Müllerian inhibiting substance inhibits an ovarian cancer cell line via ß-catenin interacting protein deregulation of the Wnt signal pathway.

Müllerian inhibiting substance/anti-Müllerian hormone (MIS/AMH) has been suggested as a biotherapeutic agent in gynecological cancers that highly express the MIS/AMH type II receptors (MISRII/AMHRII) but the anticancer mechanisms by which MIS/AMH acts are not fully understood. Our experiments show that MIS/AMH inhibits ovarian cancer by deregulating the Wnt signal pathway via the ß-catenin interacting protein (ICAT). MIS/AMH inhibition of ICAT by small interfering RNAs (siRNA) decreased ICAT driven ovarian cancer cell viability as measured by the methylthiazoltetrazolium assay, reversed cell cycle arrest and annexin V expression and diminished migration by scratch wound assay. Changes in expression of regulatory proteins were shown by western blotting. We determined that MIS/AMH upregulated ICAT in ovarian cancer cell line which caused decreased cell viability, cell cycle arrest and apoptosis. This effect, however, was blocked when ICAT was downregulated by siRNA. The present study demonstrates a role for ICAT in MIS/AMH mediated inhibition of the Wnt signaling pathway in ovarian cancer.

Transcriptome analysis reveals that Müllerian inhibiting substance regulates signaling pathways that contribute to endometrial carcinogenesis.

Müllerian inhibiting substance (MIS) has been shown to inhibit growth of a number of tumors in vitro and/or in vivo, but the downstream pathways which it regulates are not fully understood. In the present study we show that MIS type II receptor was highly expressed in AN3CA cells, a cell line derived from human endometrial cancer cell in which MIS-treatment caused a reduction of cell viability, and induced cellular apoptosis and genes involved cell cycle arrest. To understand the genome-wide effects of MIS on gene regulation, we performed serial gene expression analyses from 0 to 96 h at 24 h intervals after treating AN3CA cells with MIS. Transcriptomic analysis of molecular changes induced by MIS identified 2,688 differentially expressed genes that were significantly up- or down-regulated during the 96 h study period. When the 2,688 differentially expressed genes were mapped to known biological processes, Wnt-, cancer-, proteolysis-, cytoskeleton-, cell cycle-, apoptosis-, and MAPK-signaling pathways emerged as the functions most significantly changed by MIS in AN3CA cells. Furthermore, western blot analysis validated that protein expression of cell cycle inhibitory genes, apoptotic protease activating factor-1 (APAF-1), ß-catenin-interacting protein (ICAT), Rb related protein 130 (p130), and inhibitor of disheveled Dvl and Axin complex (IDAX), were gradually increased over the time of the study, whereas downstream cell cycle activating genes, cyclin-dependent kinase 2 (CDK2) and phospho-c-Jun were downregulated in MIS-treated AN3CA cells. These transcriptome analyses support previous observations that MIS functions as a tumor suppressor, potentially by regulating signaling pathways that could contribute to endometrial carcinogenesis, and indicating that MIS should be considered as a potential treatment for endometrial cancer.

Müllerian inhibiting substance/anti-Müllerian hormone: A novel treatment for gynecologic tumors.

Müllerian inhibiting substance (MIS), also called anti-Müllerian hormone (AMH), is a member of the transforming growth factor-ß super-family of growth and differentiation response modifiers. It is produced in immature Sertoli cells in male embryos and binds to MIS/AMH receptors in primordial Müllerian ducts to cause regression of female reproductive structures that are the precursors to the fallopian tubes, the surface epithelium of the ovaries, the uterus, the cervix, and the upper third of the vagina. Because most gynecologic tumors originate from Müllerian duct-derived tissues, and since MIS/AMH causes regression of the Müllerian duct in male embryos, it is expected to inhibit the growth of gynecologic tumors. Purified recombinant human MIS/AMH causes growth inhibition of epithelial ovarian cancer cells and cell lines in vitro and in vitro via MIS receptor-mediated mechanism. Furthermore, several lines of evidence suggest that MIS/AMH inhibits proliferation in tissues and cell lines of other MIS/AMH receptor-expressing gynecologic tumors such as cervical, endometrial, breast, and in endometriosis as well. These findings indicate that bioactive MIS/AMH recombinant protein should be tested in patients against tumors expressing the MIS/AMH receptor complex, perhaps beginning with ovarian cancer because it has the worst prognosis. The molecular tools to identify MIS/AMH receptor expressing ovarian and other cancers are in place, thus, it is possible to select patients for treatment. An MIS/AMH ELISA exists to follow administered doses of MIS/AMH, as well. Clinical trials await the production of sufficient supplies of qualified recombinant human MIS/AMH for this purpose.

Mullerian inhibiting substance induces apoptosis of human endometrial stromal cells in endometriosis.

CONTEXT: Müllerian inhibiting substance (MIS) is produced in Sertoli cells of fetal testis and causes regression of müllerian ducts in male embryos. MIS also can induce the cell cycle arrest and apoptosis in müllerian duct-derived tumors in vivo and in vitro. OBJECTIVE: Our objective was to investigate the expression of MIS type II receptor (MISR II) and whether MIS can inhibit the proliferation and induce apoptosis in primary cultures of endometrial stromal cells (ESC) of endometriosis. DESIGN AND SETTINGS: In vitro experiments were performed in the university research laboratory. PARTICIPANTS: Tissue samples from 12 patients who had undergone evisceration for ovarian endometrial cysts were included in this study. INTERVENTIONS AND MAIN OUTCOME MEASURES: The expression of MISR II in ESC was investigated by immunohistochemistry. The cell viability and apoptosis in ESC treated with MIS was measured by methylthiazoletetrazolium assay and annexin V analysis. The expression of regulatory proteins in ESC treated with MIS was shown by Western blotting. RESULTS: ESC showed specific immunostaining for the MISR II. ESC treated with MIS exhibited 32% growth inhibition (P = 0.0001). The changes in cell cycle distribution after MIS exposure at 72 h demonstrated that S and G(2)M phases were decreased; G(0)G(1) and sub-G(0)G(1) phases were increased. ESC treated with MIS showed 13.72% annexin V-fluorescein isothiocyanate positivity. In the ESCs, which contain defective p16, MIS increased the expression of pocket proteins p107 and p130 and decreased E2F transcription factor 1. CONCLUSIONS: The results support a central role for MIS in endometriosis. Although the precise mechanism of MIS-mediated inhibition of ESC growth has not been fully defined, these data suggest that MIS has activity against ESC in vitro and may also be an effective targeted therapy for endometriosis.

Expression of Müllerian inhibiting substance type II receptor and antiproliferative effects of MIS on human cervical cancer.

This study aimed to analyze expression of Müllerian inhibiting substance type II receptor (MISRII) protein and mRNA in cervical neoplasia, to demonstrate the growth inhibition of cervical cancer cells by administration of highly purified recombinant human Müllerian inhibiting substance (MIS) and, furthermore, to evaluate the clinical significance of MIS as a biological modifier for MIS receptor expressing tumors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for MISRII mRNA expression, and in situ hybridization and immunohistochemistry were used to observe expression, location of MISRII mRNA and protein, respectively. To demonstrate the effect of MIS on the viability of cervical cancer cells, methyl thiazole tetrazolium (MTT) assay was performed. Flow cytometry was used to evaluate the cell cycle distribution after exposure to MIS in cervical cancer cells, and the annexin-V-FITC staining method was performed to demonstrate apoptosis by MIS in cervical cancer cells. Expression of MISRII protein and mRNA were observed in all normal cervical and cervical carcinoma tissues. There was no significant difference in expression of MISRII protein and MISRII mRNA between normal cervical and cervical carcinoma tissues. MTT assay showed negative correlation between MIS exposure time and the viability of cervical cells (P=0.008). The changes in cell cycle distribution after MIS exposure suggest that MIS plays an important role in inducing cellular apoptosis by causing arrest at the G1 phase and increasing cells at sub-G0G1 phase. Annexin-V-FITC staining methods showed that cellular apoptosis was, respectively, 10.44 and 12.89% after 24 and 48 h of MIS exposure in cervical carcinoma cells. There was a negative correlation between cellular survival and MIS exposure time. This study demonstrates that MISRII is present on normal cervical and cervical carcinoma tissues, and MIS shows receptor-mediated antiproliferative effect on cervical cells in vitro. These data suggest that MIS may be used as a biological modifier or therapeutic modulator on MISRII-expressing tumors in the future.

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance.

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad-) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad- cells. Similarly, proliferation of the 3+Ecad- cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3-Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad- subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad- cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.

Anti-Müllerian hormone inhibits growth of AMH type II receptor-positive human ovarian granulosa cell tumor cells by activating apoptosis.

