Mutations of the AMH type II receptor in two extended families with persistent Müllerian duct syndrome: lack of phenotype/genotype correlation

Our goal was to compare phenotype and genotype in two extended Middle-Eastern families affected by persistent Müllerian duct syndrome due to mutations of the type II anti-Müllerian hormone receptor (AMHR-II). The first, consanguineous, family consisted of 6 boys and 2 girls, the second consisted of 4 girls and 2 boys. In family I, 4 boys and 1 girl were homozygous for a stop mutation in the 9th exon of AMHR-II, removing part of the intracellular domain of the protein. In family II, 1 girl and 1 boy were homozygous for a transversion changing conserved histidine 254 into a glutamine. Both homozygous girls were normal. In the homozygous males, the degree of development of Müllerian derivatives was variable. The uterus was well developed in 2 boys of family I and in the patient from family II; however, in 1 subject from family I, Müllerian derivatives were undetectable. Taken together, the diversity of clinical symptoms within the same sibship and the lack of correlation between the development of the Müllerian derivatives and the severity of the molecular defects suggest highly variable penetrance of the abnormal alleles and/or the existence of other genetic or epigenetic modifiers of gene expression.     

Müllerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications

Dr. Alfred Jost pioneered the field of reproductive endocrinology with his seminal observation that two hormones produced by the testes are required for the male embryo to develop a normal internal reproductive tract. T induces the Wolffian ducts to differentiate into epididymides, vasa deferens, and seminal vesicles. Müllerian inhibiting substance (MIS) causes regression of the Müllerian ducts, which in its absence would normally develop into the Fallopian tubes, uterus, and upper vagina as is observed in female embryos. This review will summarize our current understanding of molecular mechanisms underlying the function of MIS both as a fetal gonadal hormone that causes Müllerian duct regression and as an adult hormone, the roles for which are currently being investigated, i.e., inhibition of steroidogenesis, germ cell development, and cancer. We will also address the regulation of MIS expression as one of the first genes expressed after the commitment of the bipotential gonads to differentiate into testes under the influence of SRY, the gene on the sex-determining region of the Y chromosome. We will discuss what is known regarding MIS signal transduction, which as with other members of the TGFbeta family of growth and differentiation factors, occurs through a heteromeric complex of single transmembrane serine/threonine kinase receptors to effect downstream signaling events, including Smad, nuclear factor-kappaB, beta-catenin, and p16 activation. Finally, we will assess the clinical relevance of studying MIS in patients with persistent Müllerian duct syndrome and our efforts to determine the therapeutic value of MIS for patients with ovarian and other MIS receptor-expressing cancers.     

Anti-Müllerian hormone in disorders of sex determination and differentiation

Masculinisation of internal and external genitalia during foetal development depends on the existence of two discrete testicular hormones: Leydig cell-secreted testosterone drives the differentiation of the Wolffian ducts, the urogenital sinus and the external genitalia, whereas Sertoli cell-produced anti-Müllerian hormone (AMH) provokes the regression of Müllerian ducts. The absence of AMH action in early foetal life results in the formation of the Fallopian tubes, the uterus and the upper third of the vagina. In 46,XY foetuses, lack of AMH may result from testicular dysgenesis affecting both Leydig and Sertoli cell populations: in this case persistence of Müllerian remnants is associated with ambiguous or female external genitalia. Alternatively, defective AMH action may result from mutations of the genes encoding for AMH or its receptor: in this condition known as Persistent Müllerian Duct Syndrome, testosterone production is normal and external genitalia are normally virilised. Finally, AMH may be normally secreted in intersex patients with defects restricted to androgen synthesis or action, resulting in patients with female or ambiguous external genitalia with no Müllerian derivatives.     

Interaction between sex hormones and WNT/β-catenin signal transduction in endometrial physiology and disease

Wnt/β-catenin signalling plays a rate-limiting role in early development of many different organs in a broad spectrum of organisms. In the developing Müllerian duct, Wnt/β-catenin signalling is important for initiation, outgrowth, patterning and differentiation into vagina, cervix, uterus and oviducts. In adult life, sex hormones modulate Wnt/β-catenin signalling in the endometrium to maintain the monthly balance between estrogen-induced proliferation and progesterone-induced differentiation, and enhanced Wnt/β-catenin signalling seems to be involved in endometrial carcinogenesis. However, early in pregnancy enhanced Wnt/β-catenin signalling is prerequisite for proper implantation and invasion of trophoblast cells into endometrium and myometrium thus helping to form a placenta. Overall, it seems that tight control of Wnt/β-catenin signalling in time and space is important for initiation, development and normal function of the female reproductive tract. However, if Wnt/β-catenin signalling is not kept in check, it easily seems to initiate or contribute to development of a number of uterine disorders.     

