Comparable to 17α- methyl testosterone, dietary supplements of Tribulus terrestris and Mucuna pruriens promote the development of mono-sex, all-male tilapia fry, growth, survival rate and sex-related genes (Amh, Sox9, Foxl2, Dmrt1).

To evaluate Tribulus terrestris and Mucuna pruriens for inducing all-male tilapia, mixed-sex Nile tilapia, Oreochromis niloticus, (mean weight 0.025 ± 0.009 g; mean length 1.25 ± 0.012 cm), were given a meal supplemented with either T. terrestris powder (commercial fish feed, 40% crude protein) (TT group), M. pruriens seed extract (MP group), MP + TT (mixed group), 17α-methyl testosterone (MT, control positive), or without supplements (control negative). The MP extracts significantly increased (P < 0.05) the final weight, weight gain, weight gain rate, and specific growth rate while feed conversion ratio was significantly decreased (P < 0.05). Plant extracts markedly improved (P < 0.05) the survival rate, proportion of males, and total testosterone compared to control and MT. Estrogen levels were lower in groups with plant extract than other groups. Fifteen days post-feeding, the Amh gene was expressed in the brain of O. niloticus fries with higher levels in MP, TT, and MT groups. Additionally, the expression of the Sox9 and Dmrt1 genes as a male related genes in fish fry gonads revealed significantly (P < 0.05) higher levels in groups fed on MP, TT, and MT compared to control after 30-day post-feeding, whereas; Foxl2 gene expression as a female related gene was significantly (P < 0.05) lower in fish fed on MP, TT, and MT compared to other groups after 30 days post feeding. Histologically, MT, MP, TT, and the mixture all exhibited solely male reproductive traits without noticeable abnormalities. This study concluded that each of the TT or MP extracts can induce sex reversal in tilapia while having no negative health impact compared to MT as the growth and survival rate in the treated groups with TT and MP were higher than control and group treated with MT.

Lack of causative mutation in the AMH and AMHR2 genes in a cat (38,XY) with persistent Mullerian duct syndrome (PMDS).

A 1-year-old European shorthair male cat with a normally developed penis was subjected to genetic, endocrinological and histological studies due to unilateral cryptorchidism. The blood testosterone level was typical for males, while the level of anti-Mullerian hormone (AMH) was very low. Surgical removal of internal reproductive organs was followed by a histological study, which revealed inactive testicles with neoplastic changes and derivatives of Mullerian ducts. Cytogenetic analysis showed a normal XY sex chromosome complement and molecular analysis confirmed the presence of Y-linked genes (SRY and ZFY). Although the level of AMH was low, two normal copies of the AMH gene were found using droplet digital PCR (ddPCR). Analysis of the coding sequences of two candidate genes (AMH and AMHR2) for persistent Mullerian duct syndrome (PMDS) in the affected cat and in control male cats (n = 24) was performed using the Sanger sequencing method. In the affected cat, homozygosity was found for three novel missense variants in Exon 1 (one SNP) and Exon 5 (two SNPs) of AMH, but the same homozygous genotypes were also observed in one and two control cats, respectively, whose sex development was not examined. Three known synonymous variants with homozygous status were found in AMHR2. We conclude that the DNA variants identified in AMH and AMHR2 are not responsible for PMDS in the affected cat.

Low and High-Normal FMR1 Triplet Cytosine, Guanine Guanine Repeats Affect Ovarian Reserve and Fertility in Women Who Underwent In Vitro Fertilization Treatment? Results from a Cross-Sectional Study.

