Osterix functions downstream of anti-Müllerian hormone signaling to regulate Müllerian duct regression.

In mammals, the developing reproductive tract primordium of male and female fetuses consists of the Wolffian duct and the Müllerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct, and upper vagina. It is well established that transforming growth factor-ß family member anti-Müllerian hormone (AMH) secreted from the fetal testis and its type 1 and 2 receptors expressed in MD mesenchyme regulate MD regression. However, little is known about the molecular network regulating downstream actions of AMH signaling. To identify potential AMH-induced genes and regulatory networks controlling MD regression in a global nonbiased manner, we examined transcriptome differences in MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) by RNA-seq analysis of purified fetal MD mesenchymal cells. This analysis found 82 genes up-regulated in males during MD regression and identified Osterix (Osx)/Sp7, a key transcriptional regulator of osteoblast differentiation and bone formation, as a downstream effector of AMH signaling during MD regression. Osx/OSX was expressed in a male-specific pattern in MD mesenchyme during MD regression. OSX expression was lost in mutant males without AMH signaling. In addition, transgenic mice ectopically expressing human AMH in females induced a male pattern of Osx expression. Together, these results indicate that AMH signaling is necessary and sufficient for Osx expression in the MD mesenchyme. In addition, MD regression was delayed in Osx-null males, identifying Osx as a factor that regulates MD regression.

Induction of WNT inhibitory factor 1 expression by Müllerian inhibiting substance/antiMullerian hormone in the Müllerian duct mesenchyme is linked to Müllerian duct regression.

A key event during mammalian sexual development is regression of the Müllerian ducts (MDs) in the bipotential urogenital ridges (UGRs) of fetal males, which is caused by the expression of Müllerian inhibiting substance (MIS) in the Sertoli cells of the differentiating testes. The paracrine signaling mechanisms involved in MD regression are not completely understood, particularly since the receptor for MIS, MISR2, is expressed in the mesenchyme surrounding the MD, but regression occurs in both the epithelium and mesenchyme. Microarray analysis comparing MIS signaling competent and Misr2 knockout embryonic UGRs was performed to identify secreted factors that might be important for MIS-mediated regression of the MD. A seven-fold increase in the expression of Wif1, an inhibitor of WNT/ß-catenin signaling, was observed in the Misr2-expressing UGRs. Whole mount in situ hybridization of Wif1 revealed a spatial and temporal pattern of expression consistent with Misr2 during the window of MD regression in the mesenchyme surrounding the MD epithelium that was absent in both female UGRs and UGRs knocked out for Misr2. Knockdown of Wif1 expression in male UGRs by Wif1-specific siRNAs beginning on embryonic day 13.5 resulted in MD retention in an organ culture assay, and exposure of female UGRs to added recombinant human MIS induced Wif1 expression in the MD mesenchyme. Knockdown of Wif1 led to increased expression of ß-catenin and its downstream targets TCF1/LEF1 in the MD mesenchyme and to decreased apoptosis, resulting in partial to complete retention of the MD. These results strongly suggest that WIF1 secretion by the MD mesenchyme plays a role in MD regression in fetal males.

SMAD2/3 signaling regulates initiation of mouse Wolffian ducts and proximal differentiation in Müllerian ducts.

Male and female reproductive tracts develop from anterior intermediate mesoderm with similar differentiation processes. The anterior intermediate mesoderm develops into the mesonephros, and the Wolffian duct initiates by epithelialization in the mesonephros. The Müllerian duct invaginates from the coelomic epithelium of the cranial mesonephros for ductal formation and is then regionalized into proximal to caudal female reproductive tracts. In this study, we focused on the epithelialization of the Wolffian duct, initiation of the Müllerian duct, and the regionalization step of the Müllerian ducts as a continuous process. By using intermediate mesodermal cells from mouse pluripotent stem cells, we identified that inhibition of SMAD2/3 signaling might be involved in the differentiation into mesenchymal cells, after which mesonephric cells might be then epithelialized during differentiation of the Wolffian duct. Aggregation of coelomic epithelial cells might be related to initiation of the Müllerian duct. Transcriptomic analysis predicted that consensus sequences of SMAD3/4 were enriched among highly expressed genes in the proximal Müllerian duct. SMAD2/3 signaling to regulate differentiation of the Wolffian duct was continuously activated in the proximal Müllerian duct and was involved in proximal and oviductal regionalization. Therefore, SMAD2/3 signaling may be finely tuned to regulate differentiation from initiation to regionalization steps.