Ovarian granulosa cell tumors (GCTs) are sex cord stromal tumors that constitute 3-5% of all ovarian cancers. GCTs usually present with an indolent course but there is a high risk of recurrence, which associates with increased mortality, and targeted treatments would be desirable. Anti-Müllerian hormone (AMH), a key factor regulating sexual differentiation of the reproductive organs, has been implicated as a growth inhibitor in ovarian cancer. GCTs and normal granulosa cells produce AMH, but its expression in large GCTs is usually downregulated. Further, as the lack of specific AMH-signaling pathway components leads to GCT development in mice, we hypothesized that AMH inhibits growth of GCTs. Utilizing a large panel of human GCT tissue samples, we found that AMH type I receptors (ALK2, ALK3 and ALK6) and type II receptor (AMHRII), as well as their downstream effectors Smad1/5, are expressed and active in GCTs. AMHRII expression was detected in the vast majority (96%) of GCTs and correlated with AMH mRNA and protein expression. AMH mRNA level was low in large GCTs, confirming previous findings on low-AMH protein expression in large human as well as mouse GCTs. To study the functional role of AMH in this peculiar ovarian cancer, we utilized a human GCT cell line (KGN) and 10 primary GCT cell cultures. We found that the AMH-Smad1/5-signaling pathway was active in these cells, and that exogenous AMH further activated Smad1/5 in KGN cells. Furthermore, AMH treatment reduced the number of KGN cells and primary GCT cells, with increasing amounts of AMH leading to augmented activation of caspase-3 and subsequent apoptosis. All in all, these data support the premise that AMH is a growth inhibitor of GCTs.

Identification of characteristic molecular signature of Müllerian inhibiting substance in human HPV-related cervical cancer cells.

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone (AMH), is a member of the transforming growth factor-ß (TGF-ß) superfamily that plays an important role in the mesenchymal-epithelial interaction, cell growth and proliferation, extracellular matrix production and tissue remodeling. Previously, we demonstrated that MIS suppressed ovarian cancer cell growth and suggested large-scale genetic elements that could be responsible for anti-neoplastic effects of MIS on ovarian cancer cells. In this study, we demonstrated the expression of MIS type II receptor (MISRII) in the human papillomavirus (HPV)-16-related cervical cancer cell lines CaSki and SiHa, and a non-HPV-related cervical cancer cell line, C33A. We also showed that MIS inhibited growth of cervical cancer cells, and induced cellular apoptosis of C33A. In addition, we identified a characteristic molecular signature of MIS in CaSki cells by using whole genome expression analysis. Of the 1,690 genes that showed significant expression changes by MIS, 21 genes were related to cell cycle; 13 genes to apoptosis; and 52 genes to the cancer pathway. On performing a search for cell cycle pathways in the KEGG pathway database, several gene expressions at the G1/S checkpoint were found. In particular, the expression of p16 and p107 increased and that of E2F2 and E2F3 decreased at an early stage, whereas the expression of E2F4 and E2F5 decreased at a later stage after MIS treatment. These data suggest that MIS produces activity against HPV16-related cervical cancers in vitro, and MIS may also be an effective targeted therapy for HPV16-related cervical cancer. Genetic data obtained here could be useful in determining the treatment strategy of MISR-expressing cervical tumors in the future.

Müllerian inhibiting substance is anterogradely transported and does not attenuate avulsion-induced death of hypoglossal motor neurons.

Müllerian Inhibiting Substance (MIS, Anti-Müllerian hormone) is a gonadal hormone that contributes to the subtle sex-biases in the nervous system. Mature neurons of both sexes also produce MIS, suggesting that MIS may be a paracrine regulator of adult neural networks. We report here that murine hypoglossal motor neurons produce MIS and its receptors, MISRII and bone morphogenetic protein receptor 1A (BMPR1A, ALK3), but differentially transport them, with only MIS being detectable in axons. The production of MIS and its receptors were rapidly down regulated after axonal damage, which is a characteristic of genes involved in mature neuronal function. MIS is a survival factor for embryonic spinal motor neurons, but the rate of cell loss after hypoglossal nerve avulsion was normal in Mis(-/-) mice and was not attenuated by intraventricular administration of MIS. These observations suggest that MIS may be involved in anterograde rather than autocrine or retrograde regulation of neurons.

The rate of in vitro maturation of primary follicles from adult mice and the quality of oocytes is improved in the absence of anti-mullerian hormone.

Anti-Müllerian hormone (AMH) inhibits the recruitment of primordial follicles into the growing pool, but its role in primary and secondary follicles is not clear. We isolated primary follicles from the ovaries of 9- to 10-week old mice and examined whether AMH affected follicular development. Follicles were matured in media that was prepared using unsexed fetal bovine serum (FBS) or female FBS (FFBS) with or without added AMH for approximately 2 weeks and maturation rates to secondary follicles and metaphase II (MII) oocytes were measured by standard morphological criteria. Rates of parthenogenetic activation and in vitro fertilization (IVF) were assessed by cleavage and blastocyst development, respectively. Whereas addition of AMH blocked primary to secondary follicle transition, the primary to secondary and secondary to MII follicle maturation rates was significantly improved with FFBS. Folliculogenesis resumed once AMH was removed from the media of the arrested primary follicles. The rates of IVF and parthenogenesis of oocytes after in vitro maturation (IVM) without AMH were also improved compared to controls. The results indicate that removal of AMH from culture conditions during IVM from primary follicular stages should be considered to improve outcome.

Mullerian inhibiting substance inhibits invasion and migration of epithelial cancer cell lines.

OBJECTIVE: Given the fact that Mullerian Inhibiting Substance (MIS) causes complex remodeling of the urogenital ridge and regression of the Mullerian ducts during male embryonic development, we examined whether MIS could affect similar cell properties such as migration and invasion that could contribute ultimately to micro-metastasis of cancers arising from Mullerian tissues. MIS receptor expressing cell lines found to be invasive and migratory in vivo are examined in an in vivo assay that is cost-effective. METHODS: We designed in vitro and in vivo experiments to determine if MIS inhibited the movement of cancer lines IGROV-1, HEp3, MDA-MB-231, and HT1080 in cell culture invasion/migration chamber assays and in chick embryo metastasis assays. RESULTS: MIS, at concentrations below those that inhibit cell proliferation, blocked in vitro invasion and in vivo migration of epithelial cancer cells that express the MIS receptor. CONCLUSIONS: While our laboratory has previously established MIS as an inhibitor of cancer cell proliferation using in vitro assays and in vivo xenografts, we now show that MIS can also inhibit in vivo tumor migration.

Mullerian inhibiting substance preferentially inhibits stem/progenitors in human ovarian cancer cell lines compared with chemotherapeutics.

Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited self-renewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three--CD44, CD24, and Epcam--which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Müllerian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer.

Müllerian inhibiting substance/anti-Müllerian hormone: a potential therapeutic agent for human ovarian and other cancers.

According to the 2008 American Cancer Society statistics, cancer remains the second leading cause of death in American today. Early detection, innovative surgery, new drugs and increased public education regarding avoidable risk factors, such as smoking, have had significant impact on the incidence and survival rates of many cancers, while overall death rates from all cancers have declined a modest 5% over the past 50 years. Ovarian cancer statistics, however, have not been as encouraging. Despite recent advances in the management of this disease, 5-year survival has not improved, and the search continues for rationally designed new treatments. Müllerian Inhibiting Substance is a strong candidate because it addresses many of the deficiencies of existing treatments. Namely, Müllerian Inhibiting Substance has little demonstrated toxicity, it complements the activity of known anticancer drugs, it is highly specific against cancers expressing its receptor and it inhibits the proliferation of drug-resistant tumors.

Development of an efficiently cleaved, bioactive, highly pure FLAG-tagged recombinant human Mullerian Inhibiting Substance.

Mullerian Inhibiting Substance (MIS), a member of the TGF-beta family, causes regression of the Mullerian duct in male embryos, after binding to Mullerian Inhibiting Substance Receptor II (MISRII). It has also been extensively demonstrated that it can inhibit proliferation of various cancer cell lines such as ovarian, prostate, and breast cancer in vitro and in vivo. Hence, the availability of a recombinant, epitope tagged, bioactive MIS is important for the selection of patients for treatment and for probing novel molecular targets for MIS in various tissues. To this end, we have expressed a recombinant, internally FLAG-tagged form of hMIS with the tag (DYKDDDDK) immediately after the cleavage site (427-428) of MIS at the C-terminus with a modified dibasic cleavage motif sequence. We show that this construct results in a highly pure, endogenously processed (cleaved) FLAG MIS, that causes complete regression of the Mullerian Duct in an organ culture assay. In addition, purified FLAG MIS was able to bind and affinity purify both transfected and endogenous MIS type II receptor. The availability of this fully functional, epitope tagged form of MIS should facilitate scale-up for preclinical and clinical use and should also be used for the study of MIS binding proteins and for tracking in pharmacokinetic studies.