Amphibian melanotrophs as a model to analyze the secretory plasticity of endocrine cells

Precise regulation of hormone secretion from endocrine cells is of critical importance for the maintenance of animal homeostasis. This is partly enabled through the ability of endocrine cells to adapt dynamically their secretory activity to the physiological demands through complex functional changes. Such a secretory plasticity results from coordinated adaptive changes at several levels of cell function, including hormonal gene expression and biosynthesis, hormone processing, trafficking, storage and release, expression of membrane receptors, activation of signaling pathways, etc. Integration of all these processes at the single cell level defines the secretory status of each of the individual cells producing a given hormone, whose coordinated activity ultimately determines the response of the whole endocrine gland. This short review summarizes our most recent findings on the cellular and molecular elements and mechanisms underlying the secretory plasticity of endocrine cells, obtained from the analysis of distinct aspects of melanotroph cell function.     

Urinary retention secondary to müllerian duct remnant in an infant

To date only 2 cases of urinary retention secondary to a müllerian duct cyst have been reported in infancy. We report a third case of bladder outlet obstruction caused by massive dilatation of persistent müllerian remnant in a 2 month old boy, the youngest to date. Surgical intervention was done because patient continued to have retention following conservative management. The cyst was large, located in the midline, and was associated with an absent left kidney.     

Molecular pathogenesis of granulosa cell tumors of the ovary

Granulosa cell tumors of the ovary (GCT) comprise a distinct subset of ovarian cancers that account for approximately 5% of all ovarian malignancies. They are thought to arise from normal proliferating granulosa cells of the late preovulatory follicle and exhibit many morphological and biochemical features of these cells. GCT are distinct from other ovarian carcinomas in their hormonal activity; their ability to secrete estrogen, inhibin, and Müllerian inhibiting substance accounts for some of the clinical manifestations of the disease and also provides useful tumor markers for disease surveillance. Although considered to be of low malignant potential, GCT are commonly associated with slow, indolent disease progression, and frequent yet long delays to tumor recurrence are characteristic of this disease. Unlike the more intensely investigated epithelial ovarian tumors, relatively little is known about the molecular and genetic changes that give rise to GCT. To date, many investigations have centered around pathways known to be involved in normal granulosa cell proliferation, including those activated by FSH receptor stimulation. Most recently, the finding that approximately 97% of adult GCT harbor a somatic missense mutation in the FOXL2 gene (c.402C→G; p.C134W) represents an exciting advancement in the field of GCT research. The high frequency with which the mutation occurs in adult GCT, along with its absence from juvenile GCT and other human malignancies is suggestive of an oncogenic or gain-of-function mutation and, indeed, that the mutation is pathognomonic for adult GCT. In this review, we explore the implications of this finding and the most recent work characterizing molecular pathways of potential pathogenetic significance in GCT.     

Genetic studies of the AMH/MIS signaling pathway for Müllerian duct regression

Anti-Müllerian hormone (AMH)/Müllerian-inhibiting substance (MIS) is a member of the transforming growth factor-beta (TGF-beta) superfamily. Like other TGF-beta family members, AMH is likely to signal through two transmembrane serine/threonine kinase receptors. Whereas the AMH type II receptor has been clearly defined, only recently has there been evidence about the identity of the AMH type I receptor for Müllerian duct regression in vivo. We generated a new cre mouse line expressing the recombinase in AMH target cells. This line was then used to conditionally inactivate the Bmpr1a gene in the Müllerian duct, resulting in males with a uterus. Thus, Bmpr1a plays an essential role in the process of Müllerian duct regression. To investigate the role of Bmpr1a in granulosa cells, we took advantage of transgenic mice overexpressing human AMH. Surprisingly, these transgenic females that were also conditionally mutant for Bmpr1a in the Müllerian duct had no uterus. These results suggest that when AMH is overexpressed, other TGF-beta family type I receptors can potentially transduce AMH signals.     