Dynamic mutations in the 5' untranslated region of FMR1 are associated with infertility. Premutation alleles interfere with prenatal development and increase infertility risks. The number of CGG repeats that causes the highest decrease in ovarian reserves remains unclear. We evaluated the effect of FMR1 CGG repeat lengths on ovarian reserves and in vitro fertilization (IVF) treatment outcomes in 272 women with alleles within the normal range. FMR1 CGG repeat length was investigated via PCR and capillary electrophoresis. Alleles were classified as low-normal, normal, and high-normal. Serum levels of follicle-stimulating hormone and anti-Mullerian hormone (AMH) in the follicular phase of the menstrual cycle were measured, and antral follicles (AFC) were counted. IVF outcomes were collected from medical records. Regarding FMR1 CGG repeat length alleles, 63.2% of women presented at least one low-normal allele. Those carrying low-normal alleles had significantly lower AMH levels than women carrying normal or high-normal alleles. Low-normal/low-normal genotype was the most frequent, followed by low-normal/normal and normal/normal. A comparison of ovarian reserve markers and reproductive outcomes of the three most frequent genotypes revealed that AFC in the low-normal/normal genotype was significantly lower than the low-normal/low-normal genotype. The low number of FMR1 CGG repeats affected AMH levels and AFC but not IVF outcomes per cycle of treatment.

Expression and localization of anti-Müllerian hormone and its receptors in bovine corpus luteum.

Although anti-Müllerian hormone (AMH) is involved in the regulation of granulosa cell function in female animals, its role in tissues other than ovarian follicles remains poorly understood. It has also been suggested that cows with high circulating AMH concentrations have increased fertility; however, the mechanism has not been elucidated. This study was conducted to identify the presence of the AMH-signaling system and its target cells in the bovine corpus luteum formed from an ovulated follicle. Immunoblotting revealed that the proteolytically cleaved C-terminal region in AMH (AMHC), a biologically active peptide, was present in trace amounts in the early corpus luteum and significantly increased during the mid to regressed stages. AMHC and cleaved N-terminal region (AMHN) in AMH generate a noncovalent isoform that improves the activity of AMH signaling. An immunohistochemical analysis revealed that AMHC, AMHN, and type II AMH receptor (AMHR2) were localized to luteal cells during the entire estrous cycle. AMH in the corpus luteum seemed to be newly synthesized since AMH expression was detected. These findings suggest that AMH signaling is involved in the regulation of luteal cell function through an autocrine and post-translational processing mechanism. The level of AMHR2 and mRNA expression of AMHR2 and type I AMH receptors (activin-like kinase 2, 3, and 6) were highest in the mid stage. Thus, AMH signaling in the corpus luteum may also be regulated by changes in the receptor levels. Since the transforming growth factor-beta superfamily, to which AMH belongs, is a multifunctional polypeptide growth factor, further studies are needed to evaluate whether AMH signaling has a role in facilitating or inhibiting luteal cell functions.

DNA Methylation in the Anti-Mullerian Hormone Gene and the Risk of Disease Activity in Multiple Sclerosis.

OBJECTIVE: Multiple sclerosis (MS) has a complex pathobiology, with genetic and environmental factors being crucial players. Understanding the mechanisms underlying heterogeneity in disease activity is crucial for tailored treatment. We explored the impact of DNA methylation, a key mechanism in the genetics-environment interplay, on disease activity in MS. METHODS: Peripheral immune methylome profiling using Illumina Infinium MethylationEPIC BeadChips was conducted on 249 untreated relapsing-remitting MS patients, sampled at the start of disease-modifying treatment (DMT). A differential methylation analysis compared patients with evidence of disease activity (EDA) to those with no evidence of disease activity (NEDA) over 2 years from DMT start. Utilizing causal inference testing (CIT) and Mendelian randomization (MR), we sought to elucidate the relationships between DNA methylation, gene expression, genetic variation, and disease activity. RESULTS: Four differentially methylated regions (DMRs) were identified between EDA and NEDA. Examining the influence of single nucleotide polymorphisms (SNPs), 923 variants were found to account for the observed differences in the 4 DMRs. Importantly, 3 out of the 923 SNPs, affecting DNA methylation in a DMR linked to the anti-Mullerian hormone (AMH) gene, were associated with disease activity risk in an independent cohort of 1,408 MS patients. CIT and MR demonstrated that DNA methylation in AMH acts as a mediator for the genetic risk of disease activity. INTERPRETATION: This study uncovered a novel molecular pathway implicating the interaction between DNA methylation and genetic variation in the risk of disease activity in MS, emphasizing the role of sex hormones, particularly the AMH, in MS pathobiology. ANN NEUROL 2024;96:289-301.

