A mesenchymal perspective of Müllerian duct differentiation and regression in Amhr2-lacZ mice

The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding beta-galactosidase (beta-gal), into the AMHR-II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2-lacZ heterozygotes express beta-gal activity in an Amhr2-specific pattern. In the gonads, beta-gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. beta-gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, beta-gal activity was restricted to one side of the ductal mesoepithelium, whereas in males beta-gal expression encircled the duct. beta-gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal beta-gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2-expressing mesenchyme.     

Müllerian inhibiting substance is present in testes of dogs with persistent müllerian duct syndrome

Breeding studies in a strain of miniature schnauzer dogs with Persistent Müllerian Duct Syndrome (PMDS) indicate this syndrome is inherited as an autosomal recessive trait, as it is in man. Testes of neonatal dogs affected with PMDS and normal male littermates were examined for Müllerian Inhibiting Substance (MIS) production by immunohistochemistry and bioassay. MIS immunoactivity was detected in Sertoli cells of normal and affected pups using an avidin-biotin complex-enhanced method. Rat embryonic Müllerian ducts regressed when cocultured with testis fragments of both normal and affected pups in a graded organ culture bioassay, demonstrating that the MIS produced was bioactive. These findings indicate that Müllerian duct persistence in affected dogs is not due to a mutation in the structural gene for MIS, but rather, by inference, to a failure of response to MIS at the receptor level.     

The serine/threonine transmembrane receptor ALK2 mediates Müllerian inhibiting substance signaling

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-beta family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.     

Müllerian inhibiting substance in reproduction and cancer.

During embryogenesis normal male phenotypic development requires the action of Müllerian Inhibiting Substance (MIS) which is secreted by Sertoli cells of the fetal testis. As testes differentiate in genetic (XY) males, they produce MIS which causes regression of the Müllerian ducts, the anlagen of the female reproductive tract. Soon thereafter, testicular androgens stimulate the Wolffian ducts. In females, on the other hand, MIS is not produced by grandulosa cells until after birth, before which, estrogens induce Müllerian duct development, while the Wolffian ducts passively atrophy in the absence of androgenic stimulation. High serum MIS levels in males are maintained until puberty, whereupon they fall to baseline levels. In females MIS is undetectable in serum until the peripubertal period when values approach the baseline levels of males. This distinct pattern of sexual and ontogenic expression presupposes and requires tight regulation. MIS may play a role in gonadal function and development. Our laboratory has shown that an important role for ovarian MIS is to inhibit oocyte meiosis, perhaps providing maximal oocyte maturation prior to selection for ovulation and subsequent fertilization. Furthermore, Vigier et al. (Development 100:43-55) have recently obtained evidence that MIS may influence testicular differentiation, coincident with inhibition of aromatase activity. Current structure-function studies demonstrate that MIS, like other growth regulators in its protein family, requires proteolytic cleavage to exhibit full biological activity. MIS can be inhibited by epidermal growth factor. This antagonism, which is common to all MIS functions so far investigated, is associated with inhibition of EGF receptor autophosphorylation. We have provided evidence that bovine MIS can inhibit female reproductive tract tumors arising in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contacts between Wolffian and Müllerian cells at the tip of the outgrowing Müllerian duct in rat embryos

The developing Müllerian duct was studied at the light microscopic as well as the electron microscopic level in rat embryos, especially in the section of the terminal bud and its tip, where Wolffian and Müllerian duct are enclosed by a common basal membrane. In this zone desmosomes can be found among Wolffian cells and also among Müllerian cells. In addition, we found cell contacts between Müllerian and Wolffian cells, namely short electron-dense segments on adjacent surfaces or disc-shaped thickenings within opposite plasma membranes, as well as fusions of the plasmalemmata over short distances. Until now, these cell contacts have not been described in rat embryos.     

Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells

We have isolated the bovine and human genes for Müllerian inhibiting substance (MIS), a testicular glycoprotein that causes regression of the Müllerian duct during development of the male embryo. The mRNA sequence of bovine MIS, determined from an analysis of cDNA and genomic clones, codes for a protein of 575 amino acids containing a 24 amino acid leader peptide. The human gene has five exons that code for a protein of 560 amino acids. A comparison of the bovine and human MIS proteins reveals a highly conserved C-terminal domain that shows marked homology with human transforming growth factor-beta and the beta chain of porcine inhibin. Animal cells transfected with the human gene secrete biologically active MIS, which causes regression of the rat Müllerian duct in vitro.     

Biosynthesis and secretion of fibronectin in the cultured mesenchymal cells of the developing chick müllerian duct

We have developed a method to separate and isolate the mesenchymal cells from the epithelial cells in the left Müllerian duct of the developing chick. We then cultured the mesenchymal cells in a serum-free medium. Through an enzyme-linked immunosorbent assay, we detected fibronectin synthesis and release into the medium at stages of Müllerian duct development. Our results demonstrate that the amount of fibronectin secreted by cultured cells gradually decreased in accordance with Müllerian duct differentiation. Similar observations found in the developing embryonic intestine indicate that the highest fibronectin synthesis occurs during early stages of development, when morphogenetic movement and mesenchymal-epithelial interaction are prominent features of embryonic organ differentiation and growth.     

Identification of differentially expressed genes involved in the regression and development of the chicken Müllerian duct

Müllerian ducts of male chickens undergo regression around day 12 of incubation, but the underlining mechanisms remain unclear. The purpose of this study was to identify factors that contribute to regression of the Müllerian duct in the chicken. We first employed annealing control primer-based RT-PCR to screen candidate genes differentially expressed in the Müllerian ducts between male and female. Four differentially expressed genes (MSX2, GAL10, VCP and PLCH1) were partially sequenced. The expression of mRNA of the latter genes and MSX1 in the male and female Müllerian ducts were compared at 7.5, 8 and 9 days of incubation using semi-quantitative RT-PCR. The results indicated that both MSX1 and MSX2 mRNA was highly expressed in the male Müllerian duct at day 9 of incubation, whereas, PLCH1 mRNA was lower in the male duct at day 9 of incubation compared to that of the female duct. Although VCP mRNA was expressed in both left and right female Müllerian ducts, no expression was detected in the male duct. Whole mount in situ hybridyzation analysis showed that the expression of MSX1 and MSX2 mRNA were localized specifically in the mesenchymal cells of the male Müllerian duct at day 9 of incubation. In contrast, VCP mRNA expression was observed in both mesenchymal and epithelial cells of the female Müllerian duct but not detected in the male duct. These results suggest that both up-regulation of MSX1 and MSX2 mRNA expression is involved in the regression of the Müllerian duct in male chicken embryo, whereas VCP expression is involved in development of the female duct.     

New approaches for high-yield purification of Müllerian inhibiting substance improve its bioactivity

We have established a new method to purify Müllerian inhibiting substance (MIS) with higher purity and recovery over existing procedures. Recombinant human MIS was expressed in Chinese hamster ovary cells and secreted into chemically defined serum-free media. The secreted products were concentrated by either precipitation with ammonium sulfate or lectin-affinity chromatography, each of which was followed by anion-exchange chromatography. Further separation of the active carboxy-terminal domain of MIS was achieved after cleavage by plasmin followed by lectin-affinity chromatography. This method may be applicable to other members of the transforming growth factor beta family with which MIS shares sequence homology.     

Müllerian inhibiting substance production and testicular migration and descent in the pouch young of a marsupial

The ontogeny of Müllerian inhibiting substance (MIS) production by the developing testis of an Australian marsupial, the tammar wallaby (Macropus eugenii), was determined during pouch life using an organ-culture bioassay of mouse fetal urogenital ridge. This information was related to the morphological events during testicular migration and descent. MIS biological activity was found in testes (but not ovaries or liver) of pouch young from 2 to 85 days of age. MIS production had commenced by day 2, which is within a day of the first gross morphological signs of testicular differentiation. Müllerian duct regression occurred between 10 and 30 days, which partly coincided with testicular migration to the inguinal region and enlargement of the gubernacular bulb (15 to 30 days). These observations are consistent with the hypothesis that MIS may be involved in testicular transabdominal migration. The epididymis commenced development and growth only after the testis had descended through the inguinal ring. This provides no support for the suggestion that the epididymis is involved in testicular descent into the scrotum. The basic sequence of events in post-testicular sexual differentiation in the wallaby is sufficiently similar to that seen in eutherian mammals to make it an excellent experimental model for future studies of testicular differentiation, migration and descent.     

