Influence of gonadal hormones on endocrine activity of gonadotroph cells in the adenohypophysis of male lambs during the postnatal transition to puberty.

Using histomorphological and functional criteria we describe the feedback mechanisms which could play a role in the regulation of the gonadotrophic axis during the postnatal transition to puberty in male lambs. The working hypothesis was that the testicular factors change the peripheral levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by influencing the synthesis rate and storage of LH and FSH in adenohypophyseal gonadotroph cells of weanling and weaned pubertal lambs. The examination was made in (i) 9-week-old infantiles, suckling lambs undergoing weaning, testis-intact (TEI) and orchidectomised (ORCHX) at the 6th week of age, and (ii) 16-week-old pubertal lambs TEI and ORCHX at the 12th week of age (n=5 per group). Changes in gonadotrophs were assayed with hybridohistochemistry, immunohistochemistry and radioimmunoassay. The percentage of the adenohypophyseal area (PA) occupied by cells containing LHß-mRNA and FSHß-mRNA and peripheral levels of both gonadotrophins were lower (P<0.01) in the 16-week-old TEI lambs in comparison with the 9-week-old ones. The PA occupied by cells immunoreactive for LHß was lower (P<0.01), whereas in the case of FSH was greater (P<0.001) in the 16-week-old lambs. After orchidectomy the PA occupied by gonadotrophs stained for LHß-mRNA was greater (P<0.01) in 16-week-old lambs. The PA occupied by LHß-labelled cells was lower (P<0.05) in the 9-week-old ORCHX lambs, whereas in 16-week-old ones was higher (P<0.05) in comparison with the TEI lambs. The circulating LH was greater (P<0.01) in the ORCHX 9- and 16-week-old lambs compared to the TEI ones. The PA occupied by cells containing FSHß-mRNA and the plasma FSH concentration were greater (P<0.001) after orchidectomy in lambs from both age stages. The PA occupied by FSHß-labelled cells was greater (P<0.01) in the 9-week-old ORCHX lambs, whereas in 16-week-old ones was lower (P<0.05) compared to the lambs from TEI groups. In conclusion, in infantile lambs testicular factors may play inhibitory role in regulating FSH synthesis rate, storage and release in contrast to the stimulatory role in regulating LH storage reflected by the inhibitory role in regulating LH release. In lambs at the beginning of puberty, testicular factors may play inhibitory role in regulating LH synthesis rate, storage and release in contrast to the stimulatory role in regulating FSH storage reflected by the inhibitory role in regulating FSH synthesis rate and release. The effects of testicular hormones on the gonadotrophin storage, i.e. releasable pools in adenohypophyseal cells, are specific for both LH and FSH in lambs during the postnatal transition to puberty. Thus, the initiation of puberty in male sheep is a function of change of the inhibitory role of gonadal factors in regulating FSH storage to the stimulatory one and the stimulatory role of gonadal factors in regulating LH storage to the inhibitory one.

p-MAPK1/3 and DUSP6 regulate epididymal cell proliferation and survival in a region-specific manner in mice.

A fully developed, functional epididymis is important for male fertility. In particular, it is apparent that without the most proximal region, the initial segment (IS), infertility results. Therefore, it is important to understand the development and regulation of this crucial epididymal region. We have previously shown that many functions of the IS are regulated by luminal fluid factors/lumicrine factors from the testis. This study provides evidence that lumicrine factors activated the ERK pathway only in epithelial cells of the IS from Postnatal Day (P) 14 to P19 and sustained this activation into adulthood. The activated ERK pathway promoted cell proliferation and differentiation in the developing IS, although in the adult, its role was switched to maintain cell survival. To understand further the regulation of cell proliferation in the IS, we examined the role of DUSP6, an MAPK1/3 (ERK1/2) preferred phosphatase that is also regulated by lumicrine factors in the IS. Utilizing Dusp6(-/-) mice, our studies, surprisingly, revealed that Dusp6 was a major regulator of cell proliferation in the caput and corpus regions, whereas components of the ERK pathway, together with PTEN and SRC, were the major regulators of cell proliferation in the IS. We hypothesize that region-specific regulation of cell proliferation is caused by differences in the balance of activities between pro- and antiproliferation signaling pathway components for each epididymal region. An understanding of the mechanisms of cell proliferation may provide clues as to why the epididymis rarely succumbs to cancer.

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.

The Anti-Mullerian hormone and ovarian cancer.

