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.

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.

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.

[Hormonal and genetic causes of cryptorchidism].

Cryptorchidism is the most frequent congenital disorders of the reproductive system, is present in 2-3% of term newborn boys. Genes involved in embryonic testicular migration are known but their role in cryptorchidism development are not investigated enough. Genetical causes of cryptorchidism are identified in 5-7% of patients. The article contains data on the role of insulin-like peptide 3 and its receptor, anti-Müllerian hormone, gonadotropins, androgens in embryonic testicular migration. INSL3 and AMH are presented as markers of testicular dysfunction associated with cryptorchidism. Hypogonadotropic hypogonadism is also associated with cryptorchidism and can be diagnosed based on it. Results of modern investigations determine the necessary of hormonal and genetical examination of patients with isolated cryptorchidism to detect causes of cryptorchidism and manage of patients.

SMAD signaling pathway is disrupted by BPA via the AMH receptor in bovine granulosa cells†.

Significant events that determine oocyte competence occur during follicular growth and oocyte maturation. The anti-Mullerian hormone, a positive predictor of fertility, has been shown to be affected by exposure to endocrine disrupting compounds, such as bisphenol A and S. However, the interaction between bisphenols and SMAD proteins, mediators of the anti-Mullerian hormone pathway, has not yet been elucidated. AMH receptor (AMHRII) and downstream SMAD expression was investigated in bovine granulosa cells treated with bisphenol A, bisphenol S, and then competitively with the anti-Mullerian hormone. Here, we show that 24-h bisphenol A exposure in granulosa cells significantly increased SMAD1, SMAD4, and SMAD5 mRNA expression. No significant changes were observed in AMHRII or SMADs protein expression after 24-h treatment. Following 12-h treatments with bisphenol A (alone or with the anti-Mullerian hormone), a significant increase in SMAD1 and SMAD4 mRNA expression was observed, while a significant decrease in SMAD1 and phosphorylated SMAD1 was detected at the protein level. To establish a functional link between bisphenols and the anti-Mullerian hormone signaling pathway, antisense oligonucleotides were utilized to suppress AMHRII expression with or without bisphenol exposure. Initially, transfection conditions were optimized and validated with a 70% knockdown achieved. Our findings show that bisphenol S exerts its effects independently of the anti-Mullerian hormone receptor, while bisphenol A may act directly through the anti-Mullerian hormone signaling pathway providing a potential mechanism by which bisphenols may exert their actions to disrupt follicular development and decrease oocyte competence.

Mechanisms of DHEA-induced AMH increase in the ovarian tissues of PCOS rat.

The present study aimed to build a DHEA-induced polycystic ovary syndrome (PCOS) rat model to evaluate the potential mechanism of DHEA-induced AMH rise in these rat ovarian tissues. A total of 36 female 3-week-old rats were allocated into two groups at random. The control group received merely the same amount of sesame oil for 20 days while the experimental group received 0.2 mL of sesame oil Plus DHEA 6 mg/100 g daily. Both groups' vaginal opening times were noted, and vaginal smears were taken. By using RT-qPCR and Western blot, the mRNA and protein expression of AMH, GATA4, SF1, and SOX9 in the ovarian tissues of the two groups was investigated.The rats in the experimental group appeared to have obvious disorders of the estrus cycle, as evidenced by the ratio of estrus being significantly higher than that in the control group (P < 0.05); HE staining revealed that the ovarian volume, follicular vacuoles, and follicular lumen of the rats in the experimental group increased significantly.The ELISA results revealed that T and AMH in the experimental group were higher than those in the control group at day 15 and 20. AMH、GATA4 and SF1 mRNA and protein expression were higher in the experimental group than in the control group on day 15 and 20 (P < 0.05). On day 20, the experimental group outperformed the control group (P < 0.05). In the DHEA-induced PCOS rat model, androgen may have enhanced AMH expression via increasing the expression of genes associated to the AMH promoter binding site (GATA4, SF1, SOX9).

Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis.

Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human in vitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2-/- CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2-/- tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer.

Sexually dimorphic expression of AMH facilitates testis differentiation pathway in the tropical lizard, Calotes versicolor (Daud.).