Cellular mechanisms of Müllerian duct formation in the mouse

Regardless of their sex chromosome karyotype, amniotes develop two pairs of genital ducts, the Wolffian and Müllerian ducts. As the Müllerian duct forms, its growing tip is intimately associated with the Wolffian duct as it elongates to the urogenital sinus. Previous studies have shown that the presence of the Wolffian duct is required for the development and maintenance of the Müllerian duct. The Müllerian duct is known to form by invagination of the coelomic epithelium, but the mechanism for its elongation to the urogenital sinus remains to be defined. Using genetic fate mapping, we demonstrate that the Wolffian duct does not contribute cells to the Müllerian duct. Experimental embryological manipulations and molecular studies show that precursor cells at the caudal tip of the Müllerian duct proliferate to deposit a cord of cells along the length of the urogenital ridge. Furthermore, immunohistochemical analysis reveals that the cells of the developing Müllerian duct are mesoepithelial when deposited, and subsequently differentiate into an epithelial tube and eventually the female reproductive tract. Our studies define cellular and molecular mechanisms for Müllerian duct formation.     

Steroidogenic activities in MA-10 Leydig cells are differentially altered by cAMP and Müllerian inhibiting substance

In addition to causing Müllerian duct regression in fetal males, Müllerian inhibiting substance (MIS) inhibits the expression of the bifunctional cytochrome P450, C17 hydroxylase/C(17-20) lyase (Cyp17), the enzyme that catalyzes the committed step in sex steroid synthesis. To investigate the paracrine effects of MIS on steroidogenic activity, we have performed assays with microsomes from mouse MA-10 Leydig cells. With microsomes from untreated MA-10 cells, progesterone was largely metabolized by 5alpha-reductase and subsequently converted by 3-keto steroid reductases to allopregnanolone and epiallopregnanolone. Addition of cAMP to the cells shifted microsomal steroid production to the Cyp17 product androstenedione and its 5alpha,3beta-reduced form, epiandrosterone. Microsomes from MIS-treated cells were less active with the progesterone substrate than those of untreated cells but co-treatment of the cells with both MIS and cAMP mitigated the cAMP-induced shift of the microsomes to androstenedione production. Quantitative analyses of steroid production by Cyp17 showed that cAMP decreased the amount of 17-hydroxyprogesterone produced relative to the androstenedione, suggesting that cAMP signaling lowers the efficiency of the Cyp17 hydroxylase activity or else increases the efficiency of its lyase activity. Addition of MIS to the cAMP-treated cells partially reversed this effect, as well. These results indicate that cAMP induces MA-10 cells to switch from producing 5alpha-reduced progesterone metabolites to producing androstenedione and its metabolites by increasing Cyp17 expression and its relative lyase activity, both of which are inhibited by MIS.     

Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/β-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/β-catenin signaling were determined, suggested down-regulation of Wnt/β-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3α can inhibit the epithelial differentiation of MSCs. A loss of β-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated β-catenin expression and subsequently decreased β-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/β-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.     

