse testicular ectopia. Abnormalities of male excretory ducts are frequent. Testicular malignant degeneration occurs in 33% of adults with PMDS. Cancer of Müllerian derivatives is less frequent. Fertility is rare but possible if at least one testis is scrotal and its excretory ducts are intact. Up to January 2019, 81 families with 65 different mutations of the AMH gene, mostly in exons 1, 2 and 5, have been identified. AMHR2 gene mutations comprising 64 different alleles have been discovered in 79 families. The most common mutation, a 27-bp deletion in the kinase domain, was found in 30 patients of mostly Northern European origin. In 12% of cases, no mutation of AMH or AMHR2 has been detected, suggesting a disruption of other pathways involved in Müllerian regression.
Asunto(s)
Hormona Antimülleriana/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Mutación , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Exones , HumanosRESUMEN
Homozygous loss of function mutations in genes encoding anti-Müllerian hormone (AMH) or its receptor (AMHRII) lead to persistent Müllerian duct syndrome (PMDS). PMDS is characterized by the presence of a uterus, fallopian tubes, cervix, and upper vagina in fully virilised 46,XY males. Both surgical management and long-term follow-up of these patients are challenging. Four cases with PMDS presented with cryptorchidism and inguinal hernia, and laparoscopic inguinal exploration revealed Müllerian remnants. Three of the patients had homozygous mutations in the AMH gene, one with a novel c.1673G>A (p.Gly558Asp) mutation, and one patient had an AMHRII mutation. All patients underwent a single-stage laparotomy in which the fundus of the uterus was split along the midline to release testes and to avoid damaging the vas deferens or the deferential artery. Biopsy of Müllerian remnants did not reveal any malignancy. The cases presented here expand the clinical and molecular presentation of PMDS. Cryptorchidism and inguinal hernia in the presence of Müllerian structures in an appropriately virilised 46,XY individual should suggest PMDS. Long-term reproductive and endocrinological surveillance is necessary.
RESUMEN
Male sex differentiation is driven by 2 hormones, testosterone and anti-müllerian hormone (AMH), responsible for the regression of müllerian ducts in male fetuses. Mutations inactivating AMH or its receptor AMHRII lead to the persistent müllerian duct syndrome (PMDS) in otherwise normally virilized 46,XY males. Our objective was to review the clinical, anatomical, and molecular features of PMDS based upon a review of the literature and upon 157 personal cases. Three clinical presentations exist: bilateral cryptorchidism, unilateral cryptorchidism with contralateral hernia, and transverse testicular ectopia. Abnormalities of male excretory ducts are frequent. Testicular malignant degeneration occurs in 33% of adults with the disorder, while cancer of müllerian derivatives is less frequent. Fertility is rare but possible if at least one testis is scrotal and its excretory ducts are intact. Eighty families with 64 different mutations of the AMH gene have been identified, mostly in exons 1, 2, and 5. AMHRII gene mutations representing 58 different alleles have been discovered in 75 families. The most common mutation, a 27-bp deletion in the kinase domain, was found in 30 patients of mostly Northern European origin. In 12% of cases, no mutation of AMH or AMHRII has been detected, suggesting a disruption of other pathways involved in müllerian regression.
