anti-Müllerian Hormone Promoter Research Articles

AMH Regulation by Steroids in the Mammalian Testis: Underlying Mechanisms and Clinical Implications

Anti-Müllerian hormone (AMH) is a distinctive biomarker of the immature Sertoli cell. AMH expression, triggered by specific transcription factors upon fetal Sertoli cells differentiation independently of gonadotropins or sex steroids, drives Müllerian duct regression in the male, preventing the development of the uterus and Fallopian tubes. AMH continues to be highly expressed by Sertoli until the onset of puberty, when it is downregulated to low adult levels. FSH increases testicular AMH output by promoting immature Sertoli cell proliferation and individual cell expression. AMH secretion also showcases a differential regulation exerted by intratesticular levels of androgens and estrogens. In the fetus and the newborn, Sertoli cells do not express the androgen receptor, and the high androgen concentrations do not affect AMH expression. Conversely, estrogens can stimulate AMH production because estrogen receptors are present in Sertoli cells and aromatase is stimulated by FSH. During childhood, sex steroids levels are very low and do not play a physiological role on AMH production. However, hyperestrogenic states upregulate AMH expression. During puberty, testosterone inhibition of AMH expression overrides stimulation by estrogens and FSH. The direct effects of sex steroids on AMH transcription are mediated by androgen receptor and estrogen receptor α action on AMH promoter sequences. A modest estrogen action is also mediated by the membrane G-coupled estrogen receptor GPER. The understanding of these complex regulatory mechanisms helps in the interpretation of serum AMH levels found in physiological or pathological conditions, which underscores the importance of serum AMH as a biomarker of intratesticular steroid concentrations.

Association between an AMH promoter polymorphism and serum AMH levels in PCOS patients.

STUDY QUESTIONDo polymorphisms in the anti-Müllerian hormone (AMH) promoter have an effect on AMH levels in patients with polycystic ovary syndrome (PCOS)?SUMMARY ANSWERWe have identified a novel AMH promoter polymorphism rs10406324 that is associated with lower serum AMH levels and is suggested to play a role in the mechanism of regulation of AMH gene expression in women.WHAT IS KNOWN ALREADYFollicle number is positively correlated with serum AMH levels, reflected by elevated AMH levels in women with PCOS. In addition, it is suggested that AMH production per follicle is higher in women with PCOS than in normo-ovulatory women, implying an altered regulation of AMH in PCOS.STUDY DESIGN, SIZE, DURATIONA discovery cohort of 655 PCOS women of Northern European ancestry and both an internal and external validation PCOS cohort (n = 458 and n = 321, respectively) were included in this study. Summary-level data of an AMH genome-wide association study meta-analysis including 7049 normo-ovulatory women was included as a control cohort. A genetic approach was taken through association analysis and in silico analysis of the associated variants in the AMH promoter. In vitro analysis was performed to investigate the functional mechanisms.PARTICIPANTS/MATERIALS, SETTING, METHODSAll common two-allelic single-nucleotide polymorphisms (SNPs) in the region Chr19:2 245 353–2 250 827 bp (Build 37) were selected for the analysis. Linear regression analyses were performed to determine the association between SNPs in the AMH promoter region and serum AMH levels. For the in silico analysis, the webtools ‘HaploReg’ v4.1 for ENCODE prediction weight matrices and ‘atSNP’ were used. In vitro analysis was performed using KK1 cells, a mouse granulosa cell line and COV434 cells, a human granulosa tumor cell line. Cells were transfected with the reference or the variant human AMH promoter reporter construct together with several transcription factors (TFs). Dual-Glo® Luciferase Assay was performed to measure the luciferase activity.MAIN RESULTS AND THE ROLE OF CHANCEPolymorphism rs10406324 was significantly associated with serum AMH levels in all three PCOS cohorts. Carriers of the minor allele G had significantly lower log-transformed serum AMH levels compared to non-carriers (P = 8.58 × 10−8, P = 1.35 × 10−3 and P = 1.24 × 10−3, respectively). This result was validated in a subsequent meta-analysis (P = 3.24 × 10−12). Interestingly, rs10406324 was not associated with follicle count, nor with other clinical traits. Also, in normo-ovulatory women, the minor allele of this variant was associated with lower serum AMH levels (P = 1.04 × 10−5). These findings suggest that polymorphism rs10406324 plays a role in the regulation of AMH expression, irrespective of clinical background. In silico analysis suggested a decreased binding affinity of the TFs steroidogenenic factor 1, estrogen-related receptor alpha and glucocorticoid receptor to the minor allele G variant, however in vitro analysis did not show a difference in promoter activity between the A and G allele.LIMITATIONS, REASONS FOR CAUTIONFunctional analyses were performed in a mouse and a human granulosa cell line using an AMH promoter reporter construct. This may have limited assessment of the impact of the polymorphism on higher order chromatin structures. Human granulosa cells generated from induced pluripotent stem cells, combined with gene editing, may provide a method to elucidate the exact mechanism behind the decrease in serum AMH levels in carriers of the −210 G allele. We acknowledge that the lack of follicle number in the external validation and the control cohort is a limitation of the paper. Although we observed that the association between rs10406324 and AMH levels was independent of follicle number in our discovery and internal validation PCOS cohorts, we cannot fully rule out that the observed effects on serum AMH levels are, in part, caused by differences in follicle number.WIDER IMPLICATIONS OF THE FINDINGSThese results suggest that variations in serum AMH levels are not only caused by differences in follicle number but also by genetic factors. Therefore, the genetic context should be taken into consideration when assessing serum AMH levels in women. This may have clinical consequences when serum AMH levels are used as a marker for the polycystic ovarian morphology phenotype.STUDY FUNDING/COMPETING INTEREST(S)No external funding was used. J.S.E.L. has received consultancy fees from the following companies: Ferring, Roche Diagnostics and Ansh Labs and has received travel reimbursement from Ferring. J.A.V. has received royalties from AMH assays, paid to the institute/lab with no personal financial gain. The other authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A.