The Anti-mullerian hormone (AMH), also known as Mullerian inhibiting substance (MIS), is a glycoprotein that belongs to transforming growth factor ß superfamily. The significance of AMH during gonadal differentiation is not clearly deciphered in reptiles. Hence, current study aims to know the onset of AMH secretion and its functional role in Mullerian duct regression gonadal differentiation in tropical lizard, Calotes versicolor which exhibits a novel Female-Male-Female-Male (FMFM) pattern of temperature-dependent sex determination (TSD). The Immunohistochemistry and qRT-PCR techniques were employed to analyze the gonadal expression profile of AMH during different stages of embryonic development. The eggs of the lizard were incubated at both male-producing temperature (MPT: 25.5 ± 0.5 °C) and female-producing temperatures (FPT: 31.5 ± 0.5 °C). The results reveal that the onset of AMH gene expression was observed as early as oviposition prior to the immunolocalization of AMH protein at early-TSP (Temperature-sensitive period). The substantial rise in the intensity of the immunoreaction of AMH protein in the cytoplasm confining to Sertoli cells of seminiferous cords at MPT with low level of expression at FPT during gonadal sex differentiation, specify sexually dimorphic expression of AMH protein. Further, with the onset of sexual differentiation, the developing testis immensely expresses AMH gene which is 7-fold greater than that of transcripts levels in female embryos; signifies its conserved role in Mullerian duct regression thereby promoting testis differentiation. The robust immunnoexpression of AMH protein during post-gonadal differentiation coincides with the onset of the regression of Mullerian duct point out a positive correlation between testis differentiation and Mullerian duct regression, thus facilitating testis differentiation pathway. Based on the immunoexpression pattern of AMH protein and transcript levels of AMH gene, it is inferred that AMH plays a significant role in Mullerian duct regression, favoring testis differentiation.

The roles of anti-Müllerian hormone in breast cancer.

Anti-Müllerian hormone (AMH) is produced and secreted by granulosa cells of growing follicles, and its main role is to inhibit the recruitment of primordial follicles, reduce the sensitivity of follicles to follicle-stimulating hormone (FSH), and regulate FSH-dependent preantral follicle growth. It has become an effective indicator of ovarian reserve in clinical practice. Research on AMH and its receptors in recent years has led to a better understanding of its role in breast cancer. AMH specifically binds to anti-Müllerian hormone receptor II (AMHRII) to activate downstream pathways and regulate gene transcription. Since AMHRII is expressed in breast cancer cells and triggers apoptosis, AMH/AMHRII may play an important role in the occurrence, treatment, and prognosis of breast cancer, which needs further research. The AMH level is a potent predictor of ovarian function after chemotherapy in premenopausal breast cancer patients older than 35 years, either for ovarian function injury or ovarian function recovery. Moreover, AMHRII has the potential to be a new marker for the molecular typing of breast cancer and a new target for breast cancer treatment, which may be a link in the downstream pathway after TP53 mutation.

Durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene.

Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

Personalized prediction of the secondary oocytes number after ovarian stimulation: A machine learning model based on clinical and genetic data.

Controlled ovarian stimulation is tailored to the patient based on clinical parameters but estimating the number of retrieved metaphase II (MII) oocytes is a challenge. Here, we have developed a model that takes advantage of the patient's genetic and clinical characteristics simultaneously for predicting the stimulation outcome. Sequence variants in reproduction-related genes identified by next-generation sequencing were matched to groups of various MII oocyte counts using ranking, correspondence analysis, and self-organizing map methods. The gradient boosting machine technique was used to train models on a clinical dataset of 8,574 or a clinical-genetic dataset of 516 ovarian stimulations. The clinical-genetic model predicted the number of MII oocytes better than that based on clinical data. Anti-Müllerian hormone level and antral follicle count were the two most important predictors while a genetic feature consisting of sequence variants in the GDF9, LHCGR, FSHB, ESR1, and ESR2 genes was the third. The combined contribution of genetic features important for the prediction was over one-third of that revealed for anti-Müllerian hormone. Predictions of our clinical-genetic model accurately matched individuals' actual outcomes preventing over- or underestimation. The genetic data upgrades the personalized prediction of ovarian stimulation outcomes, thus improving the in vitro fertilization procedure.

Functional analysis of rare anti-Müllerian hormone protein-altering variants identified in women with PCOS.