Asunto(s)
Trastorno del Desarrollo Sexual 46,XY/patología , Hormona Antimülleriana/química , Hormona Antimülleriana/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Hormonas/metabolismo , Humanos , Patrón de Herencia/genética , Modelos Moleculares , Mutación/genéticaRESUMEN
CONTEXT: Anti-Müllerian hormone (AMH) is produced by the granulosa cells (GCs) of growing follicles and inhibits follicular development. OBJECTIVE: This study aimed to investigate the regulation of the AMH-specific type 2 receptor (AMHR2) gene expression in GCs by bone morphogenetic protein (BMP)15, BMP4 and growth differentiation factor (GDF)9. DESIGN, SETTING, AND PATIENTS: Their effects on AMHR2 and AMH mRNAs were studied in luteinized human GCs and in ovine GCs (oGCs) from small antral follicles. The effects of BMPs on human AMHR2 and AMH promoter reporter activities were analyzed in transfected oGCs. The in vivo effect of BMP15 on GCs AMHR2 and AMH expression was investigated by using Lacaune and Rasa Aragonesa hyperprolific ewes carrying loss-of-function mutations in BMP15. MAIN OUTCOME MEASURES: mRNAs were quantified by real-time RT-PCR. Promoter reporter constructs activities were quantified by the measurement of their luciferase activity. RESULTS: BMP15 and BMP4 enhanced AMHR2 and AMH expression in human GCs and in oGCs, whereas GDF9 had no effect. In oGCs, GDF9 increased BMP15 effect on AMH expression. Consistent with these results, BMP15 and BMP4, but not GDF9, enhanced AMHR2 promoter activity in oGCs, whereas GDF9 increased BMP15 effect on AMH promoter activity. Moreover, oGCs from both BMP15 mutant ewes had reduced AMHR2 mRNA levels but unchanged AMH expression compared with wild-type ewes. CONCLUSIONS: Altogether, these results suggest that the mechanisms of action of BMP15 on AMHR2 and AMH expression are different, and that by stimulating AMHR2 and AMH expression in GCs BMP15 enhances AMH inhibitory actions in GCs.
Asunto(s)
Proteína Morfogenética Ósea 15/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Células de la Granulosa/efectos de los fármacos , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Adulto , Animales , Proteína Morfogenética Ósea 4/farmacología , Femenino , Células de la Granulosa/metabolismo , Factor 9 de Diferenciación de Crecimiento/farmacología , Humanos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Ovinos , Adulto JovenRESUMEN
Plasma anti-Müllerian hormone (AMH) concentrations have been recently found to be predictive of the number of embryos recovered after FSH superovulatory treatment in the cow. However, the sensitivity of the Active Müllerian-inhibiting substance/AMH ELISA (ref. 10-14400; DSL-Beckman-Coulter) used to make these measurements in bovine plasma samples is low because it was developed to measure human AMH levels. To overcome this limitation, we developed an immunoassay specific for the bovine (B), ovine (O), and caprine (C) species, the bovine-ovine-caprine (BOC) ELISA. For this purpose, we produced recombinant bovine AMH for standardization, and we used monoclonal antibodies raised against bovine AMH, previously prepared by our laboratory. We evaluated the precision, accuracy, specificity, limit of detection, and functional sensitivity of the assay. The intra-assay coefficient of variation ranged between 3.4% and 11.3% for AMH concentrations between 23.68 and 1.74 ng/mL, and the interassay coefficient of variation ranged between 4.8% and 20.5% for concentrations between 25.53 and 1.42 ng/mL, respectively. The assay displayed a good linearity, had a detection limit of 0.4 ng/mL and a functional sensitivity of 1.4 ng/mL. It also cross-reacted with ovine and caprine AMHs. Both the mean and median AMH levels measured in 40 cow plasma samples using the BOC ELISA were approximately 44 fold higher than the mean and median AMH levels measured with the Active Müllerian-inhibiting substance/AMH ELISA. Moreover, a higher correlation was observed between the average number of embryos recovered from each cow after superovulatory treatment and AMH concentrations measured with the BOC ELISA. This BOC ELISA provides a very efficient tool for evaluating the ovarian follicular reserve of cows and predicting their embryo production capacity.