Molecular mechanisms underlying AMH elevation in hyperoestrogenic states in males

Anti-Müllerian hormone (AMH) is secreted by Sertoli cells of the testes from early fetal life until puberty, when it is downregulated by androgens. In conditions like complete androgen insensitivity syndrome (CAIS), AMH downregulation does not occur and AMH increases at puberty, due in part to follicle-stimulating hormone (FSH) effect. However, other conditions like Peutz-Jeghers syndrome (PJS), characterised by low FSH, also have increased AMH. Because both CAIS and PJS may present as hyperoestrogenic states, we tested the hypothesis that oestradiol (E2) upregulates AMH expression in peripubertal Sertoli cells and explored the molecular mechanisms potentially involved. The results showed that E2 is capable of inducing an upregulation of endogenous AMH and of the AMH promoter activity in the prepubertal Sertoli cell line SMAT1, signalling through ERα binding to a specific ERE sequence present on the hAMH promoter. A modest action was also mediated through the membrane oestrogen receptor GPER. Additionally, the existence of ERα expression in Sertoli cells in patients with CAIS was confirmed by immunohistochemistry. The evidence presented here provides biological plausibility to the hypothesis that testicular AMH production increases in clinical conditions in response to elevated oestrogen levels.

The regulation and signalling of anti-Müllerian hormone in human granulosa cells: relevance to polycystic ovary syndrome.