Requirement of Bmpr1a for Müllerian duct regression during male sexual development.

Elimination of the developing female reproductive tract in male fetuses is an essential step in mammalian sexual differentiation. In males, the fetal testis produces the transforming growth factor beta (TGF-beta) family member anti-Müllerian hormone (Amh, also known as Müllerian-inhibiting substance (Mis)), which causes regression of the Müllerian ducts, the primordia of the oviducts, uterus and upper vagina. Amh induces regression by binding to a specific type II receptor (Amhr2) expressed in the mesenchyme surrounding the ductal epithelium. Mutations in AMH or AMHR2 in humans and mice disrupt signaling, producing male pseudohermaphrodites that possess oviducts and uteri. The type I receptor and Smad proteins that are required in vivo for Müllerian duct regression have not yet been identified. Here we show that targeted disruption of the widely expressed type I bone morphogenetic protein (BMP) receptor Bmpr1a (also known as Alk3) in the mesenchymal cells of the Müllerian ducts leads to retention of oviducts and uteri in males. These results identify Bmpr1a as a type I receptor for Amh-induced regression of Müllerian ducts. Because Bmpr1a is evolutionarily conserved, these findings indicate that a component of the BMP signaling pathway has been co-opted during evolution for male sexual development in amniotes.

Molecular characteristics and alterations during early development of the human vagina.

Unresolved questions remain concerning the derivation of the vagina with respect to the relative contributions from the Müllerian ducts, the urogenital sinus, and the Wolffian ducts. Recent molecular and cellular studies in rodents have opened up a large gap between the level of understanding of vaginal development in mice and understanding of human vaginal development, which is based on histology. To compare the findings in mice with human vaginal development and to address this gap, we analysed molecular characteristics of the urogenital sinus, Wolffian ducts, and Müllerian ducts in 8-14-week-old human specimens using immunohistochemical methods. The monoclonal antibodies used were directed against cytokeratin (CK) 14, CK19, vimentin, laminin, p63, E-cadherin, caspase-3, Ki67, HOX A13, and BMP-4. The immunohistochemical analysis revealed that, during weeks 8-9, the epithelium of the Müllerian ducts became positive for p63 as p63-positive cells that originated from the sinus epithelium reached the caudal tip of the fused Müllerian ducts via the Wolffian ducts. The lumen of the fused Müllerian ducts was closed by an epithelial plug that contained both vimentin-positive and vimentin-negative cells. Subsequently, the resulting epithelial tube enlarged by proliferation of basal p63-positive cells. The first signs of squamous differentiation were detected during week 14, with the appearance of CK14-positive cells. According to our results, all three components, namely, the urogenital sinus, Wolffian ducts, and Müllerian ducts, interacted during the formation of the human vagina. The sinus epithelium provided p63-positive cells, the Wollfian ducts acted as a 'transporter', and the Müllerian ducts contributed the guiding structure for the vaginal anlagen. Epithelial differentiation began at the end of the period studied and extended in a caudo-cranial direction. The present study is one of the first to provide up-to-date molecular correlates for human vaginal development that can be compared with the results of cell biological studies in rodents.

Lineage infidelity of epithelial ovarian cancers is controlled by HOX genes that specify regional identity in the reproductive tract.

Although epithelial ovarian cancers (EOCs) have been thought to arise from the simple epithelium lining the ovarian surface or inclusion cysts, the major subtypes of EOCs show morphologic features that resemble those of the müllerian duct-derived epithelia of the reproductive tract. We found that HOX genes, which normally regulate mullerian duct differentiation, are not expressed in normal ovarian surface epithelium (OSE), but are expressed in different EOC subtypes according to the pattern of mullerian-like differentiation of these cancers. Ectopic expression of Hoxa9 in tumorigenic mouse OSE cells gave rise to papillary tumors resembling serous EOCs. In contrast, Hoxa10 and Hoxa11 induced morphogenesis of endometrioid-like and mucinous-like EOCs, respectively. Hoxa7 showed no lineage specificity, but promoted the abilities of Hoxa9, Hoxa10 and Hoxa11 to induce differentiation along their respective pathways. Therefore, inappropriate activation of a molecular program that controls patterning of the reproductive tract could explain the morphologic heterogeneity of EOCs and their assumption of müllerian-like features.