Asunto(s)
Hormona Antimülleriana/sangre , Desarrollo Embrionario , Ensayo de Inmunoadsorción Enzimática/veterinaria , Animales , Bovinos , Femenino , Pruebas de Función Ovárica/métodos , Pruebas de Función Ovárica/veterinaria , Sensibilidad y EspecificidadRESUMEN
The levels and intracellular localization of wild-type transforming growth factor ß superfamily (TGFß-SF) receptors are tightly regulated by endocytic trafficking, shedding and degradation. In contrast, a main regulatory mechanism of mutation-bearing receptors involves their intracellular retention. Anti-Müllerian hormone receptor II (AMHRII, also known as AMHR2) is the type-II receptor for anti-Müllerian hormone (AMH), a TGFß-SF ligand that mediates Müllerian duct regression in males. Here, we studied AMHRII processing and identified novel mechanisms of its constitutive negative regulation. Immunoblot analysis revealed that a significant portion of AMHRII was missing most of its extracellular domain (ECD) and, although glycosylated, was unfolded and retained in the endoplasmic reticulum. Exogenous expression of AMHRII, but not of type-II TGF-ß receptor (TßRII, also known as TGFR2), resulted in its disulfide-bond-mediated homo-oligomerization and intracellular retention, and in a decrease in its AMH-binding capacity. At the plasma membrane, AMHRII differed from TßRII, forming high levels of non-covalent homomeric complexes, which exhibited a clustered distribution and restricted lateral mobility. This study identifies novel mechanisms of negative regulation of a type-II TGFß-SF receptor through cleavage, intracellular retention and/or promiscuous disulfide-bond mediated homo-oligomerization.
Asunto(s)
Procesamiento Proteico-Postraduccional , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Hormona Antimülleriana/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Unión Proteica , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Péptidos/química , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
In the ovary, anti-Müllerian hormone (AMH) is produced by the granulosa cells of growing follicles and can modulate the recruitment of primordial follicles and the FSH-dependent development of follicles. However, the regulation of its production remains poorly understood. Recently, a stimulating effect of the bone morphogenetic proteins (BMPs) on AMH production by granulosa cells has been shown in vitro, but the molecular mechanisms implicated in this regulation and its physiological importance in ovarian function have not yet been established. In the hyperprolific Booroola ewes carrying the FecB(B) partial loss-of-function mutation in the fecundity gene encoding the FecB/BMP receptor, type 1B, the granulosa cells of antral follicles expressed and secreted low AMH amounts, resulting in low AMH concentrations in blood, despite high numbers of AMH-secreting follicles in ovaries. The presence of the FecB(B) mutation impaired the granulosa cell response to the stimulating action of BMP4 on AMH production, indicating a crucial role of the BMP receptor, type 1B in AMH regulation. In ovine granulosa cells, BMP4 enhanced the transcriptional activity of the human AMH promoter, and this action depended on the presence of SMAD1, acting on a promoter sequence located between -423 and -202 bp upstream of the AMH transcription start site. SMAD1 and SF1 acted in concert to mediate BMP4 action on the AMH promoter. Among the 2 SF1 binding sites present on the AMH promoter, the most proximal site, located at -92 bp upstream of the AMH transcription start site, was found to be critical for ensuring the response of the AMH promoter to BMP4. In conclusion, AMH could mediate the actions of BMPs in regulating follicular development and contributing to the determination of ovulation numbers. A molecular model of regulation of the AMH promoter transactivation by BMP signaling is proposed.
Asunto(s)
Hormona Antimülleriana/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/metabolismo , Ovinos/fisiología , Animales , Hormona Antimülleriana/genética , Sitios de Unión , Proteína Morfogenética Ósea 4/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Células Cultivadas , Femenino , Genotipo , Humanos , Mutación , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , TransfecciónRESUMEN
Cell ablation technology is useful for studying specific cell lineages in a developing organ in vivo. Herein, we established a novel anti-Müllerian hormone (AMH)-toxin receptor-mediated cell knockout (Treck) mouse line, in which the diphtheria toxin (DT) receptor was specifically activated in Sertoli and granulosa cells in postnatal testes and ovaries respectively. In the postnatal testes of Amh-Treck transgenic (Tg) male mice, DT injection induced a specific loss of the Sertoli cells in a dose-dependent manner, as well as the specific degeneration of granulosa cells in the primary and secondary follicles caused by DT injection in Tg females. In the testes with depletion of Sertoli cell, germ cells appeared to survive for only several days after DT treatment and rapidly underwent cell degeneration, which led to the accumulation of a large amount of cell debris within the seminiferous tubules by day 10 after DT treatment. Transplantation of exogenous healthy Sertoli cells following DT treatment rescued the germ cell loss in the transplantation sites of the seminiferous epithelia, leading to a partial recovery of the spermatogenesis. These results provide not only in vivo evidence of the crucial role of Sertoli cells in the maintenance of germ cells, but also show that the Amh-Treck Tg line is a useful in vivo model of the function of the supporting cell lineage in developing mammalian gonads.