The Anti-Mullerian hormone (AMH), which is produced by fetal Sertoli cells, is responsible for regression of Mullerian ducts, the anlagen for uterus and Fallopian tubes, during male sex differentiation. Ovarian granulosa cells also secrete AMH from late in fetal life. The patterns of expression of AMH and its type II receptor in the post-natal ovary indicate that AMH may play an important role in ovarian folliculogenesis. Recent advances in the physiological role of AMH has stimulated interest in the significance of AMH as a diagnostic marker and therapeutic agent for ovarian cancer. Currently, AMH has been shown to be a circulating marker specifically for granulosa cell tumour (GCT). Its diagnostic performance seems to be very good, with a sensitivity ranging between 76 and 93%. In patients treated for GCT, AMH may be used post-operatively as marker for the efficacy of surgery and for disease recurrence. Based on the physiological inhibitory role of AMH in the Mullerian ducts, it has been proposed that AMH may inhibit epithelial ovarian cancer cell both in vitro and in vivo. These observations will be the basis for future research aiming to investigate the possible clinical role of AMH as neo-adjuvant, or most probably adjuvant, therapy for ovarian cancer.

Anti-Müllerian hormone inhibits initiation of growth of human primordial ovarian follicles in vitro.

BACKGROUND: Anti-Müllerian hormone (AMH) inhibits the initiation of the development and early growth of mouse ovarian follicles. Furthermore, the ovarian follicle pool diminishes prematurely in AMH-knockout mice. In this study, we examined whether AMH plays a similar role in humans, controlling ovarian follicle growth. METHODS: Human ovarian cortical tissue biopsy specimens were cut into small pieces and cultured for 7 days in medium containing rat recombinant AMH at 0, 10, 30 or 100 ng/ml. The developmental stages and viability of the follicles were evaluated from histological sections. RESULTS: Similar to previous studies, significant initiation of follicle growth was observed in almost all culture media, as demonstrated by a significantly smaller proportion of primordial follicles (14-26%) compared with non-cultured control tissue (56%). The exception was tissue in medium supplemented with AMH at 100 ng/ml. Here, the proportion of primordial follicles was not significantly different from that in non-cultured tissue; furthermore, it was significantly greater than that in vehicle control cultures and cultures containing AMH at 10 ng/ml, indicating the inhibition of growth initiation. Viability was unaffected by the presence of AMH when compared with tissues in control media. CONCLUSIONS: Recombinant AMH at a concentration of 100 ng/ml has an inhibitory effect on early human ovarian follicular development in vitro, suppressing the initiation of primordial follicle growth.

Pathways of endocrine disruption during male sexual differentiation and masculinization.

After testis formation, further development of a male phenotype (masculinization) is driven by three hormones from the foetal testis: anti-Müllerian hormone, insulin-like factor 3, and testosterone. These hormones divert the development of reproductive and other organs from female to male and also play a role in testis development. The hormone dependence of masculinization renders this process inherently susceptible to disruption by factors that interfere with hormone production, bioavailability, metabolism, or action. This susceptibility is illustrated by the high prevalence of congenital masculinization disorders (cryptorchidism, hypospadias) and disorders in young adult men (low sperm counts, testis cancer), which may also stem from maldevelopment (dysgenesis) of the foetal testis. Testicular dysgenesis occurring in humans, or which is induced in animal models by foetal exposure to certain phthalates, is associated with impaired hormone production by the foetal testis. There is currently no definitive evidence that exposure of humans to environmental chemicals can induce testicular dysgenesis and/or impair masculinization, though pathways via which this could potentially occur are established.

Anti-Müllerian hormone initiates growth of human primordial follicles in vitro.

Survival and growth of follicles in human ovarian tissue is presently only performed with limited success. We evaluated the effect of anti-Müllerian hormone (AMH) and/or testosterone on follicular growth during a 4-week culture period using ovarian cortical tissue from six women in their reproductive years. The cortex of each biopsy was isolated and immediately cryopreserved upon collection and stored in liquid nitrogen. After thawing the tissue was placed in culture. After the culture period all follicles were counted on histological sections and classified for viability and stage of development. Based on evaluation of 6603 follicles it was found that the number of growing follicles significantly increased during the culture period as compared to the uncultured control, irrespective of the composition of the culture medium. Furthermore, significantly more follicles advanced to the primary and secondary stage (p<0.05) in tissue cultured with AMH (54%) as compared to tissue cultured in control medium (41%). The mean diameter of follicles classified as primary follicles was significantly enhanced in tissue cultured in the presence of AMH (p=0.002) and AMH plus testosterone (p<0.001) as compared to that observed in tissue cultured with control medium and medium containing testosterone alone. In contrast the mean diameter of the oocyte and its nucleus remained similar irrespective of culture medium. In conclusion, AMH seems to affect early stages of human follicular development by enhancing recruitment, survival and/or growth during a 4-week culture period.