Müllerian inhibiting substance as a novel biomarker of cisplatin-induced ovarian damage

Müllerian inhibiting substance (MIS) has been investigated as a possible serum biomarker in human aging to estimate the number of female germ cells remaining. Cisplatin is an effective chemotherapeutic agent that is associated with ovarian injury. In this study, we tested the hypothesis that decreasing serum MIS can serve as a biomarker of ovarian damage after cisplatin. Adult female rats were treated with saline, 4.5, or 6.0 mg/kg cisplatin. The serum MIS levels were lower in both cisplatin groups, in a dose-related fashion. The ovarian lysates of both cisplatin groups had less MIS than control. Immunofluorescence analysis showed that the percentage of MIS-positive follicles was lower in the 6.0 mg/kg group. TUNEL assays showed that there was a dose related increase in the number of apoptotic follicles in the cisplatin groups. In summary, a decrease in serum MIS could serve as a biomarker to discriminate the degree of ovarian damage after cisplatin. These data are the first to establish in the rat that ovarian injury due to a chemotherapeutic agent could be monitored with the non-invasive serum biomarker MIS.     

Timing and irreversibility of Müllerian duct inhibition in the embryonic reproductive tract of the human male

A study was undertaken to determine (1) the effects of endogenous Müllerian inhibiting substance (MIS) on the developing human fetal genital tract; (2) the time in fetal life when MIS is first capable of inhibiting the growth of the embryonic Müllerian ducts; and (3) the reversibility of the effects of MIS on the developing male Müllerian ducts. Human fetal reproductive tracts were transplanted and grown for sustained periods in vivo in athymic nude mice. The genital tracts from 12 male human fetuses, ages 51 to 68 days postovulation, were grafted without their associated gonads into castrated murine hosts and grown for 30 to 70 days. Controls consisted of genital tracts from 8 female human fetuses, ages day 53 to 70 that were grown under identical conditions. Male specimens grew to approximately one-half the size of female specimens and disclosed varying degrees of inhibition of the Müllerian duct system from absence of the Müllerian ducts in older specimens (after Day 63) to poorly segregated segments of stroma as the mildest defect (less than Day 61). It is concluded that (1) MIS secretion by the embryonic testes probably begins before Day 51 of gestation; (2) the effects of MIS are progressive during the so-called critical window; (3) the effects of MIS are permanent; and (4) the mesenchyme is an important target of MIS.     

Müllerian inhibiting substance signaling uses a bone morphogenetic protein (BMP)-like pathway mediated by ALK2 and induces SMAD6 expression

Signal reception of Müllerian inhibiting substance (MIS) in the mesenchyme around the embryonic Müllerian duct in the male is essential for regression of the duct. Deficiency of MIS or of the MIS type II receptor, MISRII, results in abnormal reproductive development in the male due to the maintenance of the duct. MIS is a member of the transforming growth factor-beta (TGFbeta) superfamily of secreted protein hormones that signal through receptor complexes of type I and type II serine/threonine kinase receptors. To investigate candidate MIS type I receptors, we examined reporter construct activation by MIS. The bone morphogenetic protein (BMP)-responsive Tlx2 and Xvent2 promoter-driven reporter constructs were stimulated by MIS but the TGFbeta/activin-induced p3TP-lux or CAGA-luc reporter constructs were not. The induction of Tlx2-luc was dependent upon the kinase activity of MISRII and was blocked by a dominant negative truncated ALK2 (tALK2) receptor but not by truncated forms of the other BMP type I receptors ALK1, ALK3, or ALK6. MIS induced activation of a Gal4DBD-Smad1 but not a Gal4DBD-Smad2 fusion protein. This activation could also be blocked by tALK2. The BMP-induced inhibitory Smad, Smad6, was up-regulated by MIS endogenously in Leydig cell-derived lines and is expressed in male but not female Müllerian duct mesenchyme. ALK6 has been shown to function as an MIS type I receptor. Investigation of the pattern of ALK2, MISRII, and ALK6 in the developing urogenital system demonstrated overlapping expression of ALK2 and MISRII in the mesenchyme surrounding the duct while ALK6 was observed only in the epithelium. Examination of ALK6 -/- male animals revealed no defect in duct regression. The reporter construct analysis, pattern of expression of the receptors, and analysis of ALK6-deficient animals suggest that ALK2 is the MIS type I receptor involved in Müllerian duct regression.     

AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells

Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis.