Absence of anti-Mullerian hormone (AMH) and M2A immunoreactivities in Sertoli cell-only syndrome and maturation arrest with and without AZF microdeletions.
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BACKGROUND: Some genes identified in the AZF locus are expressed only in germinal cells; others are ubiquitous. AZF microdeletions seem to occur at the earliest stages of ontogenetic development, and one might therefore assume that Sertoli cells preserve some immature characteristics and that their immunophenotype may be modified by the existence of a molecular defect. MATERIALS AND METHODS: Two immunohistological markers of Sertoli cell immaturity [anti-Mullerian hormone (AMH) and M2A] were tested in two histopathological groups (maturation arrest at spermatocyte I stage and Sertoli cell-only syndrome). We analysed 68 testicular samples obtained from 39 patients with non-obstructive azoospermia associated or not with AZF microdeletions. RESULTS: The absence of M2A and AMH immunoreactivity in adult gonads was observed without any correlation to spermatogenetic impairment or molecular deficit in the AZF region. In the samples of these two series, Sertoli cells showed a mature phenotype for AMH and M2A markers. CONCLUSIONS: In patients with AZF microdeletions, the genotype-phenotype correlations seem to be more complex than has been suggested previously; more detailed characterization of the immunohistochemical phenotype associated with the molecular defect may be useful in understanding the spermatogenic failure mechanism. (+info)
Highly purified mullerian inhibiting substance inhibits human ovarian cancer in vivo.
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PURPOSE: Mullerian inhibiting substance (MIS) causes Mullerianduct regression in mammalian, avian, and reptilian embryos; MIS also inhibits growth in vitro of Mullerian-derived cell lines and primary cells from human ovarian carcinomas. We hypothesize that highly purified MIS delivered parenterally inhibits ovarian cancer in vivo. EXPERIMENTAL DESIGN: To test the efficacy of highly purified MIS against ovarian cancer cell lines in vivo, we treated immunosuppressed mice with MIS after implanting OVCAR 8 or IGROV 1 human ovarian cancer cells beneath the renal capsules and measured tumor volume over time. Animals were treated with daily injections of 10 micro g of purified exogenous recombinant human MIS or by endogenous MIS secreted from cells growing on biodegradable mesh. RESULTS: The average graft size ratio (change in size compared with starting size) of the OVCAR 8 tumor implants was larger in the control animals than in animals treated for 2 weeks (P < 0.019) or 3 weeks (P < 0.001) with parenteral MIS, or after treating with MIS produced from transfected cells, which impregnated the biodegradable mesh (P = 0.02). The average graft size ratio of the IGROV 1 tumors was also larger in the control animals than in those treated with injected MIS (P = 0.0174). CONCLUSIONS: Highly purified recombinant human MIS, delivered parenterally, or MIS produced endogenously causes inhibition of human ovarian cancer cell lines in vivo, providing convincing preclinical evidence to support the use of MIS as a parenteral agent for the treatment of ovarian cancer. (+info)
The role of insulin 3, testosterone, Mullerian inhibiting substance and relaxin in rat gubernacular growth.
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Transabdominal testicular descent is influenced by various anatomical and hormonal factors and is mediated by gubernacular enlargement and regression of the cranial suspensory ligament, but its mechanism remains controversial. The aim of this study was to determine which hormones have a direct effect on the proliferation of cells in the day 17 fetal rat gubernaculum in vitro, using an organ culture system. The effects of synthetic rat insulin 3 (INSL3), inactive INSL3, dihydrotestosterone (DHT), DHT+INSL3, human Mullerian inhibiting substance (hMIS), hMIS+INSL3 and human gene 2 relaxin were tested, together with co-culture with fetal rat testis. Cell proliferation was assessed using a bromodeoxyuridine labelling index. The results showed that MIS and relaxin have a mild effect on gubernacular growth, whilst INSL3 and DHT have a more marked effect. The combination of MIS+INSL3 showed an effect close to that of co-culture with testis. However, the most pronounced effect was caused by DHT+INSL3. RT-PCR analysis indicated that the fetal rat gubernaculum strongly expresses putative INSL3 receptors, weakly expresses MIS type II receptors and does not express relaxin receptors. In conclusion, a number of different hormones directly influence growth of the gubernaculum in vitro, including the recently reported hormone INSL3. INSL3 shows a direct stimulatory effect on the swelling reaction, while DHT and MIS may have roles in augmenting this growth. (+info)
Different patterns of anti-Mullerian hormone expression, as related to DMRT1, SF-1, WT1, GATA-4, Wnt-4, and Lhx9 expression, in the chick differentiating gonads.