Asunto(s)
Hormona Antimülleriana/genética , Toxina Diftérica/farmacología , Células de la Granulosa/efectos de los fármacos , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Ovario/citología , Células de Sertoli/efectos de los fármacos , Testículo/citología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Linaje de la Célula , Supervivencia Celular/efectos de los fármacos , Trasplante de Células , Relación Dosis-Respuesta a Droga , Femenino , Células de la Granulosa/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Células de Sertoli/citología , Espermatogénesis/fisiologíaRESUMEN
Using measurements of circulating anti-Müllerian hormone (AMH) in diagnosing and managing reproductive disorders in pediatric patients requires thorough knowledge on normative values according to age and gender. We provide age- and sex-specific reference ranges for the Immunotech assay and conversion factors for the DSL and Generation II assays. With this tool in hand, the pediatrician can use serum concentrations of AMH when determining the presence of testicular tissue in patients with bilaterally absent testes or more severe Disorders of Sex Development (DSD). Furthermore, AMH can be used as a marker of premature ovarian insufficiency (POI) in both Turner Syndrome patients and in girls with cancer after treatment with alkylating gonadotoxic agents. Lastly, its usefulness has been proposed in the diagnosis of polycystic ovarian syndrome (PCOS) and ovarian granulosa cell tumors and in the evaluation of patients with hypogonadotropic hypogonadism.
RESUMEN
Anti-Müllerian hormone (AMH) is a member of the TGF-ß superfamily secreted by the gonads of both sexes. This hormone is primarily known for its role in the regression of the Müllerian ducts in male fetuses. In females, AMH is expressed in granulosa cells of developing follicles. Like other members of the TGF-ß superfamily, AMH transduces its signal through two transmembrane serine/threonine kinase receptors including a well characterized type II receptor, AMHR-II. The complete signalling pathway of AMH involving Smads proteins and the type I receptor is well known in the Müllerian duct and in Sertoli and Leydig cells but not in granulosa cells. In addition, few AMH target genes have been identified in these cells. Finally, while several co-receptors have been reported for members of the TGF-ß superfamily, none have been described for AMH. Here, we have shown that none of the Bone Morphogenetic Proteins (BMPs) co-receptors, Repulsive guidance molecules (RGMs), were essential for AMH signalling. We also demonstrated that the main Smad proteins used by AMH in granulosa cells were Smad 1 and Smad 5. Like for the other AMH target cells, the most important type I receptor for AMH in these cells was BMPR-IA. Finally, we have identified a new AMH target gene, Id3, which could be involved in the effects of AMH on the differentiation of granulosa cells and its other target cells.
Asunto(s)
Hormona Antimülleriana/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Células de la Granulosa/citología , Células de la Granulosa/metabolismo , Animales , Hormona Antimülleriana/farmacología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/deficiencia , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Femenino , Fibrinolisina/metabolismo , Técnicas de Inactivación de Genes , Células de la Granulosa/efectos de los fármacos , Humanos , Ratones , Transporte de Proteínas/efectos de los fármacos , Proteolisis , Transducción de Señal/efectos de los fármacos , Proteína Smad1/metabolismo , Proteína Smad5/metabolismoRESUMEN
Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-ß family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.