Recently, rare heterozygous AMH protein-altering variants were identified in women with polycystic ovary syndrome (PCOS), causing reduced anti-Müllerian hormone (AMH) signaling. However, the exact functional mechanism remains unknown. Here, we analyzed the processing, secretion, and signaling of these AMH variants. Functional analysis of six PCOS-specific AMH variants (V12G, P151S, P270S, P352S, P362S, H506Q) and one control-specific variant (A519V) was performed in the mouse granulosa cell-line KK-1. Human (h) AMH-151S and hAMH-506Q have ∼90% decreased AMH signaling compared to wild-type (wt) AMH signaling. Coexpression of hAMH-151S or hAMH-506Q with wt-hAMH dose-dependently inhibited wt-hAMH signaling. Western blotting revealed that hAMH-151S and hAMH-506Q proteins were detected in the cell lysate but not in the supernatant. Confocal microscopy showed that HEK293 cells expressing hAMH-151S and hAMH-506Q had higher cellular AMH protein levels with endoplasmic reticulum (ER) retention compared to cells expressing wt-hAMH. Using two AMH ELISA kits, hAMH-151S was detected in the cell lysate, while only very low levels were detected in the supernatant. Both hAMH-362S and hAMH-519V were detectable using the automated AMH ELISA but showed severely reduced immunoactivity in the manual ELISA. Surprisingly, hAMH-506Q was undetectable in both the cell lysate and supernatant using either ELISA. However, in PCOS cases, heterozygous carriers of the P151S and H506Q variants still had detectable AMH in both assays. Thus, P151S and H506Q disrupt normal processing and secretion of AMH, causing ER retention. Additionally, AMH variants can impair the AMH immunoactivity. An AMH variant may be considered when serum AMH levels are relatively low in PCOS cases.

Potential Role of Anti-Müllerian Hormone in Regulating Seasonal Reproduction in Animals: The Example of Males.

Seasonal reproduction is a survival strategy by which animals adapt to environmental changes to improve their fitness. Males are often characterized by a significantly reduced testicular volume, indicating that they are in an immature state. Although many hormones, including gonadotropins, have played a role in testicular development and spermatogenesis, research on other hormones is insufficient. The anti-Müllerian hormone (AMH), which is a hormone responsible for inducing the regression of Müllerian ducts involved in male sex differentiation, was discovered in 1953. Disorders in AMH secretion are the main biomarkers of gonadal dysplasia, indicating that it may play a crucial role in reproduction regulation. A recent study has found that the AMH protein is expressed at a high level during the non-breeding period of seasonal reproduction in animals, implying that it may play a role in restricting breeding activities. In this review, we summarize the research progress on the AMH gene expression, regulatory factors of the gene's expression, and its role in reproductive regulation. Using males as an example, we combined testicular regression and the regulatory pathway of seasonal reproduction and attempted to identify the potential relationship between AMH and seasonal reproduction, to broaden the physiological function of AMH in reproductive suppression, and to provide new ideas for understanding the regulatory pathway of seasonal reproduction.

miR-140-3p promotes follicle granulosa cell proliferation and steroid hormone synthesis via targeting AMH in chickens.

Granulosa cells (GCs) are the ovary's most critical cells since they undergo cell differentiation and hormone synthesis changes closely associated with follicle development. While micro RNA 140-3p (miRNA-140-3p) has an apparent cell signaling role, particularly in cell proliferation, its biological role in chicken ovarian follicle growth and development remains elusive. This study explored miR-140-3p's effects on chicken GC proliferation and steroid hormone synthesis. MiR-140-3p dramatically increased GC proliferation, prevented apoptosis, increased progesterone synthesis, and enhanced gene expression related to steroid hormone synthesis. In addition, the anti-Müllerian hormone (AMH) gene was identified as a direct miR-140-3p target. MiR-140-3p abundance correlated negatively with AMH mRNA and protein levels in GCs. Our findings show that miR-140-3p influences chicken GC proliferation and steroid hormone synthesis by suppressing AMH expression.

Expression pattern analysis of anti-Mullerian hormone in testis development of pearlscale angelfish (Centropyge vrolikii).

In vertebrates, anti-Mullerian hormone (Amh) secreted by Sertoli cells (SC) performs a pivotal function in male sex differentiation. Compared with that of higher vertebrates, the expression pattern of Amh is more diversified in fish. In this study, the full-length complementary DNA (cDNA) of Amh in Centropyge vrolikii (Cv-Amh) was cloned and analysed, which was 2,470 bp, including a 238 bp 5'UTR, a 1,602 bp ORF and a 633 bp 3'UTR; the similarity of Amh between Cv-Amh and other fish is relatively high. The quantitative real-time PCR (qRT-PCR) results of healthy tissues and gonads at sex reversal stages in C. vrolikii showed that the expression level of Amh in the testis was significantly higher than that in other tissues (P < 0.05). Amh was weakly expressed in the vitellogenic stage ovary and perinucleolus stage ovary, but its expression significantly increased in the gonads at the hermaphroditic stage, and finally reached the highest in the pure testis after sexual reversal. The results of in situ hybridization indicated that the positive signal of Amh was strongly concentrated in SCs of testis. After Amh knockdown in the gonads, the effect on sex-related genes was tested using qRT-PCR. Among these, the expression of Dmrt1, Cyp11a, Hsd11b2, Sox8 and Sox9 significantly decreased, whereas that of Cyp19a, Sox4, Foxl2 and Sox3 increased. These results suggested that Amh could be the pivotal gene in reproductive regulation in C. vrolikii, and the data will contribute to sex-related research of C. vrolikii in the future.