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In mammals, anti-Mullerian hormone (AMH) is produced by Sertoli cells from the onset of testicular differentiation and by granulosa cells after birth. In birds, AMH starts to be expressed in indifferent gonads of both sexes at a similar level and is later up-regulated in males. We previously demonstrated that, unlike in mammals, the onset of AMH expression occurs in chick embryo in the absence of SOX9. We looked for potential factors that might be involved in regulating AMH expression at different stages of chick gonad differentiation by comparing its expression pattern in embryos and young chicken with that of DMRT1, SF-1, WT1, GATA-4, Wnt-4, and Lhx9, by in situ hybridization. The results allowed us to distinguish different phases. (1) In indifferent gonads of both sexes, AMH is expressed in dispersed medullar cells. SF-1, WT1, GATA-4, Wnt-4, and DMRT1 are transcribed in the same region of the gonads, but none of these factors has an expression strictly coincident with that of AMH. Lhx9 is present only in the cortical area. (2) After this period, AMH is up-regulated in male gonads. The up-regulation is concomitant with the beginning of SOX9 expression and a sex dimorphic level of DMRT1 transcripts. It is followed by the aggregation of the AMH-positive cells (Sertoli cells) into testicular cords in which AMH is coexpressed with DMRT1, SF-1, WT1, GATA-4, and SOX9. (3) In the females, the low level of dispersed medullar AMH expression is conserved. With development of the cortex in the left ovary, cells expressing AMH accumulate in the juxtacortical part of the medulla, whereas they remain dispersed in the right ovary. At this stage, AMH expression is not strictly correlated with any of the studied factors. (4) After hatching, the organization of left ovarian cortex is characterized by the formation of follicles. Follicular cells express AMH in conjunction with SF-1, WT1, and GATA-4 and in the absence of SOX9, as in mammals. In addition, they express Lhx9 and Wnt-4, the latter being also found in the oocytes. (5) Moreover, unlike in mammals, the chicken ovary retains a dispersed AMH expression in cortical interstitial cells between the follicles, with no obvious correlation with any of the factors studied. Thus, the dispersed type of AMH expression in indifferent and female gonads appears to be bird-specific and not controlled by the same factors as testicular or follicular AMH transcription. (+info)
Regulation of ovarian function: the role of anti-Mullerian hormone.
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Anti-Mullerian hormone (AMH), also known as Mullerian inhibiting substance, is a member of the transforming growth factor beta superfamily of growth and differentiation factors. In contrast to other members of the family, which exert a broad range of functions in multiple tissues, the principal function of AMH is to induce regression of the Mullerian ducts during male sex differentiation. However, the patterns of expression of AMH and its type II receptor in the postnatal ovary indicate that AMH may play an important role in ovarian folliculogenesis. This review describes several in vivo and in vitro studies showing that AMH participates in two critical selection points of follicle development: it inhibits the recruitment of primordial follicles into the pool of growing follicles and also decreases the responsiveness of growing follicles to FSH. (+info)
Expression levels of Mullerian-inhibiting substance, GATA4 and 17alpha-hydroxylase/17,20-lyase cytochrome P450 during embryonic gonadal development in two diverse breeds of swine.