Effect of diethylstilbestrol on cell proliferation and expression of epidermal growth factor in the developing female rat reproductive tract

To evaluate mechanisms of cell proliferation in the fetal female rat reproductive tract, diethylstilbestrol (DES) effects on cell division and estrogen receptor (ER), epidermal growth factor (EGF) and EGF receptor (EGF-R) expressions were determined from gestational day (GD) 15.5 to 21.5. Reproductive tracts were evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. In fetuses from non-treated dams, epithelial and mesenchymal proliferation, as evaluated by 5-bromo-2'-deoxyuridine incorporation, was decreased with development in all regions of the Müllerian duct. EGF levels were determined by immunohistochemistry. Müllerian epithelial EGF immunoreactivity was intense in the proximal and middle regions on GDs 15.5 and 17.5. EGF staining remained intense only in the proximal epithelia by GD 19.5 and was weak in the caudal epithelium, but substantially reduced throughout epithelia in all regions by GD 21.5. Thus, decreased cell proliferation correlated with decreased EGF expression in the developing Müllerian duct. DES (100 microg/kg body weight) was injected from GD 15 to 19 and female fetuses were collected on GD 19.5. DES increased Müllerian duct cell proliferation in the proximal epithelium and mesenchyme but decreased it in the caudal epithelium compared with oil-treated controls. No proliferative DES effect was observed in any cell type in the middle region. Müllerian duct EGF immunoreactivity was suppressed by DES compared with oil. Competitive RT-PCR indicated DES also decreased mRNAs for EGF, ERbeta1 and ERbeta2, but not ERalpha and EGF-R. These results indicate EGF may be an important regulatory factor of Müllerian duct cell proliferation, and that DES may alter cell proliferation by disrupting normal EGF, ERbeta1 and ERbeta2 expression in the developing female rat reproductive tract.     

Genetic studies reveal the role of the endocrine and metabolic systems in aging

Aging is a natural process that involves a general decline in many physiological functions, resulting in loss of function and eventually death. Extensive research is being performed in order to elucidate the biology of aging, especially with the advent of newer molecular and genetic methodologies. The endocrine system plays a major role in orchestrating cellular interactions, metabolism, growth, and senescence. Thus, researchers traditionally used hormones as tools to induce and examine specific biological effects that are associated with aging. Furthermore, because our recent knowledge on hormonal action expanded significantly, downstream pathways and genetic determinants currently prevail in aging research. In this review, we will summarize the effects of several hormones on human aging and longevity and present recent data from the Longevity Genes Study performed at Albert Einstein College of Medicine, looking at the phenotype and genotype of centenarians and their offspring. We will demonstrate that genetic factors that are associated with human longevity are heritable and may contribute not only to quantitative longevity but also to protection from age-dependent disease and exceptional good health.     

Changes in ontogenetic expression of estrogen receptor alpha and not of estrogen receptor beta in the female rat reproductive tract

To evaluate ontogenetic expression and localization of estrogen receptor (ER) alpha and beta in fetal female rat reproductive tract, competitive RT-PCR and immunohistochemistry were performed. Expression levels for Müllerian ERalpha, ERbeta1 and ERbeta2 mRNAs were determined by competitive RT-PCR. ERalpha expression on gestational day (GD) 15 x 5 increased 4 x 4-fold by GD 21 x 5, whereas both ERbeta1 and ERbeta2 gene expression were maintained at lower constant levels compared with ERalpha during development. ER immunolocalization was evaluated within three regions along the Müllerian duct axis; these were proximal, middle and caudal, which differentiate into oviduct, uterus and upper vagina respectively. Nuclear ERalpha was localized predominantly in proximal Müllerian epithelium, and middle and caudal Müllerian mesenchyme on GDs 15 x 5-21 x 5. Staining intensity for ERalpha increased with development in all regions. However, ERbeta immunoreactivity was not detected in any region during prenatal life after separate staining with three different polyclonal anti-rat ERbeta antibodies. These findings provide fundamental information critical for clarifying the species-specific physiological roles of ER subtypes during fetal development and for investigating the tissue-specific mechanisms underlying the prenatal response to estrogen and estrogen receptor agonists.     