What prevents the fall in anti-Müllerian hormone (AMH) levels in polycystic ovary syndrome (PCOS) and what are the consequences of this for follicle progression in these ovaries? Exposure of granulosa cells (GCs) to high levels of androgens, equivalent to that found in PCOS, prevented the fall in AMH and was associated with dysregulated AMH-SMAD signalling leading to stalled follicle progression in PCOS. In normal ovaries, AMH exerts an inhibitory role on antral follicle development and a fall in AMH levels is a prerequisite for ovulation. Levels of AMH are high in PCOS, contributing to the dysregulated follicle growth that is a common cause of anovulatory infertility in these women. Human KGN-GC (the cell line that corresponds to immature GC from smaller antral follicles (AF)) were cultured with a range of doses of various androgens to determine the effects on AMH production. KGN-GC were also treated with PHTPP (an oestrogen receptor β (ERβ) antagonist) to examine the relationship between AMH expression and the ratio of ERα:ERβ. The differential dose-related effect of AMH on gene expression and SMAD signalling was investigated in human granulosa-luteal cells (hGLC) from women with normal ovaries, with polycystic ovarian morphology (PCOM) and with PCOS. KGN-GC were also cultured for a prolonged period with AMH at different doses to assess the effect on cell proliferation and viability. AMH protein production by cells exposed to androgens was measured by ELISA. The effect of PHTPP on the mRNA expression levels of AMH, ERα and ERβ was assessed by real-time quantitative PCR (qPCR). The influence of AMH on the relative mRNA expression levels of aromatase, AMH and its receptor AMHRII, and the FSH and LH receptor (FSHR and LHR) in control, PCOM and PCOS hGLCs was quantified by qPCR. Western blotting was used to assess changes in levels of SMAD proteins (pSMAD-1/5/8; SMAD-4; SMAD-6 and SMAD-7) after exposure of hGLCs from healthy women and women with PCOS to AMH. The ApoTox-Glo Triplex assay was used to evaluate the effect of AMH on cell viability, cytotoxicity and apoptosis. Testosterone reduced AMH protein secreted from KGN-GC at 10-9-10-7M (P< 0.05; P< 0.005, multiple uncorrected comparisons Fishers least squares difference), but at equivalent hyperandrogenemic levels no change was seen in AMH levels. 5α-DHT produced a significant dose-related increase in AMH protein secreted into the media (P= 0.022, ANOVA). Increasing the mRNA ratio of ERα:ERβ produced a corresponding increase in AMH mRNA expression (P= 0.015, two-way ANOVA). AMH increased mRNA levels of aromatase (P< 0.05, one-way ANOVA) and FSHR (P< 0.0001, one-way ANOVA) in hGLCs from women with PCOM, but not from normal cells or PCOS (normal n= 7, PCOM n= 5, PCOS n= 4). In contrast to hGLCs from ovulatory ovaries, in PCOS AMH reduced protein levels (cell content) of stimulatory pSMAD-1/5/8 and SMAD-4 but increased inhibitory SMAD-6 and -7 (P< 0.05, normal n= 6, PCOS n= 3). AMH at 20 and 50ng/ml decreased KGN-GC cell proliferation but not viability after 8days of treatment (P< 0.005, two-way ANOVA). N/A. Luteinised GC from women undergoing IVF have a relatively low expression of AMH/AMHRII but advantageously continue to display responses inherent to the ovarian morphology from which they are collected. To compensate, we also utilised the KGN cell line which has been characterised to be at a developmental stage close to that of immature GC. The lack of flutamide influence on testosterone effects is not in itself sufficient evidence to conclude that the effect on AMH is mediated via conversion to oestrogen, and the effect of aromatase inhibitors or oestrogen-specific inhibitors should be tested. The effect of flutamide was tested on testosterone but not DHT. Normal folliculogenesis and ovulation are dependent on the timely reduction in AMH production from GC at the time of follicle selection. Our findings reveal for the first time that theca-derived androgens may play a role in this model but that this inhibitory action is lost at levels of androgens equivalent to those seen in PCOS. The AMH decline may either be a direct effect of androgens or an indirect one via conversion to oestradiol and acting through the upregulation of ERα, which is known to stimulate the AMH promoter. Interestingly, the ability of GCs to respond to this continually elevated AMH level appears to be reduced in cells from women with PCOS due to an adaptive alteration in the SMAD signalling pathway and lower expression of AMHRII, indicating a form of 'AMH resistance'. This study was funded by the Thomas Addison Scholarship, St Georges Hospital Trust. The authors report no conflict of interest in this work and have nothing to disclose. N/A.