Mullerian inhibiting substance suppresses tumor growth in the C3(1)T antigen transgenic mouse mammary carcinoma model.

Mullerian inhibiting substance (MIS) inhibits breast cancer cell growth in vitro. To extend the use of MIS to treat breast cancer, it is essential to test the responsiveness of mammary tumor growth to MIS in vivo. Mammary tumors arising in the C3(1) T antigen mouse model expressed the MIS type II receptor, and MIS in vitro inhibited the growth of cells derived from tumors. Administration of MIS to mice was associated with a lower number of palpable mammary tumors compared with vehicle-treated mice (P=0.048), and the mean mammary tumor weight in the MIS-treated group was significantly lower compared with the control group (P=0.029). Analysis of proliferating cell nuclear antigen (PCNA) expression and caspase-3 cleavage in tumors revealed that exposure to MIS was associated with decreased proliferation and increased apoptosis, respectively, and was not caused by a decline in T antigen expression. The effect of MIS on tumor growth was also evaluated on xenografted human breast cancer cell line MDA-MB-468, which is estrogen receptor- and retinoblastoma-negative and expresses mutant p53, and thus complements the C3(1)Tag mouse mammary tumors that do not express estrogen receptor and have functional inactivation of retinoblastoma and p53. In agreement with results observed in the transgenic mice, MIS decreased the rate of MDA-MB-468 tumor growth and the gain in mean tumor volume in severe combined immunodeficient mice compared with vehicle-treated controls (P=0.004). These results suggest that MIS can suppress the growth of mammary tumors in vivo.

Anti-Müllerian hormone in disorders of sex determination and differentiation

A masculinização dos genitais internos e externos durante o desenvolvimento fetal depende da existência de dois hormônios testiculares distintos: a testosterona secretada pelas células de Leydig conduz à diferenciação dos dutos de Wolff, do seio urogenital e dos genitais externos, enquanto que o hormônio anti-Mülleriano (HAM) produzido pelas células de Sertoli provoca a regressão dos dutos de Müller. A falta de ação do HAM no início da vida fetal resulta na formação de tubas uterinas, útero e terço superior da vagina. Em fetos 46,XY, a falta do HAM pode resultar de disgenesia testicular, afetando tanto as células de Leydig quanto as de Sertoli: nesse caso, a presença de remanescentes Müllerianos está associada a genitais externos femininos ou ambíguos. Por outro lado, distúrbios na ação do HAM podem resultar de mutações em genes que codificam o HAM ou seu receptor: nessa afecção, conhecida como síndrome da Persistência dos Dutos de Müller, a produção de testosterona é normal e os genitais externos são virilizados normalmente. Finalmente, o HAM costuma ser secretado normalmente em pacientes com intersexo decorrente de defeitos restritos à síntese ou à ação de andrógenos, resultando em indivíduos com genitais externos femininos ou ambíguos com ausência de derivados de Müller.

Transforming Growth Factor-beta superfamily: evaluation as breast cancer biomarkers and preventive agents.

The Transforming Growth Factor-beta (TGFbeta) superfamily of cytokines is comprised of a number of structurally-related, secreted polypeptides that regulate a multitude of cellular processes including proliferation, differentiation and neoplastic transformation. These growth regulatory molecules induce ligand-mediated hetero-oligomerization of distinct type II and type I serine/threonine kinase receptors that transmit signals predominantly through receptor-activated Smad proteins but also induce Smad-independent pathways. Ligands, receptors and intracellular mediators of signaling initiated by members of the TGFbeta family are expressed in the mammary gland and disruption of these pathways may contribute to the development and progression of human breast cancer. Since many facets of TGFbeta and breast cancer have been recently reviewed in several articles, except for discussion of recent developments on some aspects of TGFbeta, the major focus of this review will be on the role of activins, inhibins, BMPs, nodal and MIS-signaling in breast cancer with emphasis on their utility as potential diagnostic, prognostic and therapeutic targets.

[Differentiation and development of internal sexual organs, and müllerian inhibiting substance].