Proteolytically activated, recombinant anti-mullerian hormone inhibits androgen secretion, proliferation, and differentiation of spermatogonia in adult zebrafish testis organ cultures

Anti-Müllerian hormone (Amh) is in mammals known as a TGFβ type of glycoprotein processed to yield a bioactive C-terminal homodimer that directs regression of Müllerian ducts in the male fetus and regulates steroidogenesis and early stages of folliculogenesis. Here, we report on the zebrafish Amh homologue. Zebrafish, as all teleost fish, do not have Müllerian ducts. Antibodies raised against the N- and C-terminal part of Amh were used to study the processing of endogenous and recombinant Amh. The N-terminally directed antibody detected a 27-kDa protein, whereas the C-terminally directed one recognized a 32-kDa protein in testes extracts, both apparently not glycosylated. The C-terminal fragment was present as a monomeric protein, because reducing conditions did not change its apparent molecular mass. Recombinant zebrafish Amh was cleaved with plasmin to N- and C-terminal fragments that after deglycosylation were similar in size to endogenous Amh fragments. Mass spectrometry and N-terminal sequencing revealed a 21-residue N-terminal leader sequence and a plasmin cleavage site after Lys or Arg within Lys-Arg-His at position 263-265, which produce theoretical fragments in accordance with the experimental results. Experiments using adult zebrafish testes tissue cultures showed that plasmin-cleaved, but not uncleaved, Amh inhibited gonadotropin-stimulated androgen production. However, androgens did not modulate amh expression that was, on the other hand, down-regulated by Fsh. Moreover, plasmin-cleaved Amh inhibited androgen-stimulated proliferation as well as differentiation of type A spermatogonia. In conclusion, zebrafish Amh is processed to become bioactive and has independent functions in inhibiting both steroidogenesis and spermatogenesis.     

Estrogen target gene regulation and coactivator expression in rat uterus after developmental exposure to the ultraviolet filter 4-methylbenzylidene camphor

Because the estrogen receptor (ER) ligand type influences transactivation, it is important to obtain information on molecular actions of nonclassical ER agonists. UV filters from cosmetics represent new classes of endocrine active chemicals, including the preferential ER beta ligands 4-methylbenzylidene camphor (4-MBC) and 3-benzylidene camphor. We studied estrogen target gene expression in uterus of Long Evans rats after developmental exposure to 4-MBC (0.7, 7, 24, and 47 mg/kg x d) administered in feed to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. 4-MBC altered steady-state levels of mRNAs encoding for ER alpha, ER beta, progesterone receptor (PR), IGF-I, androgen receptor, determined by real-time RT-PCR in uterus of 12-wk-old offspring. Western-blot analyses of the same tissue homogenates indicated changes in ER alpha and PR but not ER beta proteins. To assess sensitivity to estradiol (E2), offspring were ovariectomized on d 70, injected with E2 (10 or 50 microg/kg sc) on d 84, and killed 6 h later. Acute up-regulation of PR and IGF-I and down-regulation of ER alpha and androgen receptor by E2 were dose-dependently reduced in 4-MBC-exposed rats. The reduced response to E2 was accompanied by reduced coactivator SRC-1 mRNA and protein levels. Our data indicate that developmental exposure to 4-MBC affects the regulation of estrogen target genes and the expression of nuclear receptor coregulators in uterus at mRNA and protein levels.     

Maternal micronutrient restriction programs the body adiposity, adipocyte function and lipid metabolism in offspring: A review

Fetal growth is a complex process which depends both on the genetic makeup and intrauterine environment. Maternal nutrition during pregnancy is an important determinant of fetal growth. Adequate nutrient supply is required during pregnancy and lactation for the support of fetal/infant growth and development. Macro- and micronutrients are both important to sustain pregnancy and for appropriate growth of the fetus. While macronutrients provide energy and proteins for fetal growth, micronutrients play a major role in the metabolism of macronutrients, structural and cellular metabolism of the fetus. Discrepancies in maternal diet at different stages of foetal growth / offspring development can have pronounced influences on the health and well-being of the offspring. Indeed intrauterine growth restriction induced by nutrient insult can irreversibly modulate the endocrine/metabolic status of the fetus that leads to the development of adiposity and insulin resistance in its later life. Understanding the role of micronutrients during the development of fetus will provide insights into the probable underlying / associated mechanisms in the metabolic pathways of endocrine related complications. Keeping in view the modernized lifestyle and food habits that lead to the development of adiposity and world burden of obesity, this review focuses mainly on the role of maternal micronutrients in the foetal origins of adiposity.     