MON-208 Association Study of AMH Promoter Polymorphisms and Serum AMH Levels in PCOS Patients

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women, is diagnosed based on three criteria, including a polycystic ovarian morphology (PCOM). Although the etiology of PCOS is not fully understood, a genetic component has been demonstrated. Women with PCOS have elevated serum Anti-Müllerian Hormone (AMH) levels, a hormone known to reflect the ovarian reserve. Furthermore, the AMH production per follicle is suggested to be higher in PCOS patients and this combined implies an aberrant regulation of ovarian AMH expression. Currently, some transcription factors, such as FOXL2 and SF1, have been demonstrated to play a role in the ovarian regulation of AMH promoter activity. However, still little is known about AMH gene regulation, particularly in women with PCOS. Hence, the aim of this study was to investigate the association of single nucleotide polymorphisms (SNPs) in the AMH promoter on serum AMH levels in a cohort of PCOS patients. A candidate gene association study was conducted. All two-allelic SNPs located in or nearby the AMH promoter (Chr19:2,245,353 – 2,250,827bp) with a minor allele frequency > 1% and an imputation quality r2 threshold above 0.75 were included. We included Caucasian PCOS women, diagnosed based on the Rotterdam criteria. AMH levels were measured with the picoAMH assay (Ansh Labs®, Houston, Texas, USA). The association between SNPs and serum AMH levels was assessed by linear regression with Wald test of significance, allele carrier model analysis with one-way analysis of covariance (ANCOVA). All statistical models were adjusted for age, BMI and total follicle count (TFC). AMH levels were natural log transformed to obtain a normal distribution. A p-value below 8.51e-03 was considered as statistically significant, based on Matrix Spectral Decomposition with an effective number of independent variables (VeffLi) of 6. All analyses were performed using R studio. We assessed 11 SNPs in 700 Caucasian PCOS women. Human AMH promoter polymorphism rs10406324 (-220 A/G) was associated with serum levels of AMH in the linear regression analysis (β = 0.52, p=6.08e-07). This association was also present in the dominant carrier model (AA: 9.85 ng/ml ± 0.27 (n=645) vs AG/GG: 7.60ng/ml ± 0.84 (n=54/n=1), p=6.48e-05, F = 16.2). The association of TFC and the polymorphism rs10406324, corrected for Age, BMI and AMH, showed an inverse trend compared to serum AMH levels (β=-8.00, p=3.75e-03), however this result was not significant in the carrier model (AA: 42.2 ± 0.88 (n=645) vs AG/GG: 43.6 ± 2.41 (n=54/n=1), p=0.60, F = 0.281). Moreover, this SNP was not in LD with any other SNP in the selected region. The human AMH promoter polymorphism rs10406324 is associated with serum AMH levels in Caucasian PCOS women. Replication and functional analyses are necessary to further elucidate the mechanisms by which this SNP affects the AMH promoter activity.

Androgens downregulate anti-Müllerian hormone promoter activity in the Sertoli cell through the androgen receptor and intact steroidogenic factor 1 sites.

Testicular anti-Müllerian hormone (AMH) production is inhibited by androgens around pubertal onset, as observed under normal physiological conditions and in patients with precocious puberty. In agreement, AMH downregulation is absent in patients with androgen insensitivity. The molecular mechanisms underlying the negative regulation of AMH by androgens remain unknown. Our aim was to elucidate the mechanisms through which androgens downregulate AMH expression in the testis. A direct negative effect of androgens on the transcriptional activity of the AMH promoter was found using luciferase reporter assays in the mouse prepubertal Sertoli cell line SMAT1. A strong inhibition of AMH promoter activity was seen in the presence of both testosterone and DHT and of the androgen receptor. By site-directed mutagenesis and chromatin immunoprecipitation assays, we showed that androgen-mediated inhibition involved the binding sites for steroidogenic factor 1 (SF1) present in the proximal promoter of the AMH gene. In this study, we describe for the first time the mechanism behind AMH inhibition by androgens, as seen in physiological and pathological conditions in males. Inhibition of AMH promoter activity by androgens could be due to protein-protein interactions between the ligand-bound androgen receptor and SF1 or by blockage of SF1 binding to its sites on the AMH promoter.

FOXL2 Is an Essential Activator of SF-1-Induced Transcriptional Regulation of Anti-Müllerian Hormone in Human Granulosa Cells.