Internal sexual organs are differentiated and developed by androgens and regressed by müllerian inhibiting substance(anti-müllerian hormone). The role of 5 alpha-dihydrotestosterone, reduced form of testosterone by 5 alpha-reductase, in terms of development of Wolffian duct is discussed with soluble mesenchymal factor responsible for the epithelial branching morphogenesis of mouse seminal vesicle on the basis of experimental results using organ culture assay of mouse new-born seminal vesicle. An update of müllerian inhibiting substance, a fetal regressor of female internal organs such as uterus, fallopian tubes and upper third vagina, is also discussed.

AMH induces mesonephric cell migration in XX gonads.

Migration of mesonephric cells into XY gonads is a critical early event in testis cord formation. Based on the fact that anti-Müllerian hormone (AMH) can induce testis cord formation in XX gonads, we investigated whether AMH plays a role in the induction of cell migration. Addition of recombinant AMH induced mesonephric migration into XX gonads in culture. AMH-treated XX gonads displayed increased vascular development and altered morphology of the coelomic epithelium, both features of normal testis differentiation. AMH did not induce markers of Sertoli or Leydig cell differentiation. We examined early testis development in Amh-deficient mice, but found no abnormalities, suggesting that any function AMH may have in vivo is redundant. Other transforming growth factor (TGF-beta) family proteins, bone morphogenetic proteins (BMP2 and BMP4) show similar inductive effects on XX gonads in culture. Although neither BMP2 nor BMP4 is expressed in embryonic XY gonads, our findings suggest that a TGF-beta signalling pathway endogenous to the XY gonad may be involved in regulation of mesonephric cell migration. The factors involved in this process remain to be identified.

AMH/MIS: what we know already about the gene, the protein and its regulation.

(AMH/MIS) was first suggested by Jost, more than Four decades before this gonadal glycoprotein was purified and its gene and promoter sequenced. In mammals, AMH expression is triggered by SOX9 in Sertoli cells at the onset of testicular differentiation, and regulated by SF1, GATA factors, WT1, DAX1 and FSH. Ovarian granulosa cells also secrete AMH from late foetal life. In males, AMH is secreted into the bloodstream at high levels until puberty when it is down-regulated by androgens and meiotic germ cells and its directional secretion switches from the basal compartment to the seminiferous tubule lumen. In birds and reptiles, AMH expression shows particular features. Serum AMH determination is useful to study testicular function in boys and in patients with gonadal tumours. AMH levels in seminal and follicular fluid may also be of clinical use.

Inhibition of steroidogenesis in Leydig cells by Müllerian-inhibiting substance.

Müllerian-inhibiting substance (MIS), a member of the transforming growth factor-beta family of cytokines that signal through a heteromeric complex of single-transmembrane serine/threonine kinase receptors, is required for Müllerian duct regression and normal reproductive tract development in the male embryo. However, the continued expression of MIS at high levels in males until puberty and its induction in females after birth suggested other roles for MIS. Additionally, Leydig cell development and steroidogenic capacity and ovarian follicle recruitment were abnormal in MIS-knockout or MIS-overexpressing mice. We have shown that MIS inhibits the cAMP-induced expression of cytochrome P450 C17alpha-hydroxylase/C17-20 lyase (Cyp17) mRNA both in vitro and in vivo. Our current efforts are to understand the molecular mechanisms regulating both MIS type II receptor (MISRII) expression and its signaling in rodent Leydig cell lines. MISRII expression in R2C cells requires both steroidogenic factor-1 and an unknown protein to bind to its proximal promoter in the context of 1.6 kb 5'-flanking DNA. When bound by MIS, signaling by the receptor in MA-10 cells blocks the protein kinase A-mediated induction of Cyp17 expression by a cAMP regulatory element-binding protein independent mechanism. We continue to investigate the molecular mechanisms of MISRII expression and possible interactions between MIS-regulated SMAD activation and cAMP signaling. These studies will provide a better understanding of the role played by MIS during postnatal life.

Anti-Müllerian hormone, beta-catenin and Müllerian duct regression.

The embryo is initially sexually indifferent, and correct sexual development is dependent on gonadal hormone production. Thus, in the male embryo, anti-Müllerian hormone (AMH), secreted by the Sertoli cells of the testis, induces regression of the Müllerian duct, the anlagen of female reproductive tract. This hormone causes ductal epithelial regression through a paracrine mechanism originating in periductal mesenchyme and the cross-talk between the mesenchymal and epithelial layers accounts for the cranial-to-caudal pattern of Müllerian regression. Here, we review and discuss recent developments concerning the relationship of apoptosis of Müllerian duct to tissue remodeling, mesenchymal-epithelial interactions, and involvement of beta-catenin in AMH signaling in periductal mesenchyme. Determining the role of beta-catenin/LEF-1 signaling is critical for understanding AMH action during Müllerian duct regression.