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Sexual differentiation and early embryonic/fetal gonad development is a tightly regulated process controlled by numerous endocrine and molecular signals. These signals ensure appropriate structural organization and subsequent development of gonads and accessory organs. Substantial differences exist in adult reproductive characteristics in Meishan (MS) and White Composite (WC) pig breeds. This study compared the timing of embryonic sexual differentiation in MS and WC pigs. Embryos/fetuses were evaluated on 26, 28, 30, 35, 40 and 50 days postcoitum (dpc). Gonadal differentiation was based on morphological criteria and on localization of GATA4, Mullerian-inhibiting substance (MIS) and 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450(c17)). The timing of testicular cord formation and functional differentiation of Sertoli and Leydig cells were similar between breeds. Levels of GATA4, MIS and P450(c17) proteins increased with advancing gestation, with greater levels of MIS and P450(c17) in testes of MS compared with WC embryos. Organization of ovarian medullary cords and formation of egg nests was evident at similar ages in both breeds; however, a greater number of MS compared with WC embryos exhibited signs of ovarian differentiation at 30 dpc. In summary, despite breed differences in MIS and P450(c17) levels in the testis, which may be related to Sertoli and Leydig cell function, the timing of testicular differentiation did not differ between breeds and is unlikely to impact reproductive performance in adult boars. In contrast, female MS embryos exhibited advanced ovarian differentiation compared with WC embryos which may be related to the earlier reproductive maturity observed in this breed. (+info)
Novel genes regulated by Sonic Hedgehog in pluripotent mesenchymal cells.
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Sonic Hedgehog is a secreted morphogen involved in patterning a wide range of structures in the developing embryo. Disruption of the Hedgehog signalling cascade leads to a number of developmental disorders and plays a key role in the formation of a range of human cancers. The identification of genes regulated by Hedgehog is crucial to understanding how disruption of this pathway leads to neoplastic transformation. We have used a Sonic Hedgehog (Shh) responsive mouse cell line, C3H/10T1/2, to provide a model system for hedgehog target gene discovery. Following activation of cell cultures with Shh, RNA was used to interrogate microarrays to investigate downstream transcriptional consequences of hedgehog stimulation. As a result 11 target genes have been identified, seven of which are induced (Thrombomodulin, GILZ, BF-2, Nr4a1, IGF2, PMP22, LASP1) and four of which are repressed (SFRP-1, SFRP-2, Mip1-gamma, Amh) by Shh. These targets have a diverse range of putative functions and include transcriptional regulators and molecules known to be involved in regulating cell growth or apoptosis. The corroboration of genes previously implicated in hedgehog signalling, along with the finding of novel targets, demonstrates both the validity and power of the C3H/10T1/2 system for Shh target gene discovery. (+info)
Serum anti-Mullerian hormone levels: a novel measure of ovarian reserve.
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BACKGROUND: Anti-Mullerian hormone (AMH) is produced by the granulosa cells of preantral and small antral follicles and its levels can be assessed in serum. Since the number of ovarian follicles declines with increasing age, AMH levels might be used as a marker for ovarian ageing. Therefore, we studied the relationship between AMH levels and ovarian response during ovarian stimulation for IVF. METHODS: A total of 130 patients undergoing their first IVF treatment cycle using a long protocol with GnRH agonist was prospectively included. Blood withdrawal was performed and the number of antral follicles was assessed by ultrasound on day 3 of a spontaneous cycle. Poor response and the number of oocytes were used as primary outcome measures. In a random subset of 23 patients a GnRH agonist stimulation test was performed to investigate whether a rise in FSH and LH would affect AMH levels. RESULTS: The data of 119 patients were analysed. Serum AMH levels were highly correlated with the number of antral follicles (r = 0.77; P < 0.01) and the number of oocytes retrieved (r = 0.57, P < 0.01). A negative association was found between AMH levels and poor ovarian response (fewer than 4 oocytes or cycle cancellation; OR 0.82, 95% CI 0.75-0.90, P < 0.01). Inclusion of inhibin B and FSH concentrations to AMH in a multivariate model improved the prediction of ovarian response. The post GnRH agonist rise in FSH and LH levels did not influence AMH values. CONCLUSIONS: Poor response in IVF, indicative of a diminished ovarian reserve, is associated with reduced baseline serum AMH concentrations. In line with recent observations it appears that AMH can be used as a marker for ovarian ageing. (+info)