Mammalian uterine morphogenesis and variations.

In eutherian and marsupial mammals, the site of embryo implantation and gestation is the uterus. Uterine morphologies vary between mammalian species. For example, laboratory mice have a bipartite uterus with two uterine horns and a single cervix, whereas humans have a simplex uterus with a single chamber and single cervix. The precursor tissue of the uterus, oviducts, and upper vagina is the Müllerian duct epithelium and its adjacent mesenchyme. Morphological variation between species is established during embryogenesis by species-specific differences in Müllerian duct fusion at the midline, growth, and differentiation. In humans, alterations in Müllerian duct development can lead to variations in uterine morphology that correlate with increased risks of miscarriage and infertility. Here we review the developmental genetic factors that regulate Müllerian duct development, uterine morphogenesis, and human uterine variation.

Establishment of long-term monolayer cultures of somatic cells from human fetal testes and expansion of peritubular myoid cells in the presence of androgen.

The somatic (Sertoli cell (SC), Leydig cell (LC), and peritubular myoid (PTM) cell) cells play key roles in development of the fetal testis. We established monolayer cultures from second trimester human testes and investigated the pattern of expression of cell-lineage characteristic mRNAs. Expression of some SC-associated genes (SRY, SOX9, WT1, GATA4, and SF1) was detectable up to and including passage 3 (P3), while others (anti-Müllerian hormone; desert hedgehog) present prior to dissociation were not expressed in the cultured cells. Transcripts encoding the androgen receptor were expressed but addition of dihydrotestosterone (DHT) had no impact on expression of mRNAs expressed in SC or LC. Total concentrations of mRNAs encoding smooth muscle actin (ACTA2) and desmin increased from P1 to P3; an increasing proportion of the cells in the cultures were immunopositive for ACTA2 consistent with proliferation/differentiation of PTM cells. In conclusion, somatic cell monolayer cultures were established from human fetal testes; these cultures could form the basis for future studies based on isolation of purified populations of somatic cells and manipulation of gene expression that is difficult to achieve with organ culture systems. Our results suggest that fetal SC do not maintain a fully differentiated phenotype in vitro, yet PTM (ACTA2 positive) cells readily adapt to monolayer culture conditions in the presence of DHT. This culture system provides an opportunity to study the impact of regulatory factors on gene expression in PTM cells, a population thought to play a key role in mediating androgen action within the developing testis.

Müllerian duct regression in the embryo correlated with cytotoxic activity against human ovarian cancer.

A significant cytotoxicity index was obtained when human ovarian cancer cells in a microcytotoxicity assay were exposed during the S (DNA-synthesizing) phase of the cell cycle to purified fractions of testis exhibiting high Müllerian inhibiting substance bioactivity. The same effect was not observed when these fractions were tested against human glioblastoma or fibroblast lines. Most human ovarian cancers are said to resemble Müllerian tissues histologically. Müllerian inhibiting substance may thus deserve further study as a potential chemotherapeutic agent.

Feminizing effect of mesonephros on cultured differentiating mouse gonads and ducts.

Gonads were removed from fetal mice at about the time that gonadal sex differentiation occurs. The gonads were cultured in vitro with or without their mesonephric tissue. When gonads and ducts removed from sexually undifferentiated fetuses were cultured together, the gonads of both sexes developed female characteristics, whereas gonads cultured without mesonephros developed according to the sex of the fetus from which they were removed. Gonads of sexually differentiated fetuses developed whether they were cultured with or without the mesonephros.

Phosphorylation events during Müllerian duct regression.

Regression of the fetal rat Müllerian duct in vitro was stimulated by sodium fluoride in the absence of Müllerian inhibiting substance. The action of Müllerian inhibiting substance was inhibited by sodium vanadate, adenosine 5'-triphosphate, and several related nucleotides in the presence of manganese ions. Epidermal growth factor specifically inhibited the substance, but only with manganese ions present. Insulin, platelet-derived growth factor, and nerve growth factor had no effect. These results suggest that dephosphorylation of membrane proteins mediates the action of Müllerian inhibiting substance.

Intersex, Hermaphroditism, and Gonadal Plasticity in Vertebrates: Evolution of the Müllerian Duct and Amh/Amhr2 Signaling.