[Inhibins, activins and anti-Müllerian hormone: structure, signalling pathways, roles and predictive value in reproductive medicine]. / Inhibines, activines et hormone anti-müllerienne : structure, signalisation, rôles et valeur prédictive en médecine de la reproduction.

Anti-Müllerian hormone (AMH), inhibins and activins are members of the transforming growth factor (TGFbeta) superfamily and are known to have a variety of actions concerning reproduction, hormonogenesis, development processes and differentiation. Inhibins and activins are dimeric glycoproteins that are defined by their actions on the pituitary gonadotroph cells. AMH, inhibins and activins have a vast array of actions usually exerted through paracrine and endocrine mechanisms. The recent availability of specific inhibin assays has demonstrated that inhibin B is the relevant circulating inhibin form in the human male. Inhibin B seems to be a useful marker of spermatogenesis, but serum and seminal inhibin B levels are not predictive parameters for the selection of azoospermic men as candidates for testicular sperm extraction (TESE). AMH in seminal plasma may be important for sperm production, and is a good marker for sertoli cell development. It might be the only one seminal marker of spermatogenesis in non-obstructive azoospermia. Nevertheless, many of these studies were carried out with small patient numbers, and consequently must be interpreted with caution. In women ongoing assisted reproductive therapy (ART), day 3 inhibin B and AMH levels predict the number of oocytes retrieved, but cannot predict likelihood of pregnancy. Further studies are needed to determine if AMH and inhibin predict ART outcomes better than classical parameters (age, FSH levels and follicular ultrasonography). AMH and inhibin are also specific markers of Sertoli- and granulosa-cell origin in gonadal tumors.

[The genetics of sex determination]. / La génétique du déterminisme du sexe.

The male sex determination controls the testis determination and differentiation. So far more than a dozen of gene have been characterised, including the primary Y male sex determinant: SRY gene. All the genes encode transcription factor except FDF9 which encodes a growth factor allowing mesonephritic cell to divide and migrate to the mesenchyme. All these gene products are sensitive in man (but not in the mouse model) to gene dosage: haplo-insufficiency and or duplication of these genes all lead to sex reversal. Today, we still do not know how their products are epistatic in the sex determining cascade. So far no ovary sex determining gene has been characterised.

Regulation of ovarian function: the role of anti-Müllerian hormone.

Anti-Müllerian hormone (AMH), also known as Müllerian 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 Müllerian 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.

Mullerian-inhibiting substance regulates NF-kappa B signaling in the prostate in vitro and in vivo.

Mullerian-inhibiting substance (MIS) is a member of the transforming growth factor beta superfamily, a class of molecules that regulates growth, differentiation, and apoptosis in many cells. MIS type II receptor in the Mullerian duct is temporally and spatially regulated during development and becomes restricted to the most caudal ends that fuse to form the prostatic utricle. In this article, we have demonstrated MIS type II receptor expression in the normal prostate, human prostate cancer cell lines, and tissue derived from patients with prostate adenocarcinomas. MIS induced NF-kappaB DNA binding activity and selectively up-regulated the immediate early gene IEX-1S in both androgen-dependent and independent human prostate cancer cells in vitro. Dominant negative IkappaBalpha expression ablated both MIS-induced increase of IEX-1S mRNA and inhibition of growth, indicating that activation of NF-kappaB signaling was required for these processes. Androgen also induced NF-kappaB DNA binding activity in prostate cancer cells but without induction of IEX-1S mRNA, suggesting that MIS-mediated increase in IEX-1S was independent of androgen-mediated signaling. Administration of MIS to male mice induced IEX-1S mRNA in the prostate in vivo, suggesting that MIS may function as an endogenous hormonal regulator of NF-kappaB signaling and growth in the prostate gland.

[The role of transforming growth factor beta superfamily in male germ cell development].

Transforming growth factor beta (TGF beta) superfamily can regulate the development of primordial germ cell (PGC) and gonocyte. TGF beta, bone morphogenetic protein (BMP), activin, inhibin, and anti-Müllerian hormone (AMH), all of which belong to the TGF beta superfamily, can play important roles in male germ cell development. Their downstream signaling molecules, Smads proteins are involved in the signal transduction pathway. In addition, TGF beta and AMH contribute to the apoptosis during development. Understanding this effect will help to elucidate the molecular mechanism of the early development of male reproductive system and the pathogenesis of testicular cancer.