Anti-Müllerian hormone induces autophagy to preserve the primordial follicle pool in mice.

The reserve pool of primordial follicles (PMFs) is finely regulated by molecules implicated in follicular growth or PMF survival. Anti-Müllerian hormone (AMH), produced by granulosa cells of growing follicles, is known for its inhibitory role in the initiation of PMF growth. We observed in a recent in vivo study that injection of AMH into mice seemed to induce an activation of autophagy. Furthermore, injection of AMH into mice activates the transcription factor FOXO3A which is also known for its implication in autophagy regulation. Many studies highlighted the key role of autophagy in the ovary at different stages of folliculogenesis, particularly in PMF survival. Through an in vitro approach with organotypic cultures of prepubertal mouse ovaries, treated or not with AMH, we aimed to understand the link among AMH, autophagy, and FOXO3A transcription factor. Autophagy and FOXO3A phosphorylation were analyzed by western blot. The expression of genes involved in autophagy was quantified by RT-qPCR. In our in vitro model, we confirmed the decrease in FOXO3A phosphorylation and the induction of autophagy in ovaries incubated with AMH. AMH also induces the expression of genes involved in autophagy. Interestingly, most of these genes are known to be FOXO3A target genes. In conclusion, we have identified a new role for AMH, namely the induction of autophagy, probably through FOXO3A activation. Thus, AMH protects the ovarian reserve not only by inhibiting the growth of PMFs but also by enabling their survival through activation of autophagy.

Brca2(p.T1942fs/+) dissipates ovarian reserve in rats through oxidative stress in follicular granulosa cells.

Pathogenic variants of BRCA1/2 constitute hereditary breast and ovarian cancer (HBOC) syndrome, and BRCA1/2 mutant is a risk for various cancers. Whereas the clinical guideline for HBOC patients has been organized for the therapy and prevention of cancer, there is no recommendation on the female reproductive discipline. Indeed, the role of BRCA1/2 pathogenic variants in ovarian reserve has not been established due to the deficiency of appropriate animal models. Here, we used a rat model of Brca2(p.T1942fs/+) mutant of Sprague-Dawley strain with CRISPR-Cas9 editing to evaluate ovarian reserve in females. Fertility and ovarian follicles were evaluated and anti-Müllerian hormone (AMH) was measured at 8-32 weeks of age with a comparison between the wild-type and the mutant rats (MUT). MUT revealed a significantly smaller number of deliveries with fewer total pups. Furthermore, MUT showed a significant decrease in primordial follicles at 20 weeks and a low AMH level at 28 weeks. RNA-sequencing of the ovary at 10 weeks detected acceleration of the DNA damage repair pathway, which was accompanied by oxidative stress-induced DNA double-strand breaks, a decrease in PTEN, and an increase in mTOR in follicular granulosa cells. In conclusion, Brca2(p.T1942fs/+) dissipates primordial follicles via early activation of granulosa cells through oxidative stress, leading to earlier termination of fertility.

Estrogen Signaling Inhibits the Expression of anti-Müllerian hormone (amh) and gonadal-soma-derived factor (gsdf) during the Critical Time of Sexual Fate Determination in Zebrafish.