Amh/Amhr2 Signaling Causes Masculinization by Inhibiting Estrogen Synthesis during Gonadal Sex Differentiation in Japanese Flounder (Paralichthys olivaceus).

The anti-Müllerian hormone (Amh) is a protein belonging to the TGF-ß superfamily, the function of which has been considered important for male sex differentiation in vertebrates. The Japanese flounder (Paralichthys olivaceus) is a teleost fish that has an XX/XY sex determination system and temperature-dependent sex determination. In this species, amh expression is up-regulated in genetic males and in temperature-induced masculinization during the sex differentiation period. However, to the best of our knowledge, no reports on the Amh receptor (Amhr2) in flounder have been published, and the details of Amh signaling remain unclear. In this study, we produced amhr2-deficient mutants using the CRISPR/Cas9 system and analyzed the gonadal phenotypes and sex-related genes. The results revealed that the gonads of genetically male amhr2 mutants featured typical ovaries, and the sex differentiation-related genes showed a female expression pattern. Thus, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. Moreover, the treatment of genetically male amhr2 mutants with an aromatase inhibitor fadrozole, which inhibits estrogen synthesis, resulted in testicular formation. These results strongly suggest that Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in the flounder.

A Surgical and Clinical Approach to Persistent Müllerian Duct Syndrome: Laparoscopic, Histological, and Molecular Findings.

BACKGROUND: Persistent müllerian duct syndrome (PMDS) is characterized by the persistence of müllerian duct derivatives in otherwise normally virilized 46,XY males. Biallelic mutations of the anti-müllerian hormone (AMH) and AMH receptor type 2 (AMHR2) genes lead to PMDS type 1 and 2, respectively. AIM: The aims of the study were to report the clinical, hormonal, and genetic findings in a patient with PMDS and discuss surgical strategies to achieve successful orchidopexy. RESULTS: A 4-year-old boy was evaluated after the incidental finding of müllerian derivates during laparoscopy for nonpalpable gonads. Karyotype was 46,XY and laboratory tests revealed normal serum gonadotropin and androgen levels but undetectable serum AMH levels. PMDS was suspected. Molecular analysis revealed a novel variant c.902_929del in exon 5 and a previously reported mutation (c.367C>T) in exon 1 of the AMH gene. Successful orchidopexy was performed in two sequential surgeries in which the müllerian duct structure was preserved and divided to protect the vascular supply to the gonads. Histological evaluation of the testicular biopsy showed mild signs of dysgenesis. Doppler ultrasound showed blood flow in both testes positioned in the scrotum 1.5 years after surgery. CONCLUSION: PMDS is a rare entity that requires a high index of suspicion (from surgeons) when evaluating a patient with bilateral cryptorchidism. Surgical treatment is challenging and long-term follow-up is essential. Histological evaluation of the testis deserves further investigation.

BMP6 regulates AMH expression via SMAD1/5/8 in goat ovarian granulosa cells.

Anti-Müllerian hormone (AMH) is produced by ovarian granulosa cells (GCs)and plays a major role in inhibiting the recruitment of primordial follicles and reducing the sensitivity of growing follicles to follicle-stimulating hormone (FSH). Bone morphogenetic protein 6 (BMP6) has similar spatiotemporal expression to AMH during follicular development, suggesting that BMP6 may regulate AMH expression. However, the specific mechanism by which BMP6 regulates AMH expression remains unclear. The objectives of this study were to examine the molecular pathway by which BMP6 regulates AMH expression. The results showed that BMP6 promoted the secretion and expression of AMH in goat ovarian GCs. Mechanistically, BMP6 upregulated the expression of sex-determining region Y-box 9 (SOX9) and GATA-binding factor 4 (GATA4), which was associated with the transcriptional initiation of AMH. AMH expression was significantly decreased by GATA4 knockdown. Moreover, BMP6 treatment promoted the phosphorylation of SMAD1/5/8, whereas inhibiting the SMAD1/5/8 signaling pathway significantly abolished BMP6-induced upregulation of AMH and GATA4 expression. Interestingly, the activation of SMAD1/5/8 alone did not affect the expression of AMH or GATA4. The results suggested that BMP6 upregulated GATA4 through the SMAD1/5/8 signaling pathway, which in turn promoted AMH expression.