The expression of Müllerian inhibiting substance/anti-Müllerian hormone type II receptor protein and mRNA in benign, borderline and malignant ovarian neoplasia.

This study investigated the expression patterns of Müllerian inhibiting substance/anti-Müllerian hormone type II receptor (MIS/AMHRII) and mRNA in various types of ovarian neoplasia and evaluated the clinical significance of MIS/AMH as a biological response modifier for MIS/AMHR-positive tumors. Reverse transcriptase polymerase chain reaction was used to detect MIS/AMHRII mRNA expression and in situ hybridization and immunohistochemistry were used to localize MIS/AMHRII mRNA and protein expression. The degree of expression was scored from 0 (no staining) to 3 (strong staining). There was no significant difference in expression intensity between MIS/AMHRII protein and mRNA on all ovarian samples whether benign or malignant. MIS/AMHRII protein and mRNA were weakly expressed on 45.45% of benign ovarian tumors. In borderline tumors, expression rates of MIS/AMHRII protein and mRNA were 77.78% with score 1.22 and 55.56% with score 1, respectively. In malignant ovarian tumors, expression rates of MIS/AMHRII protein and mRNA were 70% with score 1.23 and 75% with score 1.43, respectively. Among malignant ovarian tumors, sex cord stromal tumors showed the highest expression rate and the strongest intensity of MIS/AMHRII protein and mRNA followed by germ cell tumor and epithelial ovarian tumor. Non-epithelial malignant tumors showed stronger expression than that of epithelial tumors (P<0.05, P<0.001, respectively). In serous borderline malignant and malignant tumors, MIS/AMHRII protein and mRNA expression was 63.64 and 81.82% with expression intensity of 1.27 and 1.46, respectively, which were not statistically different from non-epithelial malignant tumors. MIS/AMHRII and MIS/AMHRII mRNA demonstrate significantly variable expression among different ovarian tumor types. Non-epithelial cell tumors show higher expression than those of epithelial cell tumors. The highest expression rate and intensity were observed on sex cord stromal tumors. MIS/AMHRII expression was not different according to the differentiation, but showed tissue-type specificity. These data support that MIS/AMH may be used as a biological modifier or therapeutic modulator in MIS/AMHRII-expressed ovarian tumors.

Identification of large-scale characteristic genes of Müllerian inhibiting substance in human ovarian cancer cells.

The purpose of this study was to investigate the large-scale characteristic molecular signature of Müllerian inhibiting substance (MIS) in human ovarian cancer cells through expression genomics. To understand the comprehensive molecular mechanisms by which MIS inhibits ovarian cancer cell growth, we identified the large-scale characteristic molecular changes elicited by MIS in the human ovarian cancer cell line OVCAR-8, using DNA microarray analysis. Combined serial gene expression analysis from 0 to 96 h after MIS treatment of OVCAR-8 cells resulted in 759 genes which showed at least a 2-fold change in overexpression or underexpression compared to non-treatment groups. Of the 759 outlier genes, 498 genes were mapped to known biological cellular processes, and the resultant major pathways included metabolism, signal transduction, cell growth and apoptosis. Among these pathways, 68 genetic elements were dissected as cell cycle-related genes induced by MIS. Although cellular phenotypic changes by MIS were observed after 24 h of treatment, the characteristic large-scale molecular changes were observed from 48 to 96 h of exposure to MIS. This finding may imply that the suppressive role of MIS on ovarian cancer cells could be cumulative in that the metabolic disturbance of MIS is followed by arrest at the G1/S cell cycle checkpoint. We suggest 759 outlier genes comprise the characteristic molecular signature of MIS, which may be responsible for the suppressive effect on OVCAR-8 cells. Although the precise biological mechanisms underlying these outlier genes should be validated, the genetic elements described herein provide promising therapeutic interventions for ovarian cancer.

Serum Müllerian Inhibiting Substance/anti-Müllerian hormone levels in patients with adult granulosa cell tumors directly correlate with aggregate tumor mass as determined by pathology or radiology.