The mechanism of fish gonadal sex differentiation is complex and regulated by multiple factors. It has been widely known that proper steroidogenesis in Leydig cells and sex-related genes in Sertoli cells play important roles in gonadal sex differentiation. In teleosts, the precise interaction of these signals during the sexual fate determination remains elusive, especially their effect on the bi-potential gonad during the critical stage of sexual fate determination. Recently, all-testis phenotypes have been observed in the cyp17a1-deficient zebrafish and common carp, as well as in cyp19a1a-deficient zebrafish. By mating cyp17a1-deficient fish with transgenic zebrafish Tg(piwil1:EGFP-nanos3UTR), germ cells in the gonads were labelled with enhanced green fluorescent protein (EGFP). We classified the cyp17a1-deficient zebrafish and their control siblings into primordial germ cell (PGC)-rich and -less groups according to the fluorescence area of the EGFP labelling. Intriguingly, the EGFP-labelled bi-potential gonads in cyp17a1+/+ fish from the PGC-rich group were significantly larger than those of the cyp17a1-/- fish at 23 days post-fertilization (dpf). Based on the transcriptome analysis, we observed that the cyp17a1-deficient fish of the PGC-rich group displayed a significantly upregulated expression of amh and gsdf compared to that of control fish. Likewise, the upregulated expressions of amh and gsdf were observed in cyp19a1a-deficient fish as examined at 23 dpf. This upregulation of amh and gsdf could be repressed by treatment with an exogenous supplement of estradiol. Moreover, tamoxifen, an effective antagonist of both estrogen receptor α and ß (ERα and Erß), upregulates the expression of amh and gsdf in wild-type (WT) fish. Using the cyp17a1- and cyp19a1a-deficient zebrafish, we provide evidence to show that the upregulated expression of amh and gsdf due to the compromised estrogen signaling probably determines their sexual fate towards testis differentiation. Collectively, our data suggest that estrogen signaling inhibits the expression of amh and gsdf during the critical time of sexual fate determination, which may broaden the scope of sex steroid hormones in regulating gonadal sex differentiation in fish.

Investigations of the function of AMH in granulosa cells in hens.

Anti-mullerian hormone (AMH) plays a crucial role in follicle regulation in mammals by preventing premature primordial follicle activation and restricting follicle development through reduction of FSH sensitivity and inhibition of FSH-induced increase of steroidogenic enzymes. AMH is produced by granulosa cells from growing follicles and expression declines at the time of selection in both mammalian and avian species. The role of AMH in chicken granulosa cells remains unclear, as research is complicated because mammalian AMH is not bioactive in chickens and there is a lack of commercially available chicken AMH. In the current experiments, we used RNA interference to study the role of AMH on markers of follicle development in the presence and absence of FSH. Cultured chicken granulosa cells from 3-5 mm follicles and 6-8 mm follicles, the growing pool from which follicle selection is thought to occur, were used. Transfection with an AMH-specific siRNA significantly reduced AMH mRNA expression in granulosa cells from 3-5 mm and 6-8 mm follicles. Genes of interest were only measured in granulosa cells of 3-5 mm follicles due to low expression of AMH mRNA at the 6-8 mm follicle stage. Knockdown of AMH mRNA did not affect markers of follicle development (follicle stimulating hormone receptor, FSHR; steroidogenic acute regulatory protein, STAR; cytochrome P450 family 11 subfamily A member 1, CYP11A1; bone morphogenetic protein receptor type 2, BMPR2) or FSH responsiveness in granulosa cells from 3-5 mm follicles, indicating that AMH does not regulate follicle development directly by affecting markers of steroidogenesis, FSHR or BMPR2 at this follicle stage in chickens.

Characterization of the molecular mechanisms that govern anti-Müllerian hormone synthesis and activity.

The roles of anti-Müllerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic-pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor-binding interface, we generated a series of human AMH variants and assessed bioactivity. AMHSCUT potency (EC50 4 ng/mL) was increased 5- or 10-fold by incorporating Gln484 Met/Leu535 Thr (EC50 0.8 ng/mL) or Gln484 Met/Gly533 Ser (EC50 0.4 ng/mL) mutations, respectively. Furthermore, the Gln484 Met/Leu535 Thr double mutant displayed enhanced efficacy, relative to AMHSCUT . Finally, we identified residues within the wrist pre-helix of AMH (Trp494 , Gln496 , Ser497 , and Asp498 ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain- (Trp494 Phe or Gln496 Leu) or loss- (Ser497 Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step-wise receptor-complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.