Anorchia and persistent Müllerian duct: a variant of the embryonic testicular regression syndrome.

A 20-yr-old phenotypical male with a 46, XY chromosome complement, a hernia uteri inguinale, and bilateral anorchia was studied. Eunochoidal body proportions, infantile type of male external genitalia with empty scrotum, underdeveloped sexual characteristics, and delayed bone age suggested the existence of inadequate testicular function. Extremely low levels of circulating testosterone and a lack of response to hCG stimulation was found. Persistently elevated blood levels of LH and FSH with an adequate pituitary response to an iv bolus of synthetic LRH was demonstrated, thus indicating inadequate endocrine gonadal function as well as functional integrity of the hypothalamic-pituitary unit. At the time of an inguinal hernioplasty, a small but well developed uterus was removed. No gonads were found within the true pelvis, inguinal canals, or along the anatomical pathways of testicular descent. A cord-like structure found in the left inguinal canal contained only fibrous tissue without gonadal elements. It is proposed that the occurrence of two altered events during embryogenesis, failure of Müllerian duct regression and late testicular regression, may explain the underlying defect in this unusual abnormality of sexual differentiation.     

Endocrine disruptor vinclozolin induced epigenetic transgenerational adult-onset disease

The fetal basis of adult disease is poorly understood on a molecular level and cannot be solely attributed to genetic mutations or a single etiology. Embryonic exposure to environmental compounds has been shown to promote various disease states or lesions in the first generation (F1). The current study used the endocrine disruptor vinclozolin (antiandrogenic compound) in a transient embryonic exposure at the time of gonadal sex determination in rats. Adult animals from the F1 generation and all subsequent generations examined (F1-F4) developed a number of disease states or tissue abnormalities including prostate disease, kidney disease, immune system abnormalities, testis abnormalities, and tumor development (e.g. breast). In addition, a number of blood abnormalities developed including hypercholesterolemia. The incidence or prevalence of these transgenerational disease states was high and consistent across all generations (F1-F4) and, based on data from a previous study, appears to be due in part to epigenetic alterations in the male germ line. The observations demonstrate that an environmental compound, endocrine disruptor, can induce transgenerational disease states or abnormalities, and this suggests a potential epigenetic etiology and molecular basis of adult onset disease.     

GUT ENDOCRINOLOGY: CORRELATION OF PHYSIOLOGY AND MORPHOLOGY

The gastrointestinal tract is a large and complex endocrine organ. Elucidation of its nature, and the actions of its individual hormonal components, has advanced in recent years following the purification and sequencing of six probable hormones and the subsequent development of radioimmunoassay techniques for their measurement and immunocytochemistry for the determination of their cellular origin. Immunoassay and immunocytochemistry are clearly complimentary to each other in providing a fuller understanding of the dynamics of endocrine function at the cellular level. This is illustrated in the examples described in this paper.     

Steroidogenic factor-1 (SF-1, NR5A1) and human disease

Steroidogenic factor-1 (SF-1, Ad4BP, encoded by NR5A1) is a key regulator of adrenal and reproductive development and function. Based upon the features found in Nr5a1 null mice, initial attempts to identify SF-1 changes in humans focused on those rare individuals with primary adrenal failure, a 46,XY karyotype, complete gonadal dysgenesis and Müllerian structures. Although alterations affecting DNA-binding of SF-1 were found in two such cases, disruption of SF-1 is not commonly found in patients with adrenal failure. In contrast, it is emerging that variations in SF-1 can be found in association with a range of human reproductive phenotypes such as 46,XY disorders of sex development (DSD), hypospadias, anorchia, male factor infertility, or primary ovarian insufficiency in women. Overexpression or overactivity of SF-1 is also reported in some adrenal tumors or endometriosis. Therefore, the clinical spectrum of phenotypes associated with variations in SF-1 is expanding and the importance of this nuclear receptor in human endocrine disease is now firmly established.