Asunto(s)
Proteínas Anfibias/metabolismo , Hormona Antimülleriana/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ovario/metabolismo , Pleurodeles/fisiología , Testículo/metabolismo , Proteínas Anfibias/biosíntesis , Proteínas Anfibias/química , Proteínas Anfibias/genética , Animales , Hormona Antimülleriana/biosíntesis , Hormona Antimülleriana/química , Hormona Antimülleriana/genética , Femenino , Hibridación in Situ , Larva/crecimiento & desarrollo , Larva/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Metamorfosis Biológica , Conductos Paramesonéfricos/crecimiento & desarrollo , Conductos Paramesonéfricos/metabolismo , Técnicas de Cultivo de Órganos , Ovario/crecimiento & desarrollo , Filogenia , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Células de Sertoli/citología , Células de Sertoli/metabolismo , Diferenciación Sexual , Testículo/citología , Testículo/crecimiento & desarrolloRESUMEN
Trophoblast cell adhesion and migration are carefully coordinated during normal placental development. We have compared the expression of three adhesion molecules, E-cadherin, ß-catenin, and Lewis x, by immunohistochemistry during normal trophoblast differentiation, and in hydatidiform moles and choriocarcinomas. Both E-cadherin and ß-catenin were expressed in normal placenta cytotrophoblast, and this expression decreased with trophoblast maturation. E-cadherin was mainly localized along the contact between cytotrophoblast and syncytiotrophoblast, which indicates its role in the differentiation of the syncytial layer. Lewis x disappeared progressively during differentiation of normal villous vessels, and was expressed in molar pregnancies. Interestingly, whereas choriocarcinomas were not, or poorly, stained, invasive hydatidiform moles (invHMs) strongly expressed Lewis x in vascular structures. This observation correlated well with E-cadherin and ß-catenin expression and suggests that these three markers are associated with the invasive transformation. The presence of robust endothelial structures in invHMs could also explain their ability to maintain organized villous architecture (contrary to metastatic choriocarcinomas) during their invasion of extrauterine tissues such as the lung or the brain after dissemination through the blood flow. In our hands, Lewis x appeared to be a new, reliable marker that can be used to clearly distinguish invHMs from choriocarcinomas.
Asunto(s)
Cadherinas/metabolismo , Coriocarcinoma/diagnóstico , Mola Hidatiforme Invasiva/diagnóstico , Antígeno Lewis X/metabolismo , Neoplasias Uterinas/diagnóstico , beta Catenina/metabolismo , Cariotipo Anormal , Adulto , Coriocarcinoma/metabolismo , Diagnóstico Diferencial , Femenino , Edad Gestacional , Humanos , Mola Hidatiforme Invasiva/metabolismo , Hibridación Fluorescente in Situ , Embarazo , Trofoblastos/metabolismo , Trofoblastos/patología , Neoplasias Uterinas/metabolismoRESUMEN
STUDY QUESTION: Are anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR-II) mRNAs similarly regulated by gonadotrophins in lutein granulosa cells (GCs) from control, normo-ovulatory and oligo/anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: AMH mRNA expression was induced by LH only in lutein GC of oligo/anovulatory PCOS women; down-regulation of AMHR-II, induced by LH in control and normo-ovulatory PCOS women, was absent in oligo/anovulatory women. WHAT IS KNOWN ALREADY: It was suggested that AMH could be responsible for the blockade of follicles at the small antral stage in PCOS women. In keeping with this hypothesis, both AMH and AMHR-II are overexpressed in lutein GCs from oligo/anovulatory PCOS women. STUDY DESIGN, SIZE, DURATION: Women undergoing IVF were included in this prospective study, either in the control group (30 women) or in the PCOS group (21 normo-ovulatory and 19 oligo/anovulatory patients) between January 2010 and July 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human lutein GCs were isolated from follicular fluid during IVF protocols. Twenty-four hours after seeding, lutein GCs from each woman were serum starved and cultured for 48 h ± FSH, LH or cAMP. Then AMH and AMHR-II mRNAs were quantified by quantitative RT-PCR and AMH protein concentration was measured in the culture