In vertebrates, sex organs are generally specialized to perform a male or female reproductive role. Acquisition of the Müllerian duct, which gives rise to the oviduct, together with emergence of the Amh/Amhr2 system favored evolution of viviparity in jawed vertebrates. Species with high sex-specific reproductive adaptations have less potential to sex reverse, making intersex a nonfunctional condition. Teleosts, the only vertebrate group in which hermaphroditism evolved as a natural reproductive strategy, lost the Müllerian duct during evolution. They developed for gamete release complete independence from the urinary system, creating optimal anatomic and developmental preconditions for physiological sex change. The common and probably ancestral role of Amh is related to survival and proliferation of germ cells in early and adult gonads of both sexes rather than induction of Müllerian duct regression. The relationship between germ cell maintenance and sex differentiation is most evident in species in which Amh became the master male sex-determining gene.

Mouse Dach1 and Dach2 are redundantly required for Müllerian duct development.

dachshund/Dach gene family members encode transcriptional cofactors with highly conserved protein interaction domains and are expressed in the developing eyes, brains, and limbs in insects and vertebrates. These observations suggest that the developmental roles of dachshund/Dach in these tissues have been conserved since the divergence of arthropods and chordates. However, while Drosophila dachshund mutants have abnormalities in eye, brain, limbs, mouse Dach1 or Dach2 knockout mutants do not exhibit gross anatomical malformations in these tissues. In addition, Dach1/2 double homozygotes have intact eyes and limbs. Here we show that in Dach1/Dach2 double mutants, female reproductive tract (FRT) development is severely disrupted. This defect is associated with the Müllerian duct (MD) and not the Wolffian duct (WD), which normally differentiate into either the FRT or male reproductive tract (MRT), respectively. Dach1 and Dach2 are expressed in the MD, and in Dach1/2 double mutants, MD expression of Lim1 and Wnt7a is abnormal and MD development is disrupted. In contrast, WD and MRT development are not grossly affected. We propose that Dach1 and Dach2 proteins may redundantly control FRT formation by regulating the expression of target genes required for development of the MD. This vertebrate Dach1/2 function may have been conserved during arthropod evolution, as Drosophila dachshund mutants also exhibit an FRT phenotype.

[Anti-mullerian hormone and its role in the regulation of ovarian function. Review of the literature]. / Le rôle de l'hormone anti-muillerienne dans la régulation du fonctionnement de l'ovaire. Revue de la littérature.

Anti-mullerian hormone, also called AMH, belongs to the large family of transforming growth factor P. Its role in the sexual differentiation of male fetus is now well known. Recently, AMH has been demonstrated to play an important role in the ovarian function. In fact, AMH seems to regulate the kinetics of follicular development, inhibiting the follicular recruitment and the follicular growth. Thus, this intra-gonadic cybernin could be a decisive determinant of the rapidity of follicular pool exhaustion. Today, some experimental data from the literature suggest that AMH could be a reliable marker of ovarian reserve. This review summarizes the present knowledge about AMH and its role in physiology but also in ovarian pathology.

Analysis of gene expression induced by diethylstilbestrol (DES) in human primitive Mullerian duct cells using microarray.

The Mullerian ducts are strongly influenced by natural estrogen, estradiol (E2) and diethylstilbestrol (DES) in their development. We screened E2 and DES responsive genes using a microarray analysis in human primitive Mullerian duct cell line, EMTOKA cells expressed estrogen receptor (ER) beta. c-myc oncogene and other target genes expression was detected in cells treated by high-dose DES, but ER antagonist ICI 182,780 could not prevent c-myc induction above. Results of our present study suggested the presence of ER independent pathway in oncogenes induction process by high-dose DES treatment in a human primitive Mullerian duct cell line.

Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo.

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.

A genome-wide screen for copy number alterations in an adolescent pilot cohort with müllerian anomalies.