OBJECTIVES: Granulosa cell tumors (GCTs) comprise 2-5% of ovarian tumors. Serum Müllerian Inhibiting Substance (MIS, also known as anti-Müllerian hormone, or AMH) levels have been validated as a marker of GCT recurrence and progression. There has been little correlation between serum MIS/AMH levels and several clinical parameters in GCTs, including tumor burden. We have performed a retrospective review correlating aggregate tumor mass as reported by pathologic examination or by radiology with serum MIS/AMH levels drawn on the date of examination. METHODS: We retrospectively identified 32 GCT patients at our institution over the last 15 years who had serum MIS/AMH measurements. Patients who had serum MIS/AMH measurements within three days of surgery or on the same day as abdominal computerized tomography scan (CT) or magnetic resonance imaging (MRI) were further evaluated. RESULTS: We found a significant direct correlation between patient serum MIS/AMH levels and gross aggregate tumor mass determined by pathology (slope=15.4+/-6.06, r=0.65, p<0.04) or by radiographic aggregate tumor mass for all data points identified (slope=0.07+/-0.03, r=0.33, p<0.04) and after correcting for selection bias (slope=1.45+/-0.17, r=0.93, p<0.01). We also identified a significant difference between serum MIS/AMH levels between samples drawn the same day as negative and positive abdominal CT or MRI scans (8.16+/-1.54 vs. 158.7+/-32.2 ng/ml, p<0.0001). CONCLUSIONS: These data indicate a significant direct correlation between serum MIS/AMH levels and both gross and radiographic aggregate tumor mass in GCT patients. Together with the current literature, the present data argue for a more prominent role for serum MIS/AMH in the management of GCTs.

Cell migration and activated PI3K/AKT-directed elongation in the developing rat Müllerian duct.

In vertebrates, the Müllerian duct elongates along the Wolffian duct, a mesonephric structure that is required for Müllerian duct formation. Recently, several genes required for initial Müllerian duct formation have been identified. However, the precise mechanism of Müllerian duct elongation remains to be elucidated. In this study, we investigated dynamic morphological changes in the elongating Müllerian duct in rat urogenital ridges in organ culture manipulated by microincision and/or chemical inhibitors. Mechanical division of the developing Müllerian duct showed that epithelial cells of the Müllerian duct actively migrate along the anterior-posterior axis independent of the proliferative expansion of the anterior portion of the duct. We found that the PI3K/AKT signaling pathway is activated in the Müllerian duct epithelium and is required for elongation of the tip of the duct; however, migration of Müllerian duct epithelial cells proximal to the tip remains intact when PI3K/AKT is inactivated. Although much is known about the molecular and cellular mechanisms leading to Müllerian duct regression, the present findings provide a fuller understanding of the mechanisms contributing to Müllerian duct formation and to the general process of early tubulogenesis.

Mutations in LRP2, which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes.

Donnai-Barrow syndrome is associated with agenesis of the corpus callosum, congenital diaphragmatic hernia, facial dysmorphology, ocular anomalies, sensorineural hearing loss and developmental delay. By studying multiplex families, we mapped this disorder to chromosome 2q23.3-31.1 and identified LRP2 mutations in six families with Donnai-Barrow syndrome and one family with facio-oculo-acoustico-renal syndrome. LRP2 encodes megalin, a multiligand uptake receptor that regulates levels of diverse circulating compounds. This work implicates a pathway with potential pharmacological therapeutic targets.

Mullerian Inhibiting Substance is an ovarian growth factor of emerging clinical significance.

OBJECTIVE: To examine Mullerian Inhibiting Substance (MIS) as an emerging diagnostic marker of ovarian function. DESIGN: Medline review of published studies pertaining to the role of MIS in assessing ovarian aging, predicting response to ovulation induction in preparation for in vitro fertilization, assessing risk of developing ovarian hyperstimulation (OHSS) before ovulation induction, and diagnosis of polycystic ovarian disease (PCOS). RESULT(S): The majority of published studies to date support a role for MIS as a marker of ovarian reserve. Specific cut-off values are dependent upon the particular assay used. Mullerian Inhibiting Substance may offer value in assessing risk of OHSS and diagnosis of PCOS. CONCLUSION(S): Potential advantages of MIS compared with other conventional markers of ovarian reserve include: 1) MIS is the earliest marker to change with age; 2) it has the least intercycle variability; 3) it has the least intracycle variability; and 4) it may be informative if randomly obtained during the cycle. Widespread clinical use of MIS may await the availability of an international standard for MIS so that results using different assays may be reliably compared.