medium by ELISA. Experimental results were analyzed, within each group of women, by the non-parametric Wilcoxon test for paired comparisons between cells cultured in control medium and FSH, LH or cAMP treated cells. Clinical comparisons between the three groups of women were performed on log values using the ANOVA test with Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE: FSH up-regulated both AMH expression and secretion by lutein GCs from the three groups of women (P < 0.05). LH had no effect on AMH mRNAs levels in lutein GCs from controls and normo-ovulatory PCOS women, but increased AMH expression in oligo/anovulatory PCOS women (P < 0.05). Interestingly, LH and cAMP treatments reduced AMHR-II expression by lutein GCs from controls and normo-ovulatory PCOS women (P < 0.05), but had no effect on AMHR-II mRNA levels in oligo/anovulatory PCOS women. LIMITATIONS, REASONS FOR CAUTION: The lutein GCs are not the best model to study AMH and AMHR-II regulation by gonadotrophins. Indeed, AMH and AMHR-II are down-regulated in luteinized cells. Furthermore, these cells have been exposed to non-physiological levels of gonadotrophins and hCG. However, AMH and AMHR-II mRNAs are quantifiable by real-time RT-PCR, and the cells are still responsive to FSH and LH. The age of patients is significantly different between control and oligo/anovulatory PCOS women: this may be a bias in the interpretation of results but older women in the control group had a good ovarian reserve. WIDER IMPLICATIONS OF THE FINDINGS: The overexpression of AMH and AMHR-II in oligo/anovulatory PCOS women could be due to increased LH levels and/or inhibition of its repressive action. The fact that this dysregulation is observed in oligo/anovulatory, but not in normo-ovulatory, PCOS women emphasizes the role of LH in the follicular arrest of PCOS women and suggests that this involves the AMH/AMHR-II system. STUDY FUNDING/COMPETING INTEREST(S): The Assistance-Publique Hôpitaux de Paris provided a Contrat d'Interface and the Agence de Biomédecine provided a grant to Nathalie di Clemente. Schering-Plough provided an FARO grant to Alice Pierre. The authors have nothing to disclose.
Asunto(s)
Anovulación/etiología , Células de la Granulosa/metabolismo , Fase Luteínica/metabolismo , Hormona Luteinizante/metabolismo , Síndrome del Ovario Poliquístico/metabolismo , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Regulación hacia Arriba , Adulto , Hormona Antimülleriana/biosíntesis , Hormona Antimülleriana/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Regulación hacia Abajo , Femenino , Hormona Folículo Estimulante/metabolismo , Líquido Folicular , Células de la Granulosa/patología , Humanos , Síndrome del Ovario Poliquístico/fisiopatología , Estudios Prospectivos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de Péptidos/antagonistas & inhibidores , Receptores de Péptidos/genética , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/genética , Índice de Severidad de la EnfermedadRESUMEN
Anti-Müllerian hormone (AMH), secreted by immature Sertoli cells, provokes the regression of male fetal Müllerian ducts. FSH stimulates AMH production; during puberty, AMH is downregulated by intratesticular testosterone and meiotic germ cells. In boys, AMH determination is useful in the clinical setting. Serum AMH, which is low in infants with congenital central hypogonadism, increases with FSH treatment. AMH is also low in patients with primary hypogonadism, for instance in Down syndrome, from early postnatal life and in Klinefelter syndrome from midpuberty. In boys with nonpalpable gonads, AMH determination, without the need for a stimulation test, is useful to distinguish between bilaterally abdominal gonads and anorchism. In patients with disorders of sex development (DSD), serum AMH determination helps as a first line test to orientate the etiologic diagnosis: low AMH is indicative of dysgenetic DSD whereas normal AMH is suggestive of androgen synthesis or action defects. Finally, in patients with persistent Müllerian duct syndrome (PMDS), undetectable serum AMH drives the genetic search to mutations in the AMH gene, whereas normal or high AMH is indicative of an end organ defect due to AMH receptor gene defects.