OBJECTIVE: To examine whether pathogenic copy number changes (CNCs) can be identified in deoxyribonucleic acid from females with different classes of müllerian anomalies. DESIGN: We conducted array-based copy number variant (CNV) analysis using an oligonucleotide array from deoxyribonucleic acid in 12 adolescent females with various müllerian anomalies. SETTING: University-affiliated tertiary care institution. PATIENT(S): Twenty adolescent females with clinically confirmed müllerian anomalies. INTERVENTION(S): Array-based CNV analysis. MAIN OUTCOME MEASURE(S): Copy number changes and/or regions with absence of heterozygosity. RESULT(S): A total of 192 CNVs identified in these samples were previously annotated as polymorphic. Three CNCs that were identified in regions with minimal to no overlap with annotated polymorphisms failed significance criteria with detailed inspection. One subject harbored a 5.1-Mb region of absence of heterozygosity at Xq23 that is of unknown significance. CONCLUSION(S): We did not identify pathogenic CNCs in this small pilot cohort of patients with various müllerian anomalies, but larger studies will be needed to further investigate whether CNCs are associated with all classes of müllerian anomalies.

Pretreatment of chick embryos with estrogen in ovo prevents mullerian duct regression in organ culture.

Diethylstilbestrol (DES) feminizes chick embryos in ovo, preventing regression of the Mullerian ducts (MDs). The feminized testis, however, despite its ovarian-like appearance, continues to produce Mullerian inhibiting substance (MIS). This study was designed to test whether exogenous DES preserved the MD against endogenous MIS by affecting the target organ (feminization). DES (0.1 mg) was injected into the air sacs of 5-day-old chick embryos. Testes were obtained from 15-day-old chick embryos and MDs from 8-day-old chick embryos and combined in organ culture for 72 h. MD regression was examined macroscopically and microscopically after 3 days in vitro in medium without DES (group A: n = 155) and with 10(-6) M DES (group B: n = 169). Four types of coculture were set up as follows: 1) Control testis with control MD; 2) DES-treated testis with DES-treated MD; 3) Control testis with DES-treated MD; 4) DES-treated testis with control MD. In medium without DES (group A), MD regression was inhibited significantly when the duct was pretreated with DES [2) and 3) vs. 1) and 4)]. The pretreated testis produced slightly less regression [4) vs. 1)], but this was not significant. Previous studies in ovo had suggested that estrogen prevented MD regression directly as well as causing feminization of the testis, because MIS secretion was not inhibited from the feminized gonad. These results show that pretreatment of the MD with DES blocks regression in vitro by a normal testis, confirming that the primary site of action of estrogen is on the duct itself.

Cellular localization of müllerian inhibiting substance in the developing rat ovary.

The ontogeny of Müllerian Inhibiting Substance (MIS) production was studied in the immature developing rat ovary using immunohistochemistry and bioassay. In a graded organ culture assay, in which regression of the Müllerian duct of the 14 1/2-day rat fetus was used as a measure of bioactivity, MIS could not be detected in ovarian fragments from fetal rats. After birth, however, MIS bioactivity first became detectable at 4 days of age. Fragments from ovaries of rats 7 days of age and older contained moderate levels of MIS activity which remained detectable throughout the prepubertal period, although extreme individual variability was characteristic. A rabbit polyclonal antibody against human recombinant MIS was used to localize MIS in rat ovarian tissue. Avidin-biotin enhanced immunoperoxidase staining could not detect MIS in the 15-day fetal or 1 day postnatal ovary, where no follicular growth was noticed. In ovaries from rats 1 week or older, where follicular growth was apparent, MIS could be localized specifically and exclusively in the cytoplasm of granulosa cells. MIS was found more in the innermost layers of granulosa cells than in the peripheral layers in preantral follicles. In antral follicles, MIS was found predominantly in the cumulus oophorus cells and periantral cells. In these developing ovaries, MIS could not be found in follicles with features of atresia.

Production of anti-Müllerian hormone: another homology between Sertoli and granulosa cells.

Anti-Müllerian hormone (AMH) has been detected by RIA in the follicular fluid of mature bovine ovaries and in incubation medium of bovine granulosa cells. Purification of AMH from two independent batches of follicular fluid was achieved with a yield of 11% and 15% respectively. Both ovarian and control testicular AMH produced near-complete regression of fetal rat Müllerian ducts exposed to it in culture at a final concentration of 200-300 mU/ml and were recognized by the same monoclonal and polyclonal antibodies. These findings indicate that adult mammalian granulosa cells are capable of producing immunoreactive and bioactive AMH at a rate apparently similar to that already demonstrated for mature Sertoli cells and add yet another item to the homologies reported between male and female somatic gonadal cells.