Anti-Müllerian hormone (AMH) is required for proper sexual differentiation by regulating the regression of the Müllerian ducts in males. Recent studies indicate that AMH could be an important factor for maintaining the ovarian reserve. However, the mechanisms of AMH regulation in the ovary are largely unknown. Here, we provide evidence that AMH is an ovarian target gene of steroidogenic factor-1 (SF-1), an orphan nuclear receptor required for proper follicle development. FOXL2 is an evolutionally conserved transcription factor, and its mutations cause blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), wherein affected females display eyelid defects and premature ovarian failure (POF). Notably, we found that functional FOXL2 is essential for SF-1-induced AMH regulation, via protein–protein interactions between FOXL2 and SF-1. A BPES-inducing mutant of FOXL2 (290–291delCA) was unable to interact with SF-1 and failed to mediate the association between SF-1 and the AMH promoter. Therefore, this study identified a novel regulatory circuit for ovarian AMH production; specifically, through the coordinated interplay between FOXL2 and SF-1 that could control ovarian follicle development.

The Bone Morphogenetic Protein 15 Up-Regulates the Anti-Müllerian Hormone Receptor Expression in Granulosa Cells.

Anti-Müllerian hormone (AMH) is produced by the granulosa cells (GCs) of growing follicles and inhibits follicular development. 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. 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. mRNAs were quantified by real-time RT-PCR. Promoter reporter constructs activities were quantified by the measurement of their luciferase activity. 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. 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.

Anti-Müllerian hormone regulation by the bone morphogenetic proteins in the sheep ovary: deciphering a direct regulatory pathway.

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.

Differential Regulation of Ovarian Anti-Müllerian Hormone (AMH) by Estradiol through α- and β-Estrogen Receptors

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. The aim of this study was to investigate the regulation of human ovarian AMH by estradiol and FSH. 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. 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. 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.

SOX9 and SF1 are involved in cyclic AMP-mediated upregulation of anti-Müllerian gene expression in the testicular prepubertal Sertoli cell line SMAT1

In Sertoli cells, anti-Müllerian hormone (AMH) expression is upregulated by FSH via cyclic AMP (cAMP), although no classical cAMP response elements exist in the AMH promoter. The response to cAMP involves NF-κB and AP2; however, targeted mutagenesis of their binding sites in the AMH promoter do not completely abolish the response. In this work we assessed whether SOX9, SF1, GATA4, and AP1 might represent alternative pathways involved in cAMP-mediated AMH upregulation, using real-time RT-PCR (qPCR), targeted mutagenesis, luciferase assays, and immunocytochemistry in the Sertoli cell line SMAT1. We also explored the signaling cascades potentially involved. In qPCR experiments, Amh, Sox9, Sf1, and Gata4 mRNA levels increased after SMAT1 cells were incubated with cAMP. Blocking PKA abolished the effect of cAMP on Sox9, Sf1, and Gata4 expression, inhibiting PI3K/PKB impaired the effect on Sf1 and Gata4, and reducing MEK1/2 and p38 MAPK activities curtailed Gata4 increase. SOX9 and SF1 translocated to the nucleus after incubation with cAMP. Mutations of the SOX9 or SF1 sites, but not of GAT4 or AP1 sites, precluded the response of a 3,063-bp AMH promoter to cAMP. In conclusion, in the Sertoli cell line SMAT1 cAMP upregulates SOX9, SF1, and GATA4 expression and induces SOX9 and SF1 nuclear translocation mainly through PKA, although other kinases may also participate. SOX9 and SF1 binding to the AMH promoter is essential to increase the activity of the AMH promoter in response to cAMP.

DNA sequence variations of the entire anti-Müllerian hormone (AMH) gene promoter and AMH protein expression in patients with the Mayer–Rokitanski–Küster–Hauser syndrome