Novel homozygous mutation in a colombian patient with persistent müllerian duct syndrome: expanded phenotype

ABSTRACT The anti-Müllerian hormone triggers the regression of uterus and fallopian tubes in male embryos; if there are problems in the synthesis or action of this protein, Müllerian structures persist in an otherwise phenotypic male. The most frequent clinical presentation of Persistent Mullerian Duct syndrome is cryptorchidism and inguinal hernia. The few cases reported in adults are incidental findings or inguinal hernias. However, we present an adult male with history of bilateral cryptorchidism with unsuccessful orchidopexy, who presents with a large abdominal mass with the finding of a seminomatous tumor and persistence of Müllerian structures, in whom the variant c.916delC (p.Leu306Cysfs*29) in the AMHR2 gene not previously reported was documented.

Síndrome de persistencia del conducto de Müller debido a mutación en el gen del receptor de la hormona antimülleriana (AMHR2) / Persistent Müllerian duct syndrome due to a mutation in the anti-Müllerian hormone receptor gene (AMHR2)

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Analysis of anti-Müllerian hormone (AMH) and its receptor (AMHR2) genes in patients with persistent Müllerian duct syndrome / Pesquisa de mutações nos genes do hormônio antiMülleriano (AMH) e do seu receptor (AMHR2) em pacientes com síndrome de persistência dos ductos Müllerianos

OBJECTIVE: To screen for mutations in AMH and AMHR2 genes in patients with persistent Müllerian duct syndrome (PMDS). PATIENTS AND METHOD: Genomic DNA of eight patients with PMDS was obtained from peripheral blood leukocytes. Directed sequencing of the coding regions and the exon-intron boundaries of AMH and AMHR2 were performed. RESULTS: The AMH mutations p.Arg95*, p.Arg123Trp, c.556-2A>G, and p.Arg502Leu were identified in five patients; and p.Gly323Ser and p.Arg407* in AMHR2 of two individuals. In silico analyses of the novel c.556-2A>G, p.Arg502Leu and p.Arg407* mutations predicted that they were harmful and were possible causes of the disease. CONCLUSION: A likely molecular etiology was found in the eight evaluated patients with PMDS. Four mutations in AMH and two in AMHR2 were identified. Three of them are novel mutations, c.556-2A>G, and p.Arg502Leu in AMH; and p.Gly323Ser in AMHR2. Arq Bras Endocrinol Metab. 2012;56(8):473-8.

OBJETIVO: Analisar os genes AMH e AMHR2 em indivíduos com síndrome de persistência dos ductos de Müller (SPDM). PACIENTES E MÉTODO: Amostras de DNA genômico de oito pacientes com SPDM foram obtidas de leucócitos de sangue periférico. Sequenciamento direto da região codificadora e das áreas intrônicas próximas aos éxons dos genes AMH e AMHR foi realizado. RESULTADOS: As mutações p.Arg95*, p.Arg123Trp, c.556-2A>G e p.Arg502Leu no gene AMH foram identificadas em cinco pacientes e as mutações p.Gly323Ser e p.Arg407* no gene AMHR2, em dois indivíduos. As análises in silico das mutações c.556-2A>G, p.Arg502Leu e p.Arg407*, não descritas anteriormente na literatura, previram que elas são deletérias e possivelmente a causa da doença. CONCLUSÃO: Uma provável etiologia molecular foi encontrada nos oito pacientes portadores de SPDM avaliados. No gene do AMH foram identificadas quatro mutações e no AMHR2, duas mutações. Três das seis mutações encontradas são mutações novas, c.556-2A>G e p.Arg502Leu no gene AMH; e p.Gly323Ser no AMHR2. Arq Bras Endocrinol Metab. 2012;56(8):473-8.