The etiology of the Mayer-Rokitanski-Kuster-Hauser (MRKH) syndrome, where congenitally the Mullerian ducts fail to develop into the uterus, cervix and upper vagina, along with other malformations, is unresolved. Anti-Mullerian hormone (AMH) signal transduction inducing the degradation of Mullerian ducts in males is implicated in the MRKH syndrome. This study examined if DNA sequence variations are responsible for the activation of AMH and aberrant hormone levels in MRKH patients. The entire AMH promoter and 3' regulatory elements of the constitutively expressed splicing factor SF3a2 were sequenced in 30 MRKH patients and genotyped in 48 control individuals using matrix-assisted laser desorption/ionization-time-of-flight mass spectronomy. Ovarian AMH promoter function was correlated with protein expression in plasma and peritoneal fluid of MRKH patients. Of six identified AMH promoter variations, two at positions -639 (SP1-binding site) and -210 [steroidogenic factor (SF)1-binding site] were homozygote in 73% of patients, and 69% of control individuals, destroying the SP1-binding site. AMH protein levels in plasma and peritoneal fluid from patients were equivalent to control individuals, however in three patients plasma levels were abnormally high. AMH is an important indicator for ovarian function. AMH promoter sequence variations or the previously proposed SF3a2-AMH fusion co-transcripts cannot be responsible for aberrant AMH expression leading to Mullerian duct degradation.

Subcellular and molecular mechanisms regulating anti-Müllerian hormone gene expression in mammalian and nonmammalian species.

Anti-Müllerian hormone (AMH) is best known for its role as an inhibitor of the development of female internal genitalia primordia during fetal life. In the testis, AMH is highly expressed by Sertoli cells of the testis from early fetal life to puberty, when it is downregulated by the action of testosterone, acting through the androgen receptor, and meiotic spermatocytes, probably acting through TNFalpha. Basal expression of AMH is induced by SOX9; GATA4, SF1, and WT1 enhance SOX9-activated expression. When the hypothalamic-pituitary axis is active and the negative effect of androgens and germ cells is absent, for example, in the fetal and neonatal periods or in disorders like androgen insensitivity, FSH upregulates AMH expression through a nonclassical cAMP-PKA pathway involving transcription factors AP2 and NFkappaB. The maintenance and hormonal regulation of AMH expression in late fetal and postnatal life requires distal AMH promoter sequences. In the ovary, granulosa cells express AMH from late fetal life at low levels; DAX1 and FOG2 seem to be responsible for negatively modulating AMH expression. Particular features are observed in AMH expression in nonmammalian species. In birds, AMH is expressed both in the male and female fetal gonads, and, like in reptiles, its expression is not preceded by that of SOX9.

GATA-1 is a potential repressor of anti-Müllerian hormone expression during the establishment of puberty in the mouse.

Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS), is one of the earliest and best-known markers of Sertoli cell differentiation and is expressed until around puberty. The present study is aimed at the better understanding of the molecular pathways involved in testicular development and establishment of adult functions with regards to AMH regulation. We found, within the mouse AMH promoter, putative GATA motifs (A/T)GATA(A/G), known to be specifically bound by members of the GATA transcription factor family. We then carried out RNase protection assays and immunohistochemical techniques aimed at comparing precisely the chronological expression patterns of AMH and GATA-1, this latter being expressed in the testis after birth. Using both approaches we found an inverse and close relationship between AMH and GATA-1 mRNA and protein expression during the pre-pubertal period. These results allowed us to define a transitory 4-5-day period, starting from 3 dpp when both proteins are heterogeneously expressed in Sertoli cells and showed that the appearance of GATA-1 is associated with the decrease of AMH expression in these cells. Furthermore DNA-protein interaction in in vitro studies showed first that GATA-1 binds with various affinities on sites found in the AMH promoter and second that the proximity of the two strongest affinity sites leads to a synergistic binding effect. Altogether, the present study suggests that GATA-1 participates in AMH gene repression during the pre-pubertal period.

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

Early expression of AMH in chicken embryonic gonads precedes testicular SOX9 expression.

In mammals, anti-Müllerian hormone (AMH) is produced by Sertoli cells from the onset of testicular differentiation and by granulosa cells only after birth. SOX9, a transcription factor related to the testis-determining factor SRY, is expressed in mouse testis 1 day before AMH. To determine the relationship between AMH and SOX9 in birds, we cloned the AMH promoter in search of SOX9 response elements, and we compared the expression of AMH and SOX9 in the gonads of chick embryos using in situ hybridization. Potential SOX response elements were found in the AMH promoter; however, AMH is expressed in both sexes at stage 25, 1 day before the first SOX9 transcripts appear in the male gonads. SOX9 is never expressed in the female. These results do not support the hypothesis that SOX9 could trigger the expression of testicular AMH in the chick but does not exclude a later role in testis development.